CN101203231A

CN101203231A - Methods, compositions and articles of manufacture for enhancing survivability of cells, tissues, organs, and organisms

Info

Publication number: CN101203231A
Application number: CNA2006800221968A
Authority: CN
Inventors: M·B·洛斯; M·莫里森; E·布莱克斯通; D·米勒
Original assignee: Fred Hutchinson Cancer Research Center
Current assignee: Fred Hutchinson Cancer Center
Priority date: 2005-04-20
Filing date: 2006-04-20
Publication date: 2008-06-18

Abstract

The present invention concerns the use of oxygen antagonists and other active compounds for inducing stasis or pre-stasis in cells, tissues, and/or organs in vivo or in an organism overall, in addition to enhancing their survivability. It includes compositions, methods, articles of manufacture and apparatuses for enhancing survivability and for achieving stasis or pre-stasis in any of these biological materials, so as to preserve and/or protect them. In specific embodiments, there are also therapeutic methods and apparatuses for organ transplantation, hyperthermia, wound healing, hemorrhagic shock, cardioplegia for bypass surgery, neurodegeneration, hypothermia, and cancer using the active compounds described.

Description

Strengthen method, compositions and the manufacture of the viability of cell, tissue, organ and biology

Background of invention

The application relates to the U.S. Provisional Patent Application of submitting on April 20th, 2,005 60/673,037 and 60/673,295, and the U.S. Provisional Patent Application 60/713 of submission on August 31st, 2005,073, the U.S. Provisional Patent Application of submitting on October 28th, 2,005 60/731, the U.S. Provisional Patent Application 60/762,462 that on January 26th, 549 and 2006 submitted to is incorporated them into this paper in full by reference.

According to (government may have the right among the present invention for National Institute of GeneralMedical Sciences, NIGMS) fund assistance GM048435 from state-run general medical science research institute.

1. invention field

Present invention relates in general to cytobiology and physiology field.More particularly say, the present invention relates to be used to strengthen cell, tissue, organ and biological viability (survivability) and/or reduce method, compositions and instrument their damage, particularly under unfavorable conditions, (include but not limited to anoxia or anaerobic state), use one or more materials (comprising material) with the oxygen competition.In certain embodiments; the present invention includes by oxygen antagonist, the protectiveness metabolism agent that the experimenter is exposed to realize its described target or other chemical compound of discussing at this paper; or its precursor (being referred to as " reactive compound "), treat, prevent and diagnose the illness and method, compositions and the instrument of disease.

2. description of related art

Stasis is a Latin term, and the meaning is meant " stagnation ".In the background of stagnating in living tissue, the most common form of stagnation relates to the preservation that is used to the tissue transplanting or put again.Typically, this class tissue is immersed in the physiological fluid (for example saline), and places the biochemical process that causes cell injury under the cold environment with minimizing.This stagnation is incomplete and can not relies on for a long time.In fact, the success of putting again of organ transplantation and limbs and organ or limbs break away from the time negative correlation of complete biology.

A kind of more extreme form of stagnating relates to makes whole biology be in popular alleged " stagnate and the give birth to " state of.Although still mainly think in the field of science fiction, when the rich seek after death by cold preservation, following medical science of expectation breaks through in the time of will allowing their resurrection and cure their fatal disease, has produced disrepute.It is said, since 1967 attempt for the first time, had over one hundred people, and people more than 1,000 has carried out cryonics's law and financial arrangement with one of several tissues (for example Alcor LifeExtension Foundation) by cold preservation.These class methods relate to uses anti--ischemia medicine, cryopreservation and with the method for the cold outstanding whole biology of (cryosuspension) fluid perfusion.The biology stagnation that does not also confirm this form is reversible.

As feature be in the related effectiveness of in biological substance, inducing stagnation of compositions described herein, method or manufacture; induce in whole or in part or begin and stagnate; keep stagnating one period subsequently; return back to normal then or near normal physiological status, or those skilled in the art think to be better than the state of state of the state of the biological substance of never stagnating.Also stagnation can be defined as non-above-mentioned implication (what it is Not).The biological stagnation is not any in the following state: sleep, stupor, dead, anaesthetized or epilepsy grand mal (grand malseizure).

Exist many after being exposed to cryogenic conditions (normally being in the cold water submergence), the report of the individuality that stops to survive from obvious P﹠R.Although scientist understands fully, the ability of surviving from this situation might be derived from so-called " mammal diving reflex ".This reflection is considered to the vagus nerve stimulation system, and vagal system control lung, heart, larynx and esophagus are so that the protection vital organ.By inference, the neuroreceptor on the cold water stimulating skin causes blood shunt to brain and be diverted to heart, and leaves skin, gastrointestinal tract and extremity.Simultaneously, the guarding reflex bradycardia, or heartbeat slows down, and saved the oxygen supply that reduces in the body.Unfortunately, the performance of this reflection all is not identical in everyone, and is considered to the only factor in the 10-20% of cold water submergence case.

The compositions and the method that not exclusively rely on or do not rely on low temperature and/or oxygen may be applicable to Organ Preservation, and tissue or cell preservation.At present with cryopreservation cell and tissue, normally be in and be lower than freezing temperature basically, for example in liquid nitrogen.Yet temperature dependent may cause problem, may be not easy when needed to obtain because be used to produce this cryogenic instrument and reagent, and perhaps they may need to replace.For example, tissue culture cells is preserved a period of time usually in filling the jar of liquid nitrogen; Yet, the liquid nitrogen periodic replacement in the common claimed apparatus of these jars, otherwise liquid nitrogen can exhaust and can not keep temperature.And, owing to freeze/melt process, the damage of pair cell and tissue can take place.Therefore, the technology that needs improvement.

And, control be subjected to wound for example the shortage of the cell in amputation and the cryogenic whole biology and the metabolic ability of physiological be a critical defect in the medical domain.On the other hand, anecdotal evidence discussed above is pointed out consumingly, if correct understanding and control might be induced stagnation in cell, tissue and whole biology.Therefore, the modification method that is used for particularly control metabolic process under traumatic condition is existed huge needs.

Brief summary of the invention

Therefore, the invention provides and be arranged in the cell of biology, tissue and organ or the cell that obtains from biology, tissue and organ, and at biological method, compositions, manufacture and instrument of inducing stagnation in itself.These methods, compositions, manufacture and instrument can be used to protect biological substance, and the disease and the disease that are used for preventing, treating or diagnose biology.And, these methods itself can directly be induced stagnation, and perhaps they can not induce stagnation by itself, but by strengthening the ability that biological substance response damage or disease condition enter stagnation, for example obtain to stagnate the required damage or the time or the level of disease, work indirectly by reducing.This situation can be described as pre-stagnation (pre-stasis).The details of this application and other purposes is described below.

The present invention part has defencive function according to surveying and determination based on using, and thereby as the research of protectant chemical compound.In addition, the total result that relates to the research of different chemical compounds shows that the chemical compound with available electron donor center is effective especially in inducing stagnation or pre-the stagnation.And these chemical compounds are induced reversible stagnation, mean that they are not so toxic and cause this material dead or decompose to special biological substance.Further consider that the present invention can be used for strengthening and accept possibly or be in the viability of the biological substance under the unfavorable conditions and/or prevention or reduce damage it.

In special embodiment; method of the present invention is used for after damage (for example traumatic damage) back or seizure of disease or progress; at biological substance; for example induce in cell, tissue, organ and/or the biology and stagnate or pre-the stagnation, so as the protection biological substance before treatment damage or disease, during or avoid injury with damage or disease association afterwards.In other embodiments; method of the present invention is used for before accepting preceding or seizure of disease of injury event (for example elective surgery) or progress inducing or promoting to stagnate or pre-the stagnation at biological substance, so as to protect described biological substance avoid and unfavorable conditions as damaging or the injury of disease association.These class methods are commonly referred to reactive compound " pretreatment ".Pretreatment comprises wherein and (for example is subjected to unfavorable conditions at biological substance, the damage or the outbreak or the progress of disease) preceding and during, and before, during and offer the method for this biological substance reactive compound and the method that wherein only before biological substance is subjected to unfavorable conditions, offers this biological substance reactive compound afterwards.

The different embodiments of the method according to this invention, stagnation can be handled biological substance by the reactive compound of directly inducing stagnation with self and induce, or alternately, by not inducing stagnation with itself, but, promote or strengthen the other stimulation of biological substance response, for example, but be not limited to damage, disease, anoxia, excessive hemorrhage or handle and reach the reactive compound processing biological substance of stagnating required ability or reducing the required like this time and induce with other reactive compound.

In special embodiment, induce " the pre-stagnation " with the processing of reactive compound, pre-stagnation refers to that biological substance must transition and reach the hypometabolic state of stagnation.The pre-feature of stagnating is that the amplitude that reduces of the metabolism in the biomaterial is less than the amplitude that is defined as stagnation.In order to stagnate with reactive compound, biological substance is necessary must be by hypometabolic status transition step by step, oxygen consumption and CO in the biological substance in hypometabolic state ₂The minimizing that produces is lower than twice.A kind of like this continuous process (wherein metabolism or Cellular respiration are reduced to the degree that is lower than twice by reactive compound) can be described as " pre--as to stagnate " state.

Comprise CO in stagnation ₂Produce or O ₂When consume reducing twice (promptly be reduced to 50% or still less), stagnate in advance with these parameters (wherein detect and be lower than 2 times minimizing) expression that method known to those skilled in the art is directly measured in the biological substance.Therefore, some measurement of the labelling of other metabolic rate that carbon dioxide in the blood and oxygen level and those skilled in the art are familiar with includes but not limited to blood pO ₂, VO ₂, pCO ₂, pH and lactate level, can be used for pre-beginning or the progress of stagnating of monitoring among the present invention.When the active index of metabolism, for example via the CO of Cellular respiration ₂Produce and O ₂Consume, when comparing minimizing with normal condition less than twice, pre-stagnation may with at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% CO ₂Emit to reduce and be correlated with described CO ₂Emit and refer to CO ₂Amount from lung release.And, in a plurality of embodiments, the pre-feature of stagnating is the minimizing of one or more indexs of metabolic activity, and described minimizing is compared with the normal physiological state and is less than or equals 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 49%.In other embodiments; the pre-stagnation is characterised in that it responds the ability that another stimulates (wherein this another stimulation can comprise the extended treatment with identical activating agent) and enhancing or enters stagnation; or it strengthens the viability of biological substance or protection biological substance and avoids outbreak or progress by damage, disease; or hemorrhage, particularly can cause irreversible tissue injury, hemorrhagic shock or the lethal hemorrhage and ability of the damage that causes.

Though may relate in this inventive method that offers some clarification on by way of example and to induce " stagnation ", should be appreciated that these methods can be easy to be applicable to induces " the pre-stagnation ", and this to induce the method for pre-stagnation be that the present invention considers.And, be used to induce the identical reactive compound of stagnation also can be used to induce pre-stagnation, this can be by being used to induce the time of stagnation to realize to be lower than to induce to stagnate that employed dosage offers biological substance and/or the time that they offer biological substance is shorter than them.

In certain embodiments, the present invention relates to biological substance is exposed to a certain amount of reagent, so that realize the stagnation of biological substance.In some embodiments, the present invention relates to be used for the method that biological substance is in vivo induced stagnation, this method comprises: identify that a) wherein stagnating is the biology of expectation; And, b) described biology is exposed to the oxygen antagonist of effective dose or other reactive compound and is used in vivo biological substance and induces stagnation.Induce " stagnation " to represent that this material is alive in biological substance, but it has following one or more feature: the speed or the amount of the carbon dioxide that is produced by biological substance are reduced by at least twice; Be reduced by at least twice (promptly 50%) by the speed of the oxygen of biological substance consumption or amount; And motion or energy reduce at least 10% (cell or tissue that is only applicable to move, for example spermatid or heart or extremity, or when inducing stagnation in whole biology) (being referred to as " Cellular respiration index ").In certain embodiments of the invention, the speed of consideration biological substance oxygen consumption is had an appointment, at least about or about at most 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 60-, 70-, 80-, 90-, 100-, 150-, 200-, 250-, 300-, 350-, 400-, 450-, 500-, 600-, 700-, 800-, 900-, 1000-, 1100-, 1200-, 1300-, 1400-, 1500-, 1600-, 1700-, 1800-, 1900-, 2000-, 2100-, 2200-, 2300-, 2400-, 2500-, 2600-, 2700-, 2800-, 2900-, 3000-, 3100-, 3200-, 3300,3400-, 3500-, 3600-, 3700-, 3800-, 3900-, 4000-, 4100-, 4200-, 4300-, 4400-, 4500-, 5000-, 6000-, 7000-, 8000-, 9000-or 10000-doubly or more reduce, and maybe can come from any scope wherein.Alternately, consider embodiment of the present invention can with approximately, at least about or about at the most 50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99% or the reduction of more biological substance oxygen consumption rate discuss.Consideration can be adopted any algoscopy that is used to measure oxygen consumption, and typical algoscopy will be referred to utilize the environment of sealing and oxygen that environment is put in measurement and poor between the remaining oxygen of environment after a period of time.Further consider and to measure the amount that carbon dioxide generating is measured the biological substance oxygen consumption.Therefore, may have the reduction of carbon dioxide generating, this is corresponding to the reduction of oxygen consumption discussed above.

In the method for the invention, stagnate or pre-the stagnation is temporary transient and/or reversible, this expression biological substance is in after a while the feature that no longer embodies stagnation sometime.In some embodiments of the present invention, use chemical compound rather than the oxygen antagonist that is not suitable as the oxygen antagonist.The method that consideration is discussed about the oxygen antagonist goes for as any chemical compound of oxygen antagonist, protectiveness metabolism agent, has the chemical compound of formula I, II, III or IV structure, at any other chemical compound of this discussion, or its salt or precursor.Realize that any method of the present invention and suitable lattice will think " reactive compound " as the chemical compound of oxygen antagonist, protectiveness metabolism agent, the chemical compound with formula I, II, III or IV structure or its salt or precursor.In special embodiment, inducing of stagnation expects that in this case, chemical compound can be called " the active chemical compound of stagnating ".Consider that in some embodiments of the present invention method realizes by inducing to stagnate.For example, Therapeutic Method may relate to induces stagnation, and reactive compound is the active chemical compound of stagnating in this case.Special consideration is discussed in the embodiment of reactive compound therein, the present invention includes, and may be limited to the oxygen antagonist.

In certain embodiments of the invention, biological substance is handled with a kind of reactive compound, this reactive compound itself is not induced stagnation (not inducing stagnation on the level that provides and/or on the time durations at least), has the pre-dead state for the treatment of benefit but induce biological substance to enter, and strengthen biological substance and respond other stimulation, if for example damage, morbid state are or with other reactive compound or longer time or reach the ability of stagnation with the processing of the identical reactive compound of more heavy dose of usefulness.

Term " biological substance " refers to the biomaterial (being mammiferous biomaterial in preferred embodiments) of any work, comprises cell, tissue, organ and/or biology, and their combination in any.Consider to stagnate and in the part (for example cell, tissue and/or one or more organ) of biology, to induce,, perhaps allow whole biology stay cool no matter that part of remaining in the described biology or from this biology shifted out.And, consider that at cell and organizational aspects homology and allos cell colony can be the themes of embodiment of the present invention.Term " body in biological substance " refers in vivo, promptly still is positioned at biological or is connected biological substance on the biology.And term " biological substance " will be interpreted as the synonym of term " biomaterial ".In certain embodiments, consider one or more cells, tissue or organ and bio-separation.Term " isolating " can be used for describing this biological substance.Consideration can be induced stagnation in isolating biological substance.

Biological or other biological substance that needs to stagnate is biology or the biological substance that wherein biological all or part of stagnation can produce direct or indirect physiology's benefit.For example, having the patient of hemorrhagic shock risk to think needs to stagnate, and the patient that perhaps will carry out coronary bypass can benefit from the protection heart and avoid ischemia/reperfusion injury.Other is applied among whole the application and discusses.In some cases, can be by stagnating the condition of illness or the disease of preventing or treating based on showing, or one or more detections, screening or the assessment of the risk of condition of illness or disease, identify or determine biological or other biological substance needs to stagnate.Alternately, consider that patient medical history or family's medical history (inquiry patient) can obtain the information that biology or other biological substance need be stagnated.It should be apparent to those skilled in the art that an application of the present invention will be to stagnate the overall energy requirement that reduces biomaterial by inducing.

Alternately, biological or other biological substance will need reactive compound to strengthen viability.For example, the patient may need to damage or treatment of diseases or in any other application of this discussion.Based on the method for discussing in the paragraph in front, for example by considering patient's medical history or family's medical history, they may be confirmed as needing to strengthen viability or treatment.

Term " oxygen antagonist " refers to be required the material that biological substance (" oxygen-usability biological substance ") that oxygen lives utilizes this respect and oxygen competition at oxygen.Oxygen is that the various kinds of cell process that produces the main source of the energy that biological substance can utilize is easily used usually or needed.The oxygen antagonist reduces or eliminates the amount of the oxygen that can be utilized by oxygen-usability biological substance effectively, and/or the amount of the oxygen that can be used by oxygen-usability biological substance.In one embodiment, the oxygen antagonist can directly be realized its oxygen antagonism.In another embodiment, the oxygen antagonist can be realized its oxygen antagonism indirectly.

Directly the competition of oxygen antagonist and molecular oxygen is bonded to the molecule (for example, protein) with oxygen binding site or oxygen binding ability.Known to pharmacology or biochemical field, antagonism can be emulative, noncompetitive or uncompetitive.Directly the example of oxygen antagonist includes but not limited to, carbon monoxide (CO), and itself and oxygen competition are bonded to hemoglobin and are bonded to cytochrome c oxidase.

Lacking when oxygen is bonded to the direct competitive of oxygen associativity molecule, indirect oxygen antagonist influences the utilizability of oxygen or is delivered to and uses oxygen to come the cell of produce power (for example in Cellular respiration).The example of indirect oxygen antagonist includes but not limited to, (i) carbon dioxide, it is by being called the process of Bo Er (Bohr) effect, reduce hemoglobin (or other globulin, as Myoglobin) be bonded to the ability of blood or the oxygen in the hemolymph of oxygen-usability animal, thereby reduced the amount of the oxygen that is delivered to biological oxygen-usability cell, tissue and organ, thereby reduced the utilizability of oxygen to the cell that utilizes oxygen; (ii) carbonic anhydrase inhibitors (Supuran etc., 2003, it is incorporated herein by reference by full text), it is by suppressing the hydration of carbon dioxide in lung or other respiratory apparatus, increased concentration of carbon dioxide, thereby reduced hemoglobin (or other globulin, as Myoglobin) be bonded to the ability of blood or the oxygen in the hemolymph of oxygen-usability animal, thereby reduced the amount of the oxygen that is delivered to biological oxygen-usability cell, tissue and organ, thereby reduced the utilizability of oxygen to the cell that utilizes oxygen; And, include but not limited to oxygen chelating agen, antibody etc. (iii) in conjunction with oxygen and shield oxygen and combine or make oxygen to be not useable for and the bonded molecule of oxygen binding molecule with oxygen associativity molecule.

In some embodiments, the oxygen antagonist be direct oxygen antagonist be again indirect oxygen antagonist.Example includes but not limited to, direct competitive oxygen be bonded to cytochrome C oxidase and also can in conjunction with and suppress chemical compound, medicine or the reagent of the enzymatic activity of carbonic anhydrase.Therefore, in some embodiments, the oxygen antagonist suppresses or reduces the amount of the Cellular respiration that takes place in the cell, for example by in conjunction with the site on the cytochrome C oxidase, and this site otherwise will be in conjunction with oxygen.The cytochrome C oxidase specificity is in conjunction with oxygen and be translated into water then.In some embodiments, combine preferably releasable and reversible combination with cytochrome C oxidase this and (for example have at least 10 ^-2, 10 ^-3Or 10 ^-4The external dissociation constant K of M _d, and have and be not more than 10 ^-6, 10 ^-7, 10 ^-8, 10 ^-9, 10 ^-10Or 10 ^-11The external dissociation constant K of M _d).In some embodiments, estimate the oxygen antagonist by measuring ATP and/or carbon dioxide output.

Term " effective dose " expression can obtain described result's amount.In some method of the present invention, " effective dose " is, for example, induces the amount of stagnation in the biological substance that needs are stagnated.In other method, " effective dose " is, for example, induces the amount of pre-stagnation in the biological substance of needs stagnation or the enhanced viability of needs.In other embodiments, " effective dose " can refer to increase the amount of the viability of biology or other biological substance.This can be based on the biological substance of handling with undressed biological substance or with the different various dose that does not cause viability or scheme relatively or relatively come to determine (or supposition) in advance.

Should be appreciated that when inducing stagnation in tissue or organ, effective dose is the amount of determining by the set amount of the Cellular respiration of tissue or organ, induce stagnation in tissue or organ.Therefore, for example, if certain oxygen antagonist that is exposed to specified quantitative or other are active stagnate chemical compound after, the level of heart oxygen consumption (set of the cell of heart) reduces at least about 2 times (promptly 50%), should be appreciated that this specified quantitative is the effective dose of inducing stagnation in heart.Similarly, in biology, induce the effective dose of the reagent of stagnation to be, the amount of with regard to common or total level of a kind of special parameter of stagnating, assessing.Should be appreciated that also when inducing when stagnating, effective dose is the amount of inducing the stagnation of whole biology usually in biology, unless the special part of target biology only.And should be appreciated that effective dose can be the amount that itself is enough to induce stagnation, perhaps it can be and other reagent or stimulation, for example another kind of reactive compound, damage or morbid state combination and be enough to induce the amount of stagnation.

The notion of the specific compound of effective dose, in some embodiments, including how many available oxygen can be utilized by biological substance.Usually, exist approximately 100 in the presence of any oxygen antagonist when lacking, 000ppm or still less during oxygen (it is about 210 that room air has, 000ppm oxygen) can induce stagnation.How much oxygen the oxygen antagonist is used for changing can effectively be utilized.When the oxygen concentration of 10ppm, induced stagnant life.Therefore, because the oxygen antagonist combines the proteinic competitive effect of oxygen metabolism essential in the biological substance with oxygen,, can induce stagnation although the actual oxygen concentration that biological substance exposes may be higher than even be much higher than 10ppm.In other words, the oxygen antagonist of effective dose reduces the point that available oxygen concentration can not be utilized to the oxygen that exists.Be lower than oxygen and be bonded to the proteinic K of essential oxygen metabolism when the amount of oxygen antagonist reduces available oxygen concentration _mThe time (oxygen that is equivalent to 10ppm), just will this thing happens.Therefore, in some embodiments, the valid density that the oxygen antagonist reduces oxygen is approximately or at least about 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 60-, 70-, 80-, 90-, 100-, 150-, 200-, 250-, 300-, 350-, 400-, 450-, 500-, 600-, 700-, 800-, 900-, 1000-, 1100-, 1200-, 1300-, 1400-, 1500-, 1600-, 1700-, 1800-, 1900-, 2000-, 2100-, 2200-, 2300-, 2400-, 2500-, 2600-, 2700-, 2800-, 2900-, 3000-, 3100-, 3200-, 3300,3400-, 3500-, 3600-, 3700-, 3800-, 3900-, 4000-, 4100-, 4200-, 4300-, 4400-, 4500-, 5000-, 6000-, 7000-, 8000-, 9000-or 10000-times or more, can come from any scope wherein.Alternately, consideration embodiment of the present invention can be discussed with regard to the minimizing of available oxygen concentration, described available oxygen concentration reduce be approximately, at least about or about at the most 50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99% or more, maybe can come from any scope wherein.Should be appreciated that this is the another kind of mode that the expression Cellular respiration reduces.

And in some embodiments, the reduction of the core temperature that stagnation can be by biology is measured indirectly.Consider can to observe in the method for the invention core temperature reduce approximately, at least about or about at the most 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50_ or more, maybe can come from any scope wherein.In some embodiments of the present invention, can induce hypothermia, for example moderate hypothermia (10_ reduces at least) or severe hypothermia (20_ reduces at least).

And, effective dose can be expressed as to open-assembly time length carry out qualification or do not carry out concentration under the qualification.In some embodiments, usually consider, in order to induce the target of stagnating or reaching other statement of the present invention, biological substance is exposed to oxygen antagonist or other reactive compound pact, at least about or about at the most 5,10,15,20,25,30,35,40,45,50,55,60 seconds, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60 minutes, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 hours, 1,2,3,4,5,6,7 days, 1,2,3,4,5 weeks, 1,2,3,4,5,6,7,8,9,10,11, December, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 years or more for many years, and can come from wherein combination in any or scope.Further consider that the amount of time can be uncertain, it depends on reason or the purpose of using oxygen antagonist or other reactive compound.After this, biological substance can continue to be exposed to oxygen antagonist or other reactive compound, and perhaps, in other embodiments of the present invention, biological substance can no longer be exposed to oxygen antagonist or other reactive compound.Remove or effectively remove deoxidation antagonist or other reactive compound this step of back can exist by the biological substance of stagnating from expectation therein and finish, perhaps biological substance can be shifted out biological substance from the environment that contains aerobic antagonist or other reactive compound and finish.And, (expose unremitting a period of time) continuously, intermittently (expose) or periodically (expose in a plurality of periods) and expose or any reactive compound is provided for biological substance by rule in a plurality of periods.Based on this difference, the dosage of reactive compound can be identical or they can change.In certain embodiments, by per 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60 minutes, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 hours, 1,2,3,4,5,6,7 days, 1,2,3,4,5 weeks, 1,2,3,4,5,6,7,8,9,10,11, December, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or maybe can come from wherein any scope more for many years, offer biological substance or expose biological substance with

reactive compound

1,2,3,4,5,6,7,8,9 or 10 times, periodically provide reactive compound.

And, in some embodiments of the present invention, biological substance is exposed to or to a period of time that biological substance provides reactive compound to continue, wherein " continue " to represent a period of time at least about 2 hours.In other embodiments, biological substance can be exposed in the mode that continues or provide reactive compound to surpass one day to biological substance.In this case, provide reactive compound in the mode that continues continuously to biological substance.Sustainably in certain embodiments,, intermittently (expose) in a plurality of periods or periodically (mode exposes to repeat clocklike) biological substance is exposed to or provides reactive compound about 2 to biological substance, 3,4,5,6,7,8,9,10,11,12 or more hours (maybe can come from any scope wherein), 2,3,4,5,6,7 days and/or 1,2,3,4,5 weeks and/or 1,2,3,4,5,6,7,8,9,10,11, December and/or 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or (maybe can come from any scope wherein) more for many years.

In some embodiments, can before special damage, wound or treatment (for example surgical operation), unfavorable conditions or other dependent event or the situation and during; Before, and afterwards; During this time and afterwards; Or only after it, biological substance is exposed to or provides reactive compound to it.This exposure can or can not continue.

The dosage of the reactive compound of using in these different modes can be identical or they can change.

And, in certain embodiments, can provide reactive compound in the mode that continues continuously with the level of thinking " low ", the level of " low " is represented less than causing for example visible CBT of metabolism flexibility, heart rate or CO ₂Or O ₂Consume or produce the level of the amount that descends.

In certain embodiments, unfavorable physiology branch for example asphyxia (" in its process, breathe obviously reduce so that the experimenter carry out 10% or time period of the breathing of number of times still less "), lack observable skeletal muscle movement, dystonia and/or superfunction before, biological substance is exposed to or provides and surpass the reactive compound that before had been interpreted as the amount of maximum tolerated dose, for example metabolism agent to it.Such amount possibility is special and increase viability in some embodiments of the present invention, and for example, it is relevant to be used to be increased in the chance of surviving under the unfavorable conditions (for example will induce those dead conditions from hemorrhagic shock).

Reactive compound of the present invention can be induced a kind of physiological status, strengthen the viability that needs in the enhanced biology of viability, and the physiological change that comprises the reactive compound of one group of observable response effective dose, described variation can comprise a kind of, multiple or whole in the Autonomous Control of hyperpnea, asphyxia and forfeiture neuromuscular tonicity that follow or subsequently or motion and the lasting heartbeat.Also may observe temporary transient and measurable arterial blood change color.Hyperpnea refers to shallow breathing fast.Asphyxia refers to respiratory arrest or reduces as described above.

In certain embodiments, the experimenter becomes asphyxia, and this shows as respiratory arrest and the asphyxiating after the short time (apnic) breathing subsequently.In rat, this took place after about 20 seconds.Therefore consider, after being exposed to reactive compound, inducing into apneic experimenter and can show 0,1,2,3,4,5,6,7,8,9,10% frequency of respiration.Thereafter, the experimenter can have breathing once in a while, and this can think the asphyxiating breathing.In certain embodiments of the invention, asphyxia continues no longer to be exposed to reactive compound until the experimenter.

In some embodiments of the present invention, effective dose can be expressed as LD ₅₀, LD ₅₀Refer to " half lethal dose " dosage that kills half animal population (causing 50% mortality rate) that its expression is used.And in other embodiments, effective dose can not rely on the weight (" weight is ind ") of biological substance.For example, in rodent and people, before unfavorable physiological effect occurs, H ₂The LD of S gas ₅₀Be about 700ppm.And in some embodiments of the present invention, it is longer that the viability of increase is often referred to survival, and this is one embodiment of the invention.

The present invention also relates to be used for induce apneic method at biology, this method comprises the reactive compound that is administered to biologic effective dose.In certain embodiments, the biological any skeletal muscle movement that causes by described reactive compound that also do not show.Special consideration, biology can be a mammal, comprises the people.In other embodiments, effective dose surpasses the amount that is considered to lethasl concentration.In further embodiment, concentration can be lethal dose, however the time that exposes can be approximately, at least about or about at the most 10,15,20,25,30,35,40,45,50,55,60 seconds, 1,2,3,4,5 minute or more of a specified duration (maybe can come from wherein any scope or specified in this disclosure other the time to the time limit).In special embodiment, mammal is exposed to the active gases chemical compound at least about 600ppm, for example H ₂S.

And, in certain embodiments, there is the step of identifying the animal that needs processing.In other embodiments, there is the apneic step of observing in the biology.In embodiment further, method relates to from biology to be obtained blood sample and/or assesses the color of this biological blood.Observe, be exposed to H ₂S has changed the color from mammiferous blood; Color becomes darker red wine color from cerise, and becomes brick-red then.The assessment color can be finished and not need instrument or machine by range estimation, and in other embodiments, can use instrument, for example spectrophotometer.And, can obtain blood sample from biology, and can carry out the analysis of other type thereon.Alternately, may not need blood sample, come evaluating blood but can need not sample.For example, can adopt the pulse oximeter of the improvement of irradiation IR or visible light transmissive finger to come the change color of monitoring of blood.

What in certain embodiments, biological substance is exposed to effective dose can not cause the reactive compound stagnating or stagnate in advance.In some embodiments, when having reactive compound, may there be the sign of the minimizing of oxygen consumption or carbon dioxide generating.

In other embodiments, biology can be exposed to reactive compound when sleeping.And as discussed above, exposure can be clocklike, for example every day (expression exposes at least once every day).

Special consideration offers the experimenter by aerosol apparatus with reactive compound in some embodiments.This can use with any embodiment of the present invention.In some case, aerosol apparatus is used to handle hemorrhagic shock.In further embodiment, reactive compound is offered the experimenter as single dose.In specific case, single dose or multidose are to induce apneic dosage in the experimenter.In some embodiments, give the experimenter at least about 1,000,2,000,3,000,4,000,5,000,6,000,7,000,8,000,9,000,10,000,11,000,12,000 or the H of more ppm ₂S gas.Open-assembly time can be any time in this discussion, comprises approximately or 10,9,8,7,6,5,4,3,2,1,0.5,0.1 minutes or shorter (maybe can come from any scope wherein) approximately at the most.

In further embodiment, may change in the metabolic rate that is exposed to reactive compound artifact material.In certain embodiments, be exposed to the RQ ratio (CO of reactive compound artifact material ₂Generation/O ₂Consume) change.This can or take place after acute exposure after exposing for the first time or exposing repeatedly.In some embodiments, exposing back RQ ratio reduces.Described reduction can be approximately, at least about or about at the most 5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80% or more, maybe can come from the reduction of this any scope.Described reduction can be O ₂Consume to increase, with respect to O ₂Consume CO ₂The result of the minimizing that produces.

In some embodiments, in being exposed to the biological substance of reactive compound, do not observe physiological change, except its RQ ratio changes after exposure.Therefore, in some embodiments of the present invention, method relates to the variation of measuring RQ ratio among the experimenter.This can take place before being exposed to reactive compound and/or afterwards.

Therefore, in some embodiments of the present invention, induced stagnation, and the further step in the inventive method is that the biological substance that keeps relevant stays cool.This can finish by this biological substance being continued to be exposed to oxygen antagonist or other reactive compound and/or this biological substance being exposed to non-physiological temp or other oxygen antagonist or other reactive compound.Alternately, biological substance can be placed antiseptic or solution, or be exposed to the normal or hypoxic condition of oxygen that contains.Consider, biological substance can be remained in dead state approximately, at least about or about at the most 30 seconds, 1,2,3,4,5,10,15,20,25,30,35,40,45,50,55 minutes, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 hours, 1,2,3,4,5,6,7 days, 1,2,3,4,5 weeks, 1,2,3,4,5,6,7,8,9,10,11, December, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more for many years and can come from wherein any combination or scope.And consider, or replace changing temperature except changing temperature, can carry out other change of environment, for example pressure change or realization cryoprotection or cryopreservation environment (environment that for example contains glycerol).

Should be appreciated that, may need different length dead time with " stagnation " about cell or tissue about " stagnation " of whole animal.Therefore,, for example carry out the experimenter or the trauma patient of surgical operation therapy, the treatment of pernicious hyperpyrexia, consider 12,18 or 24 hours dead time at the most usually for people experimenter.For non-human animal experimenter, the non-human animal who transports for commercial object or preserve for example considers the stagnation in 2 or 4 days, 2 or 4 weeks or longer period.

Term " exposure " uses according to its ordinary meaning, is used for expression and allows biological substance accept oxygen antagonist or other reactive compound.In some embodiments, this can finish by allowing biological substance contact with oxygen antagonist or reactive compound.In other embodiments, this by allow biological substance with can be or can not be that the reactive compound of oxygen antagonist contacts and finishes.In the situation of cell, tissue or organ, " exposure " can further mean " opening " these materials so that it can contact with oxygen antagonist or other reactive compound in vivo.For example, this can finish by surgical operation.Expose biological substance in oxygen antagonist or other reactive compound can by in antagonist or with antagonist (comprising submergence), hatch, with the antagonist perfusion or inject, inject biological substance with oxygen antagonist or other reactive compound, or oxygen antagonist or other reactive compound be applied to biological substance finish.And, if the stagnation of whole biology is ideal, consider to suck or take in oxygen antagonist or other reactive compound, or the medicament administration of any other approach uses oxygen antagonist or other reactive compound.And it is to mean " supply " according to its common and common implication that term " provides ".Consider, chemical compound can be offered biological substance and can be translated into its form as reactive compound by chemical reaction with a kind of form.According to the present invention, term " provides " term " exposure " that comprises under term " effective dose " background.

In some embodiments, effective dose is characterized by the sublethal dose of oxygen antagonist or other reactive compound.In the situation of the stagnation of inducing cell, tissue or organ (not being whole biology), " sublethal dose " expression single administration fewer than half will cause that cell in most of at least biological substance is at the oxygen antagonist or the reactive compound of the amount of using oxygen antagonist dead in 24 hours or reactive compound.If expect the stagnation of whole biology, then " sublethal dose " expression single administration fewer than half will cause oxygen antagonist or the reactive compound of this biology in the amount of using in 24 hours dead oxygen antagonist.In some embodiments, effective dose is characterized by nearly (near-lethal) dosage that causes death of oxygen antagonist or reactive compound.Similarly, in the situation of the stagnation of inducing cell, tissue or organ (not being whole biology), " near fatal dose " expression single administration 25% will cause oxygen antagonist or the reactive compound of most of at least cell in the amount of using inhibitor dead in 24 hours with interior.If expect the stagnation of whole biology, then " near fatal dose " expression single administration 25% will cause oxygen antagonist or the reactive compound of this biology in the amount of using inhibitor dead in 24 hours with interior.In some embodiments, use sublethal dose by oxygen antagonist or reactive compound to the biological substance of using scheduled volume.Special consideration, this can realize with any reactive compound.

And consider, in some embodiments, effective dose is characterized by the supralethal dose of oxygen antagonist or other reactive compound.In the situation of the stagnation of inducing cell, tissue or organ (not being whole biology), " supralethal dose " expression single administration at least 1.5 times (1.5x) will cause that cell in most of at least biological substance is at the reactive compound of the amount of using reactive compound dead in 24 hours.If expect the stagnation of whole biology, then at least 1.5 times of single administration of " supralethal dose " expression will cause the reactive compound of this biology in the amount of using reactive compound dead in 24 hours.Special consideration, supralethal dose can be approximately, at least about or about at the most 1.5x, 2x, 3x, 4x, 5x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x, 200x, 250x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x, 1100x, 1200x, 1300x, 1400x, 1500x, 1600x, 1700x, 1800x, 1900x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, 10,000x or more, what maybe can come from wherein any scope will cause that cell (or whole biology) in most of at least biological substance is in the amount of using reactive compound dead in 24 hours.

The amount that offers the reactive compound of biological substance can be approximately, at least about, or about at the most 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,441,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980,990,1000mg, mg/kg or mg/m2 maybe can come from any scope wherein.Alternately, described amount can be expressed as 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,441,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980,990,1000mM or M maybe can come from any scope wherein.

In some embodiments, effective dose is the oxygen antagonist used by independent monitoring or the amount of other reactive compound, or monitor this with the combination of the following parameter of monitoring and measure and use: the persistent period that monitoring oxygen antagonist or other reactive compound are used, the monitoring bio material is to the physiological reaction of using oxygen antagonist or other reactive compound (pulse for example, breathe, pain reaction, motion or energy, metabolizing parameters such as cellular energy generation or redox state etc.), and minimizing, interrupting or stopping is used described chemical compound, when the lower limit that measures described reactions change or in limited time last, or the like.And, in any method of the present invention, can adopt these steps extraly.

The tissue that stays cool or stood to stagnate can use in many application.For example, they can be used for infusion or transplanting (therapeutic is used, and comprises organ transplantation); Be used for research purpose; Be used to screen detect and differentiate, characterize or produce other chemical compound of inducing stagnation; Be used to detect the sample (diagnostic application) that obtains tissue from it; Be used for preserving or prevent damage (prophylactic use) the tissue that will put back to tissue-derived biology; Be used for preserving or prevent in the damage of transportation or storing process to them.This class is used and the details of other purposes is described below.Term " isolating tissue " expression is organized and is not positioned in the biology.In some embodiments, tissue is all or part of of organ.Term " tissue " and " organ " are to use according to their common and common meaning.Although tissue is made up of cell, should be appreciated that term " tissue " finger-type becomes to determine the cytoid aggregation of class of the structural material of type.And organ is the tissue of specific type.

The present invention relates to be used for inducing at isolating tissue the method for stagnation, this method comprises: a) identify the tissue that wherein expectation is stagnated; And b) the oxygen antagonist that this tissue is exposed to effective dose is induced stagnation.

Compositions of the present invention, method and manufacture can be used on the biological substance, and this biological substance will be transferred among biological or different acceptance (allogenic) experimenter of (from body) donor of getting back to biological substance source.In some embodiments, biological substance directly obtains from donor is biological.In other embodiments, before being exposed to oxygen antagonist or other reactive compound, biological substance is placed culture.In some cases, biological substance obtains from the donor tissue of having implemented the ECMO before taking out this biological substance, and the ECMO is the technology that a kind of enforcement helps preserve biological substance.And method comprises and will wherein induce the biological substance of stagnating to use or migrate to acceptance biology alive.

In some embodiments, will fetch and then transplanted organ or tissue be exposed to oxygen antagonist or other reactive compound, simultaneously still in the donor experimenter.Consideration is used for the vascular system of donor organ or tissue is exposed to oxygen antagonist or other reactive compound in some cases.If heart is still beaten or pump, conduit or syringe can be used for using the oxygen antagonist or other reactive compound is delivered to organ or tissue to vascular system, can carry out this method.

Method of the present invention also relates in isolating tissue induces stagnation, comprises that stagnating chemical compound with the activity of oxygen antagonist or generation hypoxia condition hatches the time of organizing effective dose, is used for this tissue and enters stagnation.

The cell that stays cool or stagnated can use in many application.For example, they can be used for infusion or transplanting (therapeutic application); Be used for research purpose; Be used to screen detect and differentiate, characterize or produce other chemical compound of inducing stagnation; Be used to detect the sample (diagnostic application) that obtains cell from it; Be used for preserving or prevent cells injury (prophylactic use) the biology that will put back to the cell source; Be used for preserving or prevent damage at transportation or storing process pair cell.The details of this application and other purposes is described below.

The present invention relates to be used for inducing the method for stagnation with one or more cells of bio-separation, this method comprises: a) identify the cell that wherein expectation is stagnated; And b) described cellular exposure is induced stagnation in oxygen antagonist or other active chemical compound of stagnating of effective dose.

Consider that cell can be any cell that utilizes oxygen.Cell can be eucaryon or protokaryon.In certain embodiments, cell is an eucaryon.More particularly, in some embodiments, cell is a mammalian cell.Consider that being used for mammalian cell of the present invention includes but not limited to from those following cells: people, monkey, mice, rat, rabbit, hamster, goat, pig, Canis familiaris L., cat, ferret, milch cow, sheep and horse.

And cell of the present invention can be a diploid, but in some cases, cell is monoploid (sexual cell).And thin portion can be polyploid, aneuploid or akaryote.Cell can be from special organization or organ, for example from following tissue or organ: the heart, lung, kidney, liver, bone marrow, pancreas, skin, bone, vein, tremulous pulse, cornea, blood, small intestinal, large intestine, brain, spinal cord, smooth muscle, skeletal muscle, ovary, testis, uterus and umbilical cord.And cell also can be characterized by one of following cell type: platelet, myelocyte, erythrocyte, lymphocyte, adipose cell, fibroblast, epithelial cell, endotheliocyte, smooth muscle cell, Skeletal Muscle Cell, endocrine cell, neurogliocyte, neuron, secretory cell, the barrier function cell, contractive cell (contractile cell), absorptive cell, mucomembranous cell, marginal cell (from cornea), stem cell (totipotency, versatility (pluripotent) or multipotency (multipotent)), unfertilized or be fertilized oocyte or spermatid.

The present invention also is provided for by reducing metabolic demand, oxygen demand, temperature or their combination in any in the target organism material, is increased in the viability of biological substance under the unfavorable conditions and/or reduces method, compositions and instrument to its damage.In some embodiments of the present invention, the viability of biological substance strengthens by the protectiveness metabolism agent that offers its effective dose.This reagent is by preventing or reduce damage to biological substance, stop all or part of biological substance dead or old and feeble, and/or the life-span that prolongs all or part of biological substance strengthen viability, with respect to the biological substance that is not exposed to this reagent.Alternately, in some embodiments, reagent prolongs tissue and/or biological survival, otherwise described tissue and/or biologically when this reagent not, then will can not survive.

Consider that " protectiveness metabolism agent " is the material or the chemical compound that can reversibly change the metabolism of the biological substance that is exposed to or is in contact with it or strengthen the viability of these biomass.

In certain embodiments, protectiveness metabolism agent is induced stagnation in the biological substance that is subject to processing; Simultaneously, in other embodiments, protectiveness metabolism agent itself is not directly induced stagnation in the biological substance that is subject to processing.Protectiveness metabolism agent and other reactive compound can strengthen the viability of biological substance and/or reduce to it damage and this does not induce stagnation in described biological substance, but by reducing Cellular respiration and corresponding metabolic activity degree to the reduction that is less than about 50% oxygen consumption or carbon dioxide generating.And this compounds can cause biological substance response damage or morbid state and enter more fast, easily or effectively or reach stagnation, for example by inducing biological substance to reach pre-dead state.

Viability comprises when material and is in unfavorable conditions-promptly, in the viability that has condition following time of disadvantageous and irreversible infringement of biological substance or damage.Disadvantageous condition includes but not limited to: when oxygen concentration in the environment reduces (hypoxia or anaerobic, for example at high height above sea level or under water); When biological substance can not receive those oxygen (for example when ischemia), this can be caused by following factor: i) since angiemphraxis (for example, myocardial infarction and/or apoplexy) the minimizing blood flow that causes is to organ (for example, heart, brain and/or kidney), the extracorporeal blood shunting that ii) takes place in heart/lung by-pass operation process (for example, " pump head (pumphead) syndrome " that its cardiac or cerebral tissue sustain damage owing to cardiopulmonary bypass surgery) or iii) because lose blood (for example, hemorrhagic shock or operation) that wound causes; Hypothermia, wherein biological substance stands to be lower than physiological temperature, and this is owing to be exposed to cold environment or because the low temperature state of biomaterial, it can not keep the oxygenate of enough biomaterials like this; High temperature, wherein biomaterial stands to surpass physiological temperature, and this is owing to be exposed to thermal environment or the condition of high temperature of the biomaterial that for example caused by pernicious heating; The condition of illness of excessive heavy metal, for example ferrum obstacle (genetic and environment) is as hemochromatosis, acquired iron overload, sicklemia, juvenile hemochromatosis, diet hemosiderosis disease, thalassemia, porphyria cutanea tarda, sideroblastic anemia, iron deficiency anemia and ACD.Consider that protectiveness metabolism agent is the oxygen antagonist in the certain embodiments of the invention.Consider that also in some other embodiment, the oxygen antagonist is not protectiveness metabolism agent.In other embodiments of the present invention, one or more chemical compounds can be used for increasing or strengthening the viability of biological substance; The metabolism and/or the activity that reversibly suppress biological substance; Reduce the oxygen demand of biological substance; Reduce or prevent under unfavorable conditions damage biological substance; Prevent or reduce infringement or damage biological substance; Prevent the aging or old and feeble of biological substance, and the treatment benefit about the oxygen antagonist as describing in the whole application is provided.Consider, relate to the embodiment of inducing stagnation and also can be applicable to these other embodiments.Therefore, can implement according to these other embodiments about stagnating any embodiment of discussing.

Be used to induce the reactive compound of stagnation or arbitrarily these other embodiments can cause or provide their Expected Results, in some embodiments, 24 hours after time in their environment at biological substance provides Expected Results (do not have continue effect) and/or them can provide these effects to surpass this biological substance no longer to be exposed to them only.And when using the combination of reactive compound, this also can be this situation.

In certain embodiments, biological substance is exposed to a certain amount of oxygen antagonist or other reactive compound, it reduces biological substance and produces the speed of carbon dioxide or measure at least 2 times, and be approximately, at least about or about at the most 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 100-, 200-, 300-, 400-, 500-times or more, maybe can come from any scope wherein.Alternately, consider embodiment of the present invention can with approximately, at least about or about at the most 50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99% or biological substance more, that maybe can come from any scope wherein produces the speed of carbon dioxide or the minimizing of amount is discussed.In embodiment further, biological substance is exposed to a certain amount of oxygen antagonist or other reactive compound, it reduces the speed of biological substance oxygen consumed or measures at least 2 times, and be approximately, at least about or about at the most 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 100-, 200-, 300-, 400-, 500-times or more, maybe can come from any scope wherein.Alternately, consider embodiment of the present invention can with approximately, at least about or about at the most 50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99% or more, maybe can come from the speed of biological substance oxygen consumed of any scope wherein or the minimizing of amount and discuss.In embodiment further, biological substance is exposed to reduction at least 10%, and reduce approximately, at least about or about at the most 15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,99 or 100% maybe can come from the motion of any scope wherein or amount oxygen antagonist or other reactive compound of energy.The same with other embodiment, these features and parameter are relevant with any biological substance of dead state into of inducing.Therefore, if induce stagnation at the heart of biology, these parameters will be assessed according to heart rather than whole biology.About biology, the minimizing of the oxygen consumption of general 8 times of magnitudes is stagnations of a kind of being called " hibernation ".And, should be appreciated that in this application the minimizing of the oxygen consumption of about 1000-times magnitude can be thought " stagnate and give birth to ".Should be appreciated that the embodiment of the present invention that relates to stagnation can be as required, on hibernation or stagnant unboiled water are flat, finish.The amount that should be appreciated that " demultiplication is few " minimizing is relevant; For example, if non-hibernator consumes the oxygen of 800 units, then hibernator consumes the oxygen of 100 units.

And, in some embodiments of the present invention, provide method to be used to reduce Cellular respiration, described Cellular respiration can be or can not be with reach stagnate required the same high.Provide in the method for the invention approximately, at least about or the minimizing of about at the most 1,2,5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100% oxygen consumption.This also can represent and assess with any Cellular respiration index.

Consideration can be exposed to biological substance one or more oxygen antagonisies or other reactive compound more than once.Consider that biological substance can be exposed to one or more

reactive compounds

1,2,3,4,5,6,7,8,9,10 times or more times, this represents when exposing biological substance repeatedly there is the intermission (with regard to being exposed to reactive compound) therebetween.

Also consider, can before the beginning or progress of harmful infringement or morbid state, during, afterwards or their combination in any use reactive compound.In certain embodiments, be enough to strengthen the damage that viability and/or the harmful infringement of minimizing or disease infringement cause with reactive compound pretreatment biological substance.Pretreatment is defined as before harmful infringement or disease infringement beginning or detecting, biological substance is exposed to reactive compound.After the pretreatment, can when beginning injury or near the time stop exposing, or after the injury beginning, continue exposure.

In certain embodiments, will comprise that the method that is exposed to reactive compound (being pretreatment) in advance is used to handle such situation, harmful infringement or disease infringement are 1 in this situation) predetermined or select in advance, or 2) prediction in advance might take place.Eligible 1 example includes but not limited to, the big surgical operation of wherein may be spontaneously or being caused occurring losing blood by operation, wherein the Oxygenation of blood may vascular delivery undermined or wherein blood may reduce the cardiopulmonary bypass surgery of (as in the environment that coronary artery bypass grafting (CABG) operation is installed), or is used for carrying and transplanting into needing the receiver of organ transplantation pre-treatment organ donor in that donor organ is shifted out.Eligible 2 example comprises, but be not limited to, wherein the risk of damage or disease progression is that inherent medical conditions is (for example at unsettled angina pectoris, postangioplasty, hemorrhagic aneurysm, hemorrhagic apoplexy, after the big wound or lose blood), or wherein risk can detect the medical conditions of diagnosis with medical diagnosis.

When being exposed to harmful infringement or disease infringement beginning or detecting the back when obtaining therapeutic effect, be exposed to reactive compound and can strengthen viability or reduce and damage.It can be of short duration or secular being exposed to reactive compound.Exposing the persistent period can be only for reaching stagnation activity or pre-index (for example, the blood pCO that stagnates ₂, pO ₂, pH, lactic acid or sulfhemoglobin level or body temperature) required time is equally long, or can be longer.In certain embodiments, be exposed to biology and be subjected to the generation of traumatic damage (comprising iatrogenic and/or non-iatrogenic injury) back, and be used for whole biology or wherein the tissue of damaged induce and stagnate or pre-the stagnation, so as before treatment, the treatment during and/or the treatment after, prevent damage, for example ischemia and reperfusion injury or it is minimized.

In one embodiment, the present invention includes the method that the protection mammal avoids suffering the cell injury that caused by operation, it comprises that offering mammal is enough to induce mammal to advance into hydrogen sulfide or other reactive compound of the amount of pre-stagnation in operation.Operation can be selectable, through plan or emergency surgery operation, for example cardiopulmonary surgery.Hydrogen sulfide can comprise by any means available in the art, for example uses by intravenous or by sucking.

In another embodiment; the present invention includes the protection mammal and avoid suffering the method for the cell injury that caused by disease or unfavorable medical condition, it comprises that offering mammal is enough to induce mammal to begin or make progress hydrogen sulfide or other reactive compound of the amount that advances into pre-stagnation or stagnation at disease or disadvantageous medical condition.This embodiment can be used for multiple various disease and disadvantageous medical condition, comprises, for example unsettled angina pectoris, postangioplasty, aneurysm, hemorrhagic apoplexy or shock, wound or lose blood.

In special embodiment, the present invention relates to is enough to prevent hydrogen sulfide or other reactive compound of the amount of the hemorrhage death of animal by offering mammal, prevent biology, for example the hemorrhage death of mammal or suffer method by the hemorrhage irreversible tissue injury that causes.In other the embodiment, biology can enter hemorrhagic shock at some, but can be owing to excessive hemorrhage death.Term " is bled " and " losing blood " commutative making is used to refer to any outflow of blood from blood vessel.It includes but not limited to, internal hemorrhage and external haemorrhage, by damage (it may be from inside sources or from the external physical source, for example from gunslinging, stab, physical trauma etc.) cause hemorrhage.

And other embodiment of the present invention relates to and preventing by losing blood or the lacking of other pair cell or tissue oxygenation, and for example lacks the dead or irreversible tissue injury that enough blood supplies cause.This for example can be, actual blood loss causes, maybe may be by preventing that cell or tissue from obtaining perfusion (for example reperfusion injury), causing that blood is to obstruction, part of cell or tissue or integrally reduce the situation of the number of oxygen Portability cell in the blood pressure in the biology, the amount that reduces the oxygen that carries in the blood or the minimizing blood or the result of disease.The situation and the disease that may relate to include but not limited to, blood clot and thromboembolism, cyst, growth (growth), tumor, anemia (comprising sicklemia), hemophilia, other hemopexis disease are (for example, Feng's Willibrand disease, ITP) and atherosclerosis.These situations and disease comprise also that owing to damage, disease or situation pair cell or tissue produce those of basic hypoxia or anaerobic situation.

In some cases, deadly accumulated dose in Asia or non-deadly accumulated dose are administered to biological substance.As discussed above, about in the biological substance that is not whole biology, inducing stagnation, the amount of reactive compound is repeatedly used in " inferior cause death accumulated dose " expression, and it totally is less than and will causes half of amount of most of at least cell dead reactive compound in 24 hours of applied once.In other embodiments, effective dose is characterized by the nearly fatal dose of oxygen antagonist or other reactive compound.Equally, the amount of oxygen antagonist or other reactive compound is repeatedly used in " closely cause death accumulated dose " expression, its amount that will cause reactive compound dead in most of at least cell 24 hours at applied once 25% in.And the amount of reactive compound is repeatedly used in " super deadly accumulated dose " expression, and it is at least and will causes 1.5 times of amount of most of at least cell (or whole biology) dead reactive compound in 24 hours of applied once.Consideration can be used multidose so that induce stagnation in whole biology.The definition of " inferior cause death accumulated dose ", " accumulated dose closely causes death " and " super deadly accumulated dose " can be inferred based on be used for that whole biology stagnates individually dosed previously discussed.

Biological substance can be exposed to more than a kind of oxygen antagonist or other reactive compound or be in contact with it.Biological substance can be exposed at least a reactive compound, comprise 2,3,4,5,6,7,8,9,10 or more polyoxy antagonist or other reactive compound, maybe can come from any scope wherein.For the various active chemical compound, term " effective dose " refers to the total amount of reactive compound.For example, biological substance can be exposed to first kind of reactive compound and also be exposed to second kind of reactive compound then.Alternately, biological substance can be exposed to simultaneously or in eclipsed mode more than a kind of reactive compound.In addition, consideration can will comprise more than a kind of reactive compound or mix, and for example comprises or is mixed in the single compositions that biological substance exposes.Therefore consider, in some embodiments, in compositions of the present invention, method and manufacture, adopt the combination of reactive compound.

Can provide or be exposed to reactive compound to biological substance by suction, injection, conduit insertion, dipping, lavation, perfusion, topical application, absorption, absorption or dosage forms for oral administration.And, can pass through intravenous, Intradermal, intra-arterial, intraperitoneal; in the pathological changes; intracranial; intraarticular; in the prostate; in the pleura; in the trachea; intranasal; in the sheath; in the vitreous body; intravaginal; internal rectum; surface; in the tumor; intramuscular; intraperitoneal; ophthalmic; subcutaneous; under the conjunctiva; in the capsule; through mucous membrane; in the pericardium; in the umbilicus; ophthalmic; per os; surface; part; by sucking; by injection; pass through infusion; pass through continuous infusion; pass through regional perfusion; be applied to biological substance, provide or be exposed to reactive compound to biological substance via conduit or via lavation.

Method and apparatus of the present invention relates to protective agent, and this protective agent is the oxygen antagonist in some embodiments.In embodiment further, the oxygen antagonist is a Reducing agent.And the oxygen antagonist can be characterized as being the chalcogenide chemical compound.Should be appreciated that reactive compound also can be a protective agent.And as long as it realizes target of the present invention, any chalcogenide chemical compound can be thought reactive compound, and no matter whether it is the oxygen antagonist.

In certain embodiments, the chalcogenide chemical compound comprises sulfur, and in other embodiments, it comprises selenium, tellurium or polonium.In certain embodiments, the chalcogenide chemical compound contains the sulfide group of one or more exposures.Consider that this chalcogenide chemical compound contains 1,2,3,4,5,6 or the more sulfide group that exposes, and maybe can come from any scope wherein.In special embodiment, this sulfur-containing compound is CS ₂(Carbon bisulfide).

And, in certain methods of the present invention, by cellular exposure is induced stagnation in the Reducing agent with following chemical constitution (being called formula I) in cell:

Wherein X is N, O, Po, S, Se or Te;

Wherein Y is N or O;

R wherein ₁Be H, C, low alkyl group, lower alcohol or CN;

R wherein ₂Be H, C, low alkyl group, lower alcohol or CN;

Wherein n is 0 or 1;

Wherein m is 0 or 1;

Wherein k is 0,1,2,3 or 4; And,

Wherein p is 1 or 2.

Term " low alkyl group " and " lower alcohol " are to use according to their ordinary meaning, and symbol is the symbol that is used in reference to chemical element.This chemical constitution will be called " Reducing agent structure ", and any chemical compound with this structure will be called the Reducing agent structural compounds.In other embodiments, k is 0 in the Reducing agent structure.And, in other embodiments, R ₁And/or R ₂Group can be amine or low-grade alkylamine.In other embodiments, R ₁And/or R ₂Can be short chain alcohol or chain ketones.And, R ₁And R ₂Bridge and/or this chemical compound that can be linearity or side chain can be cyclic compounds.In embodiment further, X also can be a halogen.Term " rudimentary " meaning is meant that 1,2,3,4,5 or 6 carbon atom maybe can come from any scope wherein.And, R ₁And/or R ₂Can be other little organic group, comprise C ₂-C ₅Ester, amide, aldehyde, ketone, carboxylic acid, ether, nitrile, acid anhydride, halogenide, acyl halide, sulfide, sulfone, sulfonic acid, sulfoxide and/or mercaptan.About R ₁And/or R ₂, consider these replacements clearly.At some in other the embodiment, R ₁And/or R ₂It can be the discussed above little organic group of short chain form.1,2,3,4,5,6,7,8,9,10,11 or 12 carbon molecule of " short chain " expression maybe can come from any scope wherein.

Consideration is in some cases, and described Reducing agent structural compounds can be the chalcogenide chemical compound.In certain embodiments, the chalcogenide chemical compound has alkyl chain, and it contains the chalcogenide of exposure.In other embodiments, the chalcogenide that becomes and expose in case described chalcogenide chemical compound has for biological material absorbing.In this respect, described chalcogenide compounds is similar to the prodrug as the oxygen antagonist.Therefore, after biological substance being exposed to described chalcogenide chemical compound, one or more sulfur, selenium, oxygen, tellurium, polonium or ununhexium (ununhexium) molecular change on this chemical compound gets and can utilize.In this case, " can utilize " expression sulfur, selenium, oxygen, tellurium, polonium or ununhexium will keep negative charge.

In certain embodiments, chalcogenide is a salt, and preferably wherein chalcogen is the salt of-2 oxidation state.The sulphide salt that embodiment of the present invention comprise includes but not limited to, sodium sulfide (Na ₂S), NaHS (NaHS), Potassium monosulfide. (K ₂S), potassium bisulfide (KHS), lithium sulfide (Li ₂S), rubidium sulfide (Rb ₂S), cesium sulfide (Cs ₂S), ammonium sulfide ((NH ₄) ₂S), sulfur hydrogenation ammonium ((NH ₄) HS), sulfuration beryllium (BeS), magnesium sulfide (MgS), calcium sulfide (CaS), strontium sulfide (SrS), barium sulfide (BaS) etc.Similarly, embodiment of the present invention comprise, but are not limited to corresponding selenides and tellurides salt.Special consideration the present invention includes the compositions that contains chalcogenide salt (being the chalcogenide chemical compound of salt) that has pharmaceutically suitable carrier or be prepared as pharmaceutically acceptable preparation.In further embodiment, the Reducing agent structural compounds is selected from H ₂S, H ₂Se, H ₂Te and H ₂Po.In some cases, it is the X of S that the Reducing agent structure of formula (I) has.In other case, X is Se, or X is Te, or X is Po, or X is O.In addition, in some embodiments, the k in the Reducing agent structure is 0 or 1.In certain embodiments, the Reducing agent structural compounds is dimethyl sulfoxine (DMSO), dimethyl sulphide (DMS), carbon monoxide, methanthiol (CH ₃SH), mercaptoethanol, rhodanate, Blausure (German), methanthiol (MeSH) or CS ₂In special embodiment, the oxygen antagonist is H ₂S, H ₂Se, CS ₂, MeSH or DMS.The chemical compound of these bulk of molecule magnitudes be special consider (that is, they the molar mass average value 50% in).

In certain embodiments, employing contains for example H of selenium compound ₂Se.In some embodiments of the present invention, H ₂The amount of Se can be in the scope of 1,000,000,000/1-1000 part.Further consider that any embodiment of discussing can realize with containing selenium compound in the content of sulfur-containing compound.This comprises with the one or more sulphur atoms in the alternative sulfur-containing molecules of corresponding selenium atom.

Further aspect of the present invention comprises the chemical compound of being represented by formula IV:

Wherein:

X is N, O, P, Po, S, Se, Te, O-O, Po-Po, S-S, Se-Se or Te-Te;

N and m are 0 or 1 independently; And

R wherein ²¹And R ²²Be hydrogen independently, halogen, cyano group, phosphate ester, sulfenyl, alkyl, alkenyl, alkynyl, alkoxyl, aminoalkyl, the cyano group alkyl, hydroxy alkyl, haloalkyl, the hydroxy halogeno alkyl, alkyl sulfonic acid, thiosulfonic acid, alkyl thiosulfonic acid, alkylthio, alkylthio group, alkyl-thio-alkyl, alkylaryl, carbonyl, alkyl-carbonyl, halogenated alkyl carbonyl, alkyl thiocarbonyl, amino carbonyl, amino thiocarbonyl, thio-alkyl amino-carbonyl, halogenated alkyl carbonyl, alkoxy carbonyl, amino alkylthio group, the hydroxyl alkylthio group, cycloalkyl, cycloalkenyl group, aryl, aryloxy group, heteroaryloxy, heterocyclic radical, heterocyclic oxy group, sulfonic acid, alkyl sulfonate esters, thiosulfates or sulfonamido; And

Y is a cyano group; isocyano group; amino; alkyl amino; amino carbonyl; the amino carbonyl alkyl; alkyl-carbonyl-amino; amidino groups; guanidine; diazanyl; hydrazides; hydroxyl; alkoxyl; aryloxy group; heteroaryloxy; cycloalkyloxy; the ketonic oxygen base; the alkyl-carbonyl oxygen base; halogenated alkyl carbonyl oxygen base; aryl carbonyl oxygen base; the carbonyl peroxy; the alkyl-carbonyl peroxy; the aryl carbonyl peroxy; phosphate ester; alkyl phosphate; sulfonic acid; alkyl sulfonate esters; thiosulfates; the sulfo-sulfenyl; sulfonamide;-R ²³R ²⁴, R wherein ²³Be S, SS, Po, Po-Po, Se, Se-Se, Te or Te-Te, and R ²⁴Be as at this about R ²¹Definition, perhaps Y is

Wherein X, R ²¹And R ²²As definition in this article.

And, consider that in some embodiments of the present invention provide precursor compound to biological substance, these precursor compounds for example become the activity form of formula I or IV chemical compound by being exposed to biological substance by chemistry or enzymatic means.And, can be with chemical compound as the salt of this chemical compound, with the form of free radical or electronegative, positively charged or offer biological substance with the form of a plurality of electric charges.Some chemical compounds not only meet formula I but also meet formula IV structure, and in this case, the use of phrase " formula I or formula IV " has no intention to hint this compounds of eliminating.

In certain embodiments, the chemical compound of being determined by the structure of formula I or formula IV also is characterized as being oxygen antagonist, protectiveness metabolism agent or its precursor, prodrug or salt.Further consider that described chemical compound itself needn't characterize or itself prove and be used for chemical compound of the present invention, as long as it realizes a special method of the present invention.In other the embodiment, chemical compound can be thought the chalcogenide chemical compound at some.Special consider, in method of the present invention, compositions and instrument, determine or the anyization thing that proposes in this disclosure can replace the oxygen antagonist to use or is used in combination with the oxygen antagonist by the structure of formula I or formula IV; Any embodiment that discuss about the arbitrary structures with formula I or formula IV similarly, or that propose in addition in the disclosure can replace the oxygen antagonist to use or be used in combination with the oxygen antagonist.And, can make up with any oxygen antagonist described here or any other reactive compound by the structure of formula I or formula IV anyization thing definite or statement in this disclosure.Also consider, in method of the present invention, compositions and other manufacture, the combination in any of these chemical compounds can be together, (overlapping or not overlapping) and/or (begin to use a kind of chemical compound and before it is finished continuously with eclipsed continuation mode, begin to use another chemical compound again) provide or prepare, obtain desired effects in this statement.

In certain embodiments, provide the chemical compound that has formula I or formula IV structure more than a kind of.In certain embodiments, adopt the multiple different chemical compounds with phase cotype (being formula I or formula IV) structure, and in other embodiments, when adopting multiple different chemical compound, they are from different formulas.

In special embodiment, to consider to use the various active chemical compound, wherein a kind of chemical compound is carbon dioxide (CO ₂).Consider that at least a other chemical compound also is formula I and/or is the IV chemical compound in some embodiments.In some case, with carbon dioxide and H ₂S or H ₂The S combination of precursors offers biological substance (together, continuously or with eclipsed continuation mode).

The amount of the carbon dioxide that biological substance can expose be approximately, at least about or about at the most 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30% or more, maybe can come from any scope wherein.In certain embodiments, described amount is represented with ppm, for example about, at least about or about at the most 350,400,500,600,700,800,900,1000,1100,1200,1300,1400,1500,1600,1700,1800,1900,2000,2100,2200,2300,2400,2500,2600,2700,2800,2900,3000,3100,3200,3300,3400,3500,3600,3700,3800,3900,4000,4100,4200,4300,4400,4500,4600,4700,4800,4900,5000,6000,7000,8000,9000,10000,11000,12000,13000,14000,15000,16000,17000,18000,19000,20000,21000,22000,23000,24000,25000,26000,27000,28000,29000,30000,31000,32000,33000,34000,35000,36000,37000,38000,39000,40000,41000,42000,43000,44000,45000,46000,47000,48000,49000,50000,60000,70000,80000,90000,100000,110000,120000,130000,140000,150000,160000,170000,180000,190000,200000,210000,220000,230000,240000,250000,260000,270000,280000,290000,300000 or more ppm, maybe can come from any scope wherein, and represent with molar equivalent.Consider that these concentration can be applicable to any other reactive compound of gas form.

In other embodiments, consider that especially reactive compound is sodium sulfide, sulfo-Feldalat NM, mercaptamine, sodium rhodanate, mercaptamine-S-sodium ascorbyl phosphate or tetrahydric thiapyran-4-alcohol.In other embodiments, reactive compound is dimethyl sulfoxine, thiacetic acid., selenourea, 2-(3-aminopropyl)-aminoothyl mercaptan-dihydro-phosphate ester, 2-sulfydryl-ethanol, sulfydryl ether (thioglycolicether), sodium selenide, methyl-sulfinic acid sodium, thiourea or dimethyl sulphide.Special consideration, these chemical compounds or anyly have a formula I in comprising of this discussion, II, the chemical compound of III or IV can be with about at interior any other chemical compound, at least about or about at the most 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392,393,394,395,396,397,398,399,400,401,402,403,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,429,430,431,432,433,434,435,436,437,438,439,440,441,442,443,444,445,446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,485,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500,501,502,503,504,505,506,507,508,509,510,511,512,513,514,515,516,517,518,519,520,521,522,523,524,525,526,527,528,529,530,531,532,533,534,535,536,537,538,539,540,541,542,543,544,545,546,547,548,549,550,551,552,553,554,555,556,557,558,559,560,561,562,563,564,565,566,567,568,569,570,571,572,600,700,800,900,1000,1100,1200,1300,1400,1500,1600,1700,1800,1900,2000,2100,2200,2300,2400,2500,2600,2700,2800,2900,3000,3100,3200,3300,3400,3500,3600,3700,3800,3900,4000,4100,4200,4300,4400,4500,4600,4700,4800,4900,5000,5100,5200,5300,5400,5500,5600,5700,5800,5900,6000,6100,6200,6300,6400,6500,6600,6700,6800,6900,7000,7100,7200,7300,7400,7500,7600,7700,7800,7900,8000,8100,8200,8300,8400,8500,8600,8700,8800,8900,9000,9100,9200,9300,9400,9500,9600,9700,9800,9900, the amount that 10000mM or mmol/kg (biological substance) maybe can come from any scope wherein offers or is applied to biological substance.

Special consider that the random subset of the reactive compound of being determined by title or structure can be used in method, compositions and the manufacture.Consider especially that also can abandon the random subset of these chemical compounds, it does not constitute embodiment of the present invention.The present invention also relates to contain the pharmaceutical composition that one or more reactive compounds of effective dose are gone up in treatment.Certainly, this class pharmaceutical composition is mixed with pharmaceutically acceptable compositions.For example, described compositions can comprise pharmaceutically acceptable diluent.

In certain embodiments, pharmaceutical composition contains the reactive compound of effective dose, with when being administered to the patient, provides the reactive compound plasma concentration of Cmax or stable state, goes up effective benefit to produce treatment.In certain embodiments, Cmax that reaches or Cpss are approximately, at least about or about at the most 0.01,0.1,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,441,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980,990,1000,1100,1200,1300,1400,1500,1600,1700,1800,1900,2000,3000,4000,5000,6000,7000,8000,9000,10000 (M or more maybe can come from any scope wherein.In certain embodiments, for example for H ₂S, the Cmax of expectation or Cpss be about 10 μ M between about 10mM, about 100 μ M are between about 1mM or about 200 μ M extremely between about 800 μ M.Can consider suitable means and the assessment chemical compound in blood, for example level of sulfur.

In certain embodiments, pharmaceutical composition provides the H of effective dose ₂S, with when being administered to the patient, be provided at about 10 μ M between about 10mM, about 100 μ M are between about 1mM, or about 200 μ M Cmax or Cpss between about 800 μ M extremely.Aspect giving hydrogen sulfide and giving the relation of sulphide salt, in typical embodiment, the administration of this salt is based on uses and H ₂The sulfur equivalent that the administration of S is roughly the same.To consider suitable means and be used for assessing the level of the sulfur in blood.

In certain embodiments, compositions contains specified reactive compound above one or more of gas form.In another embodiment, compositions comprises the salt of one or more these chemical compounds.In a special embodiment, the salt of the formula I of pharmaceutical composition air inclusion form or IV or formula I or IV.Aspect more of the present invention, consider H especially ₂The gas form of S or salt.The amount that consideration offers the gas of biological substance is approximately, at least about or about at the most 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980,990,1000,1100,1200,1300,1400,1500,1600,1700,1800,1900,2000,2100,2200,2300,2400,2500,2600,2700,2800,2900,3000,3100,3200,3300,3400,3500,3600,3700,3800,3900,4000,4100,4200,4300,4400,4500,4600,4700,4800,4900,5000,6000,7000,8000,9000,10000,11000,12000,13000,14000,15000,16000,17000,18000,19000,20000,21000,22000,23000,24000,25000,26000,27000,28000,29000,30000,31000,32000,33000,34000,35000,36000,37000,38000,39000,40000,41000,42000,43000,44000,45000,46000,47000,48000,49000,50000,60000,70000,80000,90000,100000,110000,120000,130000,140000,150000,160000,170000,180000,190000,200000,210000,220000,230000,240000,250000,260000,270000,280000,290000,300000,310000,320000,330000,340000,350000,360000,370000,380000,390000,400000 or more ppm, maybe can come from any scope wherein.Alternately, the effective dose of gas can be expressed as the airborne concentration that exposes about at biological substance, for about, at least about or about at the most 0.001,0.002,0.003,0.004,0.005,0.006,0.007,0.008,0.009,0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99 or 100%, maybe can come from any scope wherein.And, consider that for some embodiments the amount that offers the gas of biological substance is approximately, at least about or about at the most 5/1000000000ths, 10,15,20,25,30,35,40,45,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980,990,1000,1100,1200,1300,1400,1500,1600,1700,1800,1900,2000,2100,2200,2300,2400,2500,2600,2700,2800,2900,3000,3100,3200,3300,3400,3500,3600,3700,3800,3900,4000,4100,4200,4300,4400,4500,4600,4700,4800,4900,5000,6000,7000,8000,9000,10000 parts (ppb) maybe can come from any scope wherein.In special embodiment, the amount that offers the Selenium hydride. of biological substance is on this magnitude.

In some embodiments of the present invention, pharmaceutical composition is a liquid.As discussing elsewhere, compositions can be to have the liquid that dissolving or bubbling advance the related compound in the said composition.In some cases, pharmaceutical composition is a medical gas.According to U.S. food and drug administration, " medical gas " is such gas: this gas is the interior medicine of § 201 (g) (1) implication of federal food drug and cosmetic act, medicine and cosmetics law (" bill ") (21U.S.C. § 321 (g)), and, require prescription to make up a prescription according to the § 503 (b) (1) of this bill (A) (21U.S.C. § 353 (b) (1) (A)).Like this, these medical gases need suitable FDA label.Medical gas comprises at least a reactive compound.

The present invention further comprises instrument and manufacture, and it comprises packaging material and the activity that is contained in the packaging material is stagnated chemical compound, and wherein packaging material comprise and show that it can be used for the label that in vivo biological substance is induced stagnation.

In some embodiments, instrument or manufacture further comprise medicinal diluent.In special other embodiment, instrument or manufacture have buffer agent.Described reactive compound provides in the container of first kind of sealing, and described pharmaceutically acceptable diluent provides in the container of second kind of sealing.In other embodiments, described device or article further have the description that is used for mixed active chemical compound and diluent.In addition, reactive compound reconstruct can be realized any method of the present invention, the biological substance that for example is used for is in vivo induced stagnation.Consider that any label will specify the application that the result that will obtain and chemical compound need to be used to this result's patient.

The present invention also relates to manufacture, it comprises following material packaging together: reactive compound, the described active description of stagnating chemical compound of use, and described description comprises: the in-vivo tissue of (a) differentiating needs stagnation processing; (b) use the reactive compound of effective dose to the interior biological substance of described body.

In further embodiment of the present invention, there is the manufacture contain medical gas, it comprises reactive compound and label, label comprises being used for inducing at biological substance to be stagnated or is used for the details or the usage of any other method of the present invention and uses.

The present invention also relates to the method for test kit and these test kits of use.In some embodiments, exist and be used for the test kit of delivery of active compounds to the tissue site of any other processing of needs stagnation processing or claimed invention, described test kit comprises: covering (drape) is applicable to the bag (envelope) that tissue site is formed sealing; The container that contains the aerobic antagonist; And an inlet in the covering, this container that wherein contains reactive compound is communicated with this inlet.In certain embodiments, test kit comprises an outlet in covering, wherein should outlet be communicated with negative pressure source.In some cases, this covering comprises elastomeric material and/or has the contact adhesive that covers this blanket edge.Can allow this outlet and negative pressure source be in fluid and be communicated with, described negative pressure source can be or can not be vacuum pump.Between outlet and negative pressure source, also can there be flexible conduit to be communicated with.In some embodiments, test kit comprises a canister (canister), and it can be dismountable or can not be dismountable, is communicated with fluid between outlet and the negative pressure source.Consider that container comprises the reactive compound that carries out gas communication with inlet.In certain embodiments, container comprises it being the reactive compound of gas or liquid gas.Test kit also can comprise with the container that contains the aerobic antagonist and the vaporizer that is communicated with between entering the mouth.And it can have the bypass outlet that is communicated with the container that contains reactive compound.

In special embodiment, the reactive compound in the test kit is carbon monoxide, carbon dioxide, H ₂Se and/or H ₂S.In certain embodiments, the tissue site of test kit or method application comes to harm.

And, usually should be appreciated that, can offer biological substance with the form of prodrug at this any chemical compound as the discussion of oxygen antagonist, other material in this expression biological substance or this biological substance environment becomes prodrug into its activity form, promptly becomes the oxygen antagonist.The chemical compound that is considered to " prodrug " contained in consideration term " precursor ".

Oxygen antagonist or other reactive compound can be or can be used as gas, semi-solid liquid (example gel or paste), liquid or solid and provide.Consideration can be exposed to biological substance more than a kind of this class reactive compound and/or more than this reactive compound of a kind of state.And, can prepare reactive compound and be used for special mode of administration, as in this discussion.In certain embodiments, reactive compound is to be used for the pharmaceutically acceptable preparation that intravenous is sent.

In certain embodiments, reactive compound is a gas.In special embodiment, the gas reactive compound comprises carbon monoxide, carbon dioxide, nitrogen, sulfur, selenium, tellurium or polonium, or its mixture.And, special consideration, reactive compound is the chalcogenide chemical compound of gas form.In some embodiments, reactive compound is in the admixture of gas that contains more than a kind of gas.In some embodiments, other gas is nontoxic and/or non-active gas.In some embodiments, other gas is noble gas (helium, neon, argon, krypton, xenon, radon or ununoctium (ununoctium)), nitrogen, nitrous oxide, hydrogen or its mixture.For example, non-active gas can be the mixture that constitutes " room air " simply, and it is nitrogen, oxygen, argon and carbon dioxide, and other molecule of trace mixture of neon, helium, methane, krypton and hydrogen for example.Although typical sample may contain 78% the nitrogen of having an appointment, 21% oxygen, 0.9% argon and 0.04% carbon dioxide, the accurate amount of every kind of gas changes.Consideration is in environment of the present invention, and " indoor gas " is the mixture of carbon dioxide of oxygen, about 0.7 to about 1.1% argon and the about 0.02%-about 0.06% of the nitrogen that contains the 75-that has an appointment about 81%, about 18-about 24%.Can before using or being exposed to biological substance, at first the gas reactive compound be diluted with avirulence and/or non-reactive gas.Extraly or alternately, can before using or being exposed to biological substance, any gas reactive compound be mixed with room air, maybe can or be exposed to biological substance in the room air compound administration.

In some cases, admixture of gas also contains oxygen.In other embodiments of the present invention, reactive compound gas and oxygen mix form oxygen (O ₂) mixture.It is special that what consider is the oxygen mixture that the amount of oxygen in the wherein said oxygen mixture is less than the total amount of all other gases in this mixture.

In some embodiments, reactive compound is a carbon monoxide, and the amount of carbon monoxide approximately equals or exceeds any amount of oxygen in the oxygen mixture.In special embodiment, carbon monoxide is used for the depletion of blood biological substance.Term " depletion of blood biological substance " refers to that its Oxygenation does not rely on or be no longer dependent on the cell and the organ of vascular system, for example is used for transplanted organ.Preferably, atmosphere will be 100% CO, but the amount that it is apparent to those skilled in the art that CO can make the amount of available oxygen be reduced to the level that stops Cellular respiration with the gas balance that is not oxygen.Aspect this, the ratio of carbon monoxide and oxygen preferably 85: 15 or higher, 199: 1 or higher or 399: 1 or higher.In certain embodiments, this ratio is approximately, at least about or about at the most 1: 1,2: 1,2.5: 1,3: 1,4: 1,5: 1,6: 1,7: 1,8: 1,9: 1,10: 1,15: 1,20: 1,25: 1,30: 1,35: 1,40: 1,45: 1,50: 1,55: 1,60: 1,65: 1,70: 1,75: 1,80: 1,85: 1,90: 1,95: 1,100: 1,110: 1,120: 1,130: 1,140: 1,150: 1,160: 1,170: 1,180: 1,190: 1,200: 1,210: 1,220: 1,230: 1,240: 1,250: 1,260: 1,270: 1,280: 1,290: 1,300: 1,310: 1,320: 1,330: 1,340: 1,350: 1,360: 1,370: 1,380: 1,390: 1,400: 1,410: 1,420: 1,430: 1,440: 1,450: 1,460: 1,470: 1,480: 1,490: 1,500: 1 or more, maybe can come from any scope wherein.

In embodiment further, top numeral is suitable for the ratio of the mixture of carbon monoxide and oxygen and one or more other gases.In some cases, consider that described other gas is non-active gas, for example nitrogen (N ₂).Therefore, in other embodiments of the present invention, top digital application is in the combination (O of the carbon monoxide that can be used for method of the present invention and instrument and oxygen and nitrogen ₂/ N ₂) ratio.Therefore, should be appreciated that and to exist or may not have other gas.In some embodiments, CO: the ratio of oxygen is with one or more other gases (non-carbon monoxide and non-oxygen) balance.In special embodiment, CO: the ratio nitrogen balance of oxygen.In embodiment further, the amount of CO is the ratio of CO with respect to room air, as what described by top numeral.

In some cases, the amount of carbon monoxide is the amount with respect to oxygen, and in other situation, it is an absolute magnitude.For example, in some embodiments of the present invention, the amount of oxygen is to be unit with " 1,000,000/umber (ppm) ", it is the measurement of oxygen volume parts in 1,000,000 parts of air under 20 ℃ and the atmospheric standard temperature and pressure (STP), and wherein the balance of gas volume is supplied with carbon monoxide.In this respect, in the umber of the equilibrated oxygen of per hundred general-purpose carbon monoxides, carbon monoxide is relevant with the amount of oxygen.Consider that biological substance exposure or the atmosphere of hatching can be the equilibrated oxygen of usefulness carbon monoxide of at least hundred 0/10000th, 50,100,200,300,400,500,1000 or 2000 (ppm), and be to use and non-toxicity and/or the equilibrated oxygen of the blended carbon monoxide of non-active gas in some cases.Term " environment " refers to the direct environment of biological substance, i.e. its environment that directly contacts.Therefore, biological substance must directly be exposed to carbon monoxide, and one jar of carbon monoxide of sealing and biological substance are in the identical chamber and think to hatch in " environment " according to the present invention is inadequate.Alternately, atmosphere can be represented with kPa.It has been generally acknowledged that 100 ten thousand umbers=101kPa under 1 atmospheric pressure.In embodiments of the invention, biological substance is hatched or the environment that exposes is therein approximately, at least about or about at the most 0.001,0.005,0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.10,0.11,0.12,0.13,0.14,0.15,0.16,0.17,0.18,0.19,0.20,0.21,0.22,0.23,0.24,0.25,0.26,0.27,0.28,0.29,0.30,0.35,0.40,0.45,0.50,0.55,0.60,0.65,0.70,0.75,0.80,0.5,0.90,0.95,1.0kPa or more O ₂, maybe can come from any scope wherein.As described above, these levels can be used carbon monoxide and/or other non-toxicity and/or non-active gas balance.And atmosphere can be the CO level definition of unit in order to kPa.In certain embodiments, atmosphere be approximately, at least about or about at the most 1,5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,101,101.3kPaCO maybe can come from any scope wherein.In special embodiment, dividing potential drop be approximately or at least about 85,90,95,101,101.3kPa CO maybe can come from any scope wherein.

In embodiments of the invention, sample is hatched or the time quantum that is exposed to carbon monoxide also can be different.In some embodiments, sample hatched or be exposed to carbon monoxide approximately, at least about or about at the most 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60 or more minutes and/or 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 hours, and/or 1,2,3,4,5,6,7,8,9,10 or more days.

In some embodiments, the present invention relates to contain the compositions and the manufacture of one or more reactive compounds.In certain embodiments, compositions has one or more these reactive compounds as gas, makes this gas bubbling in compositions, makes that said composition provides chemical compound to this biological substance when biological substance is exposed to said composition.These chemical compounds can be gel, liquid or other semi-solid material.In certain embodiments, solution has the oxygen antagonist as the gas that passes through its bubbling.The amount of considering bubbling in the gas will provide the chemical compound of appropriate amount to the biological substance that is exposed to this solution.In certain embodiments, the amount that bubbling enters the gas in the solution be the pact that effectively offers the amount of biological substance, at least about or about at the most 0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,9.5,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100 times or more times, maybe can come from any scope wherein.

In some embodiments of the present invention, biological substance is exposed to gas in the hermetic container.In some cases, hermetic container can keep special environment or by desired adjusting environment.Described environment refers to the amount of the oxygen antagonist that biological substance exposes and/or temperature, gas composition or the pressure of this environment.In some cases, before being exposed to oxygen antagonist or other reactive compound, during or afterwards, biological substance is placed under the vacuum.And, in other situation, after being exposed to oxygen antagonist or other reactive compound, biological substance is exposed to and contains the normal environment of oxygen.In certain embodiments; the present invention includes and be used to induce stagnation or protection biological substance to avoid damaging or avoiding the method for disease; this method comprises provides a kind of reactive compound to biological substance, and uniting provides reactive compound that another kind induces stagnation or environmental condition to this biological substance.This Combined Treatment is with any order, for example simultaneously or recur.In certain embodiments, a kind of reactive compound is offered biological substance, and with this biological substance with for example being placed under the anoxia condition 5% O ₂Down, or it is exposed to subsequently increases progressively under the anoxybiotic condition, for example 5%O ₂, 4%O subsequently ₂, 3%O ₂, 2%O ₂, 1%O ₂Or there is not an O ₂Condition, or under the condition of any order of these conditions combination.

And in other embodiments, the environment circulation that contains biological substance is at least once to oxygen antagonist or other reactive compound of not commensurability or concentration, and wherein the difference of amount or concentration is the difference of at least one percent.Environment can circulation back and forth between the oxygen antagonist of one or more amounts or concentration or other reactive compound, or it can increase or reduce the amount or the concentration of the sort of chemical compound gradually.In some cases, different amount or concentration are between the amount or concentration of about 0 and 99.9% the initial oxygen antagonist that exposes of biological substance or other reactive compound.Consider, the difference of amount and/or concentration is approximately, at least about or about at the most 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99% or more, maybe can come from any scope wherein.

Method of the present invention also can comprise allows biological substance be in step under the controlled temperature environment.In certain embodiments, it is the temperature of " non-physiological temp environment " that biological substance is exposed to, and this temperature refers to that biological substance can not survive therein more than 96 hours temperature.The controlled temperature environment can have approximately, at least about or about at the most-210,-200,-190,-180,-170,-160,-150,-140,-130,-120,-110,-100,-90,-80,-70,-60,-50,-40,-30,-20,-10,-5,0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200 ℃ or higher, maybe can come from the temperature of any scope wherein.Also biological substance can be exposed to oxygen antagonist or other reactive compound under the room temperature, room temperature is illustrated in the temperature between about 20 ℃ and about 25 ℃.In addition, consider that biological substance reaches the core temperature of the scope of any amount discussed or amount.

Consideration can allow biological substance before being exposed to oxygen antagonist or other reactive compound, during or accept non-physiological temp environment or controlled temperature environment afterwards.In addition, in some embodiments, allow biological substance accept a period of time between non-physiological temp environment or the controlled temperature environment about one minute to about 1 year.Time quantum can be approximately, at least about or about at the most 30 seconds, 1,2,3,4,5,10,15,20,25,30,35,40,45,50,55 minutes, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 hours, 1,2,3,4,5,6,7 days, 1,2,3,4,5 weeks, 1,2,3,4,5,6,7,8,9,10,11, December, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more for many years and can come from wherein any combination or scope.And, the step that increases ambient temperature with respect to the temperature that reduces also can be arranged.

And, change or the described temperature that circulates during the consideration process that temperature is controlled therein.In some embodiments, before biological substance is placed the environment with oxygen antagonist or other reactive compound, can be at first its temperature be reduced, and in other embodiments, can be lower than environment its temperature, that have reactive compound by biological substance is placed, with this biological substance cooling.Biological substance and/or environment can cool off or heat gradually, and the temperature of biological substance or environment is initially located in a kind of temperature like this, but reach another temperature then.

Method of the present invention also can comprise allows biological substance be in step under the controlled pressure environment.In certain embodiments, biology is exposed under the pressure that is lower than the common residing pressure of this biological substance.In certain embodiments, allow biological substance accept " non-physical stress environment ", it refers to that biological substance can not survive therein more than 96 hours pressure.The controlled pressure environment can have approximately, at least about or about at the most 10 ^-14, 10 ^-13, 10 ^-12, 10 ^-11, 10 ^-10, 10 ^-9, 10 ^-8, 10 ^-7, 10 ^-6, 10 ^-5, 10 ^-4, 10 ^-3, 10 ^-2, 10 ^-1, 0.2,0.3,0.4 or 0.5 atmospheric pressure or bigger, maybe can come from the pressure of any scope wherein.

Consideration can allow biological substance before being exposed to reactive compound, during or accept non-physical stress environment or controlled pressure environment afterwards.In addition, in some embodiments, allow biological substance accept a period of time between non-physical stress environment or the controlled pressure environment about one minute to about 1 year.Time quantum can be approximately, at least about or about at the most 30 seconds, 1,2,3,4,5,10,15,20,25,30,35,40,45,50,55 minutes, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 hours, 1,2,3,4,5,6,7 days, 1,2,3,4,5 weeks, 1,2,3,4,5,6,7,8,9,10,11, December, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more for many years, and can come from wherein any combination or scope.

And, change or the described pressure that circulates during the consideration process that pressure is controlled therein.In some embodiments, before biological substance is placed the environment with reactive compound, can be at first the pressure of its exposure be reduced, and in other embodiments, after being exposed to reactive compound, biological substance is placed under the pressure.Pressure can reduce gradually, the pressure of environment is initially located in a kind of pressure like this, but subsequently at 10,20,30,40,50,60 seconds, 1,2,3,4,5,10,15,20,25,30,35,40,45,50,55 minute, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 hour and/or 1,2,3,4,5,6,7 day or more of a specified duration, maybe can come from wherein any combination or scope in reach another kind of pressure.In certain embodiments, method comprises the oxygen level of regulating environment or biomaterial is removed from the environment with oxygen.Operationally be, biomaterial is exposed to that oxygen wherein reduces or the environment that lacks in, can simulate biological substance is exposed to the oxygen antagonist.Consider that in some embodiments of the present invention, the environment of biological substance is under the condition of anoxia or anaerobic therein, biological substance is exposed to or provides reactive compound to it, as will be described in further detail below.This can be have a mind to or unintentionally.Therefore, in some embodiments of the present invention, intentionally biological substance is placed the environment of anaerobic or hypoxia or places the environment that causes anaerobic or hypoxia.In other embodiments, biological substance is in by under this condition that situation causes unintentionally, for example, if biological substance is to be in ischemia or potential ischemia situation following time.Therefore, consider that in some cases anoxia or oxygen free condition will damage described material when lacking reactive compound.

In some method of the present invention, also there are the oxygen antagonist be evaluated in the biological substance of wherein inducing stagnation and/or the step of oxidative phosphorylation level.And, in some embodiments of the present invention, there is the step of the level of the cellular metabolism that takes place usually in the assessment biological substance.In some cases, the reduction of temperature in the amount of reactive compound and/or the assessment biological substance in the measurement biological substance.And, in certain methods of the present invention, estimate the degree of one or more therapeutic effect.

In some other embodiment, reactive compound and/or environmental change (temperature, pressure) are monitored or are controlled any toxic effect of biological substance.Toxicity is controlled in the variation of the environment that consideration can expose by level, amount, persistent period or frequency and/or the biological substance of change reactive compound.In certain embodiments, described change is to reduce, and in some other embodiment, described change is to increase.Consider that those skilled in the art know the method for the toxic effect in many evaluation biological substances.

Other optional step of the inventive method comprises identifies suitable reactive compound; The diagnosis patient; Before using or opening reactive compound, consider the historical of patient and/or the patient is carried out one or more detections to the patient.

Compositions of the present invention, method and manufacture can be used for and will be transferred in (from body) donor biology of getting back to biological substance source or on different acceptance (allogenic) experimenters' the biological substance.In some embodiments, biological substance directly obtains from donor is biological.In other embodiments, before being exposed to oxygen antagonist or other reactive compound, biological substance is placed culture.In some cases, biological substance obtains from the donor tissue of having implemented the ECMO before taking out this biological substance, and the ECMO is the technology that a kind of enforcement helps preserve biological substance.And method comprises and will wherein induce the biological substance of stagnating to give or migrate to acceptance biology alive.

Method of the present invention also relates in vivo the biological substance induces stagnation, comprises the time of hatching the biological substance effective dose with oxygen antagonist or other reactive compound of producing hypoxia condition, is used to make this tissue to enter stagnation.

In addition, other embodiment of the present invention comprises the method that reduces the oxygen demand in the biological substance in the body, and this method comprises allows biological substance contact with oxygen antagonist or other reactive compound of effective dose, to reduce their oxygen demand.Consider that the oxygen demand is with respect to the biological substance cell that is not exposed to or no longer is exposed to oxygen antagonist or other reactive compound or from the oxygen demand of the representative cell sample of biological substance, reduce approximately, at least about or about at the most 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99 or 100%, maybe can come from any scope wherein.

Others of the present invention relate to the method that is used for biological substance in the preservation body, and this method comprises that biological substance comes preservation biological substance in the body in oxygen antagonist or other reactive compound of effective dose in the exposure body.

The present invention also relates to delay or reduce the method for the influence of upward biological or the wound in biological, this method comprises exposing oxygen antagonist or other reactive compound of the biological substance of wound risk in effective dose.

In others of the present invention, there is the method that is used in patient's treatment or prevention hemorrhagic shock, this method comprises that the exposure patient is in oxygen antagonist or other reactive compound of effective dose.Alternately, in some embodiments, method prevents by the fatality rate among hemorrhage and/or the patient that hemorrhagic shock causes.Prevent that patient's bleeding is dead or prevent in the method for the fatality rate in the bleeding patients that step comprises that the exposure patient is in oxygen antagonist or other reactive compound of effective dose in this class.In certain embodiments, consider that especially the oxygen antagonist is for example H of chalcogenide chemical compound ₂S.

The method that also comprises the heart rate that is used for reducing biology is as part of the present invention.These class methods relate to the oxygen antagonist of effective dose or other reactive compound contact biological sample or biology.

One embodiment of the invention relate to the method for inducing hibernation in mammal, and this method comprises with the oxygen antagonist of effective dose or other reactive compound contact mammal.

In another embodiment, have the biological method of anesthesia, this method comprises that the biological substance with expectation anesthesia therein is exposed to oxygen antagonist or other reactive compound of effective dose.Consider that described anesthesia can be similar to part or general anesthesia.

The present invention comprises that further the protection mammal is not subjected to the method for radiotherapy or chemotherapy injury, this method be included in before radiotherapy or the chemotherapy or during, contact described mammal with oxygen antagonist or other reactive compound of effective dose.About local application cancer therapy, special consideration also can be with oxygen antagonist or other reactive compound local application to affected organ, tissue and/or cell.In certain embodiments, method can be used for preventing or reduces alopecia in the patients undergoing chemotherapy.Consider that such patient may accept the chemotherapy or the candidate of chemotherapy.In special situation, consider reactive compound is offered the patient as topical gels, be applied to the place of expecting the appearance or having alopecia.

The present invention also covers the oxygen demand that reduces biological substance, and this meaning is meant that the amount of the oxygen that the biological substance survival needs is reduced.This can reach by one or more reactive compounds that effective dose is provided.Usually know that how much oxygen special biological substance needs survive, this also depends on time, pressure and temperature.In certain embodiments of the invention, the demand of biological substance relatively during with the reactive compound that do not have effective dose, the oxygen demand of biological substance reduces approximately, at least about or about at the most 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99 or 100%, maybe can come from any scope wherein.

In other embodiments, there is the method for the hyperplasia disease (for example cancer) among the treatment patient, this method comprises with the oxygen antagonist of effective dose or other reactive compound contact mammal and allows mammal accept heating therapy.

Be used for transplanted organ though method of the present invention can be applied to preservation, others of the present invention relate to receiver's biology.In some embodiments, exist in the method that suppresses the repulsion of organ transplantation in the mammal, this method comprises provides the oxygen of mammal effective dose antagonist or other reactive compound.

Can in biology, finish thermoregulation by adopting oxygen antagonist or other reactive compound.In some embodiments, exist treatment to have hypothermic experimenter's method, this method comprises (a) oxygen antagonist contact experimenter with effective dose, and (b) allows the experimenter accept to be higher than experimenter's ambient temperature then.In other embodiments, the present invention includes the method that treatment has the experimenter of high fever, this method comprises (a) oxygen antagonist or other reactive compound contact experimenter with effective dose.In some cases, the treatment of high fever comprises that also (b) allows described experimenter accept to be lower than at least experimenter's the about 20 ℃ ambient temperature of temperature.As discussed above, expose the experimenter and can be used for other embodiments in non-physiology or controlled temperature environment.Consider that this method can finish with reactive compound usually.

In some cases, the present invention relates to be used for inducing the patient who carries out by-pass operation the method for cardioplegia, this method comprises oxygen antagonist or other reactive compound of using this patient's effective dose.Considering to use can be the heart part, so that protect it.

Others of the present invention relate to the method that is used in patient's prevention hemorrhagic shock, and this method comprises oxygen antagonist or other reactive compound that is administered to patient's effective dose.

And, there is the method be used for promoting wound healing at biology, this method comprises and is administered to biology or wound oxygen antagonist or other reactive compound with effective dose.

And the present invention's covering is used in the mammal prevention or treats neurodegenerative method, and this method comprises oxygen antagonist or other reactive compound that is administered to the mammal effective dose.

The oxygen demand that reduces biological substance is also contained in the present invention, and this meaning is meant that the amount of the oxygen that the biological substance survival needs is reduced.This can realize by one or more reactive compounds that effective dose is provided.Usually know that how much oxygen special biological substance needs survive, this also depends on time, pressure and temperature.In certain embodiments of the invention, the demand of biological substance relatively during with the reactive compound that do not have effective dose, the oxygen demand of biological substance reduces approximately, at least about or about at the most 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99 or 100%, maybe can come from any scope wherein.

Other embodiments of the present invention relate to and are used to prevent alopecia, the method for the alopecia that causes by chemotherapy for example, and this method is by being administered at least a reactive compound that maybe will carry out patient's effective dose of chemotherapy.

Protect therein biological substance avoid damaging or the situation of further damage in; the back can take place approximately in first damage (wound or wound or degeneration) in consideration; at least about or about at the most 30 seconds; 1; 2; 3; 4; 5; 10; 15; 20; 25; 30; 35; 40; 45; 50; 55 minutes; 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24 hours; 1; 2; 3; 4; 5; 6; 7 days; 1; 2; 3; 4; 5 weeks; 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12 months; 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20 or more for many years and can come from wherein the combination in any or scope, biological substance is exposed to the oxygen antagonist.Thereby in other embodiments of the present invention, method comprises the initial assessment of any damage, wound, wound or degeneration.

In certain embodiments of the invention, have the method that is used for the treatment of the patient who is subjected to the influence of hematology's obstacle, hematology's obstacle meaning is meant disease, obstacle or the condition of illness of any hematopoietic cell of influence or tissue.Example comprises drepanocytosis and thalassemia.Therefore, in some embodiments, there is the method for suffering from the patient of drepanocytosis and thalassemia with the reactive compound treatment of effective dose.In other embodiments, exist and to be used for by using or provide the reactive compound of effective dose to strengthen to suffer from cystic fibrosis the patient's of (CF) the method for viability.In other method of the present invention, there is the method that is used in experimenter's treatment cyanide poisoning, this method comprises the reactive compound of using effective dose.In certain embodiments, described chemical compound is H ₂S.

Others of the present invention relate to the method for the cell that is used for preservation one or more and bio-separation, and this method comprises with the oxygen antagonist of effective dose or other reactive compound exposing cell with one or more cells of preservation.Except top and other local cell and cell type of discussing of this application, consider that special consideration is used for the present invention with the shrimp embryo.

And, in some embodiments of the present invention, exist to be used for the hematoblastic method of preservation.Use the technology of present disclosure, reduced or eliminated the shortcoming of prior art.The embodiment that relates to platelet and hydrogen reduction is widely used, and includes but not limited to benefit from the hematoblastic any application of long preservation.

In one embodiment, hydrogen reduction technology can embody in test kit.For example, the selection technology here described of the test kit utilization of selling at present that provides of BectonDickinson with production number 261215.This test kit comprises no Oxygen Generator (for example hydrogen generator), palladium catalyst, no oxygen indicator and airtight, sealable " BioBag ", and top component (platelet in air-locked pocket) is placed in one and seals.

In other embodiments of the present invention, exist, reversibly suppress cell and/or biological metabolic method by the reactive compound of effective dose is provided.Special consideration rotenone is not the chemical compound that adopts in this method or possibility other method of the present invention.And, also consider in some embodiments, get rid of rotenone as reactive compound.Similarly, consideration can be got rid of nitric oxide as reactive compound.

In other embodiments of the present invention; provide method to be used for by the reactive compound of effective dose is provided; strengthen the ability that biological substance response damage or disease enter stagnation, thereby the protection biological substance is avoided infringement or damage, therefore strengthens the survival of biological substance.Relevant embodiment comprises by the reactive compound of effective dose is provided, and allows preparation of thing material or the response damage of initiation biological substance or disease enter the method for stagnation.Other relevant embodiment comprises induces biological substance to enter pre-stagnation, thereby protects biological substance to avoid the method for damaging or damaging.For example; induce the reactive compound of stagnating required dosage to handle or handle to be less than to induce to stagnate the required time with being less than with reactive compound; make biological substance can respond damage or disease and reach the favo(u)red state of stagnation more easily or more completely; and when handling without this reactive compound; biological substance will reach the stagnation of protectiveness level at it, for example be enough to give the preceding death of the anoxybiotic level of biological substance opposing lethal or sustain damage or damage.

Some damage and morbid state cause that biological substance reduces the degree that its metabolism and/or temperature extremely may not reach stagnation.For example, anoxia, ischemia and lose blood all to have reduced and can utilize and offer the amount of the oxygen of the biological substance that utilizes oxygen, thereby reduced oxygen utilization in the biological substance cell, reduced the energy that comes from oxidative phosphorylation and produced, and thereby reduced heat production, cause hypothermia.Depend on the seriousness after harmful injury or disease injury beginning or the progress or the time of experience, " stagnations " may realize maybe not realization.Reduced the threshold value (promptly reaching the seriousness or the persistent period of stagnating required infringement) of inducing stagnation with the reactive compound processing, or it can increase or cause injury sexual stimulus or disease of potentiation stimulates in the biological substance that comes under being in harmful condition and induce stagnation, if wherein said harmful condition is not used in reactive compound and handles, will can not cause stagnating.This activity of reactive compound is relatively measured by destructive stimulus or disease being stimulated the effect (size, kinetics) of inducing stagnation separately and those effects that wherein biological substance exposed in advance, expose simultaneously, expose afterwards or their combination in any is exposed to reactive compound.For example, as what describe among the embodiment 11 of present patent application, be exposed to anoxia (5%O ₂) preceding, mice is exposed to airborne 150ppm H in advance ₂S causes CO ₂Produce approximately and reduce by 2 times.Subsequently, in the anoxia process, the CO in pretreated mice ₂Produce and reduce about 50 times.By contrast, though contrast, do not use H ₂CO in the mice that S handles ₂Producing also decline, but can not reach the anoxia viability of mice, may be because mice just in the dust before reaching stagnation.

In others of the present invention, there is the method that is used at the biology induced hypnotic, this method comprises the reactive compound that biology is exposed to effective dose, wherein said effective dose is less than the amount that can induce stagnation in biology.Term " sleep " is to use according to its meaning common and common in aspect medical science.Sleep is different from unconscious other state, and automatism also is thought of as the state that available method of the present invention obtains.

The present invention also relates to be used to anaesthetize the method for biological substance, this method comprises this material of exposure in the reactive compound of effective dose, and wherein said effective dose is less than the amount that can induce stagnation in biological substance.

In the method for Tao Luning, can reduce the persistent period and/or the amount of the effective dose that is less than the amount that in biological substance, to induce stagnation in the above.Described minimizing can be to induce percent 1 of the amount of stagnation, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96, it can be 1 that 97,98,99 maybe can come from the minimizing of the amount of any scope wherein. reduce, 2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,55 minutes, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 hours, 1,2,3,4,5,6,7 days, 1,2,3,4,5 weeks and/or 1,2,3,4,5,6,7,8,9,10,11, maybe can come from the minimizing of the duration of any scope wherein in 12 months. Alternately, described minimizing can be about offering total effective dose of biological substance, it can be with respect to inducing total effective dose of stagnation in the biology of described species and/or size,

percent

1,2, and 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99 maybe can come from the minimizing of any scope wherein.

Special consider that the present invention can be used for that preservation is used to consume or the biology of laboratory research, for example fly class, batrachia, Fish, mice, rat, Canis familiaris L., shrimp and their embryo.

Method of the present invention can relate to using keeps that biological substance is placed therein or the instrument or the system of the environment that exposes.The present invention includes a kind of instrument, in this instrument, provide oxygen antagonist or other reactive compound, particularly as gas.In some embodiments, described instrument comprises the container with the sample room that holds biological substance, and wherein this container is connected to the gas supply that contains the aerobic antagonist.Special this container of consideration can be firm or it can be flexible, for example sack.

In some embodiments, the present invention is the instrument that is used for the preservation cell, and this instrument comprises: have the container that volume is not more than 775 liters sample room; Carry out first gas supply that fluid is communicated with this sample room, first gas supply comprises carbon monoxide.In further embodiment, this instrument also comprises the cooling unit of regulating the sample indoor temperature, and/or the gas regulator of the amount of the amount of the oxygen antagonist in the adjusting sample room or other active ingredient or the oxygen antagonist in the solution in the sample room or other reactive compound.

Consideration can be useful on the gas supply of second kind or extra gas or be used for second kind or extra gas supply of oxygen antagonist or other reactive compound.Second kind of gas supply can link to each other with the sample room or it can link to each other with first kind of gas supply.As discussed above, extra gas can be non-toxicity and/or non-active gas.

Gas regulator is the part of described instrument in some embodiments of the present invention.Can use one, two, three or more gas regulatoies.In some cases, gas regulator is regulated the gas that offers the sample room from first gas supply.Alternately, its regulation and control offer the gas of sample room or the supply of first kind of gas from second kind of gas supply, or exist and not only be used for first kind but also be used for second kind of gas and supply both actuators.Further consider the amount of the gas of can any gas regulator of programme-control controlling supply sample room and/or the supply of other gas.Described adjusting can be or can not be for specified a period of time.Gas regulator can be arranged, and for any gas supply that directly or indirectly is connected with sample, it can be or can not be can be programme controlled.In some cases, gas regulator is by electronic program.

In some cases, pressure in the sample room and/or temperature can be regulated with pressure regulator or thermoregulator respectively.The same with gas regulator, these actuators can pass through electronic program.Instrument of the present invention also can have the cooling and/or heating unit to obtain temperature discussed above.This unit can or can be by electronic program.

In other embodiments, described instrument comprises a wheeler (wheeled cart), and above container is placed on, or it can have one or more handles.

Special consider, the present invention includes be used for cell, tissue, organ and even the instrument of whole biology, wherein this instrument has: the container with sample room; The first kind of gas supply that is communicated with the sample room fluid, first kind of gas supply comprises oxygen antagonist or other reactive compound; But the gas regulator of electronic program, it regulates the gas of supplying with the sample room from first kind of gas supply.

In some embodiments, described instrument also has configuration provides vacuum in the sample room structure.

And, consider that any oxygen antagonist or other reactive compound described in the application uses with instrument of the present invention.In special embodiment, can use carbon monoxide with this instrument.In other situation, the chemical compound that can use the chalcogenide chemical compound or have the Reducing agent structure.In embodiment further, use reactive compound with described instrument.In special embodiment, a kind of device or its purposes are contained in the present invention.In certain embodiments, this device is the single dose delivery apparatus.In other embodiments, this device is inhaler or aerosol apparatus.In embodiment further, other device includes, but are not limited to injection device, for example pen, pump infusion pump for example, or paster.And, consider that these devices can be or can not be the single dose delivery apparatus.

In addition, the present invention relates to the Screening test method.In some embodiments, screening of candidate substances particularly comprises the ability of protectiveness metabolism agent as oxygen antagonist or reactive compound.This can for example export by the measurement carbon dioxide and finish with any algoscopy described here.Can further characterize or detect any material that embodies oxygen antagonist or other reactive compound characteristic through evaluation.And consideration can be used this material to biological substance and be induced stagnation or produce this material subsequently.

In certain embodiments, exist and be used for reactive compound, comprise the active screening technique of stagnating chemical compound.In addition, screening technique can be used for the oxygen antagonist or be used for being implemented in any other chemical compound of the method for this discussion.In some embodiments, have screening technique, this method comprises a) the Brachydanio rerio embryo is exposed to material; B) measure the heart rate of this embryo; C) in the time of will having this material the heart rate of embryo's heart rate when not having this material relatively, the wherein minimizing of heart rate for example reduces 50% or more, identifies that this material is candidate's reactive compound.Except the Brachydanio rerio embryo, consider also can use other inhuman biology, for example fish, batrachia, fly class, shrimp or their embryo.In further embodiment, measure the heart rate of embryo by the counting beats.In some cases, this can finish by observe embryo under anatomic microscope.

Other screening embodiment comprises: a) expose nematicide in material; B) one or more of the following Cellular respiration factor of mensuration: i) core temperature; Ii) oxygen consumption; Iii) energy; Or iv) carbon dioxide generating; C) in the time of will having this material the Cellular respiration factor of the Cellular respiration factor of nematicide when not having this material relatively, the minimizing of wherein said feature identifies that this material is candidate's reactive compound.In certain methods of the present invention, the special energy of considering to measure nematicide.

In some embodiments, described method at first relates to identifies suitable screening material.In certain embodiments, described material will be chalcogenide chemical compound, reducing substances or have formula I or the structure of formula IV, or at any other chemical compound of this discussion.

Further consider that screening subsequently can be carried out at those more senior or more complicated biologies that are considered to than being used for Preliminary screening or initial screening.Therefore, consideration will detect one or more Cellular respiration factors with further evaluate candidate chemical compound in these other biologies.In certain embodiments, screening subsequently relates to use mice, rat, Canis familiaris L. etc.

Consideration can be used many different biologies or biological substance (other cell or tissue) in screening technique of the present invention, and can measure many different Cellular respiration factors.And, consider in some embodiments of the present invention, carry out repeatedly this class screening simultaneously.

Certainly should be appreciated that; for material being thought candidate's reactive compound (or oxygen antagonist; or stagnate derivant or protectiveness metabolism agent etc.), this material cannot kill biology or cell and effect in algoscopy must be reversible (i.e. feature of Gai Bianing need return to it be exposed to the preceding level of described material).

It will of course be appreciated that any processing method can be used for preparing the medicine that is used for the treatment of or resists specified disease or situation.This includes but not limited to be used for the treatment of the preparation of the medicine of hemorrhagic or hematology's shock, wound and tissue injury, high fever, hypothermia, neural degeneration, sepsis, cancer and wound.And, the present invention includes but be not limited to, preparation is used for the treatment of and prevents death, shock, wound, organ or tissue repels, cancer therapy causes damage, neural degeneration and wound or the medicine of tissue injury.

As discussed above, biological stagnation is not any in the following state: sleep, stupor, dead, anesthesia or epilepsy grand mal.Yet, consider that in some embodiments of the present invention this class state is to use the present invention to arrive the expectation target of method, compositions and manufacture.Any embodiment about an aspect of of the present present invention discussion also is applied to others of the present invention.And, can merge embodiment.

Relate to " exposure " biological substance and also can implement in any embodiment of reactive compound, making provides reactive compound or uses reactive compound to biological substance to biological substance.It is to use according to its common and common meaning that term " provides "." supply or provide and use " (Oxford english dictionary), with regard to the patient, its special reactive compound or directly it is administered to patient's doctor or the behavior that other medical worker carries out of can referring to write out a prescription.

Embodiment in the embodiment part is construed as the embodiment of the present invention that can be applicable to all aspects of the invention.

Term in claims " or " use be used for expression " and/or " unless clearly to indicate be only to refer to that alternatives or alternatives repel mutually, although the present disclosure support only refer to alternatives and " and/or " definition.

In whole the application, term ' approximately " is used to represent to comprise the value of standard deviation of the error of the equipment that is used to measure this value or method.In any embodiment of discussing in the context of the numerical value that is used in combination with term " approximately ", it is about that special consideration can be omitted term.

According to long-standing Patent Law, word " a " and " an ", when in claims or description, " comprising " when being used in combination with word, refer to one or more, unless specifically note.

From following detailed description, other target, feature and advantage of the present invention will become apparent.Yet should be appreciated that, although shown particular of the present invention, this detailed description and specific embodiment only provide in illustrational mode, because will become apparent those skilled in the art from this various changes and correction of describing in detail within the spirit and scope of the present invention.

The accompanying drawing summary

Following accompanying drawing has constituted the part of this description and has been included in interior with further proof some aspect of the present invention.Can be by with reference in these accompanying drawings one or more, the detailed description that this specific embodiments that provides is provided is better understood the present invention.

Fig. 1 people's keratinocyte is survived when being exposed to 100%CO.Cell inverted phase contrast microscope perusal.The keratinocyte number of judging by trypan blue dyeing alives quantitatively, trypan blue dye be cell death indicant.

Fig. 2. the discontinuity of viability when anoxia.In the wild type embryo, be exposed to anaerobic (pure N ₂), malhypoxia ((0.01kPa O ₂, 0.05kPa O ₂Or 0.1kPa O ₂) or slight hypoxia assess the viability in adult stage after 24 hours.All data points are results of at least 3 independent trialss, and inexplicable anthelmintic is removed from sum.

Fig. 3. carbon monoxide is protected from the anoxia injury.In the wild type embryo, be exposed to pure carbon monoxide, 0.05kPa O ₂/ N ₂Or 0.05kPa O ₂Behind/the CO 24 hours, assess the viability of manhood.All data points are results of at least 3 independent trialss, and inexplicable anthelmintic is removed from sum.

Fig. 4 A is when mice is exposed to hydrogen sulfide, and metabolic rate reduces before body core temperature.Mice is exposed to 80ppm (on X-axis 0 minute time) and causes being less than CO in 5 minutes ₂Produce (black line) and reduce about 3 times.This core temperature animal reduces (gray line) before towards ambient temperature.

Fig. 4 B is exposed to the temperature of the mice of hydrogen sulfide.Every line represents to be exposed to the H of 80ppm ₂The continuous measurement of the core temperature of the individual mice of S or room air.Numeral on the vertical axis is a Celsius temperature.On transverse axis, numeral reflection is hour being the time of unit.Test has been write down recovery after carrying out 6 hours.Starting point is at 1:00, and it is about 7:00 that processing in 6 hours finishes.

Fig. 5 is exposed to the core temperature that 80ppm hydrogen sulfide causes mice and approaches ambient temperature.0:00 begins in the time, opens gas, and temperature reduces.At time 6:00, atmosphere gains to room air.Triangle is represented the core temperature by the mice of radiotelemetry mensuration.This is about 39 ℃ when time 0:00.Rhombus is represented ambient temperature, and it drops to 13 ℃ from 23 ℃ in test in initial 3 hours, and rises once more near 23 ℃ during subsequently from 6:00, stablizes when about 9:00.

The speed that Fig. 6 body core temperature descends depends on the concentration of the hydrogen sulfide that gives mice.All lines are represented the core temperature by the single mice of radiotelemetry mensuration.Accept 20ppm and 40ppm H ₂The mice of S shows little core temperature and reduces.Be exposed to 60ppm and induced declining to a great extent of the temperature that begins from about 4:00.The mice that is exposed to 80ppm has shown declining to a great extent of the temperature that begins from about 2:00.

The core temperature that Fig. 7 is minimum.The minimum core temperature of the mice that is exposed to 80ppm hydrogen sulfide of record is 10.7 ℃.Triangle is represented the core temperature of the mice measured by radiotelemetry, its 0 o'clock time from about 39 ℃.Rhombus is represented the ambient temperature with about 23 ℃ of beginnings, and is reduced to and is lower than 10 ℃ when the mid point of test, afterwards itself and then rise near room temperature.

The mice endogenous hydrogen sulfide levels that Fig. 8 A adapts to mild temperature increases.Gray rectangular histogram (left side two) expression adapts to the endogenous H of two independent mices of 4 ℃ ₂S concentration; The rectangular histogram of black (the right two) expression adapts to the endogenous H of two independent mices of 30 ℃ ₂S concentration.Concentration of hydrogen sulfide is measured by GC/MS.

The influence that Fig. 8 B ambient temperature descends to the temperature that relies on hydrogen sulfide.Owing to be exposed to the rate dependent adaptive temperature that core temperature that hydrogen sulfide causes descends.When 1:00, mice is exposed to gas.Triangle is represented the core temperature of the mice of 12 ℃ of the adaptations measured by radiotelemetry.The square expression adapts to the core temperature of 30 ℃ animal.

Fig. 9 illustrates the block chart of breathing gas delivery system according to embodiments of the present invention.

Figure 10 illustrates the sketch map of breathing gas delivery system according to embodiments of the present invention.

Figure 11 is the sketch map of explanation breathing gas delivery system of further embodiment according to the present invention.

Figure 12 illustrates the flow chart of operation according to embodiments of the present invention.

Figure 13 illustrates the sketch map of organized processing gas delivery system according to embodiments of the present invention.

Figure 14 illustrates the flow chart of operation according to embodiments of the present invention.

Figure 15 metabolism suppresses the protection nematicide and avoids the inductive death of hypothermia.The nematicide that is exposed to cold temperature (4 ℃) can not survive after 24 hours.Yet, if during hypothermia, keep oxygen free condition (and before it and kept afterwards 1 hour), the nematicide survival of vast scale.

The CO that Figure 16 is short ₂Pretreatment causes the longest anaerobic survival.The fly class of will growing up is exposed to 100%CO ₂The time that continues to indicate is by using N ₂It is anoxia that flushing causes atmosphere, and then test tube is sealed.After 22 hours, test tube is opened to room air.Before to viability marking, allow the fly class recover 24 hours.

Figure 17 CO ₂Strengthened the anoxia survival to some extent.The fly class of will growing up causes anoxia in low-flow test, directly (do not carry out pretreatment) in room air, or at the CO that is exposed to 100% ₂After 10 minutes.After the specified time, test tube is opened to room air.Before to viability marking, allow the fly class recover 24 hours.

Figure 18 adds 50ppm H in CO ₂S has increased the ratio that survives anoxybiotic fly class.The fly class of will growing up causes anoxia in low-flow test, directly (do not carry out pretreatment) in room air, or is being exposed to the equilibrated 50ppm H with CO ₂Behind the S.

Figure 19 is used for example system that oxygen is removed from platelet and according to the sketch map of the scheme of the embodiment of present disclosure.

Figure 20 A-B has shown the variation of the core temperature of rat (A) that is exposed to hydrogen sulfide and the mice (B) that is exposed to carbon dioxide.

Figure 21 has shown the gas matrix (matrix) of the substep test plan of the concentration that is used to measure reactive compound.

Figure 22 A-B has shown the negative pressure device that can be used for sending or using reactive compound.

The survival of Figure 23 mice in 5% oxygen.With mice or be exposed to 5%O ₂Before be exposed to 30 minutes room air (contrast; Black line; N=9) or be exposed to 5%O ₂Before be exposed to 10 minutes room air, be exposed to 20 minutes 150ppm H then ₂S (experiment; Red line; N=20), and measure their the survival length.Termination test in the time of 60 minutes, and if animal still survive (all survivals in experiment, and in matched group, do not have a survival), they are put back in their cage.

Figure 24 H ₂S has increased the survival under lethal oxygen tension.Figure has shown the result of the test of describing in Figure 23.The x-axle shown that mice survives under low oxygen tension minute being the time of unit.The black rectangular histogram has shown works as H ₂Time-to-live when S does not exist, and light rectangular histogram has shown and has H ₂The time-to-live of S.In the group of back, between oxygen partial pressure is reduced to 5% and 2.5% before, mice is exposed to 150ppm H ₂S.Measure the time-to-live and at all H ₂The time-to-live was at least 60 minutes in the S processed group.

The metabolic rate of the mice of Figure 25 in 5% oxygen.Mice is being exposed to 5%O ₂Before, be exposed to 10 minutes room air, be exposed to 20 minutes 150ppm H subsequently ₂S.Metabolic rate is passed through CO ₂Output measure.CO before exposure ₂Be output as about 2500ppm, at the H that is exposed to 20 minutes ₂Behind the S then metabolic rate reduce about 2 times, and be exposed to 5%O ₂Behind the several hrs, CO ₂Output has reduced about 50 times, and the level before exposing is reduced to about 50ppm.In the time of 6 hours, mice is returned to room air and allows its recovery.These data are come one of mice included among comfortable Figure 23 (experimental group).

Figure 26 is exposed to 100ppb H ₂The mice of Se.The figure illustrates with minute be unit to H ₂The decline (Celsius temperature of Xian Shiing is with showing that the line drawing that descends gradually goes out on the right) of core temperature is followed in the exposure of Se (x-axle), and is accompanied by the reduction (ppmCO that on the left side shows of breathing ₂With showing that the jaggies that reduces draws).

Figure 27 is exposed to 10ppb H ₂The mice of Se.The figure illustrates with minute be unit to H ₂The decline (Celsius temperature of Xian Shiing is with showing that the line drawing that descends gradually goes out on the right) of core temperature is followed in the exposure of Se (x-axle), and is accompanied by the reduction (ppmCO that on the left side shows of breathing ₂With showing that the jaggies that reduces draws, and minimum point occurs when exposing 5 minutes).

Figure 28 H ₂The S pretreatment strengthens the survival of mice under hypoxia condition.Mice is being exposed to 5%O ₂(5%), 4%O ₂(4%), 5%O ₂Be exposed to 4%O in 1 hour then ₂(4%+1 hour 5%) or 5%O ₂Be exposed to 3%O in 1 hour then ₂(3%+1 hour 5%) is preceding, is exposed to 30 minutes room air (no PT) or is exposed to 10 minutes room air to be exposed to 20 minutes 150ppm H subsequently ₂S (PT), and measure their time-to-live length.Termination test in the time of 60 minutes, and if animal still survive then it put back in their cage.

Figure 29 is CO during being converted to lethal hypoxia ₂Generation.In mice, measure and be converted to 5%O ₂Or 4%O ₂The time CO ₂The variation that produces, described mice is exposed to room air 30 minutes (no PT), or is exposed to room air and is exposed to 150ppm H after 10 minutes ₂S20 minute (PT).And, measured and be converted to 5%O step by step ₂1 hour, be converted to 4%O subsequently ₂The time CO ₂The variation that produces.CO has drawn ₂The percentage ratio that changes has marked standard error simultaneously.

The survival of Figure 30 people's keratinocyte when being exposed to 100% carbon monoxide (CO).With inverted phase contrast microscope naked eyes procuratorial work cell.The keratinocyte number of judging by trypan blue dyeing alives quantitatively, trypan blue dye be cell death indicant.

Figure 31 long term exposure is in low-level H ₂S causes the heat resistance in the Caenorhabditis elegans (C.elegans).Compare with the compatriot who in room air, raises separately, adapt to and in room air, contain about 50ppm H ₂The nematicide of the environment of S more has resistance to the lethal effect of rising ambient temperature to 35 ℃ significantly.

Figure 32 long term exposure is in low-level H ₂S has increased the life-span of Caenorhabditis elegans.Be adapted to contain in the room air about 50ppm H ₂The nematicide of the environment of S has the longer life-span with comparing of not being subject to processing.

The example that Figure 33 Si Pula-Dao comes core temperature temporary transient in (Sprague-Dawley) rat to reduce.Be exposed to the rat (Lycoperdon polymorphum Vitt/dotted line) of blended 0.03% hydrogen sulfide of room air or the core temperature that is exposed to the rat (black/solid line) of 15% carbon dioxide/8% oxygen/77% helium and measure.In this test, the processing stage environmental chamber temperature be 10 ℃.When gas is returned to room air, the temperature of environmental chamber is returned to room temperature (22 ℃).In every kind of situation, this is the time point of core temperature when beginning to rise (is about 2 hours for black/solid wire, and be about 7.4 hour for Lycoperdon polymorphum Vitt/dotted line).

Figure 34 under 5 ℃ of ambient temperatures in room air, the mice core temperature of Selenium hydride. during 2 hours 10 minutes that is being exposed to 1.2ppm.

Figure 35 is exposed to the rat core temperature during the room air in environmental chamber under 10 ℃ ambient temperature.Black line has been described the core temperature of rat.Gray line has been described ambient temperature.

Figure 36 is exposed to the core temperature of the rat of 80% helium, 20% oxygen under 7 ℃ ambient temperature.Time on X-axis hour being unit description.Total open-assembly time is about 5 hours (from 9:15AM to 2:15PM).Do not see that core temperature significantly descends.

Figure 37 under 7 ℃ ambient temperature, the core temperature of rat during the helium that is exposed to 15% carbon dioxide, 20% oxygen and 75%.The time that exposes approximately is 2 hours.In time point that temperature begins to rise (after being labeled as 38512.6 point soon) beginning, rat is exposed to room air.During rat is exposed to room air, ambient temperature is returned to room temperature.

Figure 38 is exposed to the rat core temperature of the helium of 15% carbon dioxide, 8% oxygen and 77% under 7 ℃ ambient temperature.Open-assembly time approximately is 4 hours.Gray line has been described ambient temperature.Black line has been described core temperature.The time point that rises at environment and core temperature is the time point of gas when being converted to room air.

Figure 39 is exposed to the core temperature of the Canis familiaris L. of carbon dioxide/helium/oxygen.Dotted line is the core temperature when gas is decontroled (about 24 minutes) and closed (about 55 minutes).Figure 40. be exposed to the core temperature of the Canis familiaris L. of the carbon dioxide that increases concentration.When dotted line is represented gas changed.In the time of about 63 minutes, gas is changed into the room air 9% carbon dioxide from room air.In the time of about 85 minutes, atmosphere carbon dioxide of 9% from room air changed in the room air 12% carbon dioxide.In the time of about 115 minutes, atmosphere carbon dioxide of 12% from room air changed in the room air 15% carbon dioxide.This test finished in the time of about 135 minutes.

Figure 41. the instrument that in screening technique, adopts.

Figure 42 is exposed to oxygen consumption (Lycoperdon polymorphum Vitt rectangular histogram) and the carbon dioxide generating (black rectangular histogram) that hydrogen sulfide carried out the animal at least 1 week in 4 hours and is exposed to the control animal of the same terms that lacks hydrogen sulfide every day.

Figure 43 is exposed to the animal (H that hydrogen sulfide carried out at least 1 week in 4 hours every day ₂S 2900 and H ₂S 2865) and be exposed to the respiratory quotient of the control animal (2893 and 2894) of the same terms that lacks hydrogen sulfide.

The description of illustrative embodiment

I. stagnate

In " stagnation " or " stagnate give birth to ", cell, tissue or organ or biology (being referred to as " biological substance ") are alives, but cell division, grow and make progress necessary cell function and/or metabolism state slows down or even stop.This state is expected in many cases.Stagnation itself can be used as method for preserving, maybe can induce its part as the cryopreservation scheme.Can the preservation biological substance be used for for example studying purposes, be used for transportation, be used for transplanting, being used for the treatment of property processing (therapy for example exsomatizes) and be used to prevent the wound outbreak.Stagnation about whole biology has similar applications.For example, if biology has entered stagnation, can help biological transportation.This may by reduce or remove stress or physical damnification, reduce physics and physiological damage to biology.These embodiments further go through below.Stagnation can be by reducing biological substance to the needs of oxygen and thereby reduced blood flow but useful.This can prolong can be with biological substance from environment separation that earns a bare living and the time cycle that is exposed to the environment of inducing death.

Though reported from recovering (Gilbert etc., 2000) the accident hypothermia relatively for a long time, be interested in recently in the stagnant life of having a mind to induce in the biology.(discussion of any list of references should not be construed as and admits that this list of references constitutes prior art.In fact, some lists of references in this discussion are not prior aries with regard to priority application).After deliberation the in check heating therapy of crossing, and use cold flow of solution to aorta (Tisherman, 2004), induce asystole (Behringer etc., 2003) or the inductive stagnant life of nitric oxide (Teodoro etc., 2004).

The biology of stagnating is different from the biology that is in general anesthesia.For example, the biology (reduction of the Cellular respiration between 2 times to 5 times) that is exposed to the slight stagnation of room air is trembled beginning, and the biology that is in anesthesia will can not.And the slight biology of stagnating of expection is extruded with reaction to toe, and is in the not reaction of biology of anesthesia.Therefore, stagnating with the narcotism that is in common practice is not identical thing.

CO ₂Generation is the direct mark of the Cellular respiration relevant with the metabolism of biology.This may with " CO ₂Emit " difference, CO ₂Emit the CO that refers to that lung is breathed out ₂Amount.Some reactive compound, for example, hydrogen sulfide can suppress the carbonic anhydrase activity in the lung, and this has suppressed carbonic acid and has been converted into CO ₂With and from the release of pulmonary's blood, thereby show the CO that follows ₂The minimizing of emitting, and do not have corresponding cell CO ₂The reduction that produces.

The present invention is based on the chemical compound of observing some type and in biological substance, induce reversible stagnation effectively.Inducing of stagnating discussed in other patent application, comprises following application: U.S. Patent application 10/971,576,10/972,063 and 10/971,575; U.S. Patent application 10/971,576; U.S. Patent application 10/972,063; With U.S. Patent application 10/971,575, all these are applied at this by being incorporated herein by reference.

A. thermoregulation

Stagnation in the homoiothermic animal will influence thermoregulation.Thermoregulation is the characteristic of so-called " homoiothermy " animal, and it makes the biological constant relatively core temperature that keeps, or even when being exposed to (cold or hot) ambient temperature of remarkable change.By inducing the thermotaxic ability of stagnation control is one aspect of the present invention, and allows to be similar to those purposes discussed above.

Thermoregulation can be by promoting in the chamber/device that places its temperature to control biology, extremity or isolating organ or tissue.For example, be similar to the equipment that the greenhouse of high-pressure chamber or chamber kind equipment can hold whole biology and can be connected to the scalable temperature.Also consider littler device for example blanket, sleeve, oversleeve (cuff) or glove (glove) (for example AVAcoreTechnologies, Palo Alto, the CORE CONTROL cooling system of CA, United States Patent (USP) 6,602,277).This chamber/device can be used for increasing or reducing ambient temperature.

B. biological substance

Consider that being used for biological substance of the present invention comprises the material that comes from invertebrates and vertebrates (comprising mammal); Biomaterial comprises biology.Except the people, the present invention also can be used for veterinary or the important mammal of agricultural, and described mammal comprises from those of following type: Canis animals, felid, equine species, bovid, sheep, murine, porcine animals, goat, rodent, lagomorph, wolf section animal and Bears.The present invention also extends to Fish and birds.Other example is open below.

And, the type difference of biological substance.It can be cell, tissue or organ and the different relevant biologies of compositions, method and instrument.Non-interim U.S. Patent application 10/971,576,10/972,063 and 10/971,575 is incorporated herein by reference by full text at this.

In some embodiments, biomaterial is or comprises cell.Consider that cell can be any cell that utilizes oxygen.Cell can be eucaryon or protokaryon.In certain embodiments, cell is an eucaryon.More particularly, in some embodiments, cell is a mammalian cell.Consider that being used for mammalian cell of the present invention includes but not limited to from those following cells: people, monkey, mice, rat, rabbit, hamster, goat, pig, Canis familiaris L., cat, ferret, milch cow, sheep and horse.

And cell of the present invention can be a diploid, but in some cases, cell is monoploid (sexual cell).And cell can be polyploid, aneuploid or akaryote.Cell can be from special organization or organ, for example from the cell with undertissue or organ: the heart, lung, kidney, liver, bone marrow, pancreas, skin, bone, vein, tremulous pulse, cornea, blood, small intestinal, large intestine, brain, spinal cord, smooth muscle, skeletal muscle, ovary, testis, uterus and umbilical cord.And cell also can be characterized as being one of following cell type: the oocyte or the spermatid of platelet, myelocyte, erythrocyte, lymphocyte, adipose cell, fibroblast, epithelial cell, endotheliocyte, smooth muscle cell, Skeletal Muscle Cell, endocrine cell, neurogliocyte, neuron, secretory cell, barrier function cell, contractive cell, absorptive cell, mucomembranous cell, marginal cell (from cornea), stem cell (totipotency, versatility or multipotency), unfertilized or fertilization.

1. different sources

Be the example that can therefrom obtain the source of biological substance below: embodiment of the present invention include but not limited to these examples.

A. mammal

Aspect some, mammal is Monotremata, Marsupialia, Insectivora, dassie rat order (Macroscelidia), Dermoptera, Chiroptera, climbs beastly order (Scandentia), Primates, Desmodonta, Pholidota, Tubulidentata, Lagomorpha, Rodentia, Cetacea, Carnivora, Proboscidea, Hyracoidea, Sirenia, Perissodactyla or Artiodactyla of the present invention.

The example of Monotremata comprises Tachyglossidae (for example echidna belongs to class) and Ornithorhynchidae (for example platypus).The example of Marsupialia comprises Didelphidae (for example didelphid class), little

Section (Microbiotheriidae) (for example Monito del Monte), Caenolestidae (for example caenolestid class), Dasyuridae (for example marsupial mouse class), Numbat section (for example Numbat), Thylacinidae (for example thylacine), Peramelidae (for example bandicoot class), rabbit bandicoot section (for example rabbit bandicoot class), Notoryctidae (for example bag mole class), phalanger section (for example phalanger class), bag Wu section (racoon class for example, bag Wu class), Wu section (for example short and small negative sub-kangaroo (Pygmy Possums)), Macropodidae (kangaroo class for example, the pouch muroid), honey ermine section (for example negative sub-kangaroo (Honey Possum) of Mel), Vombatidae (for example wombat class) and koala section (for example koala class).

Insectivora comprises, for example, ditch tooth Shrew section (for example ditch tooth muroid), Tenrecidae (for example tenrec, otter Shrew), Chrysochloridae (for example chrysochlore class), Erinaceidae (for example Rrinaceus earopaeus, groin), Shrew Murinus section (for example Shrew Murinus) and mole section (for example mole, desman).Dassie rat order order comprises Petromyidae (for example resembling Shrew).Climb beastly order and comprise tree shrew section (for example tree shrew).Dermoptera comprises flying fox section (for example speckle Wu monkey).Chiroptera comprises following section: Pteropidae (flying fox for example, flying fox), Rhinopomatidae (for example Mus tail Vulpes), hollow face Rhinopomatidae (for example hog snout Vespertilio or hornet Vespertilio), Emballonuridae (for example sheath tail bat), hollow face Rhinopomatidae at night (for example hollow face bat), Megadermatidae (for example false vampire), Rhinolophidae (for example rhinolophine), Noctilionidae (for example, bullfight Canis familiaris L. bat, fish people bat (FishermanBats)), the whisker Rhinopomatidae, Phyllostomatidae (for example leaf-nosed bat of New World (New World Leaf-NosedBats)), the long leg Rhinopomatidae, the cigarette Rhinopomatidae, Thyropteridae, suction Rhinopomatidae, Vespertilionidae (for example, common (Common) Vespertilio), the short-tail Rhinopomatidae (for example, the short-tail bat), and wrinkle nose Rhinopomatidae (for example, wrinkle nose bat).

Primates comprise following section: Lemuridae (for example mongoose lemur), mouse lemur section are (for example, the smallmouth mongoose lemur), Indriidae (for example, indri, by the fine hair mongoose lemur), Daubentoniidae (for example, the finger mongoose lemur), Lorisidae (for example, slender loris, baby monkey, galago), Tarsiidae (for example, tarsier), Cebidae (for example New World monkey, marmoset, thin,tough silk hair monkey), hylobratidae (for example Gibbon), pongidae (for example, ape) and Hominidae (for example, the mankind).

The example of Desmodonta comprises Myrmecophagidae (for example, anteater), Bradypodidae (for example, three-toed sloth), two toe Trees Lazy sections (for example, unau) and Dasypodidae (for example tatou).The example of Pholidota comprises Squama Manis section (for example, Squama Manis).The example of Tubulidentata comprises earth pig section (for example, earth pig).The example of Lagomorpha comprises Ochotonidae (for example, pika) and rabbit section (for example hare and rabbit).

Rodentia comprises following section: the mountain Castoridae (for example, mountains and rivers leopard cat (MountainBeavers)), Sciuridae (for example, Sciurus vulgaris, marmot, chipmuck), pocket mouse section (for example, pocket mouse), Heteromyidae (for example, bag mice (Pocket Mice), lattice Lu Mus (KangarooRats) more), Castoridae (for example, beaver), scaletail section (for example, scaletail), Pedetidae (for example, jerboa), Muridae (for example, rat and mice), Gliridae (for example, glirid), the desert Gliridae (for example, the desert glirid), Zapodidae (for example, jerboa (Jumping Mice)), Dipodidae (for example, jerboa), Hystricidae (for example, old world porcupine (Old WorldPorcupines)), Erethizontidae (for example, the New World porcupine), Caviidae (for example, Cavia porcellus, Patagonia hare (Maras)), Hydrochoeridae (for example, capybara), branick's rat section (for example, the long-tail Cavia porcellus), the camel Muridae (for example, paca), agouti section (for example, agouti), the lousiness Muridae (for example, chinchilla, hairy rat), the bristle Muridae (for example, hutia), the beaver Muridae (for example, nutria), the comb Muridae (for example, Tuco-Tucos), the octadentate Muridae (for example, tooth Mus (Octodonts), degus), China's hair Muridae (Abrocomidae) (for example chinchilla), sour jujube Muridae (Echimyidae) (for example, agouti (Spiny Rat)), thryonomyid section (Thryonomyidae) (for example, Caulis Sacchari sinensis Mus (Cane Rat)), Petromyidae (Petromyidae) (for example, Africa rock Mus (African Rock Rat)), Bathyergidae (Bathyergidae) (for example, Mole Rat), and comb toe porcupine section (for example, comb toe porcupine).

Cetacea (Cetacea) order comprises following section: inferior Puffer (Iniidae) (for example Amazon Neomeris phocaenoides G. Cuvier (Amazon Popoise)) Bai Remand platanistidae (Lipotidae), Henghe platanistidae (Platanistidae), general Puffer (Pontoporiidae), beak Ziphiidae (Ziphiidae) (for example, beaked whale (Beaked Whales)), Physeteridae (Physeteridae) (for example, sperm whale (Sperm Whales)), narwhal section (Monodontidae) (for example, beluga (Beluga Whale), narwhal (Narwhal)), Delphinidae (Delphinidae) (for example, dolphin (Marine Dolphin), killer whale (Killer Whale)), Phocaenidae (Phocoenidae) (for example, dolphin (Porpoises)), baleen whale section (Balaenopteridae) (for example, whale), Balaenidae (Balaenidae) (for example, the U.S. whale (Right Whale) of ridge), and gray whale (Eschrichtiidae) (for example, California gray whale (Gray Whale)).

Carnivora (Carnivora) comprises following section: Canidae (Canidae) (for example, Canis familiaris L., Vulpes, wolf, jackal, Coyote), Ursidae (Ursidae) (for example, Bears), Procyonidae (Procyonidae) (for example, racoon, coati, kinkajou, lesser panda), giant panda section (Ailuropodidae) (for example, giant panda), Mustelidae (Mustelidae) (for example, weasel, skunk, badger, Lutra lutra), Viverridae (Viverridae) (for example, Zibethum, civetta) Mongoose section (Herpestidae) (for example, mongoose), aardwolf section (Protelidae) (for example, aardwolf), Hyaenidae (Hyaenidae) (for example, hyena), cat family (Felidae) (for example, cat), Otariidae (Otariidae) (for example, lug sea dog (Eared Seal), sea lion), Odobenidae (Odobenidae) (for example, walrus), and Phocidae (Phocidae) (for example, earless sea dog (Earless Seal)).

Proboscidea (Proboscidea) comprises section: Elephantidae (Elephantidae) (for example, resembling).Hyracoidea (Hyracoidea) comprises Procaviidae (Procaviidae) (for example, hyrax).Sirenia (Sirenia) comprises Dugongidae (Dugongidae) (for example, Dugong dugon (Muller).) and Trichechidae (Trichechidae) (for example, Doris).Perissodactyla (Perissodactyla) comprises equine (Equidae) (for example, horse, donkey, zebra), tapiridae (Tapiridae) (for example, tapir) and Rhinocerotidae (Rhinocerotidae) (for example, rhinoceros).Artiodactyla (Artiodactyla) comprises following section: Suidae (Suidae) (for example, pig, wild boar), west Included-in-the-Appendices section (Tayassuidae) (for example, west Included-in-the-Appendices), Hippopotamidae (Hippopotamidae) (for example, river horse), camelidae (Camelidae) (for example, camel, Llama, vicugna), tragulid section (Tragulidae) (for example, hevrotains), Moschus moschiferous section (Moschidae) (for example, the Moschus moschiferous deer), Cervidae (Cervidae) (for example, deer, elk, elk), Giraffidae (Giraffidae) (for example, giraffe, Okapi), Antilocapridae (Antilocapridae) (for example, pronghorn Antilocapra americana), and Bovidae (Bovidae) (for example, cattle, sheep, Saigae Tataricae, goat).

B. reptile

In some embodiments, biomaterial is reptile or is derived from reptile.Reptile can be Chelonia, Pleurodira, Squamata, Rhynchocephalia or Crocodilia.The reptile of Chelonia can be, for example Carettochelyidae, Chelydridae are (for example, Trionyx sinensis Wiegmann), Cheloniidae (for example Loggerhead Testudinis, Chelomia mydas (Linnaeus).), mud Testudinidae are (for example, leatherback), Testudinidae (for example, the sliding Testudinis of brocade Testudinis, yellow abdomen, terrapin, Limax Testudinis, box tortoise), Sternotherus section (for example, the Moschus Testudinis), Lacertide (Saurotypidae), Testudinidae (for example galapagos tortoise, desert suslik Testudinis, Aldabra, European Testudo elongata, He Man Testudo elongata), Trionychidae (for example, Trionyx sinensis (Wiegmann), thorn Trionyx sinensis Wiegmann) or Plalysternidae.The reptile of Pleurodira can be, for example Plesiochelyidae section (for example, Plesiochelyidae) or Pelomedusidae (for example, marsh side neck Testudinis).

The reptile of Squamata can be, Iguanidae (pappus tree lizard for example for example, pine lion lizard, India bloodsucker (Bloodsucker), Syria thorn tail lizard), dragonfly section (Chamaeleontdidae) (for example, chameleon), the America Iguanidae (for example, peaceful and comfortable lizard, Basilisk, common have a neck Eremiatis argi, mane squama lizard, crown horned toad, Chuckwalla, thin strong rib lizard, male side speckle Eremiatis argi), Gekkonidae (for example, Gecko), Pygopodidae, the America Lacertide (for example, racerunner, the teju lizard), Lacertide (for example, prompt Eremiatis argi, simple eye lizard, viviparous Eremiatis argi, Gekko Swinhonis, the long-tail Eremiatis argi), Huang lizard section, Scincidae (for example, Eumeces), the Africa Lacertide (for example, Sungazer), Dibamidae, Xenosauridae, Anguidae (for example, the Pygopodidae lizard, alligator (Alligator) lizard, Sheltopusik, the Ophisaurus lizard), Helodermatidae (for example, Heloderma suspectum), mother-in-law sieve lizard section, Varanidae (for example, huge lizard), thin Typhlopidae, Typhlopidae, different shield Typhlopidae, tube Serpentis section (for example, the pipe Serpentis), Uropeitidae, dodge Xenopeltidae, boa section (for example, boa, anaconda, the rock boa), wart snake section (for example, wart snake), Colubridae (for example, Boiga, whip snake, sliding Serpentis, eggeater, boomslang, Ratsnake, aesculapian snake, four stricture of vagina Serpentiss, the east rat snake, muzzle You Serpentis, hognose snake, boa, (Montpelier) Serpentis, Grass Serpentis, cloubrid, Zaocys, the rattan Serpentis, the Os Draconis back of the body (Keelback) Serpentis), Elapidae (for example, death adder, Bungarus fasciatus, mamba, the Corallium Japonicum Kishinouye Serpentis, Naja, copperhead, puff adder), Viperidae (for example, poisonous snake, right adder (Right Adders), rattle snake, massasauge, adder), Hydrophiidae (for example, sea snake (Sea Brait)), Amphisbaenidae (for example, slowworm), biped Amphisbaenidae or brachycephaly Amphisbaenidae ((Burrowing) lizard for example, burrows).

The reptile of Rhynchocephalia can be, for example Sphenodontidae (for example, sphenodon).The reptile of Crocodilia can be, for example Chinese alligator section (for example, crocodile, Caiman), Crocodylidae (for example, crocodile) or Gavialidae (for example, ichthyophagy crocodile).

C. Amphibian

Biomaterial of the present invention can be Amphibian or be derived from Amphibian.Amphibian can be, for example the frog or Bufo siccus.The frog or Bufo siccus can be, (for example for example save Ranidae, the Lai Xiejie frog), tail toad section (for example, the tail toad), brachycephalia section (for example, the gold frog and shield (shield) Bufo siccus), Bufonidae (for example, true (true) Bufo siccus), attached Ranidae (for example, the glass frog and the leaf frog), the curare Ranidae (for example, the curare frog), Discoglossidae (for example, fire-bellied toad), natural pond toad section (for example, ghost (ghost) frog), shovel nose Ranidae (for example, the shovel nose frog), Hylidae (for example, New World Rhacophorus), the kevazingo Ranidae (for example, the Africa Rhacophorus), (for example slide sole of the foot toad section, the New Zealand frog), Leptodactylidae (for example, neotropical realm frog frogs), horned frog section (for example, the South Asia frog), Microhylidae (for example, Ji Wa (microhylid frogs)), Testudinis toad section (for example, Australia frog), Pelobatidae (for example, uproot sufficient toad), (for example close attached toad section, speckle shovels sufficient toad), Pipidae (for example, the aglossate frog), many dactylus toad section (for example, blaming (paradox) frog), Ranidae (for example, dwell the bank frog and its (true) frog), Rhacophoridae (for example, New World Rhacophorus), point kiss toad section (for example, Darwin frog), different tongue cave toad section (for example, mud (burrowing) Bufo siccus), plug tongue Ranidae (for example, the Seychelle frog), Caudata (for example, newt) or Caeciliformes (for example, caecilian).

Amphibian can be a newt.Newt can be, for example Ambystomatidae (for example, boring the ground newt), Amphiuma section (for example, Amphiuma), latent cheek salamander section are (for example, Andrias davidianus Blanchard and hellbender), the huge mud eel in land section (for example, the huge mud eel in sword land), Hynobiidae (for example, Asia newt), Plethodontidae (for example, plethodont), Proteidae are (for example, Necturus and speckle mud mud eel), torrent mud eel section (for example, torrent (torrent) newt), Salamandridae (for example, Cynops orientalis (David) and newt) or mud eel section (for example, sirens).Alternately, Amphibian can be a caecilian.Caecilian can be that for example true earthworm section (for example, true earthworm), fish mud eel section are (for example, there is the tail caecilian in the Asia), kiss earthworm section (for example, there is the tail caecilian neotropical realm), the earthworm section of wriggling (for example, African caecilian), blind trip earthworm section (for example, aquatic caecilian) or blind tail earthworm section (for example, India's caecilian).

D. birds

Biomaterial of the present invention can be that bird maybe can be derived from bird.Bird can be, for example Anseriformes (for example, aquatic bird), Apodiformes (for example, Hummingbird and swift), Caprimulgiformes (for example, night bird), Phasianidae (for example, bank bird (shorebirds)), Ciconiiformes (for example, stork), Coliiformes (for example, coly), Columbiformes (for example, Columba livia and turtledove Columba livia), coraciiformes (for example, kingfisher), the curassow order (for example, young guan, curassow, guan, megapod), Cuculiformes (for example, Cuculus polioephalus, hoatzins, the any of several broadleaf plants cuckoo), Falconiformes (for example, Lycoperdon polymorphum Vitt bird) round the clock, Galliformes (for example, chicken sample bird), Gaviiformes (for example, diver), Gruiformes (for example, coot, crane, Carnis Rallus aquaticus), Passeriformes (for example, roost (perching) bird), Pelecaniformes (for example, pelican), Phoenicopteriformes (for example, flamingo), Picidae (for example, Picus canus) Grebe Ti order (for example ， Grebe Grebe), the albatross order (for example, the osing seabird), Psittaciformes (for example, Psittacula alexandri fasciata), Sphenisciformes (for example, penguin), Strigeata (for example, owl), Struthioniformes (for example, cassowary (cassowaires), Dromaius novaehollandiae, apteryx, Ostriches, rhea), shape order (for example, kiwi), Trogoniformes (for example, sting cuckoo) or button quail order (for example, button quail).

E. Fish

Biomaterial of the present invention can be that fish maybe can be derived from fish.Fish can be, for example Gadiformes (for example, paddlefish, spoon fish (oonfishes) and Acipenser Sinensis), Polypteriformes (for example, polypterus bichir (bichirs), polypterus bichir (birchers), fin fish (lobed-finned pike) and phragmites communis fish (reed fishes)), hand hay cutter Chinese fish eyes (for example, rainbow fish and Silverside), chin pin argon order (for example, fish and needlefish), BERYCIFORMES, the milk fish order, tooth Cyprinus carpio order (for example, the Medaka fish) Bao Fang Fu order (for example Bao Fang Fu), perverse fish eyes (for example, Solenognathus and stickleback), Mugiliformes (for example, mullet), Pagasus natans (for example, stomiatid and Pegasus laternarius (cuvier).), Perciformes (for example, perch sample (perch-like) fish), Pleuronectiformes (for example, flatfish, Pleuronectidae and tongue sole), Rockfish shape order (for example, scorpion fiss and Cottus pollux Gunther.), strange BERYCIFORMES, (for example close gill fish eyes, Monopterus albus (Zuiew)), Tetraodontiformes (for example, the case Puffer, filefish, squama Puffer, Fugu ocellatus, bessycerka and case Puffer), zeus japonicus (for example, globefish nose fish, dory and triangular bream), the silverside catalogue, Clupeiformes (for example, long tail anchovy and Mylopharyngodon piceus), fairy maiden's fish eyes Bei Suo Fish order, Anguilliforme (for example, eel), Elopiformes (for example, A Bangyu (arpons)), Notacanthiformes (for example, thorn eel and back of the body stickleback), the bursa pharyngea fish eyes, Lampridiform (for example, opah and Trichiurus lepturus), the characin order (for example, hare fish (leporins) and Piranha), Cypriniformes (for example, Hemiculter lcucisculus (Basil.), Myxocyprinus asiaticus, Brachydanio rerio) Shu Xi order (for example, milk fish and shell ear fish (shellears)), naked back of the body electric eel order, catfish shape (for example, Silurus asotus fish), trout-perch order (Aphredoderiforme) (for example, cave fish and perch), Batrachoidiformes, Gadiformes (for example, cod and morrhua), Gobiesociformes Monkfish Angler order (for example Monkfish Angler), weasel Blenniidae order, the trout-perch order (for example, trout-perch), palpus Wei order (for example, palpus Wei), Cetoinimiformes Zhi Gan order, the pike order (for example, mudminnow and pike), the cucumber fish eyes (for example, argentines and smelt), salmon shape order (for example, salmon), Myctophiformes (for example, myctophids (Latern Fishes)), the pigtail fish eyes, the meloschisis fish eyes, the bowfin order (for example, bowfin), the semionotid order (for example, garpike), the Solenognathus order (for example, Solenognathus and Hippocampus), single fish glue Dipnoi (for example, barramunda), two fish glue Dipnois (for example, lepidosiren and Protopterus) or Coelacanthiformes (for example, coelacanth).

F. invertebrates

Biomaterial can be invertebrates or be derived from invertebrates.Invertebrates can be, for example, Phylum porifera (for example, sponge), Cnidaria (for example, Jellyfish, hydra, sea anemone, Portuguese man-of-war and Corallium Japonicum Kishinouye), Phylum platyhelminthes (for example, flatworms, comprise turbellarian worm, trematodiasis and cestode), Nemathelminthes (for example, roundworm comprises wheel animalcule and nematicide), Mollusca (for example, Mollusca, Limax, Limax, Octopus, squid), Annelida (for example, the anthelmintic of merogenesis, comprise Lumbricus, Hirudo and ocean anthelmintic), Echinodermata (for example, Asterias amurensis Lutken, Stichopus japonicus, sand dollar, Hemicentrotus seu Strongylocentrotus), Phoronida (for example, Phoronida), Tardigrada (for example, tardigrades), Acanthocephala (for example, spiny-headed worm), Ctenophora (for example, ctenophore) or arthropod (for example, Aranea, crustacean, myriapod, centipedes, insects).

Arthropod can be, for example coleoptera (for example, beetle), Diptera (for example, fly), Hymenoptera (for example, Formica fusca, Apis, wasp), Lepidoptera (for example, butterfly, moth), Mecoptera (for example, scorpion fly), Megaloptera, Neuroptera (for example, lacewing and relevant animal), Siphonaptera (for example, flea), Strepsiptera (for example, parasitic insect and twisted winged insect), Trichoptera (for example, caddis fly), Anoplura (for example, suck louse), Semiptera (for example, Semiptera snake and their relevant animal), Mallophaga (for example, sting louse), Corrodentia (for example, psocid), Thysanoptera (for example, thrips), Orthoptera (for example, locust, Cicadae), Dermaptera (for example, earwig), Dictyoptera, Embioptera (for example, embiid), Grylloblattodea, Mantidis ring order (for example, dry measure used in former times scholar (gladiators)), _ wing order (for example, perlid), Zoraptera (for example, exhausted wing order (zorapterans)), Ephemerida (for example, mayfly), Odonata (for example, Aeschna melanictera and damselfly), Phasmatodea (for example, walkingstick), Thysanoptera (for example, moth) Archaeognatha, Collembola (for example, snow fly (snow flies) and springtail), Chcilopoda (for example, centipedes), Diplopoda (for example, myriapod), lizard foot class (for example, Pauropoda, guiding principle (pauropodans) and before grow the hole class), Symphyla (for example, false Scolopendra (pseudocentipedes) and Symphyla (symphylans)), Malacostraca (for example, Eriocheir sinensis, krill, the ball shrimp, astacus), jaw foot guiding principle, Branchiopoda (for example, branchiopod animal), shrimp guiding principle, Ostracoda (for example, ostracode), oar foot guiding principle, gill tail guiding principle, Cirripedia (for example, barnacle), Arachnoidea (for example, arachnidea, comprise the whip spider, Aranea, daddy longlegs, blind spider, miniature scorpion (microscorpions), book scorpion (book scorpions), pseudo-scorpion, false scorpion, Scorpio, keep away a day worm, day Aranea and urine spider (uropygids)), Merostomata (for example, king crab) or extra large spider guiding principle (for example, sea spider).

G. fungus

Biomaterial of the present invention can be that fungus maybe can be derived from fungus.Fungus can be, for example Ascomycota (ascomycetes), Basidiomycota (Clavaria), chytrid door (chytrid), Fungi Imperfecti door or Zygomycota.Fungus can be Rhizopus (Rhizopus), Pilobolus (Pilobolus), the nodal plexus spore belongs to (Arthrobotrys), Eurotium (Aspergillus), Allomyces (Allomyces), Chytridium (Chytridium), Agaricus (Agaricus), Amanita (Amanita), Cortinarius (Cortinarius), neurospora (Neurospora), morchella (Morchella), Saccharomyces (Saccharomyces), pichia (Pichia), Candida (Candida), Schizosaccharomyces (Schizosaccharomyces) or Claviceps (Ergot).In special embodiment, fungus can be saccharomyces cerevisiae (Saccharomyces cerevisiae), schizosaccharomyces pombe (Schizosaccharomycespombe), Candida albicans (Candida albicans) or Pichia sp. (Pichia pastoris).

H. plant

Biomaterial of the present invention can be a plant or can be plant-derived.Plant can be bryophyte (for example, mosses, liverwort, hornwort), lycopod (for example, lycopods, liberty), sphenopsid (for example, equisetales), pteridophyta (for example, fern), Cycadopsida plant (for example, cycad), MATENG class are (for example, Gnetum, Ephedra, Your Highness Cymbidium), confierophyte (for example, coniferals), ginkgopsida (for example, Semen Ginkgo) or flowering plant (for example, phanerogam).Flowering plant can be monocotyledon or dicotyledon.Monocotyledonous limiting examples comprises Semen Tritici aestivi, corn, naked barley, Oryza sativa L., turfgrass, Sorghum vulgare Pers., foxtail millet, Caulis Sacchari sinensis, Bulbus Lilii, Rhizoma Iridis Tectori, Folium Agaves variegatae, Aloe, orchid, bromelia and babassu.The limiting examples of dicotyledon comprises Nicotiana tabacum L., Fructus Lycopersici esculenti, Rhizoma Solani tuber osi, Semen sojae atricolor, Helianthi, Herba Medicaginis, Semen Brassicae Campestris, Flos Rosae Rugosae, arabidopsis, coffee tree, citrus fruit, bean, Herba Medicaginis and Cotton Gossypii.

I. protista

Biomaterial of the present invention can be that protista maybe can be derived from protista.Protista can be Rhodophyta (for example, red algae), Phaeophyta (for example, Brown algae, Sargassum), Chlorophyta (for example, chlorella), Euglenophyta (for example, Euglena), myxomycota (for example, Acarasiales), Oomycete are (for example, fish molds, downy mildew, potato blight) or Bacillariophyta (for example, diatom).

J. prokaryote

Aspect some, biomaterial is prokaryote or comes from prokaryote of the present invention.In certain embodiments, prokaryote is archeobacteria (archaebacteria).Archeobacteria can be for example ancient bacterium door (Euryarchaeota) of spring, wide ancient bacterium door (Euryarchaeota), first ancient bacterium door (Korarchaeota) or the ancient bacterium door (Nanoarchaeota) of receiving.In some aspects, the ancient bacterium door of spring is bacillus guiding principle (Halobacteria), methagen guiding principle (Methanobacteria), methane coccus guiding principle (Methanococci), methane germ guiding principle (Methanomicrobia), the folded Coccaceae (Methanosarcinae) of methane, methane fire Gammaproteobacteria (Methanopyri), ancient spherical Gammaproteobacteria (Archeoglobi), pyrogen body guiding principle (Thermoplasmata) or hot-bulb Gammaproteobacteria (Thermococci).The concrete limiting examples of archeobacteria comprises: hyperthermophilic archaeon strain (Aeropyrum pernix), Methanococcus jannaschii (Methanococcus jannaschii), Dead Sea salt box bacterium (Halobacterium marismortui) and thermoplasma acidophilum (Thermoplasma acidophilum).

In certain embodiments, prokaryote is eubacteria (Eubacteria).Eubacteria for example can be, actinomycetes door (Actinobacteria), produce water bacterium door (Aquificae), Bacteroidetes (Bacteroidetes), green sulfur bacteria (Green sulfur bacteria), chlamydia door (Chlamydiae), wart germ door (Verrucomicrobia), green curved bacterium door (Chloroflexi), pan bacterium door (Chrysiogenetes), Cyanophyta (Cyanobacteria), deferrization bacillus door (Deferribacteres), abnormal cocci-hot bacterium the door (Deinococcus-Thermus) of dwelling, net group bacterium door (Dictyoglomi), cellulomonas door (Fibrobacteres)/acidfast bacilli door (Acidobacteria), Firmicutes (Firmicutes), Fusobacterium door (Fusobacteria), bud Zymomonas mobilis door (Gemmatimonadetes), nitrated spirillum door (Nitrospirae), omnivorous Bacteriophyta (Omnibacteria), floating mycete door (Planctomycetes), Proteobacteria (Proteobacteria), spirillum door (Spirochaetes), thermally desulfurizing bacillus door (Thermodesulfobacteria) or thermobacillus door (Thermotogae).The limiting examples of actinomycetes door comprises the antibacterial of actinomyces (Actinomyces), Arthrobacter (Arthrobacter), corynebacterium (Corynebacterium), Frankia (Frankia), micrococcus (Micrococcus), Micremonospora (Micromonospora), Mycobacterium (Mycobacterium), propionibacterium (Propionibacterium) and streptomyces (Streptomyces).Actinomycetic instantiation comprises Mycobacterium leprae (Mycobacterium leprae), Mycobacterium tuberculosis (Mycobacterium tuberculosise), Mycobacterium avium (Mycobacteriumavium), corynebacterium glutamicum (Corynebacterium glutamicum), propionibacterium acnes (Propionibacterium acnes) and Rhodococcus equi (Rhodococcus equi).

The limiting examples of producing water bacterium door comprises product water Pseudomonas (Aquifex), Hydrogenivirga, hydrogen Bacillus (Hydrogenobacter), hydrogen bacillus (Hydrogenobaculum), Thermocrinis, Hydrogenothermus, Persephonella, Sulfurihydrogenibium, Pasteur's silk Pseudomonas (Balnearium), removes Thiobacillus (Desulfurobacterium) and hot vibrio (Thermovibrio).The limiting examples of Firmicutes comprises the antibacterial of tooth born of the same parents Bacillus (Bacilli), fusobacterium (Clostridia) and gentle film bacterium (Molecutes).The instantiation of Firmicutes comprises: harmless listeria spp (Listeria innocua), Listeria monocytogenes (Listeriamonocytogenes), bacillus subtilis (Bacillus subtilis), anthrax bacillus (Bacillusanthracis), bacillus thuringiensis (Bacillus thuringiensis), aurococcus (Staphylococcus aureus), clostridium acetobutylicum (Clostridiumacetobutylicum), clostridium difficile (Clostridium difficile), clostridium perfringens (Clostridium perfringens), mycoplasma genitalium (Mycoplasma genitalium), mycoplasma pneumoniae (Mycoplasma pneumoniae), mycoplasma pulmonis (Mycoplasma pulmonis), streptococcus pneumoniae (Streptococcuspneumoniae), streptococcus pyogenes (Streptococcus pyogenes), Streptococcus mutans (Streptococcus mutans), lactococcus lactis (Lactococcus lactis) and enterococcus faecalis (Enterococcus faecalis).

The limiting examples of chlamydia door/wart germ door comprises antibacterial for example chlamydia trachomatis (Chlamydia trachomatis), Chlamydia pneumoniae (Chlamydia pneumoniae), chlamydia psittaci (Chlamydia psittaci).The limiting examples of the abnormal cocci-hot bacterium door of dwelling comprises that abnormal cocci belongs to and Thermus.

Proteobacteria is a gram negative bacteria.The limiting examples of Proteobacteria comprises Escherichia (Escherichia), Salmonella (Salmonella), vibrio (Vibrio), rickettsiae (Rickettsia), Agrobacterium (Agrobacterium), Brucella (Brucella), rhizobium (Rhizobium), eisseria (Neisseria), Bo Erdeshi bacillus (Bordetella), Burkholderia belongs to (Burkholderia), Buchnera, yersinia's genus (Yersinia), klebsiella (Klebsiella), Proteus (Proteus), Shigella (Shigella), haemophilus (Haemophilus), Pasteurella (Pasteurella), Actinobacillus (Actinobacillus), Legionella (Legionella), Mannheimia, Coxiella (Coxiella), Aeromonas (Aeromonas), soil draws Pseudomonas (Francisella), Moraxella (Moraxella), Rhodopseudomonas (Pseudomonas), Campylobacter (Campylobacter) and Helicobacterium (Helicobacter).The instantiation of Proteobacteria comprises: Kang Shi rickettsia (Rickettsia conorii), Rickettsia prowazekii (Rickettsia prowazekii), muricola (Rickettsia typhi), Ehrlichia bovis (Ehrlichiabovis), Agrobacterium tumdfaciens (Agrobacterium tumefaciens), brucella melitensis (Brucella melitensis), root nodule bacteria (Rhizobium rhizogenes), meningitis naphthalene plucked instrument Salmonella (Neisseria meningitides), parapertussis is won Dai Shi (bar) bacterium (Bordetella parapertussis), pertussis is won Dai Shi (bar) bacterium (Bordetellapertussis), glanders Burkholderia (Burkholderi mallei), melioidosis Burkholderia (Burkholderi pseudomallei), gonococcus (Neisseria gonorrhoeae), escherichia coli (Escherichia coli), intestinal Salmonella (Salmonella enterica), Salmonella typhimurium (Salmonella typhimurium), Yersinia pestis (Yersiniapestis), klepsiella pneumoniae (Klebsiella pneumoniae), intestinal colitis yersinia (Yersinia enterocolitica), proteus vulgaris (Proteus vulgaris), shigella flexneri (Shgella flexneri), shigella sonnei (Shigella sonnei), shigella (Shigella dysenterica), influenza (bloodthirsty) bacillus (Haemophilusinfluenzae), pasteurella multocida (Pasteurella multocida), actinobacillus actinomycetem comitans (Actinobacillus actinomycetemcomitans), Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), Haemophilus somnus (Haemophilus somnus), legionella pneumophila (Legionellapneumophila), mannheimia haemolytica (Mannheimia haemolytica), vibrio cholera (Vibrio cholerae), vibrio parahaemolyticus (Vibrio parahaemolyticus), rickettsia burneti (Coxiella burnetii), Aeromonas hydrophila (Aeromonashydrophila), aeromonas salmonicida (Aeromonas salmonicida), soil draws hot Frances Salmonella (Francisella tularesis), Moraxella catarrhalis (Moraxellacatarrhalis), Pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas putida (Pseudomonas putida), campylobacter jejuni (Campylobacter jejuni) and helicobacter pylori (Helicobacter pylori).

The limiting examples of spirillum door comprises the antibacterial of short Spirochaetaceae (Brachyspiraceae), Leptospiraceae (Leptospiraceae) and Spirochaetaceae (Spirochaetaceae).The instantiation of spirillum door comprises borrelia burgdorferi (Borrelia burgdorferi) and treponema pallidum (Treponema pallidum).

2. dissimilar biological substances

Method of the present invention and instrument can be applicable to biology.Can induce biological stagnation maybe can induce biological cell, tissue and/or intraorganic stagnation.Consider to be used for the inventive method and instrument, can induce the biological substance of stagnation to only limit to comprise the scope of utilizing the energy-producing cell of oxygen therein.

Can in the cell that comprises the heart, lung, kidney, liver, bone marrow, pancreas, skin, bone, vein, tremulous pulse, cornea, blood, small intestinal, large intestine, brain, spinal cord, smooth muscle, skeletal muscle, ovary, testis, uterus and umbilical cord, tissue or organ, induce stagnation.

And, can in the cell of following type, induce stagnation: the oocyte or the spermatid of platelet, myelocyte, erythrocyte, lymphocyte, adipose cell, fibroblast, epithelial cell, endotheliocyte, smooth muscle cell, Skeletal Muscle Cell, endocrine cell, neurogliocyte, neurocyte, secretory cell, barrier function cell, contractive cell, absorptive cell, mucomembranous cell, marginal cell (from cornea), stem cell (totipotency, versatility or multipotency), unfertilized or fertilization.

In addition, can comprise in fruit, flower, leaf, stem, seed, the cutting and induce stagnation in the part of plant or plant.Plant can be agriculture, medicinal or ornamental plant.The shelf time or the pathogen-resistance of inducing the part that can strengthen whole strain plant or plant in plant, stagnated.

Method of the present invention and instrument can be used for the stagnation of biological substance in the inductor.This can be used for protecting and/or the preservation biological substance or biological self or be used to prevent infringement or damage to them or whole biology.

3. algoscopy

Stagnation can be by many modes, comprise the amount (the indirect measurement of Cellular respiration) of the amount of the oxygen that consumes by quantitative biological sample, carbon dioxide that sample produces or measure by characterizing motoricity.

For the speed of measuring oxygen consumption or the speed of carbon dioxide generating, biological substance is placed in the chamber with two openings (being used for the gas input and output) sealing.Gas (room air or other gas) is fed in the described chamber with given flow velocity and flow out outlet in this chamber, to keep about 1 atmospheric pressure.Before being exposed to this chamber and afterwards, with this gas by carbon dioxide indicator and or the oxygen detection device measure the amount of every kind of chemical compound in (per second) this admixture of gas.More time dependent these values obtain the speed of oxygen consumption or carbon dioxide generating.

II. oxygen antagonist and other reactive compound

The present invention relates to method, compositions and manufacture, to such an extent as to relating to one or more, described method, compositions and manufacture can act on the reagent that biological substance produces many effects, described effect comprises, but be not limited to, induce stagnation, strengthen or increase viability, reversibly suppress metabolism, inducing cell or biological metabolism and activity, minimizing oxygen demand, reduce or prevent damage, prevent the damage of ischemia type, prevent agingly or old and feeble, and/or obtain multiple therapeutic and use in this discussion.In certain embodiments, the suitable lattice of described reagent are as " reactive compound ".

In some embodiments, described reagent is the oxygen antagonist, and it can directly or indirectly work.Oxygen metabolism is that a basic demand aerobic respiration of aerobic metazoa life is responsible in the most animals most energy and produces, and also is used to keep and carries out the necessary oxidation-reduction potential of important cell effect.When hypoxia, the oxygen availability of reduction causes in the final step of electron transport chain electronics to the invalid transfer of molecular oxygen.This invalid not only caused reduction that aerobic energy produces and but also cause the increase of destructive free-radical generating, this mainly is because at the too early release of composite I II place electronics and the O by cytochrome oxidase formation ₂ ^-(Semenza, 1999).Limited energy supply and radical damage can disturb basic cell processes, and for example protein synthesis and cell membrane are polar keeps (Hochachka etc., 1996), and will finally cause cell death.

In other embodiments, described reagent is protectiveness metabolism agent.Metabolism is generally understood as and refers to life needed (in cell or the biology) chemical process; They relate to multiple reaction and keep energy generation and synthetic (anabolism) and decomposition (catabolism) complicated molecule.

In certain embodiments of the invention, reactive compound has the chemical constitution of listing as formula I described here or IV, or the precursor of formula I or IV.

At this number of chemical structure and chemical compound have been described.Be applied to be used to be described in these structures of this discussion and the term of chemical compound to give a definition:

" alkyl ", using separately or for example in " aryl alkyl ", " aminoalkyl ", " alkylthio ", " the cyano group alkyl " and " hydroxy alkyl " during use, referring to have an extremely straight chain or ramose group of about 20 carbon atoms at other term.Term " low alkyl group " refers to C ₁-C ₆Alkyl group.As used herein, the term alkyl comprises and uses hydroxyl; halogen (F for example; Cl; Br; I); haloalkyl; alkoxyl; halogenated alkoxy; alkylthio group; cyano group; isocyano group; carboxyl (COOH); alkoxy carbonyl group (COOR); acyl group; acyloxy; amino; alkyl amino; urea (--NHCONHR); sulfydryl; alkylthio group; sulfur oxygen base (sulfoxy); sulfonyl; aryl sulfonyl; alkyl sulphonyl; sulfonamido; Arenesulfonyl amino; heteroaryl; heterocyclic radical; Heterocyclylalkyl; acylamino-(amidyl); the alkyl carbonimidoyl; amidino groups; guanidine radicals (guanidono); diazanyl; hydrazides; sulfonyl sodium (SO ₃Na), sulphonyl sodium alkyl (RSO ₃Those groups of replacing of group such as Na).These examples of groups include, but is not limited to methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, sec-butyl, the tert-butyl group, amyl group, isopentyl, hexyl etc.

" hydroxy alkyl " refer to replace with one or more oh groups, alkyl group as defined herein.The hydroxy alkyl examples of groups includes but not limited to methylol, 2-ethoxy, 2-hydroxypropyl, 3-hydroxypropyl, 2-hydroxyl butyl, 3-hydroxyl butyl, 4-hydroxyl butyl, 2,3-dihydroxypropyl, 1-(methylol)-2-ethoxy, 2,3-dihydroxy butyl, 3,4-dihydroxy butyl and 2-(methylol)-3-hydroxypropyl etc.

" aryl alkyl " refers to radicals R ' R-, and wherein alkyl group " R " replaces with aromatic yl group " R ' ".The example of aromatic yl alkyl group includes but not limited to benzyl, phenethyl, 3-phenyl propyl etc.

" aminoalkyl " refers to group H ₂NR '-, wherein alkyl group replaces with amino group.This examples of groups comprise aminomethyl, aminoethyl etc." alkyl amino alkyl " refers to the alkyl group with the alkylamino group replacement.

" alkyl sulfonyl amino " refers to be attached to the sulfonamido of alkyl (S (O) as defined herein ₂-NRR ').

" sulfane base " refers to that wherein alkyl replaces with one or more sulfydryls." alkyl alkylthio base " refers to that wherein alkyl replaces with one or more alkylthio groups.Example includes but not limited to methyl sulfidomethyl, ethyl sulfur isopropyl etc." sulfur alkyl aryl " refers to that wherein alkyl replaces with one or more arylthios as defined herein.

" carboxyalkyl " refers to group-RCO ₂H, wherein alkyl group carboxyl substituted.Example includes but not limited to carboxymethyl, carboxyethyl, carboxylic propyl group etc.

" alkylidene " refers to the bridging alkyl group.

Term " alkenyl " refers to undersaturated, acyclic hydrocarbon group, because it contains at least one two key.This alkenyl contains about 2 to about 20 carbon atoms.Term " low-grade alkenyl " refers to C ₁-C ₆Kiki alkenyl group.As used herein, the term alkenyl group comprises those groups that replace with alkyl group.The example of suitable alkenyl group comprise acrylic, 2-Chloroallyl, butene-1-Ji, isobutenyl, penta-1-alkene-1-base, 2-2-methyl isophthalic acid-butene-1-Ji, 3-methyl-1-butene-1-base, oneself-2-alkene-1-base, 3-hydroxyl oneself-1-alkene-1-base, heptan-1-alkene-1-base and suffering-1-alkene-1-base etc.

Term " alkynyl " refers to undersaturated, acyclic hydrocarbon group, because it contains one or more triple bonds, this group contains about 2 to about 20 carbon atoms.Term " low-grade alkynyl " refers to C ₁-C ₆Alkynyl group.As used herein, the term alkynyl comprises those groups that replace with alkyl group.The example of suitable alkynyl group comprises acetenyl, propinyl, hydroxypropyn base, fourth-1-alkynes-1-base, fourth-1-alkynes-2-base, penta-1-alkynes-1-base, penta-1-alkynes-2-base, 4-methoxyl group penta-1-alkynes-2-base, 3-methyl fourth-1-alkynes-1-base, own-1-alkynes-1-base, own-1-alkynes-2-base, own-1-alkynes-3-base, 3,3-dimethyl-ethyl acetylene-1-base etc.

" alkoxyl " refers to radicals R ' O-, and wherein R ' is an alkyl group as defined herein.Example includes but not limited to methoxyl group, ethyoxyl, propoxyl group, butoxy, isopropoxy, tert-butoxy etc." alkoxyalkyl " refers to the alkyl group with one or more alkoxyls replacements.Example includes but not limited to methoxy, ethoxyethyl group, methoxy ethyl, isopropoxy ethyl etc.

" alkoxy carbonyl group " refer to radicals R-O-C (O)-, wherein R is an alkyl group as defined herein.The example of alkoxycarbonyl group includes but not limited to methoxycarbonyl group, carbethoxyl group, the second month in a season-butoxy carbonyl, the different third oxygen carbonyl etc.The alcoxyl thiocarbonyl refer to R-O-C (S)-.

" aryl " refers to the monovalent aromatic carbon ring group be made up of an independent ring or one or more condensed ring; at least one ring in the fused rings is fragrant in nature; it can be chosen wantonly by one or more; preferred one or two following substituent group replaces; unless otherwise noted, for example: hydroxyl; halogen (F for example; Cl; Br; I); haloalkyl; alkoxyl; halogenated alkoxy; alkylthio group; cyano group; carboxyl (COOH); alkoxy carbonyl group (COOR); acyl group; acyloxy; amino; alkyl amino; urea (--NHCONHR); sulfydryl; alkylthio group; sulfur oxygen base; sulfonyl; aryl sulfonyl; alkyl sulphonyl; sulfonamido; Arenesulfonyl amino; heteroaryl; heterocyclic radical; Heterocyclylalkyl; acylamino-; alkyl imino; amidino groups; guanidine radicals; diazanyl; hydrazides; sulfonyl sodium (SO ₃Na), sulphonyl sodium alkyl (RSO ₃Na).Alternately, two of aromatic ring can replace with methylene-dioxy or ethylenedioxy in abutting connection with atom.The example of aromatic yl group includes but not limited to phenyl, naphthyl, xenyl, indanyl, anthraquinol base, tert-butyl-phenyl, 1,3-benzo dioxolyl etc.

" Arenesulfonyl amino " refers to be attached to sulfonamido aryl, as defining as definition herein herein.

" sulfur aryl " refers to the aryl with one or more sulfydryls replacements.

" alkyl amino " refers to the amino group with one or two alkyl group replacement.Example comprises mono-substituted N-alkylamino group and N, N-dialkyl amino group.Example comprises the N-methylamino, N-ethylamino, N, N-dimethylamino, N, N-diethylamino, N-methyl, N-ethyl-amino etc.

" amino carbonyl " refers to group H ₂NCO-." amino carbonyl alkyl " refers to the alkyl group as defining that one or more amino carbonyl groups replace herein.

" acylamino-" refers to RCO-NH-, and wherein R is H or alkyl, aryl or heteroaryl as define herein.

" imino group carbonyl " refers to the carbon back that two in 4 covalent bond sites are total with imino group.The example of this imino group carbonyl group comprises, for example, and C=NH, C=NCH ₃, C=NOH and C=NOCH ₃Term " alkyl imino carbonyl " refers to the imino group with the alkyl replacement.Term " amidino groups " digital is bonded to two of imino group carbonyl group can utilize amino one of key, that be substituted or be unsubstituted.This amidino groups examples of groups comprises, for example, and NH ₂-C=NH, NH ₂-C=NCH ₃, NH-C=NOCH ₃And NH (CH ₃)-C=NOH.Term " guanidine radicals " refers to be bonded to amino amidino groups as defined above, and wherein said amino can be bonded to the 3rd group.The example of this guanidino group comprises, for example NH ₂-C (NH)-NH-, NH ₂-C (NCH ₃)-NH-, NH ₂-C (NOCH ₃)-NH-and CH ₃NH-C (NOH)-NH-.Term " diazanyl " refers to-NH-NRR ' that wherein R and R ' are hydrogen, alkyl etc. independently." hydrazides " refer to-C (=O)-NH-NRR '.

Term " heterocyclic radical " refers to saturated and containing of fractional saturation of heteroatomic annular group, and it has 4 to 15 ring memberses, is called " C at this ₄-C ₁₅Heterocyclic radical ", ring members is selected from carbon, nitrogen, sulfur and oxygen, and wherein at least one annular atoms is a hetero atom.The heterocyclic radical group can contain one, two or three rings, and wherein this ring can connect in the mode of dangling, and maybe can condense.The example of saturated heterocyclic group comprises saturated 3 to 6 yuan of heteromonocyclic group groups [for example pyrrolidinyl, imidazolidinyl, piperidyl, piperazinyl etc.] of containing 1 to 4 nitrogen-atoms; Saturated 3 to 6 yuan of heteromonocyclic group groups [for example morpholinyl] of containing 1 to 2 oxygen atom and 1 to 3 nitrogen-atoms; 3 to 6 yuan of saturated heteromonocyclic group groups [for example thiazolidinyl etc.] of containing 1 to 2 sulphur atom and 1 to 3 nitrogen-atoms; The example of the heterocyclic group of fractional saturation comprises dihydro-thiophene, dihydropyran, dihydrofuran and thiazoline.The limiting examples of heterocyclic group comprises 2-pyrrolinyl, 3-pyrrolinyl, pyrrolidinyl (pyrrolindinyl), 1,3-dioxolanyl, 2H-pyranose, 4H-pyranose, piperidyl, 1,4-two _ alkyl, morpholinyl, 1,4-dithiane base, thio-morpholinyl etc.This heterocyclic group can be chosen wantonly with group such as substituent group, for example hydroxyl; halogen (F for example; Cl; Br; I); haloalkyl; alkoxyl; halogenated alkoxy; alkylthio group; cyano group; carboxyl (COOH); alkoxy carbonyl group (COOR); acyl group; acyloxy; amino; alkyl amino; urea (--NHCONHR); sulfydryl; alkylthio group; sulfur oxygen base; sulfonyl; aryl sulfonyl; alkyl sulphonyl; sulfonamido; Arenesulfonyl amino; heteroaryl; heterocyclic radical; Heterocyclylalkyl; acylamino-; the alkyl imino carbonyl; amidino groups; guanidine radicals; diazanyl; hydrazides; sulfonyl sodium (SO ₃Na), sulphonyl sodium alkyl (RSO ₃Na) replace.

" heteroaryl " refers to have one or more rings; a preferred monovalent aromatic cyclic group to three rings; each ring has 4 to 8 atoms; in ring, one or more hetero atoms have been integrated; preferred one or two (is selected from nitrogen; oxygen or sulfur); described heteroaryl can be chosen wantonly with one or more; preferred one or two is selected from following substituent group and replaces; unless otherwise noted, for example: hydroxyl; halogen (F for example; Cl; Br; I); haloalkyl; alkoxyl; halogenated alkoxy; alkylthio group; cyano group; carboxyl (COOH); alkoxy carbonyl group (COOR); acyl group; acyloxy; amino; alkyl amino; urea (--NHCONHR); sulfydryl; alkylthio group; sulfur oxygen base; sulfonyl; aryl sulfonyl; alkyl sulphonyl; sulfonamido; Arenesulfonyl amino; heteroaryl; heterocyclic radical; Heterocyclylalkyl; acylamino-; the alkyl imino carbonyl; amidino groups; guanidine radicals; diazanyl; hydrazides; sulfonyl sodium (SO ₃Na), sulphonyl sodium alkyl (RSO ₃Na).The example of heteroaryl include but not limited to imidazole radicals, _ azoles base, thiazolyl, pyrazinyl, thienyl, furyl, pyridine radicals, quinolyl, isoquinolyl, benzofuranyl, benzothienyl, benzo thiapyran base, benzimidazolyl, phenylpropyl alcohol _ azoles base, benzothiazolyl, benzopyranyl, indazolyl, indyl, isoindolyl, quinolyl, isoquinolyl, phthalazinyl, benzenesulfonyl-thienyl etc.

" heteroaryloxy " refers to be connected to the heteroaryl groups of oxygen base.This examples of groups includes, but not limited to 2-sulfo-phenoxy group, 2-2-pyrimidinyl oxy, 2-pyridyloxy, 3-pyridyloxy, 4-pyridyloxy etc.

" heteroaryloxy alkyl " refers to the alkyl group with one or more heteroaryloxies replacements.This examples of groups comprises 2-pyridyloxy methyl, 3-pyridyloxy ethyl, 4-pyridyloxy methyl etc.

" cycloalkyl " refers to by one or more rings; typically one or two encircles the saturated carbon ring group of forming of unit price; each ring has 3 to 8 carbon; described cycloalkyl can replace with one or more following substituent groups usually, unless otherwise noted: hydroxyl; halogen (F for example; Cl; Br; I); haloalkyl; alkoxyl; halogenated alkoxy; alkylthio group; cyano group; carboxyl (COOH); alkoxy carbonyl group (COOR); acyl group; acyloxy; amino; alkyl amino; urea (--NHCONHR); sulfydryl; alkylthio group; sulfur oxygen base; sulfonyl; aryl sulfonyl; alkyl sulphonyl; sulfonamido; Arenesulfonyl amino; heteroaryl; heterocyclic radical; Heterocyclylalkyl; acylamino-; the alkyl imino carbonyl; amidino groups; guanidine radicals; diazanyl; hydrazides; sulfonyl sodium (SO ₃Na), sulphonyl sodium alkyl (RSO ₃Na).The example of group of naphthene base includes but not limited to cyclopropyl, cyclobutyl, 3-ethyl cyclobutyl, cyclopenta, suberyl etc." cycloalkenyl group " refers to have the group of 3 to 10 carbon atoms and one or more carbon-to-carbon double bonds.Typical cycloalkenyl groups has 3 to 7 carbon atoms.Example comprises cyclobutane base, cyclopentenyl, cyclohexenyl group, cycloheptenyl etc." cycloalkenyl alkyl " refers to the group that wherein defined alkyl is replaced by one or more cycloalkenyl groups herein.

" cycloalkyloxy " refers to be connected to the group of naphthene base of oxygen base.Example includes but not limited to cyclohexyloxy, cyclopentyloxy etc.

" cycloalkyloxy alkyl " refers to the alkyl group with one or more cycloalkyloxies replacements.Example comprises cyclohexyloxy ethyl, cyclopentyloxy methyl etc.

" sulfinyl " refer to-S (O)-.

" sulfonyl " refers to-S (O) ₂-, wherein " alkyl sulphonyl " refers to the sulfonyl RSO with the alkyl group replacement ₂-, aryl sulfonyl refers to be connected to the aryl of sulfonyl." sulfonamido " refers to-S (O) ₂-NRR '." sulfonic acid " refers to-S (O) ₂OH." sulphonic acid ester " refers to-S (O) ₂OR, wherein R is for example alkyl in the alkyl sulfonate esters of group.

" sulfenyl " refers to-S-." alkylthio group " refers to RS-, and wherein mercapto groups replaces with alkyl R.Example comprises methyl mercapto, ethylmercapto group, butylthio etc." arylthio " refers to R ' S-, and wherein sulfenyl replaces with the aryl as definition herein.Example includes but not limited to thiophenyl etc.Example includes but not limited to benzene sulfidomethyl etc." alkyl thiosulfonic acid " refers to group HO ₃SR ' S-, wherein alkylthio group replaces with sulfonic acid group.

" sulfo-sulfenyl " refers to-S-SH.

" acyl group "; alone or in combination the time, digital is bonded to and is selected from for example carbonyl or the thiocarbonyl of following group: hydroxyl (hydrido), alkyl, alkenyl, alkynyl, haloalkyl, alkoxyl, alkoxyalkyl, halogenated alkoxy, aryl, heterocyclic radical, heteroaryl, alkyl sulfenyl alkyl, alkyl sulfonyl alkyl, aralkyl, cycloalkyl, cycloalkyl-alkyl, cycloalkenyl group, alkylthio group, arylthio, amino, alkyl amino, dialkyl amido, aralkoxy, arylthio and alkylthio alkyl.The example of " acyl group " is formoxyl, acetyl group, benzoyl, trifluoroacetyl group, phthalyl, malonyl, nicotinoyl etc.

Term " acyl sulfenyl " and " acyl group disulfide group " refer to RCOS-and RCOSS-group respectively.

Term " thiocarbonyl " refers to contain carbon compound and the part that is connected to sulphur atom with two keys ,-C (=S)-." alkyl thiocarbonyl " refer to thiocarbonyl wherein with as the alkyl group R of definition herein replace and form monoradical RC (=S)-." amino thiocarbonyl " refers to the thiocarbonyl with amino replacement, NH ₂C (=S)-.

" ketonic oxygen base " refers to-OCOR.

" alkoxy carbonyl group " refers to-COOR.

" carboxyl " refers to-COOH.

For those chemical compounds, consider its its all stereoisomers to comprise cis/trans geometric isomer, diastereomer and independent enantiomer with stereoisomer.

A. carbon monoxide

Carbon monoxide (CO) is colourless, odorless and tasteless gas, and it can comprise that the people is deleterious to animal.According to Center for Disease Control (CDC), have every year more than 450 people and unexpectedly die from carbon monoxide.

It can carry oxygen to its blood, and to keep the biology of its existence be virose.It can be by entering lung and replacing oxygen in the blood flow but deleterious via eupnea.Interrupt normal oxygen supply harm heart, the function of brain and other vital functions of health.Yet, studying carbon monoxide be used for medical application (Ryter etc., 2004).

When the amount of 50/1000000ths parts (ppm), carbon monoxide does not show symptom to the people who is exposed to it.Yet when 200ppm, in 2 to 3 hours, carbon monoxide can cause slight headache; When 400ppm, in 1 to 2 hour, it can cause metopodynia, and this can become in 3 hours respectively extensively; And when 800ppm, it can cause in 45 minutes feels dizzy, feels sick and/or twitch, and makes experimenter's numbness in 2 hours.In the level of about 1000ppm, biology can be dead after exposing more than about 1-2 minute.

Because the well-known and carbon monoxide that fully proved is to the poisonous effect of biology, thereby carbon monoxide can be used to induce the stagnation of living organism sample and/or to help its preservation be wondrous and unexpected.Thereby consider and carbon monoxide can be used at isolating biological substance for example do not have in the biological substance of blood and induce stagnations (because the effect to hemoglobin that carbon monoxide has, it is and relates to the different approach of approach of inducing stagnation).

Induce any damage of stagnating or limit or preventing to be caused by the stagnation derivant except being exposed to carbon monoxide, the present invention considers and can and help the preservation of biomaterial and/or the reagent or the method for transplanting/grafting process to be used in combination with carbon monoxide.

B. chalcogenide chemical compound

The chemical compound (but except oxide) that contains chalcogen (those in the periodic table of elements the 6th family) is commonly called " chalcogenide " or " chalcogenide chemical compound " (can exchange use mutually at this).These elements are sulfur (S), selenium (Se), tellurium (Te) and polonium (Po).Common chalcogenide also contains one or more of S, Se and Te except other element.Chalcogenide comprises for example particle of the micronized and/or nanometer granulation of S and Se of element form.Can adopt the chalcogenide chemical compound as Reducing agent.

The inventor although be not subjected to following theory constraint, thinks stagnation in the chalcogenide inducing cell, and the ability that allows to regulate the core temperature in the animal comes from these molecules and is bonded to cytochrome oxidase.So, chalcogenide suppresses or has reduced the activity of oxidative phosphorylation.The thermoregulation that the chalcogenide blocking-up is spontaneous, the ability that promptly allows the core temperature of " homoiothermy " animal to handle by the temperature that controls environment, be considered to come from and the identical mechanism of listing above-be bonded to cytochrome oxidase, and blocking-up or reduce the activity of oxidative phosphorylation.Chalcogenide can provide with liquid and gas form.

Chalcogenide can be virose to mammal, and is lethal on some levels.According to the present invention, the level of expection chalcogenide should not surpass the deadly level in the proper environment.The deadly level of chalcogenide can be for example in the material safety data sheet (Material Safety Data Sheets) of every kind of chalcogenide, or finds from the information table that can obtain from the Occupational Safety and Health Administration (OSHA) of U.S. government.

Though carbon monoxide and chalcogenide chemical compound can be by inducing stagnation as the oxygen antagonist, they have different poisonous effects, and it is isolating that the ability of stagnation is induced in this effect and they.And because the different affinitys of cytochrome oxidase, the required concentration of mediation retention effects is different.Cytochrome oxidase relatively is about 1: 1 to the affinity of oxygen with affinity to carbon monoxide, and to H ₂The affinity of S with on now about 300: 1 magnitude of the affinity comparison sheet of oxygen.This has influenced and has used the stagnation induced concentration can observe what poisonous effect.Therefore, consider the stagnation that the chalcogenide chemical compound is particularly suitable for inducing the stagnation of biological substance in whole biology and induces whole biology.

Can prove that also be useful for extra stimulation to biological substance removing the chalcogenide prerequisite.Particularly, anticipation can allow the ambient temperature that animals received increases before removing chalcogenide source.

1.H ₂The chemical compound of S and other sulfur-bearing

Hydrogen sulfide (H ₂S) be a kind of usually and the genotoxic potential gas that interrelates of petrochemistry and natural-gas, sewage, paper pulp, tanning and food processing.Main effects on cellular level shows as cytochrome oxidase or other oxidasic inhibition, causes the cell hypoxia.Be exposed to extreme level (500ppm) and cause sudden collapse and unconscious (so-called " knocking down " effect), recover then.Effect after the exposure may lasting for years, and comprise coordination can not, the loss of memory, dyskinesia, personality changes, hallucination and insomnia.

Yet, most H ₂The S contact takes place under far below this acute toxicity level.Yet, be concerned about long-term contact on subacute level usually.Exist some reports to point out, at chronic low-level H ₂After S exposed, the damage of persistent balanced capacity and memory and the sensorimotor function of change may take place in the people.Kilburn and Warshaw (1995); Kilburn (1999).Other people are reported that in perinatal stage and expose rat back 21 day every day in low (20 or 50ppm) H from gestation to producing ₂ S 7 hours causes the longer arborizations of the tree-shaped difference with minimizing (reduced aborization) of cerebellar Purkinje cell.With relative low-level H ₂Other neurologic defect that S is relevant comprises the brain neurotransmitter concentration of change and the nerves reaction of change, for example Hippocampus и EEG activity of Zeng Jiaing.

Be exposed to the H of medium level ₂Studied behavioral toxicity in the rat of S.The result shows, is exposing end back H ₂S has suppressed to distinguish avoidance response (Higuchi and Fukamachi, 1997) immediately, and has disturbed the ability (Partlo etc., 2001) of rat study bait inductive radiation arm shape labyrinth task (baitedradial arm maze task).Use 80ppm H at another ₂In the S perinatal stage research, in the rat cub that exposes, do not find motor activity, passive avoidance or the sound alarm response (coustic startleresponse) of neuro pathology's effect or change.Dorman etc., (2000).At last, Struve etc., (2001) are the gas by multiple level successive 5 day every day, allows rat be exposed to H ₂S 3 hours.Observe and be exposed to 80ppm or higher H ₂Behind the S, the remarkable minimizing of motor activity, water maze performance and body temperature.All in all, these reports show, H ₂S can have multiple effect to the biochemistry of mammalian tissues, but does not have tangible reaction pattern about behavior.

In case be dissolved in the blood plasma H ₂S will participate in series of chemical.Described chemical reaction is: (1) molecule H ₂S dissociates and forms sulfur hydrogen radical ion (bisulfide ion), (2) sulfur hydrogen radical ion dissociate formation sulphion and the self-ionization of (3) water.Provide below being reflected at:

Equilibrium constant K when utilizing pH 7.4 ₁=1.039E ^-07, K ₂=6.43E ^-16And K _w=1.019E ^-14, the different material that calculates is about 23% H with respect to the amount of total S concentration ₂S and 77% HS ^-, and S ^2-Amount trend towards 0.

The link coupled extraction alkylation of inventor's utilization and gas chromatography and quality specific detection (extractive alkylation) technology is come quantitative hydrogen sulfide (according to Hyspler etc., 2002 reorganizations).This method comprises that the reaction buffer of at first being made up of the benzalkonium chloride (BZK) of 5mM with 150 μ L adds 50 μ L blood, serum or tissue extract sample in saturated borate buffer solution, and described sample has been diluted in the deoxygenated water that nitrogen purified the concentration to 1mg/mL.At first 4-chloro-benzyl methyl sulfide (4CBMS) solution with 15 μ M in the 100 μ L ethyl acetate adds wherein, adds PFBBR bromine (PFBBr) solution of the 20mM in the 100 μ L toluene then.Seal this solution then and hatched 2 hours in 55 ℃ of following accompanying rotation or jolting.After this incubation period, add the saturated KH of 200 μ L then ₂PO ₄Solution, and shift out organic facies and according to Hyspler etc., the method for describing in 2002 is analyzed by gas chromatography and quality specific detection.Then with these measured values with above-described identical method, (prepare in the deoxygenated water that nitrogen has purified, scope is the Na of 1 μ M-1mM with known normal concentration ₂S) begin and the standard curve comparison of generation, so that measure endogenic hydrogen sulfide levels.In order to analyze the sulfide level of bonded and/or oxidation, use identical method, except having used degeneration/reduction reaction buffer, described buffer by the 1mM in 5mM BZK with tetraethylammonium hydroxide (TEAH) of 1% and the saturated borate buffer solution hydrochloric acid three (2-carboxyethyl)-phosphine (TCEP) is formed, rather than above-described reaction buffer.

Consider that typical hydrogen sulfide levels used according to the invention comprises about 1 to about 150ppm, about 10 to about 140ppm, about 20 to about 130ppm and about 40 to about 120ppm or its normal per os, intravenous or through skin dosage.Other relevant scope comprises about 10 to about 80ppm, about 20 to about 80ppm, about 10 to about 70ppm, about 20 to about 70ppm, about 20 to about 60ppm and about 30 to about 60ppm or its equivalent per os, intravenous or through skin dosage.Also consider, in the preset time section for given animal, should reduce chalcogenide atmosphere avoid among the experimenter may lethal chalcogenide accumulation.For example, the initial environment concentration of 80ppm can be reduced to 60ppm after 30 minutes, further reduced when 1 hour (40ppm) and 2 hours (20ppm) subsequently.

A.H ₂The S precursor

The present invention also relates to use can be under certain conditions, for example when being exposed to biological substance or expose in the near future, produces H ₂The chemical compound of S and reagent.Consider that this precursor produces H when one or more enzymatics or chemical reaction take place ₂S.

3. other chalcogenide

In certain embodiments, the Reducing agent structural compounds is dimethyl sulfoxine (DMSO), dimethyl sulphide (DMS), methanthiol (CH ₃SH), mercaptoethanol, Hydrogen thiocyanate, Blausure (German), methanthiol (MeSH) or CS ₂In special embodiment, the oxygen antagonist is CS ₂, MeSH or DMS.The chemical compound of these bulk of molecule magnitudes be special consider (that is, they molecular weight about 50% in).

Imagination is stagnated other useful chemical compound and is included, but are not limited to following structure for inducing, and these structures many can obtain easily and be known (determining by CAS number): 104376-79-6 (ceftriaxone sodium salt) to those skilled in the art; 105879-42-3; 1094-08-2 (dibutil); 1098-60-8 (Fluoracyzine); 111974-72-2; 113-59-7; 113-98-4 (benzylpenicillin K ⁺); 115-55-9; 1179-69-7; 118292-40-3; 119478-56-7; 120138-50-3; 121123-17-9; 121249-14-7; 1229-35-2; 1240-15-9; 1257-78-9 (prochlorperazine edisylate salt); 128345-62-0; 130-61-0 (mellaril) 132-98-9 (penicillin V K ⁺); 13412-64-1 (dicloxacillin Na ⁺Hydrate); 134678-17-4; 144604-00-2; 146-54-3; 146-54-5 (hydrochloric acid Fluphenazine); 151767-02-1; 159989-65-8; 16960-16-0 (thyroliberin fragment 1-24); 1982-37-2; 21462-39-5 (Clindamycin Hydrochloride); 22189-31-7; 22202-75-1; 23288-49-5 (probucol); 23325-78-2; 24356-60-3 (cefaloject); 24729-96-2 (clindamycin); 25507-04-4; 26605-69-6; 27164-46-1 (cefazolin sodium Na ⁺); 2746-81-8; 29560-58-8; 2975-34-0; 32672-69-8 (mesoridazine besilate); 32887-01-7; 33286-22-5 (( ⁺)-cis-diltiazem hydrochloride _); 33564-30-6 (cefoxitin sodium); 346-18-9; 3485-14-1; 3511-16-8; 37091-65-9 (Azlocillin Sodium); 37661-08-8; 3819-00-9; 38821-53-3 (cefradine); 41372-02-5; 42540-40-9 (Cefadole); 4330-99-8 (alimemazine half-( ⁺)-tartrate); 440-17-5 (two hydrochloric acid triflurins); 4697-14-7 (ticarcillin disodium); 4800-94-6 (the two sodium of carboxy benzyl penicillin); 50-52-2; 50-53-3; 5002-47-1; 51481-61-9 (cimetidine); 52239-63-1 (6-propyl group-2-thiouracil); 53-60-1 (promazine hydrochloride); 5321-32-4; 54965-21-8 (albendazole); 5591-45-7 (tiotixene); 56238-63-2 (Cefuroxime Sodium); 56796-39-5 (Cefmetazon (Sankyo)); 5714-00-1; 58-33-3 (promethazine hydrochloride); 58-38-8; 58-39-9 (fluphenazine); 58-71-9 (cephalothin sodium); 59703-84-3 (avocin); 60-99-1 (leyomepromazine maleate salt); 60925-61-3; 61270-78-8; 6130-64-9 (aquacillin salt hydrate); 61318-91-0 (sulconazole nitrate); 61336-70-7 (BRL-2333); 62893-20-3 (cefoperazone sodium); 64485-93-4 (cefotaxime sodium); 64544-07-6; 64872-77-1; 64953-12-4 (drawing tower head p0-357 sodium); 66104-23-2 (pergolide mesylate); 66309-69-1; 66357-59-3 (ranitidine hydrochloride); 66592-87-8 (Cefodroxil); 68401-82-1; 69-09-0 (chlorpromazine hydrochloride); 69-52-3 (sodium ampicillin); 69-53-4 (ampicillin); 69-57-8 (penicillin G sodium); 70059-30-2; 70356-03-5; 7081-40-5; 7081-44-9 (cloxacillin sodium H ₂O); 7177-50-6 (sodium nafcillin H ₂O); 7179-49-9; 7240-38-2 (oxacillin sodium H ₂O); 7246-14-2; 74356-00-6; 74431-23-5; 74849-93-7; 75738-58-8; 76824-35-6 (famotidine); 76963-41-2; 79350-37-1; 81129-83-1; 84-02-6 (prochlorperazine dimaleate); 87-08-1 (Phenoxymethyl .gamma.-keto-.beta.-methoxy-.delta.-methylene-.DELTA..alpha.-hexenoic acid .); 87239-81-4; 91-33-8 (benzthiazide); 91832-40-5; 94841-17-5; 99294-94-7; 154-42-7 (6-thioguanine); 36735-22-5; 536-33-4 (ethionamide); 52-67-5 (Beracilline); 304-55-2 (meso-2,3-dimercaptosuccinic acid); 59-52-9 (2, the 3-dimercapto ⁺Propanol) 6112-76-1 (6-mercaptopurine); 616-91-1 (N-acetyl group-L-cysteine); 62571-86-2 (captopril); 52-01-7 (spironolactone) and 80474-14-2 (fluticasone propionate).Be considered as other chemical compound that comes in handy for stagnation and comprise those of chemical constitution with formula I or IV.

C. other antagonist or reactive compound and relevant environmental condition

1. hypoxia and anoxia

Hypoxia is a kind ofly common naturally stress and to have several very conservative reactions, and it promotes cell adapted low-oxygen environment.In order to compensate for the reduction that aerobic energy produces ability in the hypoxia, cell must increase no oxygen energy generation or reduce energy requirement (Hochachka etc., 1996).The example of these two kinds of reactions is the amount that specific response common and that utilize depends on the oxygen that can offer cell usually in metazoa.

In slight hypoxia, oxidative phosphorylation still part is active, is possible so some aerobic energy produce.Cell effect (its part is by inducing the transcription factor HIF-1 mediation of hypoxia) to this situation is to relate to the gene that no oxygen energy produces by rise, for example glycolytic ferment and the glucose transporter aerobic energy of supplying minimizing produces (Semenza, 2001; Guillemin etc., 1997).This reaction has also promoted to prevent for example rise of catalase (catylase) and superoxide dismutase of antioxidant of the damage of free yl induction.Therefore, cell can be kept near the activity that contains the oxygen normal level in slight hypoxia.

(be called " anaerobic "-be defined herein as＜0.001kPa O at the hypoxia of extreme form ₂) in-oxidative phosphorylation stops also thereby energy-producing ability significantly reduces.In order to survive in this environment, cell must reduce energy requirement (Hochachka etc., 2001) by reducing cellular activity.For example, in having deprived the Chelomia mydas (Linnaeus). hepatocyte of oxygen, the cell restraint for example orienting response of protein synthesis, ion channel activity and metabolic pathway of synthesizing causes the ATP demand to reduce by 94% (Hochachka etc., 1996).In Brachydanio rerio (Danio rerio) embryo, be exposed to anaerobic and cause heartbeat, motion, cell cycle progress and grow the stopping fully of progress (Padilla etc., 2001).Similarly, Caenorhabditis elegans reacts to anaerobic by entering stagnant giving birth to, and in stagnant giving birth to, all observable motions comprise that cell division and growth progress stop (Padilla etc., 2002; Van Voorhies etc., 2000).Caenorhabditis elegans can keep stagnate giving birth to 24 hours or more of a specified duration, and in case to get back to oxygen content normal, will recover high viability.This reaction make beautiful latent rhabditida can by reduce the ratio of the big process of consumption aspect the energy and prevent damage, irrevocable incident (for example non-multiple state) generation and under hypoxia stress survival (Padilla etc., 2002; Nystul etc., 2003).

The reaction of a recent findings is the generation (Dulak etc., 2003) of the hypoxia inducible carbon monoxide that undertaken by Heme oxygenase-1.The carbon monoxide that endogenous produces can the activation signal cascade reaction, this cascade reaction is by anti-apoptotic (Brouard etc., 2003) and anti-inflammatory (Otterbein etc., 2000) activity alleviates hypoxia injury, and in transplantation model, can obtain similar cytoprotective effect (Otterbein etc., 2003 by pouring into ectogenic carbon monoxide; Amersi etc., 2002).Under higher concentration, carbon monoxide and oxygen competition are bonded to the protein that contains ferrum, and for example mitochondrial cytochrome and hemoglobin (Gorman etc., 2003) also do not have the research cytoprotective effect that this activity may have in hypoxia.

Although there are these complicated defense mechanisms of antagonism hypoxia injury, hypoxia still usually be a kind of damaging stress.For example, mammal has Heme oxygenase-1 and HIF-1, and the stagnant life of some evidence promptings also is possible (Bellamy etc., 1996 in mammal; Alam etc., 2002).Yet, since wound for example the hypoxia damage that causes of heart attack, apoplexy or lose blood be a dead main cause.The circumscribed understanding that survives two kinds of elementary tactics (keep active or stagnate give birth to) of hypoxia stress is hindered by such fact: it is based on the research of carrying out in the multiple systems under the multiple condition.

When the oxygen of normal physiological level is not supplied to cell or tissue, occur " hypoxia "." oxygen content is normal " refers to the oxygen of the normal physiological level of special cells type, cell state or the tissue discussed." anoxia " is not have oxygen." hypoxia condition " is those conditions that cause the cell hypoxia.These conditions depend on cell type and depend on tissue or the metabolism status of the special construction of intraorganic cell or position and cell.For the purposes of the present invention, hypoxia condition comprises that oxygen concentration wherein is or is lower than the condition of normal atmosphere situation, and it is less than 20.8,20,19,18,17,16,15,14,13,12,11,10,9,8,7,6,5,4,3,2,1,0.5,0%; Alternately, these numerals can be represented the percentage ratio of the atmosphere under 1 atmospheric pressure (101.3kPa).The oxygen concentration of 0 percentage ratio has defined anoxia condition.Therefore, hypoxia condition comprises anoxia condition, although in some embodiments, implements to be no less than 0.5% hypoxia condition.As used herein, " normoxic condition " is equal to about 20.8% or higher oxygen concentration.

Obtaining hypoxia or anoxybiotic standard method is fully to establish, and comprises that use depends on chemical catalyst and comes from wherein removing the environmental chamber of deoxidation.This class chamber can (it be GASPAK DisposableHydrogen+Carbon Dioxide Envelopes or BIO-BAG environmental chamber for Sparks, MD) the commercial acquisition from for example BDDiagnostic Systems.Alternately, oxygen can be by using non-carrier of oxygen, and for example the air in the nitrogen switch room is exhausted.Oxygen concentration can for example use FYRITE oxygen analyzer (Bacharach, Pittsburgh PA) to measure.

Considering that method of the present invention can be used is exposed to oxygen antagonist or other reactive compound and relatively changes the combination of oxygen concentration with room air.And, the oxygen concentration that contains the environment of biological substance can be approximately, at least about or about at the most 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99 or 100%, maybe can come from wherein any scope.And, the variation of considering concentration just with room air or with the reduction or the increase of contrast environment facies ratio can be any top percentage ratio or scope.

D. Mitochondrially targeted reagent

Embodiment of the present invention consider that selectivity targeting mitochondrion is so that enhanced activity in some respects.This optionally Mitochondrially targetedly realize by puting together reagent to lipotropy triphenyl _ cation, the big current potential of triphenyl _ cation by crossing over mitochondrial inner membrane (150 to-180mv) drive, be easy to pass lipid bilayer and in mitochondrial matrix, accumulate about 1000 times.Prepare the analog of vitamin E and ubiquinone and be used for successfully targeting mitochondrion.(Smith etc., 1999; Kelso etc., 2001; Dhanasekaran etc., 2004).Prepared mercaptan, bromination butyl triphen _ (thibutyltriphosphoniumbromide) (showing below) also is used for the targeting mitochondrion, and it accumulates hundreds of times of (Burns etc., 1995 in mitochondrion; Burns ﹠amp; Murphey, 1997).

This conjugate looks like the suitable candidate of reactive compound.Except free thiol reagent, the chemical compound (H-S-S-R) that the sulfo-sulfenyl replaces may be useful.Consider that described reagent has following structure in some embodiments:

Wherein Z is P or N;

R ¹, R ²And R ³Be that (that suitable is phenyl, benzyl, tolyl, pyridine radicals, cyclohexyl, C for aryl, heteroaryl, alkylaryl, cycloalkyl or alkyl ₃-C ₁₀Alkyl, optional through halogenation);

R ⁴Be-R ⁵SR ⁶, R wherein ⁵Be C ₁-C ₁₀Alkyl, R ⁶Be H or SH, SO ₃H or PO ₃H.

III. the detection of Ting Zhiing

Multiplely can be used for inducing the chemical compound of stagnation initially to estimate with multiple different detection method.Stagnation can be by many modes, comprise the amount (the indirect measurement of Cellular respiration) of the amount of the oxygen that consumes by quantitative biological sample, carbon dioxide that sample produces or measure by characterizing motility.

For the speed of measuring oxygen consumption or the speed of carbon dioxide generating, biological substance is placed in the chamber with two openings (being used for the gas input and output) sealing.Gas (room air or other gas) is fed in the described chamber with given flow velocity and flow out outlet in this chamber, to keep about 1 atmospheric pressure.Before being exposed to this chamber and afterwards, with this gas by carbon dioxide indicator and or the oxygen detection device measure the amount of every kind of chemical compound in (per second) this admixture of gas.More time dependent these are worth, and obtain the speed of oxygen consumption or carbon dioxide generating.

Set up other screening technique and identified that candidate's activity stagnates chemical compound.Can adopt these screening techniques and variant thereof as a part of the present invention or be used to realize aspect of the present invention.

A. use the algoscopy of Brachydanio rerio

The Screening test method that is used to stagnate derivant is set up with 48 hours big Brachydanio rerio (D.rerio) embryos.These embryos are transparent, and the anatomic microscope that allows human to have 4-20 times of lens is observed heartbeat and the blood that causes flows into along in the main blood vessel at the back side and flow into afterbody.Heart rate in these animals is the active index of biological metabolism, and metabolic minimizing is represented in the minimizing of heart rate like this.The embryo is dissected out and each hole flat polystyrene tissue culture plate is distributed 5 from their ovum shell, and in 1mL standard fish and water, hatch.Described fish and water is made of per 5 gallons of 1 Instant Ocean (artificial sea water mixture, Aquarium Systems company).Calcium chloride is adjusted to 150ppm and sodium bicarbonate is adjusted to～100ppm.This electrical conductivity of water is 900 little Siemens, and pH is about 6.5-7.4.Hydrogen sulfide solution is by will be with the mixture of the equilibrated hydrogen sulfide of room air with the speed of 100 cubic centimetres of per minutes in the flask that contains the 150mL fish and water bubbling 60-90 minute.This is enough to obtain saturated or nearly saturated or most of saturated hydrogen sulfide solution according to estimates.Based on the dissolubility of known hydrogen sulfide in the Ringer's mixture of pH 7 under 1 atmospheric pressure and room temperature, fish and water contains about 0.1 moles of hydrogen sulfide according to estimates.Fish is exposed to hydrogen sulfide solution and monitored their heart rate at subsequently 24 hours by the heartbeat number of times of counting per minute.Contrast fish (only being exposed to fish and water) has the about heartbeat number of times of beating for 160-200 time of per minute, and this was 24 hours not significantly change of viewing duration.To being exposed to behind the fish and water that contains hydrogen sulfide 2-3 hour, the heartbeat number of times reduces only about half ofly beats for 60-80 time to per minute.By 4 hours, the heartbeat number of times further reduced, the heartbeat number of times that comprises some examples be 0 or per minute only several times.After exposing 5 hours, replace hydrogen sulfide solution with normal fish and water, and allow the embryo recover to spend the night down at 28 degrees centigrade.Be exposed to behind the hydrogen sulfide 24 hours to initial, treated mistake also shows the normal cardiac rate that per minute is beaten for 160-200 time through the animal of flushing.Because hydrogen sulfide has caused the dormancy of heartbeat, has caused in some cases to stop, and returns to normality subsequently, think that hydrogen sulfide is accredited as stagnation derivant or other reactive compound by the standard of this Screening test method.

B. use the algoscopy of nematicide

Set up the Screening test method with nematicide (Caenorhabditis elegans).Nematicide can not well survive under 4 degrees centigrade, and under this temperature 24 hours like this, they were all dead.Anthelmintic they being exposed to 4 degrees centigrade before following 16 hours, at room temperature is exposed to atmosphere X minute that contains the Y% carbon monoxide.Compare with all dead contrast anthelmintic that is exposed to room air in advance, the anthelmintic that carbon monoxide is handled survives with high viability after being exposed to low temperature.Because carbon monoxide is the stagnation inducer in a kind of known nematicide and the neonate foreskin keratinocyte, the nematicide algoscopy can be when the anthelmintic pre-equilibration be in stagnating inducer or other reactive compounds, and the ability that increases the viability that is exposed to the cryogenic anthelmintic of lethal by them is identified the chemical compound of inducing stagnation.

IV. treat or prophylactic use

A. wound

In some embodiments, the present invention can be used for treating the patient who suffers or subject to damage.Wound can by exterior injury (for example burn, wound, excision wound, gunshot wound, or operation wound) internal injuries (for example cause circulate and sharply descend apoplexy or heart attack) or since the circulation decline that non--invasive stress (for example be exposed to low temperature or radiation) to be caused cause.On cellular level, wound usually causes cell, tissue and/or organ to be exposed to hypoxia, thereby causes inducing programmed cell death, or " apoptosis ".Systematicness ground, wound causes having induced a series of biochemical process, for example solidifies, inflammation, hypotension, and can cause shock, if shock continues, then it can cause organ dysfunction, irreversible primary cellular defect and death.Biological process is that design is used for defending body in order to avoid traumatic injury; Yet they can cause a continuous incident, and these incidents prove deleterious, and are fatal in some cases.

Therefore, the present invention consider to allow tissue, organ, extremity and even whole biology enter stagnation, they avoid the means of the illeffects of wound as protection.In the particular case that medical science nurse therein is not easy to obtain, the interior or in-vitro inducing stagnation of body, alternately, in conjunction with reducing tissue, organ or biological temperature, can be the experimenter and " race against time ", provide the medical science nurse, or transport the experimenter and go to accept the medical science nurse to the experimenter.The present invention has also considered to be used for to come by the biological process of wound healing that prevents from/delay to cause to delay and tissue regeneration the method for induced tissue regeneration and wound healing.About this point, existing therein has in the situation of big wound limbs or biology, interior or the in-vitro inducing stagnation of body, alternately, in conjunction with the temperature that reduces tissue, organ or biology can inhibition be healed and regenerated biological process be helped wound healing and tissue regeneration process by managing.

Except wound healing and the hemorrhagic shock of discussing below, can implement the inventive method and prevent or treat wound for example heart beating stops or apoplexy.The present invention is for from the emergency surgery operation technique, and for example the damage risk of thoracotomy, laparotomy and spleen crosscut has special importance.

1. wound healing

In many cases, wound and histologic lesion are refractory or need the too much time to heal.Example is chronic open wound (diabetic ulcer of foot and 3﹠amp; 4 phase pressure ulcers), acute and traumatic wound, flap and graft, and subacute wound (that is the otch that, splits).This also can be applicable to other tissue injury, for example burn and the injury of lung that sucks from cigarette/steam.

Previous test demonstration hibernation can be protected antagonism damage (for example, the pin ' s in the brain), so it may have healing effect.Therefore, this technology can be used for controlling wound healing process by the environment that allows tissue enter the control of metabolism more.More particularly, cell or tissue keeps the time length of stagnation to change according to damage.In some embodiments of the present invention, biological substance is exposed to oxygen antagonist or other reactive compound pact, at least about, or about at the most 30 seconds; 1,2,3,4,5,10,15,20,25,30,35,40,45,50,55 minute; 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 hour; 1,2,3,4,5,6,7 day; 1,2,3,4,5 weeks; 1,2,3,4,5,6,7,8,9,10,11,12 month or longer.

2. hematology shock (hemorrhagic shock)

Shock is a kind of life-threatening state that makes progress rapidly when postponing to intervene.Shock is wherein not exist sufficient perfusion to keep the state of the physiological need of organ-tissue.This is the hematodinamics and the state of metabolic of the degree of depth, it is characterized by blood circulation and can not keep abundant perfusion to vital organ.It may be also referred to as distributivity shock (neurogenic shock, septic shock, anaphylactic shock) by due to hypovolemia (hypovolemic shock), cardiac function deficiency (cardiogenic shock) or the vasomotion tonicity deficiency.This causes the patient dead rapidly usually.Many diseases comprise sepsis, lose blood, self regulate the state that impaired and autonomous tonicity forfeiture can produce shock or similar shock.Expection the present invention can prevent the unfavorable effect of all above-mentioned states of suffering a shock, and keeps the life of the biological substance of this shock of experience.

In hemorrhagic shock, the ability of losing blood and having exceeded the health compensation and sufficient perfused tissue and oxygenate are provided.This is usually owing to wound, but can also be caused by hematostaxis (for example gastrointestinal hemorrhage, childbirth), operation and other reason.Be that clinical hemorrhagic shock is to be caused by the acute hemorrhage incident of following discontinuous burst (precipitating) incident the most commonly.Seldom insight is, hemorrhagic shock can be seen in the chronic disease of subacute blood loss is arranged.

Hemorrhage physiology compensation mechanism comprises that initial periphery and mesentery vasoconstriction make the circulation of blood flowing maincenter.This strengthens by carrying out property tachycardia subsequently.The invasive monitoring can show that cardiac index increases, oxygen is carried (is DO ₂) increase, and the oxygen consumption of tissue (is VO ₂) increase.Lactate level, acid-bare status and other labelling also can provide the physiological status index of usefulness.Age, medication and common pathogenic factors all may influence the response of patient to hemorrhagic shock.

Compensation mechanism failure in the hemorrhagic shock can cause death.When not intervening, in serious hemorrhagic shock, observe the death that typical three peaks distribute.Initial dead peak takes place in hemorrhage several minutes owing to blood-letting at once.Another peak takes place after 1 to a few hours because the mistake of carrying out property is compensatory.The 3rd peak is because sepsis and organ failure and take place after a couple of days to several weeks.

In the U.S., unexpected injury be the age be 1 and 44 the step between the philtrum mortality rate and the main cause of sickness rate.In calendar year 2001,157,078 residents are owing to injury takes place dead.These philtrums, 64.6% is classified as unexpected death, and the 19.5%th, commit suiside, 12.9% for murdering, and 2.7% does not determine intention, and 0.3% relates to legal intervention or war-like action.The nocuity main causes of death are motor vehicles vehicle accident, firearm and fall.These death by accidents of vast scale result from the massive blood loss that wound causes, cause hemorrhagic shock.

In most of traumatic injury incidents, the patient who arrives hospital emergency rooms handles by the emergency treatment doctor and need not to undergo surgery or external wounds (trauma service) nursing is left hospital.Yet, have the needs of patients of major injury stable in " prime time " after damage takes place, to improve the chance of surviving and permanent disability is minimized.

Because most shock incidents are the damages that cause owing to accident, pre hospital care (pre-hosiptal care) is critical for patient's survival.Pre hospital care comprises rapid evaluation, stablizes and is transported to rapidly the nursing that suitable center is assessed and determined.In suffering from syndromic all patients of shock, keeping open airway, breathing and circulate fully fully is the primary focus of emergency treatment.Assessment is essential, because the variation of prescription on individual diagnosis person's situation shows the syndromic progress of shock.It is vital that early intervention minimizes and make permanent disability to minimize to the infringement that makes tissue and organ, and determines that in early days the main clinical reason is critical.Treatment concentrates on to be corrected the syndromic reason of shock and slows down progress.Intravenous input and fluid recovery (being generally IV saline) are standards,, have some arguements about this point.The reverse blood volume is crossed to hang down rapidly may increase clot hemorrhage, that movable part forms and dilution coagulation factors.

In case, then concentrate on the perfusion and the oxygenate optimization that make vital organ to emergency room.Potential hemorrhage diagnosis and processing must be implemented rapidly and carry out simultaneously with the processing shock.There are two Main Stage in shock: early stage compensatory stage and carrying out the sexual stage.Consider that embodiment of the present invention can be applicable to be in the patient in arbitrary stage or these two stages.

When hypovolemic shock by due to bleeding profusely the time, the substitution fluid of selection is whole blood or the erythrocyte that concentrates (packed).Crystalloid solution will temporarily improve circulation volume, but the patient also needs the erythrocyte substitute to transport oxygen to tissue.The management of shock concentrates on fluid management, Acid-Base balance, and improves myocardial contraction.Also should handle the potential cause of shock so that reduce to suffer a shock syndromic progress.In mice, induce the whole body hibernation, then exist the decline immediately of whole metabolism state (to pass through CO ₂Emit measurement).As if this is reversible, and mice works orderly, or even after exposure repeatedly.Therefore, the present invention relates to use H ₂S (or other oxygen antagonist or other reactive compound) induces whole body hibernation-like state, preserves patient's vital organ and life.This will make and can be transported in check environment (for example, operation), can solve the initial reason of shock in this environment, and the patient recover normal function in check mode then.To this indication, the very first time that is called after the damage of " prime time " is most important to the result of success.Stablizing the patient in this time period is main purpose, and the patient is transported to CC mechanism (for example, emergency room, operating room etc.), and damage can be able to suitable solution there.Therefore, the patient remained in stagnates, and solve direct problem so that can reach this purpose, for example Xiu Ke source, replenish and lose blood, and the reconstruction homeostasis is ideal.Though this will have a great difference, as a rule, be between about 72 hours of about 6-after the damage with keeping the amount of the time of stagnation.In some embodiments of the present invention, biological substance is exposed to oxygen antagonist or other reactive compound pact, at least about, or about at the most 30 seconds; 1,2,3,4,5,10,15,20,25,30,35,40,45,50,55 minute; 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 hour; 1,2,3,4,5,6,7 days or longer, and any range wherein or combination.

Lethal is hemorrhage suffers a shock and finally understands fully the biology of dead physiological event with causing.Yet, have some mechanism, by this mechanism H ₂S can reduce the lethal effect of ischemic hypoxia.Hydrogen sulfide suppresses cytochrome C oxidase and can pass through to suppress this enzyme ³Reduce oxygen demand.Oxygen demand reduces the illeffects that can reduce the hypoxia level, comprises the minimizing metabolic acidosis.In addition, organize the sulfydryl level in the shock process, to reduce (Beck etc., 1954).External H ₂S can prevent this protosulphide (hyposulfidic) state and keep the homeostasis of sulfur.

Hydrogen sulfide produces and demonstrates effective biological activity (Kamoun, 2004) naturally in animal.Most protein contain the cysteine residues that disulphide connects, and the specific enzymatic activity (Ziegler, 1985) of the reversible transition scalable from free sulfydryl to disulphide.In addition, sulfide is electronegative and shows high-affinity to transition metal.The protein that contains transition metal atoms, for example cytochrome oxidase can be subjected to H deeply ₂The influence of S.And last, H ₂S is metabolized to other molecule that contains reductive sulfur has increased the quantity that can show specific bioactive mercaptan.Except (perhaps may because) these potential binding modes, H ₂S can apply effect to cardiorespiratory system, neuroendocrine system, immune system and/or hemostasis system, and the last proof of this effect is favourable in damage and disease.

Mark B.Roth, Mike Morrison and Eric Blackstone on April 20th, 2006 application, title be that the U.S. Provisional Application of " Methods, compositions and articles ofmanufacture for treating shock " has been described the treatment of shock and so by reference as a reference.

B. hypothermia

In another embodiment, this people's suggestion is handled extremely hypothermic patient with the present invention.Method and composition of the present invention is used in to be needed to induce hypothermia in the hypothermic mammal.Hypothermia can be slight, the moderate or the degree of depth.Slight hypothermia comprises that core temperature is reached is lower than about 0.1-5 degree centigrade of mammiferous normal core temperature.Mammiferous normal core temperature is usually between 35 and 38 degrees centigrade.The moderate hypothermia comprises that core temperature is reached is lower than about 5-15 degree centigrade of mammiferous normal core temperature.Dark hypothermia comprises that core temperature is reached is lower than about 15-37 degree centigrade of mammiferous normal core temperature.

Slight hypothermia known in the art for all being useful and effective in the treatment in non-human mammal and people.Slight hypothermic treatment benefit in people's clinical trial, is observed aspect heartbeat stops outside about hospital.With normal body temperature is arranged, or the standard that lacks slight hypothermic nursing is relatively, and the people is exposed to neurological result (Bernard etc., 2002 that mild hypothermia causes survival advantage and improvement when the asystole; The Hypothermia After CardiacArrest Study Group etc., 2002).

Method and composition of the present invention can have the advantage that is better than other method known in the art, be used at mammal or people's inducing mild, moderate or deep hypothermia, described other method comprises, but be not limited to the experimenter is wrapped in the ice, or wrap up the experimenter with " the refrigeration curtain cover " of circulate cold air or liquid.In these cases, the experimenter resists and reduces core temperature and be lower than normal body temperature and attempt to produce by trembling heat.Tremble, and the body heat that wherein produces can pass through, the speed that the core temperature realized with the hypothermia abductive approach of standard of for example slowing down reduces, and have negative effect to reaching slight hypothermia.Therefore, the hypothermic people of the level of receiving treatment is also with the drug treating (Bernard etc., 2002) of suppress to tremble (by the neurotransmission at block nerves flesh contact place).

In preferred embodiments, method and composition of the present invention and invasive method known in the art or medical apparatus and instruments are combined in the hypothermia of mammal or philtrum inductive treatment.This invasive method and device include, but are not limited to insert the flexible stylet or the conduit of the vascular system that needs hypothermic experimenter, and wherein the thermoregulation of conduit causes the blood cooling that contacts with conduit to the normal body temperature that is lower than the experimenter.Refrigerative blood causes that subsequently mammiferous core temperature descends.By the feedback of combination from the thermocouple monitoring of monitoring mammal core temperature, the temperature of scalable conduit is so that keep preassigned core temperature.Thisly being used to reach and keeping slight or the hypothermic medical apparatus and instruments of moderate, be called temperature therapy in the blood vessel in the art, is known in the art and for example at www. Innercool.comAnd www. Radiantmedical.comOn description is arranged.

Described method provides, and gives extremely that hypothermic patient uses or allows it be exposed to oxygen antagonist or other reactive compound, allows it return to normal temperature gradually when removing oxygen antagonist or other reactive compound in the mode of control then.By this way, oxygen antagonist or other reactive compound have cushioned the intravital biosystem of experimenter, do not impact (or injury) experimenter so that they can begin gradually.

In one embodiment, will suffer oxygen antagonist or other reactive compound of hypothermic experimenter's per os or intravenous dosages.Since the experimenter potential non--reactive and the ability of in check dosage was provided in a period of time, it is preferred that intravenous is supplied with.Alternately, if can utilize, oxygen antagonist or other reactive compound can provide with gaseous state, for example are used to the face shield that sucks or or even can hold whole experimenter's confined chamber.

Ideally, before realizing any variation, will stablize patient's heart rate, breathing and temperature.In case stable, ambient temperature will rise once more gradually.This can be easily finishes by the experimenter is shifted out under the cryogenic conditions.The increase that temperature is more regulated can be by realizing like this: add the medicated clothing or the blanket of pantostrat, the hot wrappage that utilizes temperature to increase gradually, or if possible, the experimenter is placed indoor that its temperature can increase gradually.

Preferably in temperature increase process, monitor experimenter's vital sign.And, in conjunction with increasing temperature, oxygen antagonist or other reactive compound are removed from experimenter's environment.The processing of heat and oxygen antagonist (or other reactive compound) all stops at suitable terminal point, and this terminal point is judged by the medical worker of monitoring situation, but under any circumstance this terminal point is when experimenter's temperature returns to normal range with other vital sign.Suggestion continues to monitor a period of time of at least 24 hours after stopping to handle.

C. high fever

Under some situation that can be caused by heredity, infection, medicine or environment reason, the patient can loosen homeostatic thermoregulation, causes serious uncontrollable heating (high fever).This can cause dead or secular morbidity, especially cerebral lesion, if it is not suitably controlled.

Suck 80ppm H ₂The mice of S experiences hibernation immediately.This is included in the body temperature that can not regulate them when being reduced to room temperature under the ambient temperature.Therefore, this technology is used in the body temperature of control whole body in the state of some high fever.This may comprise by sucking or being fed into blood supply uses H ₂S (or other oxygen antagonist or reactive compound) induces hibernation-like state.Allow the patient be in to stagnate down about 6-will be useful between about 24 hours, during this period of time pyrotoxin can be resolved.In some embodiments of the present invention, allow the patient be exposed to oxygen antagonist or other reactive compound pact, at least about, or about at the most 30 seconds; 1,2,3,4,5,10,15,20,25,30,35,40,45,50,55 minute; 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 hour; 1,2,3,4,5,6,7 days or longer, and any range wherein or combination.

This can make up with some whole body thermoregulation (ice bath/blanket/cooling system).

D. cardioplegia and coronary heart disease

In some embodiments, the present invention can be used as and is used for the treatment of coronary heart disease (CHD) solution of (comprising the cardioplegia that is used for heart bypass operation (CABG)).

CHD is caused that by atherosclerosis atherosclerosis provides stricture of artery and the sclerosis that oxygen enrichment blood is given cardiac muscle.Because the accumulation of speckle on tremulous pulse inwall or the liner, tremulous pulse hardening and become narrow.The blood that flows to heart reduces owing to speckle makes CAN.This has reduced the oxygen supply to cardiac muscle.This can show as: 1) angina pectoris, its chest pain or discomfort for taking place when heart does not obtain enough blood; 2) heart attack, it can cut off suddenly the most of of cardiac component or all take place during the blood supply at clot, and does not receive enough cells of taking oxygen blood in the cardiac muscle and begin death, may cause the permanent lesion to cardiac muscle; 3) heart failure, blood pump for can not be effectively at heart taking place during to the health other parts in it; Arrhythmia, it is the change of the normal rhythm of heartbeat.

Since nineteen ninety, more people dies from CHD rather than any other reason.There are every year 3.8 hundred ten thousand male and 3.4 hundred ten thousand women to die from CHD.In 2002, only just there are 500,000 people of surpassing directly to die from heart disease in the U.S..Although improved survival rate, the male in the U.S. 1/4th, and 1/3rd women is still dead in a year of approval heart attack for the first time.

The Drug therapy that comprises the therapeutic treatment of CHD reduces the risk of heart attack, heart failure and apoplexy, changes the further accumulation that important life style prevents lipidosis in the coronary artery simultaneously.But, also usually need the surgical intervention of some type.

About 1/3rd CHD patient will experience coronary angioplasty and stenting (stenting).In the balloon angioplasty process, adopt top air bag (balloon-tipped) conduit that speckle is pushed back arterial wall so that can improve blood flow in the tremulous pulse.The coronary stent art is attended by the angioplasty operation usually.Support provides the little gauze metal tube of the injured arterial wall of stent support, and it reduces blood vessel in the postangioplasty probability of closed (restenosis) once more.In the U.S., annual enforcement near 100 ten thousand balloon angioplasties.Not every patient can both treat with this technology; This class patient must carry out operation on heart.Michaels etc., 2002.

About 10% CHD patient will carry out coronary artery bypass grafting art (CABG) operation.Suffer from the patient of total coronary stricture in a serious left side or obstruction, perhaps suffer from the candidate that those patients that relate to two or three diseases coronarius are considered to by-pass operation usually.In CABG, the surgeon is used for setting up around inaccessible detour (or bypass) partly coronarius from the healthy blood vessel of health another part (tremulous pulse or vein).In given operation, the patient accepts 1 to 5 bypass usually.In this process, allow heart be in palsy usually, be called cardioplegia (CP), circulation is kept in the heart-lung machine artificially in this process.The patient is in general paralysis in operation process, it generally continues 3-6 hour.

All patients of about 13% will be owing to relevant with CABG former thereby entered hospital once more in 30 days.Hannan etc., 2003; Mehlhorn etc., 2001.The one of the main reasons of being in hospital once more is a heart failure, may be because the ischemic lesions in the operation process.Therefore, do many work improve heart do not obtain normal perfusion during to the protection of cardiac muscle.

The nearest progress of operation on heart has concentrated on optimizes the cardioplegia parameter, and expectation can prevent postoperative ventricle malfunction and improve the long and.Cohen etc., 1999.

With cardioplegia solution perfusion by blood vessel and heart the chamber and cause its inherent beating to stop, keeping the viability of organ simultaneously.Cardioplegia (paralysis of heart) is expected in opening the operation on heart process, obtaining, transport and preserve the donor's heart process that is used for heart transplant operation.

Early stage cardioplegia technology adopts cold crystalloid solution to come initial and keeps perioperative asystole.Yet clear, BC has promoted the aerobic myocardial metabolism in the intersection pincers process and has reduced the generation of anaerobism lactic acid.And BC improves oxygen carrying capacity, increases myocardial oxygen consumption and keeps myocardium energy-rich phosphate deposit.Multiple different heart attack is available, and the different technologies of use cardioplegia solution is known in the art.For example, cardioplegia solution has not commensurability potassium, magnesium usually, and multiple other micro constitutent.Sometimes, medicine is added in the cardioplegia solution to help of flaccid muscles and to prevent ischemia.Current method also comprises has replenished suitable electrolyte, and for example glutamic acid-aspartic acid only is the preparation of blood.The instantiation of normally used solution is St Thomas hospital solution, University of Wisconsin solution, Stanford solution and Bretschneider solution.The example of the solution of other new development comprises and contains the arginic previously mentioned solution of adenosine, insulin or L-.The temperature that changes when using cardioplegia solution has favourable effect too.

Successive degeneration cardioplegia can keep ATP to lay in, and improve the metabolism recovery intersecting the myocardium lactic acid generation of pincers release back minimizing with the combination of the anterograde cardioplegia of interruption.Warm (29 ℃) cardioplegia reduces lactic acid and the sour generation in the heart paralytic stopped process, and improves postoperative ventricular function.Need 200mL/ minute at least heart attack flow velocity to wash out deleterious metabolic end product and improve ventricular function.Fully clear at present, the following direction that cardioplegia is handled will be referred to use the cardioplegia additive further to improve the protection effect.For example, attempted the fixing preregulated advantageous effects of ischemia of using adenosine.Similarly, adopted the insulin cardioplegia so that by stimulating early stage postoperative aerobic metabolism to strengthen ventricular performance.At last, proved that L-arginine, nitric oxide donors are favourable in experimental study, and representative is used for strengthening the further selection of the myocardial preservation of operation.The benefit in future that cardioplegia is replenished might be observed in the high-risk group with weak ventricular function, is not enough for the present resist technology of described high-risk group.The incidence rate of present high-risk patient is stable to be increased, and these cases, and the complication that takes place subsequently, has increased out-of-proportion burden to healthcare system.Therefore, the nursing in this field of the very hopeful development of the improvement in this field.

Although be used to induce the method for cardioplegia that the protection effect is provided by current, but still existence is to the ischemia reperfusion injury of some degree of cardiac muscle.Ischemia reperfusion injury in the heart bypass operation process causes the effect (M ﹠ M both) of difference, especially because the heart state that has weakened.Myocardial ischemia causes the anaerobic type myocardial metabolism.The end-product of anaerobic metabolism causes acidosis, mitochondrial fuctionning obstacle and myocyte's necrosis rapidly.The loss of energy-rich phosphate almost takes place immediately, has lost 50% ATP deposit in 10 minutes.Contractility reduces generation in 1 to 2 minute, and ischemic contracture and irreversible damage take place behind 30-40 minute room temperature (37 ℃) ischemia.

Reperfusion injury is a kind of well-known phenomenon after coronary circulation recovers.Reperfusion injury be characterized as unusual myocardium oxidative metabolism.Except the structural change that produces in the ischemia process, perfusion can produce Cytotoxic oxygen-derived free radicals again.Thereby these oxygen-derived free radicals also destroy the integrity of film by the sarcolemmic phospholipid of oxidation, and bring into play significant role in the pathogeny of reperfusion injury.The free fatty of oxidation is discharged in the Coronary vein blood, and is the snperoxiaized labelling of phospholipid of myocardium.Protamine is induced complement activation, and it has activated neutrophilic granulocyte.Activatory neutrophilic granulocyte and other leukocyte are the other sources of oxygen-derived free radicals and other cytotoxic substance.

The invention provides the method and composition that is used to induce cardioplegia, this will provide the heart better protection in the by-pass operation process.In certain embodiments, the invention provides to contain and be dissolved in the solution or as the H of gas bubbling in solution ₂The cardioplegia solution of S (or another kind of reactive compound).In some embodiments, the present invention further comprises at least the first kind of device, and for example conduit or intubate are used to import the cardioplegia solution of suitable dose to heart.In some aspects, the present invention further comprises at least the second kind of device, and for example conduit or intubate are used for cardioplegia solution is shifted out from heart.

By-pass operation continues 3-6 hour usually, yet it is 12 hours or longer that complication and multivessel CABG can prolong the persistent period.Consider that heart should keep stagnating in operation process.Thereby, in some embodiments of the present invention, allow heart be exposed to oxygen antagonist or other reactive compound pact, at least about, or about at the most 30 seconds; 1,2,3,4,5,10,15,20,25,30,35,40,45,50,55 minute; 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18 hours or longer, and any range wherein or combination.

E. reduce the damage that causes by the cancer treatment

Cancer is the first cause of mortality rate in the industrialized country in the world.The conventional method of treatment cancer is to give the cancer patient (or ex vivo treatment of tissue) by the dosed cells toxic agents, and described like this medicament has the bigger lethal effect of comparison normal cell to cancerous cell.Dosage lethal high more or medicament is strong more, will be effective more; Yet, by the same token, this class medicament on the sort of degree to normal cell more virose (and being fatal sometimes).Therefore, chemotherapy and radiotherapy are characterized by serious adverse usually, some side effect are life-threatening, for example oral ulcer, dysphagia, xerostomia, feel sick, diarrhoea, vomiting, tired, hemorrhage, alopecia and infection, skin irritation and energy loss (Curran, 1998; Brizel, 1998).

Nearest studies show that, temporary transient and reversible reduction core temperature, or " hypothermia " can cause anticancer improvement.The hypothermia that recent findings is 28 ℃ can reduce radiation in the mice, amycin-and cisplatin-inductive toxicity.The active anticancer of these medicine/treatments does not have impaired when being administered to cryogenic (cooled) animal; On the contrary, activity is strengthened, and especially the activity of cisplatin is strengthened (Lundgren-Eriksson etc., 2001).Based on this research and other disclosed research, the further reduction that the present inventor proposes core temperature will help the cancer patient.Therefore, the present invention considers to use oxygen antagonist or other active stagnation chemical compound to induce the stagnation of cancer patient normal structure, thereby minimizing chemotherapy or radiotherapy are to the potential impact of those tissues.This also allows to use the more chemotherapy and the radiotherapy of high dose, thereby strengthens the anti--cancer effect of these treatments.

Consider in fact any excessively proliferative disease, comprise treatment optimum and malignant tumor formation, non-tumprigenicity excessively proliferative disease, the preceding disease of tumor and precancerous lesion.This class disease comprises restenosis, cancer, multi-drug resistant cancer, constitutional psoriasis and metastatic tumo(u)r, blood vessel generation, rheumatoid arthritis, inflammatory bowel, psoriasis, eczema and secondary cataract, and oral cavity hairy leukoplakia, bronchus abnormal development, cancer in situ and last Intradermal hypertrophy.Especially, the present invention's cancer of being intended to treat the people comprises carcinoma of prostate, pulmonary carcinoma, the brain cancer, skin carcinoma, hepatocarcinoma, breast carcinoma, lymphoid system cancer, gastric cancer, carcinoma of testis, ovarian cancer, cancer of pancreas, osteocarcinoma, bone marrow cancer, human primary gastrointestinal cancers, H﹠N cancer, cervical cancer, esophageal carcinoma, cancer eye, carcinoma of gallbladder, renal carcinoma, adrenal carcinoma, heart cancer, colon cancer and leukemia.Consider that also treatment relates to the cancer of epithelial cell and endotheliocyte.

Usually, design chemotherapy and X-ray therapy reduce the tumor size, reduce growth of tumour cell, inducing tumor cell is transferred and died, reduces tumor vascular system, reduces or prevent to shift, reduces tumor growth rate, acceleration death of neoplastic cells, and the kill tumor cell.Target of the present invention there is no difference.Therefore, consider oxygen antagonist of the present invention (or other reactive compound) compositions and second kind of effectively anti--cancer medicament (second kind of medicament) combination of treatment hyperplasia disease." anti--cancer " agent can influence the cancer among the experimenter negatively, for example the speed of growth by kill cancer cell, cancer cell specific induction of apoptosis, reduction cancerous cell, reduce metastatic tumor incidence rate or quantity, reduce the tumor size, suppress tumor growth, reduce blood supply to tumor or cancerous cell, strengthen antagonism cancerous cell or tumor immunne response, prevent or suppress the cancer progress, or prolong the experimenter's who suffers from cancer life-span.

Second kind of anti--cancer agent comprises biological agent (biotherapy), chemotherapeutant and radiotherapy dose.More generally, these other compositions to be effectively to kill or anticancer or tumor cell proliferation, and reducing simultaneously or minimizing second kind of medicament provides the unitized dose of Normocellular influence.This method can relate to and cell is contacted with second kind of medicament simultaneously with oxygen antagonist (or other reactive compound) or exposes.This can realize by such: cell is contacted with the single compositions or the pharmacological preparation of this two classes medicament of comprising, or by with cell at one time with the contact of two kinds of different compositionss or preparation or expose, wherein a kind of compositions contains the aerobic antagonist and another compositions contains second kind of medicament.

Alternately, oxygen antagonist (or other reactive compound) treatment can be before or after second kind of pharmaceutical treatment, blanking time be several minutes to several weeks.Other medicament and expression construct separate administration are given in the embodiment of cell therein, should guarantee that usually effectual time can not stop between each Delivery time, and medicament and expression construct will still can be brought into play favourable synergy by pair cell like this.Under this class situation, consideration can contact two kinds of modalities of cell and this, each other in about 12-24 hour, more preferably each other in about 6-12 hour.Yet, in some cases, prolong the effectively time of treatment, it may be ideal wherein between using separately the interval of a couple of days (2,3,4,5,6 or 7) to several weeks (1,2,3,4,5,6,7 or 8) being arranged.In certain embodiments, imagination keeps biological substance to stagnate about 2-and between about 4 hours, implements the cancer treatment simultaneously.In some embodiments of the present invention, allow biological substance be exposed to oxygen antagonist or other reactive compound pact, at least about, or about at the most 30 seconds; 1,2,3,4,5,10,15,20,25,30,35,40,45,50,55 minute; 1,2,3,4,5,6 hours or longer, and any range wherein or combination.

Can adopt multiple combination; Reactive compound is " A " and second anti--cancer agent, and for example radiotherapy dose or chemotherapeutant are " B ":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/AA/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/AA/B/A/A A/A/B/A

Oxygen antagonist of the present invention or other reactive compound are administered to the patient will be according to the conventional method that is used to implement the chemotherapeutics case, if virose words are considered the toxicity of chemical compound.Expection is the repetitive therapy cycle as required.Should consider that also multiple standards therapy and surgical intervention can resist-cancer therapy use in conjunction with above-described.Further consider,, can use the use reactive compound considered and any therapeutic alliance of non-active compound (for example chemotherapy) for the various active chemical compound.

1. chemotherapy

The cancer therapy also comprises the multiple conjoint therapy of using based on the treatment of chemistry and radiation.Combined chemotherapy comprises, for example cisplatin (CDDP), carboplatin, procarbazine, chlormethine, cyclophosphamide, camptothecine, ifosfamide, melphalan, chlorambucil, busulfan, nitroso ureas, dactinomycin, daunorubicin, amycin, bleomycin, plicamycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, the estrogen receptor bonding agent, paclitaxel, gemcitabine, nvelbine, farnesyl-protein transferase inhibitor, anti-platinum (transplatinum), 5-fluorouracil, vincristine, vinblastine and methotrexate), Temazolomide (the moisture form of DTIC), or any analog of aforementioned medicine or the variant of deriving.The combination of chemotherapy and biotherapy is called biochemical therapy.

2. X-ray therapy

The other factors that causes DNA damage and extensively utilize comprises those that are commonly referred to gamma-radiation, X-ray, and/or directly sends radiosiotope to tumor cell.The DNA damage factor of other form also is considered, for example microwave and ultraviolet radiation.Probably, all of these factors taken together realizes the duplicating and repairing of DNA, DNA precursor, DNA, and chromosomal assembling and the wide model damage kept.The dosage range of X ray is from the 50-200 roentgen's of long time period (3 to 4 week) daily dose, to 2000-6000 roentgen's single dose.The dosage range of emitting isotope alters a great deal, and depends on isotopic half-life, radiating intensity and the type sent, and the absorption of neoplastic cell.

Term " contact " and " exposure ", when being applied to cell, being used herein to description is delivered to target cell with component of the present invention (for example, the antitumoral compounds of hypoxia) or chemotherapeutant or radiotherapy dose or it is placed directly and target cell method arranged side by side.In conjoint therapy, kill or stagnate in order to realize cell, can be with two kinds of preparations to be effective to cell killing or to prevent that its splitted unitized dose is delivered to cell.

3. immunotherapy

Usually, immunization therapy depends on and uses immune effector cell and targeting and tumoricidal molecule.Immunological effector can be, for example to the specific antibody of some labelling on the tumor cell surface.Antibody can be used as the effector of treatment alone, and perhaps it can be raised the next reality of other cell and realize that cell kills.Antibody also can be puted together with medicine or toxin (chemotherapeutant, radionuclide, ricin A chain, cholera toxin, pertussis toxin, PT etc.) and only as the targeting agent.Alternately, effector can be to carry directly or indirectly and the lymphocyte of the interactional surface molecular of tumor cell target.Multiple effector cell comprises cytotoxic T cell and NK cell.

Immunotherapy also can be used as the part of conjoint therapy.The conventional method that is used for conjoint therapy is discussed below.In the one side of immunization therapy, tumor cell must have some labellings that are subjected to targeting, promptly is not stored on other cell of great majority.Exist many tumor markers and arbitrarily these labellings can be suitable for targeting within the scope of the present invention.Common tumor marker comprises carcinoembryonic antigen, prostate specific antigen, urinary system tumor associated antigen, fetal antigen, tryrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155.An alternative aspect of immunotherapy is the anticarcinogenic effect with immune-stimulating effect.Also have molecules of immunization stimulus, comprising: cytokine is IL-2, IL-4, IL-12, GM-CSF, г-IFN for example; Chemotactic factor is MIP-1, MCP-1, IL-8 for example; And somatomedin FLT3 part for example.With molecules of immunization stimulus as protein or with gene delivery and tumor inhibitor for example the mda-7 combination shown and strengthened antitumous effect (Ju etc., 2000).

As previously discussed, the example of the current immunotherapy of just studying or using be immunological adjuvant (for example, Mycobacterium bovis (Mycobacterium bovis), Plasmodium falciparum (Plasmodiumfalciparum), dinitrochlorobenzene and aromatic compounds) (United States Patent (USP) 5,801,005, United States Patent (USP) 5,739,169, Hui and Hashimoto, 1998, Christodoulides etc., 1998), cytokine therapy (for example, interferon-ALPHA, β and γ; IL-1, GM-CSF and TNF) (Bukowski etc., 1998; Davidson etc., 1998; Hellstrand etc., 1998), gene therapy (for example TNF, IL-1, IL-2, p53) (Qin etc., 1998; Austin-Ward and Villaseca, 1998; United States Patent (USP) 5,830,880 and United States Patent (USP) 5,846,945) and monoclonal antibody (for example anti-Ganglioside GM2, anti--HER-2, anti--p185) (Pietras etc., 1998; Hanibuchi etc., 1998).Trastuzumab (trastuzumab) is chimeric (Mus-people) monoclonal antibody of blocking-up HER2-neu receptor.It has the active treatment (Dillman, 1999) of also having ratified to be used for malignant tumor of Anti-tumor.Use the conjoint therapy of Trastuzumab and chemotherapeutic cancer to show more effective than independent therapy.Therefore, consider that one or more anti--cancer therapies can use with Anti-tumor therapy described here.

F. neural degeneration

The present invention can be used for treating neurodegenerative disease.The degeneration that is characterized as neuronal tissue of neurodegenerative disease, and be attended by the loss of memory, motor function forfeiture usually, and dull-witted.About dementia disease, the comprehensive cognitive competence of intelligence and Geng Gao becomes more and more impaired in time.About according to estimates 15% 65 years old or more old people are slightly to moderate dementia.Neurodegenerative disease comprises the neurological damage of parkinson disease, constitutional neurodegenerative disease, hungtington's chorea, apoplexy and other hypoxia or ischemic process, neurotrauma, metabolic induction, sequela, hemorrhagic shock, Secondary cases neurodegenerative disease (metabolic or toxic), Alzheimer, other dysmnesia or vascular dementia, multi-infarct dementia, dementia with Lewy body or the neural degeneration dementia that cerebral fit produces.

Evidence show, biological health, and especially neural health depends on the circulation between oxidation and the reducing condition, itself and circadian rhythm are closely related.That is to say that the oxidative stress that in clear-headed process health is produced is circulated to the reproducibility environment in sleep procedure.Think this is why to sleep to health very important one big reason.Some neurodegenerative disease state, for example Huntington Chorea and Alzheimer, and inharmonious relevant in normal aging course and this circulation pattern.Also there are some evidences, i.e. the H of brain ₂The S level reduces (Eto etc., 2002) under these states.

The present invention can be used for regulating and controlled oxidation and reducing condition between circulation, for example in order to prevent or to reverse the influence of neurodegenerative disease and process.The control circadian rhythm can have other application, for example, is used for regulating these circulation patterns after travelling to another time zone from a time zone, so that adapt to new time zone.In addition, shown that metabolic activity reduces relevant with old animal and human's health generally.Therefore, the present invention's metabolic function that also will can be used for suppressing total increases old people's life-span and health.Consider such processing may every day at night, in sleep procedure, implemented about 6-10 individual hour.This may handle in every day in several months to several years long-time.

G. old and feeble

In addition, under some dead state, include but not limited to that wherein biological substance is in stagnant state of giving birth under the state, old and feeble itself can being subjected to fully in the period when biological substance is in described state or inhibition fully.Therefore, the present invention can suppress the aging of biological substance, proceeds to another stage of development about prolonging biological substance with the time quantum of normal existence and/or from stage of development of life.

H. hematopathy

A large amount of hematologic disease and disease can solve with the compositions and methods of the invention.These diseases include, but are not limited to thalassemia and sicklemia.

1. thalassemia

Normal hemoglobin contains two б and two в globin polypeptide (protein) chain, and every in conjunction with ferruginous blood red prime ring.Thalassemia is such one group of disease: have the imbalance of б and в chain in this disease, cause azygous chain to be deposited on the erythrocyte membrane of common fragility, cause cytoclasis.To such an extent as to this causes serious anemia bone marrow to attempt to compensate by managing to produce more erythrocyte.Unfortunately, because the toxicity of azygous chain, this process efficiency is very low, causes a large amount of expansions in marrow crack and blood to generate the other parts that are diffused into health.This and anemia cause main toxicity.Why exist severally has abrasive model like this about azygous globin chain, but majority shows that the erythrocytic early damage of radical pair of the increase that is produced by the ferrum that is connected to azygous globin chain is important.Therefore, any intervention that can reduce from the oxidative damage of these free radicals can prolong the erythrocytic life-span, improves anemia, causes reducing to producing erythrocytic demand, and reduces from the expansion of marrow crack and the damage of scattering.

Annual according to estimates above 30, suffers from serious thalassemic child's birth for 000, wherein arrived their more than 20 year old in most of work of developed country life according to estimates, and death when majority is young child in third world countries' (most patient is there living).Based on the current results in other model system of here introducing, expection will suffer from the ability that erythrocyte that thalassemic animal is exposed to sulfide and will increases them stands oxidative damage, cause erythrocyte survival prolongation.

2. sickle cell disease

Normal hemoglobin (HbA) contains two б and two в globin polypeptide (protein) chain, and every in conjunction with ferruginous blood red prime ring.Sickle cell disease (SCD; Be also referred to as sicklemia) be that the в chain that wherein suddenlys change causes hemoglobin to change a class disease of (HbS).In case deoxidation, HbS polymerizable (crystallization) and precipitation, the erythrocyte membrane that damage is fragile usually causes cytoclasis and anemia, erythrocyte (RBC) to reduce.In addition, have polymeric HbS cell change the shape (falciform) and the toughness that becomes, and activated the mechanism that causes blood flow to solidify and block.The hypoxia infringement that this can cause surrounding tissue causes pain, organ dysfunction and final premature dead.In the patient, observe the minimizing of reserves of the antioxidant of sulfur-bearing.And, the active oxygen (ROS) of oxidative damage and increase and crystallization, RBC membrane damage and relevant with the not enough relevant histologic lesion of blood flow.Sulfide and " supply " antioxidant deposit, and it is relevant that oxidative damage is minimized.Have reason to think that sulfide can prevent the problem at the several stages of meniscocyte's pathology.In addition, in view of the ability that the oxygen antagonist is resisted hypoxia in other systems, hinting that it should also protect the animal and human who suffers the unfavorable conditions that caused by this morbid state.

There are every year 120,000 children of surpassing to have SCD from birth.Patient in the developed country more than 40 year old of having arrived them and over fifty years old of living now, however have greatly about the problem of pain and organ injury, comprise apoplexy, lung, the heart and skin problem.Third world countries (most patient is there living), great majority death when young child.Our hypothesis is, will suffer from the animal of SCD and finally is that the people who suffers from SCD is exposed to sulfide, will cause healthy the improvement.

IV. preservation is used

The present invention can be used for for transportation and/or storage purpose preservation or preserves multiple biological substance, comprises cell, tissue, organ, and whole biology.In certain embodiments, the preservation biological substance is so that prevent infringement from unfavorable conditions.

In embodiments of the invention, biological substance can be exposed to reactive compound approximately, at least about, or about at the most 30 seconds; 1,2,3,4,5,10,15,20,25,30,35,40,45,50,55 minute; 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 hour; 1,2,3,4,5,6,7 day; 1,2,3,4,5 weeks; 1,2,3,4,5,6,7,8,9,10,11,12 month; Or 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more for many years, and can come from wherein combination in any or scope.Consideration can be used to reactive compound to induce stagnation, and considers other material can be used to keep and stagnate and with their preservations significant a period of time arbitrarily.Alternately, consideration can be used to reactive compound to induce and/or keep stagnation.This can make up with other factors (for example environmental change of pressure and/or temperature).

1. cell

Discuss as top, consider various kinds of cell is used for the present invention.Consideration can be with the preservation in method of the present invention, device and compositions of this cell.

A. platelet

In certain embodiments, the present invention can be applied aspect the hematoblastic preservation.Platelet is to play the little cell fragment of most important effect (～erythrocytic one 1/3 sizes) in the clot of hemorrhage position forms.Hemostasis is by realizing like this: adhere to blood vessel wall, discharge and solidify chemical substance, the formation clot is with the breach in artery-clogging wall and/or the narrow blood vessel.Normal platelet count is 150, and 000-400 is between the 000 counting/μ L.The platelet transfusion concentrated solution is used for different indications, for example: 1) prevent because thrombocytopenic hemorrhage; 2) in bleeding patients, be used to keep platelet count greater than 50,000; 3) in order to solving platelet function abnormality, described platelet function abnormality is geneogenous or owing to medication, sepsis, malignant tumor, tissue injury, obstetric complication, extracorporeal circulation or organ failure for example liver or kidney disease.

The platelet of per unit on average contains 0.8-0.85 * 10 ¹¹Individual platelet.PC also contains blood plasma (thrombin) and a spot of erythrocyte and the leukocyte of the 60mL that has an appointment.Platelet unit must maintain under the room temperature (20 ℃-24 ℃) and shake in storage.They can (Blood Center) be stored to many 5 days in the blood station.Because hematoblastic rotten, and the risk of microbial contamination, the storage of longer time is impossible at present.There are two kinds of hematoblastic sources at present:

1) the platelet preparation of the blended PC of donor at random from gathering by centrifugal U/WB.The platelet of 8 units (each unit is from different donors) at the most can be mixed in the bag and be used for infusion.Platelet lost efficacy in mixing in back 4 hours.All units are from identical ABO type.If the platelet of ABO compatibility can not obtain, then replaceable is the incompatible platelet of ABO, and very little risk is arranged.Usually adult's dosage is the mixing donor platelet at random of 4-6 unit.

2) single blood sampling composition art platelet is collected from single donor, with standard (be equivalent to～4 mix units) size and " big " (be equivalent to～6 mix units) size preparation.Single blood sampling composition art platelet concentrate contains the blood plasma of 200-400mL.They can be collected as unit (single blood sampling composition art platelet at random) at random or can be specific receiver and obtain from " orientation " donor of family member or voluntary HLA compatibility.Single blood sampling composition art platelet discharges inefficacy in back 4 hours in processing and from the blood station.

Platelet stores the undiscovered problem in the storage of whole blood or other composition that proposed.Though whole blood, erythrocyte and leukocyte can be 4 ℃ of following stored for several weeks, platelet will be assembled in cold preservation and be deposited then.Therefore, storing hematoblastic standard method is at room temperature, about 20-24 ℃, follows mild agitation.Even under these conditions, platelet only can be preserved 5 days, needed then they are abandoned.This expired problem causes the revenue losses of the annual about $500 1,000,000 of United States Hospital.Prolong if can obtain the appropriateness of storage life, this loss of about 90% can be avoided.

Another problem that platelet stores is germ contamination.Pollute mainly owing in the venotomy process from the staphylococcus of skin, perhaps owing to the donor bacteremia.Hematoblastic germ contamination has been represented with any blood transfusion operation all maximum infection risk.

The key factor that influences hematoblastic viability is the adjusting of pH.The nearly all platelet unit that stores according to present generally accepted method shows that all pH reduces from their initial values of about 7.0.This reduction is mainly owing to producing lactic acid by the human platelet glycoprotein zymolysis, and on less degree owing to CO from oxidative phosphorylation ₂Accumulation.Along with pH descends, platelet changes shape, from discoid englobement.If be reduced to 6.0 under the pH, hematoblastic morphology and physiological irreversible change make them not have vigor after transfusion.Therefore, the platelet preservation important goal is to prevent that this pH from reducing.Thought in the past that platelet must be stored in the permeable container of oxygen because glycolysis when the oxygen availability is restricted, be upset (referring to, for example United States Patent (USP) 5,569,579).Yet the present invention has proved that the hematoblastic viability that stores can be prolonged by they are stored in the anaerobic environment.

The invention provides the method and composition that increases the hematoblastic time-to-live that stores and reduce germ contamination.In one embodiment, the invention provides sealable, the impermeable container of oxygen, platelet is placed this container.After sealing, anaerobism generator (the sodium borohydride sheet that for example, has palladium catalyst) is converted into water with the aerial oxygen in the container.This container also contains an indicator, the level of its indication oxygen tension.In case be under the anoxia condition, platelet also can store under lower temperature.

Platelet can suspend and be stored in blood plasma or any platelet storage solutions known in the art.For example, United States Patent (USP) 4,828,976 and 4,447,415 disclose the solution that multiple normally used suitable platelet stores.

Typically, platelet is stored in from using in the blood plasma of donor and with the sort of form.

Usually, the present invention is made up of the environment of a sealing (container, jar, impermeable bag or chamber), in this environment oxygen tension can be reduced to and be lower than 1% (10,000ppm) also more especially in the scope of 10-100ppm, or littler.Atmospheric oxygen in this environment reduces and can realize by many methods known in the art.For example, the oxygen that reduces in the atmosphere can produce the water realization with oxygen chemical combination by producing hydrogen (having or catalyst-free).But other reacts catalysis oxygen and other chemical compound, and for example carbonization is closed and produced carbon dioxide, or the like.Equally, oxygen can replace by all indoor air being converted to the gas that contains the arbitrary gas combination that does not comprise oxygen.In addition, oxygen can be removed except that all gas by this chamber is placed under the vacuum.Alternately, oxygen can be by using another kind of gas or the chemical compound of competing with oxygen, and for example CO competes.Also can utilize combination except that deoxidation and the remaining oxygen of competition.This device also can comprise measure oxygen concentration means to guarantee to obtain suitable anaerobic state.For example, oxygen concentration can be used based on the anaerobism indicator of methylene blue and measure, and described methylene blue becomes colourless when not having oxygen from blueness.Alternately, can use oxygen meter or other to measure the device of oxygen.

Described device comprises that also some means are contained in platelet in the airtight environment, oxygen can be removed from contain hematoblastic solution like this, and they self is removed from platelet.Such example is to allow platelet be in the gas-pervious bag, and this bag places airtight environment.Platelet also can remain in the interior container that opens wide of closed environment.Alternately, platelet directly can be placed impermeable, airtight container/bag.

Bio-Bag from Becton Dickinson ^TM(article number 261215) is an example sealable, oxygen-impermeable container, and it can be used for producing anaerobic environment and is used for hematoblastic storage.Bio-Bag, it comprises sealable, an airtight sack, anaerobism indicator, anaerobism generator (hydrogen generator) and palladium catalyst for selling the test kit that is used to separate anaerobic bacteria.Platelet in the gas-pervious bag will be sealed in the Bio-Bag preserves.

Anaerobism generator among the Bio-Bag is activated device by adding water, and water arrives filter paper sliver (wick) by a series of passage.This paper slip delays and regulates water and introduce in the into described chamber, and the sustained release of hydrogen is provided.The sheet that produces gas is made up of sodium borohydride.The hydrogen and the ambient oxidation in the hermetic container that discharge from this reaction are closed generation water.This reaction is by the palladium catalysis in the container.

Puget Sound Blood Center (PSBC) adopts standardized one group of in vitro tests, has assessed the platelet of preserving under the oxygen free condition state the 0th day, the 5th day and the 8th day independently.The result shows that it is the same with the platelet of preserving good to preserve up to 8 days platelet performance, or better under standard conditions under oxygen free condition.Ongoing research is to repeat this experiment, and observation time is extended to 13 days.

Those technical staff in this area will be familiar with being used to measure the method for platelet function.For example, as United States Patent (USP) 6,790, describe in 603, platelet function can be measured by following factor: (1) responds the attack that activates-induce agonist, the expression of internal protein on cell membrane; (2) accumulative ability when attacked by agonist; And the secretion of (3) adenosine triphosphate.Can cause the example of the activated agonist of platelet function to comprise thrombin, epinephrine, ADP and collagen.

The expression of internal protein can be by puting together molecule and fluorescent dye, sorting and measuring in the fluorecyte grader afterwards.Usually, preferably use two kinds of monoclonal antibodies, a kind of cell surface molecule in conjunction with the expression of formation type, and another kind of in conjunction with a cell surface molecule of only after activation, expressing.Every kind of monoclonal antibody and dyes in different colors are puted together, and described dyestuff can be distinguished by fluorescence spectrophotometry.A limiting examples of the cell surface molecule that the formation type is expressed is GPIIbIIIa; A limiting examples of the cell surface molecule of expressing after activation is P-selectin (P-selectin).It is well known in the art producing proteinic monoclonal antibody.U.S. Patent No. 5,470,738 is examples of the monoclonal antibody method of the GPIIIa that creates antagonism.Another kind of anti--platelet monoclonal antibody is the monoclonal antibody of antagonism GP IV, and as by U.S. Patent No. 5,231,025 is disclosed.Antibody also can be from for example commercial purchase of Becton-Dickinson (Philadelphia) of company.

Another parameter of platelet function is an accumulative ability when agonist is attacked.Platelet suspension is thick and milky.The sedimentation of gathering and aggregation subsequently can be passed through visual assessment, or uses densimeter measurement.

It is the secretion of ATP that yet the another kind of platelet function is measured.Can the fine platelet that plays a role can secrete ATP, and the cell that has been activated or can not secrete ATP at the cell of others loss of function.

2. cell culture

The present invention can expand to the protection cells in culture, itself otherwise may be dead or induced to transfer and die.In background of the present invention, cell is exposed to reactive compound before cultivating and/or when cultivating.Can cultured cells comprise those cells that can be put back at last in the physiological environment according to the present invention, those cells that promptly are used for transplanting subsequently.These cells include, but are not limited to bone marrow, Skin Cell and epithelial cell.Equally, some transplantable cells will be benefited from the expansion in culture very much, thereby increase the amount that can be introduced into the material among the host.Look ahead especially from the gastrointestinal epithelial cell as benefiting from the cell that is exposed to reactive compound.

In addition, the present invention has extended to the cultivation of tumor cell.The cultivation of known cancer cell can cause the change of phenotype, and causes death in some cases.This makes that tissue culture's test of tumor cell is very unpredictable.

General cell culture technology is that those skilled in the art know.The example of this knowledge can find in Shaw (1996) and Davis (1994), with them both at this by being incorporated herein by reference.The general information of traditional cell culture technology and revise also can be at United States Patent (USP) 5,580 finds in 781, with it by being incorporated herein by reference.In addition, the technology that is used to cultivate Skin Cell is at United States Patent (USP) 6,057, describe in 148, with it by being incorporated herein by reference.Consideration will be to these technology, and known other technique complementary of those skilled in the art contains the culture medium of one or more reactive compounds, or the reactive compound of perfusion of fluid and/or gas form.

E. the preservation of cell, tissue and organ

In certain embodiments of the invention, expectation preservation biological substance is so that prevent death as much as possible or decompose infringement to this material.Although successful renal transplantation was for the first time finished in 1954 and for the first time the heart regulating liver-QI be implanted in enforcement in 1967, every year, the thousands of people's death that needs organ transplantation.For various reasons, they need heart, lung, kidney regulating liver-QI.In addition, existence can be used the patient of pancreas or cornea.Though there are the lasting needs to organ donor, it is the restriction of current organ preservation technology to another major obstacles of those patients that needs organ transplantation that organ is provided.For example, generally believe that human heart must transport in 4 hours, for subsequently transplanting has any chance success.Rager, 2004 (seeing the following form).

The longest cold ischemia time

Organ	The preservation time
Organ	The preservation time	Heart and lung Liver and kidney pancreas small intestinal	4-6 hour 12-24 hour 48-72 hour 12-24 hour 12 hours

And in initial 30 days, the main cause of the organ transplantation of the heart of transplanting failure is an ischemia reperfusion injury.

Organ acquisition and preservation, tissue matching, and immunosuppressant is the principal element of successful solid organ transplantation.The technical elements that organ obtains operation allows a plurality of team to work together to obtain all useful organs from single donor.On average, obtain 3.6 organs from single late donor.

The preservation solid organ depends on the quick blood vessel internal cooling that original position is carried out, and excises organ afterwards, organ is preserved and is transported to rapidly receiver's hospital in ice-cold preservation fluid.Cold ischemia time is organ does not have blood flow on ice a time span.But the longest cold ischemia time has limited elapsed time amount (table 5) between organ recovery and organ transplantation.The coupling of 2%-10% and organ that obtain can not use owing to delayed Ischemia Time, and this depends on the type of organ.Similarly, the organ of the acquisition of about 10-20% is because weak organ dysfunction and/or infection (not comprising HIV/CMV/ hepatitis) and can not use.

Current preservation technology comprises and utilizes ice-cold solution that it comprises electrolyte, antioxidant, hydrion buffer and sugar.Punch etc., 2001.The blood group coupling (for example, blood group A, B or O) of all organs is depended in the tissue matching that is fit to.Immunosuppressant scheme generally includes three kinds of medicines: glucocorticoid is for example azathioprine or mycophenolate of prednisone, antimetabolite for example, and neurocalcin inhibitor for example cyclosporin or tacrolimus.

Two kinds the preservation/transport heart of being used for of frequent use be cryopreservation and lasting perfusion in order to the method for transplanting.In a kind of method of pro-, heart is stopped, in the donor body, shifting out, cool off rapidly then and Refrigerated Transport.In a kind of method in back, adopt the following step usually: 1) pulsation flows; 2) low temperature; 3) film oxygenate, and 4) contain the two infusion liquid.

In order to improve the prospect of successfully transplanting, developed the technology that better preservation is used for transplanted organ that is used for.The general field that two development occurred, one is the preservative solution field, and another is the organ container field.

In some aspects, for example transplant, the unfavorable result of wound healing can weaken or stop the normal immigration of transplanted tissue.Under background of the present invention, preceding use oxygen antagonist or the processing of other reactive compound are transplanted in being organized in of tissue that imagination will offer and acceptance, relevant wound healing is discussed as mentioned, and for example inflammation, accent are died and other infringement moves into wound healing/transplanting back incident of organizing to be devoted to suppress biological process.

F. biological

This class biology can be used for research purpose, test mice (mice pile up (banking)) for example, or be used for consumption, for example fish.In these cases, but consider to stop keeping indefinite duration stagnation.And stagnation can be induced in the part (comprising fruit, flower, leaf, stem, seed, cutting) of plant or plant.Plant can be crops, medicinal plants or ornamental plant.In plant, induce stagnation can increase the storage life or the pathogen-resistance of whole plants or plant part.Therefore, in embodiments of the invention, biology or its part are exposed to oxygen antagonist or other reactive compound pact, at least about, or about at the most 30 seconds; 1,2,3,4,5,10,15,20,25,30,35,40,45,50,55 minute; 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 hour; 1,2,3,4,5,6,7 day; 1,2,3,4,5 weeks; 1,2,3,4,5,6,7,8,9,10,11,12 month; Or 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more for many years, and can come from wherein combination in any or scope.

G. preserving agent

Disclose multiple preservative solution, when transporting organ, organ has been surrounded with preservative solution or pour into preservative solution.A kind of solution that the most generally uses is ViaSpan_ (Belzer UW), and it follows cold preservation to use.Other example of the composition of this class solution or this class solution comprises St.Thomas solution (Ledingham etc., J.Thorac.Cardiobasc.Surg.93:240-246,1987); Broussais solution; UW liquid (Ledingham etc.; Circulation82 (Part 2) IV351-8,1990); Celsior solution (Menasche etc., Eur.J.Cardio.Thorax.Surg.8:207-213; 1994); Stanford University solution and B20 solution (Bernard etc., J.Thorac.Cardiovasc.Surg.90:235-242,1985); and in United States Patent (USP) 6,524,785; 6; 492,103; 6,365; 338; 6,054,261; 5; 719,174; 5,693; 462; 5,599,659; 5; 552; 267; 5,405,742; 5; 370; 989; 5,066,578; 4; 938; describe in 961 and 4,798,824 and/or claimed those.

Except solution, also the material of known other type is used to transport organ and tissue.These comprise gelatinous or other semisolid material, for example as at United States Patent (USP) 5,736, and those that describe in 397.

Some are used for the system of organ preservation and solution and are particularly related at described solution or system and pour into oxygen, so that organ is exposed to oxygen, can improve viability because it is believed that organ or tissue maintained in the environment of oxygenate.Referring to Kuroda etc., (Transplantation 46 (3): 457-460,1988) and United States Patent (USP) 6,490,880,6,046,046,5,476,763,5,285,657,3,995,444,3,881,990 and 3,777,507.It is believed that depriving isolating heart that oxygen was longer than 4 hours has lost vigor and can not use because of ischemia/reperfusion injury in the receiver.See United States Patent (USP) 6,054,261.

And, most (if not whole words) be used for the solution of organ preservation and transplanting and container relate to low temperature (temperature is lower than room temperature, usually near but can not be lower than 0 ℃), it is called as " all useful organs and organize the foundation stone of method for preserving ".United States Patent (USP) 6,492,103.

In filed of organ transplantation, it is believed that the state of some condition and organ and the prognosis that success is transplanted are relevant: 1) cellular swelling and edema are minimized; 2) prevent acidosis in the cell; 3) ischemic lesions is minimized; And 4) in refilling process, be provided for the regenerating substrate of high-energy phosphate compound and ATP.Ischemic/reperfusion injury in the organ transplantation is that problem is especially arranged, because the organ of gathering shifts out in health, separates with the blood source, and thereby loses oxygen and nutrient (United States Patent (USP) 5,912,019) in long-time.In fact, one of problem of most critical is that graft function postpones the relative high rate of (DGF) in transplanting now, and this is owing to postoperative acute tubular necrosis.Current approach is experience difficulty in these fields still, and this has given prominence to importance of the present invention.

Yet the present invention can unite use with other preservation compositions and method.As at United States Patent (USP) 5,952, discuss in 168,5,217,860,4,559,258 and 6,187,529 (especially with it by being incorporated herein by reference), can the preservation biomaterial, for example be used for the transplantable or replaceable organ of long preservation.

Can give cell, tissue/organ, or corpse strengthens or keeps the chemical compound of the state that is used for transplanted organ.This method and composition is included in United States Patent (USP) 5,752, those that describe in 929 and 5,395,314.

In addition, method of the present invention can comprise except being exposed to oxygen antagonist or other reactive compound, also biological substance is exposed to preservative solution, example as discussed those.

Consider, be used for living and will as any medicament of the biological sample of the material of living or solution must be pharmaceutically useful or the pharmacology on acceptable.Phrase " pharmaceutically acceptable " or " pharmacology is last acceptable " refer to be administered to molecular entity and the compositions that man-hour can not produce anaphylactic reaction or similar adverse effect.The preparation that contains as the proteinic Aquo-composition of active component is that this area is fully understood.Usually, this based composition can be prepared into liquid solution agent or suspensoid; Also can be prepared into and be fit to dissolve before use or be suspended in solid form in the liquid.

Can monitor and be used for transplanted organ, especially about state as graft to assess their state.This method is at United States Patent (USP) 5,699, describes in 793.

Can after accepting organ transplantation, high amount of drug be administered to the patient to promote recovering process.These medicines comprise chemical compound and the medicament that reduces or suppress to resist the immunne response of the organ that offers.

In addition; constantly study other medicine and using it for organ transplantation; for example at United States Patent (USP) 6; 552; 083 (inhibitor that comprises N-(3,4-dimethoxy cinnamoyl) ortho-aminobenzoic acid) and 6,013; those that describe in 256 (in conjunction with the antibody of IL-2 receptor, for example humanization resists-Tax antibody).

H. preservation instrument and application

Be used to transport the system of organ and tissue or container by also being developed these years.Arbitrarily these embodiments can with instrument combination of the present invention, described instrument of the present invention allows to use oxygen antagonist or other reactive compound.

Great majority relate to and are used for realizing for example at United States Patent (USP) 4,292, the cooling system of those that describe in 817,4,473,637 and 4,745,759, and it adopts the active refrigeration with cooling liquid, and described cooling liquid is pumped out by this system.Designed several complicated apparatus, it comprises a plurality of chambers or twin containers, and for example United States Patent (USP) 5,434, and 045 and 4,723,974 is described.

Some have formed the system that wherein a kind of instrument design is used for pouring into the organ or tissue of preservative solution, as United States Patent (USP) 6,490, and 880,6,100,082,6,046,046,5,326,706,5,285,657,5,157,930,4,951,482,4,502, describe in 295 and 4,186,565.

Some are used for the system of organ preservation and solution and are particularly related at this solution or system and pour into oxygen, so that organ is exposed to oxygen, can improve viability because it is believed that organ or tissue maintained in the environment of oxygenate.Referring to Kuroda etc., (Transplantation46 (3): 457-460,1988) and United States Patent (USP) 6,490,880,6,046,046,5,476,763,5,285,657,3,995,444,3,881,990 and 3,777,507.It is believed that depriving isolating heart that oxygen was longer than 4 hours has lost vigor and can not use because of ischemia/reperfusion injury in the receiver.See United States Patent (USP) 6,054,261.

And, in some embodiments of the present invention, exist and be used for the hematoblastic method of preservation, as above mentioned.Use the technology of present disclosure, reduced or eliminated the shortcoming of prior art.The embodiment that relates to the minimizing of platelet and oxygen is widely used, and includes but not limited to preserve hematoblastic any application with benefiting from longer-term.

In one embodiment, oxygen minimizing technology can embody in test kit.For example, the present sell goods that provide of BectonDickinson number are the selection technology that 261215 test kit utilization is here described.This test kit comprises anaerobism generator (for example hydrogen generator), palladium catalyst, anaerobism indicator and airtight, sealable " BioBag ", and top component (platelet in ventilative pocket) is placed in one and seals.

Anaerobism generator in this exemplary kit activates by adding water, and water arrives the filter paper bar by a series of passage.This paper slip delays and regulates water and introduce in the into described chamber, and the sustained release of hydrogen is provided.The sheet that produces gas comprises sodium borohydride.The hydrogen and the ambient oxidation in the hermetic container that discharge from this reaction are closed generation water.This reaction is by the palladium catalysis in the container.

One more general aspect, the sealed environment of the technology any number of present disclosure (for example, container such as jar, impermeable bag, or chamber) is implemented, oxygen tension can reduce in the sealing environment.In one embodiment, the oxygen level in container and/or platelet or the related solution can be reduced to and be lower than about 1% (about 10/1000000ths, 000 part).In another embodiment, oxygen can be reduced to the scope of about 1,000,000/10-100 part, or still less.In yet another embodiment, oxygen can be reduced to any percent value that the oxygen in expression container and/or platelet or the related solution reduces.In preferred embodiments, container is air-locked, and sealable.One skilled in the art will understand, and " gas-pervious " not necessarily means the impermeability of absolute or 100% level.On the contrary, " air-locked " should be as its represented in the art explaining, expression for example can be kept to less than 10ppm and (with respect to the gradient of room air, be generally 210, atmosphere 000ppm) at least 4 days.Usually, can the commercial sack that obtains in 6 weeks or longer time, be impermeable.

Sealing when container can place in it at the element of relevant minimizing oxygen.The atmosphere oxygen that reduces in this environment can close the realization of generation water with oxidation by producing hydrogen (having or catalyst-free).But other reacts catalysis oxygen and other chemical compound, for example carbon combination results carbon dioxide.Other reaction and combination will be conspicuous for persons skilled in the art.Equally, oxygen can replace by indoor gas being converted to the gas that contains the arbitrary gas combination that does not comprise oxygen.In addition, oxygen can be by placing vacuum to remove in container, and described vacuum is enough to remove gas and especially is enough to remove oxygen and reaches level expectation, that reduce.Alternately, oxygen can be by using another kind of gas or the chemical compound of competing with oxygen, and for example CO competes.Can utilize the combination of removing oxygen and the remaining oxygen of competition.

In different embodiments, available apparatus is measured the anaerobic state of oxygen level to guarantee to reach suitable.Can use the anaerobism indicator based on methylene blue, described methylene blue becomes colourless when not having oxygen from blueness.Alternately, can use can the commercial oxygen meter (for example machinery and/or electronic surveying meter) that obtains or other measure the mechanism of oxygen.

In different embodiments, platelet is contained in the environment of sealing, like this can be with oxygen from containing hematoblastic solution, and they self are removed from platelet.For example, the platelet in the venting bags can be placed the environment of sealing.Other non-limiting instance can have a unlimited container and contain platelet in the environment of sealing.Alternately, platelet can be contained in the container (for example, bag) of impermeable a, sealing and be integrated with deaerating plant.

In one embodiment, the present invention relates to wherein platelet and solution are introduced method in air-locked container.This container seals.Oxygen is removed from container or from platelet and solution.Consideration is with the pact in the gas-pervious bag, at least about, or about at the most 20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99 or 100%, or remove at the oxygen of this derivable any range.

This method also can comprise indicates remaining oxygen level in except that container behind the deoxidation.Oxygen in the container can be reduced to about 10/1000000ths, 000 part or still less.Oxygen in the container can be reduced to the level between about 1,000,000/10-100 part.The introducing platelet can relate to be added the ventilative container that fills platelet and solution in air-locked container.The introducing platelet can relate to puts platelet and solution into sealable, flexible sack, or in sealable, the inflexible chamber.Sealed container can relate to the use binding agent, and sealed container can take place in any stage of given method.

Removal oxygen can relate to be extracted oxygen out from container, and this suction can relate to roughing pump and/or turbine pump suction.Removal oxygen can relate to be introduced hydrogen in the container, and generation water is closed in itself and oxidation.Hydrogen can be introduced by chemical reaction.Chemical reaction can be by catalysis.Removal oxygen can relate to the sheet of use generation gas to be introduced hydrogen in the container.The sheet that water can be added to the generation gas that contains sodium borohydride produces hydrogen.This water can add in mode time-delay and that regulated.For example, can use the filter paper bar.Water can be introduced into the filter paper bar by one or more passages.But palladium catalysis produces the chemical reaction of hydrogen.Removing oxygen can relate to and will introduce in the container with bonded one or more reagent of oxygen.CO can be introduced in the container, itself and oxygen bonding form CO ₂Removing oxygen can relate to one or more gas displacement oxygen.

The level that marks residue oxygen can relate to the methylene blue indicator of utilization variable color when anaerobic.Alternately, can use oxygen meter.The level that marks residue oxygen in the container can relate to the level that shows the residue oxygen in platelet or the solution.

In one embodiment, the present invention relates to wherein platelet and solution are introduced method in air-locked container.This container seals.Hydrogen is by adding the chemical reaction generation of entry to sodium borohydride.This chemical reaction forms water and removes oxygen in platelet and the solution by closing with hydrogenation.Indicate the level of the residue oxygen in the container after deoxygenation.

This chemical reaction can be used palladium catalysis.The adding of water can relate to uses the filter paper core.

In one embodiment, the present invention relates to be used for oxygen from system that platelet and solution are removed.This system comprises: (a) generator of sealable, air-locked container, (b) oxygen reduction, and (c) oxygen indicator.Dispose this sealable, air-locked container and adjust size so that accept platelet and solution.The generator of oxygen reduction is connected with this container and is designed for and remove oxygen in platelet and the solution by suction or chemical reaction.Oxygen indicator is connected with container and is designed for the oxygen level in the container after deoxygenation of indicating.

This container can be sealable, flexible bag.The generator of oxygen reduction can comprise hydrogen generator, and it designs and produces hydrogen and be used for closing with oxidation and produce water.Hydrogen generator can comprise the material that produces gas, and it produces hydrogen with a kind of reagent chemical combination the time.The material of this generation gas can comprise the sodium borohydride sheet, and described reagent can comprise water.Hydrogen generator also can comprise palladium catalyst.System can comprise that also design comes the introducing by one or more compositions of control chemical reaction, delays or regulate the member of this chemical reaction.For example, this member can comprise the core that delays and regulate chemical reaction.

In one embodiment, the present invention relates to comprise the test kit of hydrogen generator, air-locked resealable container and oxygen indicator.

Hydrogen generator can comprise the material that produces gas, and it produces hydrogen with a kind of preparation chemical combination the time.The material of this generation gas can comprise the sodium borohydride sheet, and described reagent can comprise water.Test kit also can comprise palladium catalyst.Test kit also can comprise the core that design delays or regulate the chemical reaction that produces hydrogen.

As above discuss, method of the present invention can relate to be used a kind of instrument or system, described instrument or system held biological substance to be placed in one or is exposed to wherein environment.The present invention includes the instrument that reactive compound (especially as gas) wherein is provided.In some embodiments, this instrument comprises the container with the sample room that is used to contain biological substance, and wherein this container is connected with the source of supply of the gas that contains reactive compound.Special consider that this container can be that solids container or it can be flexible, for example sack.

In some embodiments, the present invention is the instrument that is used for the preservation cell, this instrument comprises: have the container that volume is no more than 775 liters sample room, and with first kind of gas supply source of this sample room fluid communication, this first kind of gas supply source comprises carbon monoxide.In other embodiments, this instrument also comprises a chiller and/or a gas regulator of regulating the sample indoor temperature, the amount of the reactive compound in this gas regulator conditioning chamber or the amount of the reactive compound in the solution in the conditioning chamber.

Consider, can have the gas supply source of second kind or additional gas, or be used for second kind or additional gas source of supply of reactive compound.Second kind of gas supply source can be connected with described sample room, and perhaps it can be connected with first kind of gas supply source.Extra gas (as above discussing) can be nontoxic and/or non-active gas.

In some embodiments of the present invention, gas regulator is the part of described instrument.Can adopt one, two, three or more gas regulatoies.In some cases, gas regulator is regulated the gas that is supplied to the sample room by first kind of gas supply source.Alternately, it regulates the gas that is supplied to sample room or first kind of gas supply source by second kind of gas supply source, maybe can have the actuator that is used for first kind and second kind gas supply source.Further consider that any gas regulator of programmable is controlled the amount of the gas that is supplied to sample room and/or another kind of gas supply source.Adjusting can be carried out or can not carry out specified a period of time.A gas regulator can be arranged, and for any gas supply source that directly or indirectly is connected with sample, it can be or can not be can be programme controlled.In some cases, gas regulator is an electronic program.

In some cases, described indoor pressure and/or temperature can be regulated with pressure regulator or thermoregulator respectively.The same with gas regulator, but these actuators can be electronic programs.Instrument of the present invention also have the cooling and/or heater to obtain temperature discussed above.This device can be or can not be electronic program.

In other embodiments, described instrument comprises a wheeler, and above container is placed on, or it can have one or more handles.

Special consideration the present invention includes the instrument that is used for cell, and wherein this instrument has: the container with sample room; With first kind of gas supply source of sample room fluid communication, this first kind of gas supply source comprises reactive compound; But and the gas regulator of electronic program, it regulates the gas that is supplied to the sample room by first kind of gas supply source.

And, consider that any oxygen antagonist of describing in the application's case uses with instrument of the present invention.In specific embodiment, carbon monoxide can be used with this instrument.In other situation, the chemical compound that can use the chalcogenide chemical compound or have the Reducing agent structure.

Figure 19 is the sketch map of an example system and has embodied the notion of discussing above that this system is used for oxygen is removed from platelet and solution.Gas-pervious sack 1920 can place sealable air-locked container 1904.Air-locked container 1904 can be connected with the generator 1906 of oxygen reduction.In one embodiment, the generator 1906 of oxygen reduction can surround sealable air-locked container 1904.In different embodiments, the generator 1906 of oxygen reduction can adopt different forms.For example, it can be pump (for example, roughing pump and/or turbine pump) or hydrogen generator.Relevant with the generator 1906 of oxygen reduction can be one or more elements such as core or other delay mechanism.What be connected with sealable air-locked container 1904 is pick off 1908 and actuator 1910.In one embodiment, pick off 1908 can be an oxygen meter, and it can take various ways.In other embodiment, pick off 1908 can be temperature or piezometer.Certainly, can use pick off more than one.In one embodiment, actuator 1901 can be temperature or pressure regulator.For example, actuator 1901 can be that heating or chiller are in order to regulate the temperature in sealable air-locked container 1904.

V. diagnostic application

Sulphite is produced by intravital all cells in the homergy process of sulfur-containing amino acid.Sulfite oxidase is removed, and thereby the level of having regulated sulphite.The different activities of these enzymes will cause the sulphite of varying level to be emitted in the tissue specificity mode.In the example of Miao Shuing, for the solid tumor under the hypoxia condition, the level that sulphite can be higher produces in the above, in order to come to provide partial guard mode for tumor cell by minimizing metabolism state and inhibition immunological surveillance.Therefore, measure the sulphite level and with its add as to several morbid states for example the part of the diagnosis of solid tumor be favourable.In addition, because our suggestion utilizes sulphite to be used for many-sided application, will be useful so adopt the imaging of some type or other monitoring method to follow this suggestion.

The sulphite level of using current technology (for example HPLC) to measure in the serum is possible to obtain total sulphite level.Worth research makes the probability of sulphite imaging.Alternately, the protein science method can make to understand how to change the adjusting that participates in the metabolic enzyme of sulphite under some morbid state, makes this method can be used for diagnosis.

VI. screening is used

In embodiment further, the invention provides and be used to identify to be similar to the oxygen antagonist that the mode of inducing stagnation works and the method for molecule and other reactive compound.In some cases, oxygen antagonist of being sought or reactive compound work at similar chalcogenide chemical compound aspect reduction core temperature or the reservation viability in hypoxia or anaerobic environment, if there is no oxygen antagonist or other reactive compound, then described hypoxia or anaerobic environment will be killed biological substance.These algoscopys can comprise the random screening to the large-scale storehouse of candidate substances; Perhaps, this algoscopy can be used for concentrating on according on the chemical compound that is conceived to the specific type that following character selects: this character is considered to make them more likely to work as oxygen antagonist or reactive compound, and a kind of candidate's reactive compound is provided;

(a) this candidate's reactive compound is mixed with biological substance;

(b) measure distinctive one or more cell effects that the oxygen antagonist is handled; And

(c) described one or more reactions are compared with the reaction that does not have the biological substance under candidate's reactive compound situation.

Algoscopy can be carried out with isolated cell, tissue/organ or complete biology.

Certainly will be appreciated that all screening techniques of the present invention itself are useful, although in fact may not can find effective material standed for.The invention provides the method that is used to screen this class material standed for, just do not find their method.Yet, should also be understood that candidate's reactive compound can be accredited as effective reactive compound according to one or more algoscopys, as if this means candidate's reactive compound and has the ability that some serve as reactive compound, for example by induce stagnation in biological substance.In some embodiments, screening comprises that the algoscopy that the embodiment that uses in the present disclosure or other places are described identifies regulator.And; except the method for in this part, describing or replace the method in this part, describe, can detect candidate's reactive compound as the oxygen antagonist or as having the activity of the another kind of chemical compound (for example protectiveness metabolism agent or therapeutic substance) of active ingredient properties.Some embodiments of screening technique are provided hereinbefore.

Can further characterize or measure effective reactive compound.In addition, effectively reactive compound animal or the middle use of animal model (as hereinafter discussing) in vivo, or can be in other body use in animal or the animal model, described other animal or animal model can relate to the animal of same species or different animal species.

In addition, consider, also can after screening, be produced according to the reactive compound that embodiment of the present invention are identified.Equally, according to the inventive method,, biological substance can be exposed to effective reactive compound or contact with it especially about the embodiment of treatment or prevention.

A. reactive compound

As used herein, term " candidate's reactive compound " refers to and can change core temperature is induced stagnation in biological substance any molecule by for example.Even candidate's reactive compound can be that protein or its fragment, micromolecule are nucleic acid molecules.The micromolecule storehouse of can also be from various commercial source, being considered to meet the basic standard of useful medicine obtains candidate's reactive compound, identifies useful chemical compound to be devoted to " violence ".Screening these storehouses, comprise the storehouse (for example peptide storehouse) of combination results, is the active quick and effective method of a large amount of relevant (with the incoherent) chemical compounds of screening.Yet combined method itself also help by produce active aspect other not the mould of the desired compounds second filial generation of building, the third generation and the 4th generation chemical compound, develop potential medicine rapidly.

Candidate's reactive compound can comprise the fragment or the part of naturally occurring chemical compound, or is found with the active combining form of known compound, itself otherwise be non-activity.Someone proposes, and for example animal, antibacterial, fungus, plant source (comprising leaf and bark) and the chemical compound of marine products sample separation can be used as candidate detection, the existence of the medical substance of detection potentially useful from natural origin.Should be appreciated that the medical substance that will screen also can come from chemical constituent or synthetic compounds or synthetic with them.Therefore, should be appreciated that candidate's reactive compound of identifying by the present invention can be from known inhibitor or stimulant, peptide, polypeptide, polynucleotide, micromolecular inhibitor or any other chemical compound of designing by rational drug.

Other suitable reactive compound comprises antisense molecule, siRNA, ribozyme and antibody (comprising single-chain antibody), and they are specific to target molecule separately.This compounds other place in presents is described in more detail.For example, be bonded to translation initiation site or transcriptional start site, or the antisense molecule of splice junction will be ideal candidate inhibitor.

Except the reactive compound of initial evaluation, the inventor also considers to prepare the key component of the chemical compound of other structural similarity with simulation reactive compound structure.This compounds (it can comprise the peptide mimics (peptidomimetics) of peptide modulators) can use in the mode identical with the initial activity chemical compound.

B. algoscopy in the body

Measure the use that relates to several animal models in the body.Because their size, processing easily, and about their physiology and the information of genetic constitution, mice is an embodiment preferred.Yet other animal is also suitable, comprises rat, rabbit, hamster, Cavia porcellus, pallasiomy, marmot, mice, cat, Canis familiaris L., sheep, goat, pig, milch cow, horse and monkey (comprising chimpanzee, Gibbon and baboon).Fish also can consider to be used for algoscopy in the body, and nematicide too.The mensuration useful source of regulator carries out from the animal model of any these species.

In this class algoscopy, one or more candidate substances are administered to animal, and with inert carrier (negative control) and H ₂S (positive control) relatively, described candidate substances is induced stagnations, is reduced core temperature, or gives the ability evaluation regulator of the ability that biological substance survives under hypoxia or anaerobic environment condition.Handle animal with test compound and will be referred to this chemical compound, the form with suitable is administered to animal.Using of candidate compound (gas or liquid) can be by being used for any approach of clinical or non--clinical purpose, includes but not limited to per os, per nasal (sucking or aerosol), through cheek or or even local application.Perhaps, use and to be undertaken by tracheal instillation, bronchus instillation, intradermal injection, subcutaneous injection, intramuscular injection, peritoneal injection or intravenous injection.The special approach of considering is the general intravenous injection, passes through the local application of blood or lymph supply, or is applied directly to affected position.

VII. mode of administration and pharmaceutical composition

The effective dose of the pharmaceutical composition of chalcogenide, oxygen antagonist or reactive compound is normally defined to be enough to can to improve with detecting, reduce, minimize or the amount of the degree of limited target disease.More strict definition be can adopt, elimination, elimination or the healing of disease comprised.

A. use

Naturally, the route of administration of chalcogenide or other reactive compound will change with the position and the character of disease to be processed, and for example comprise, suction, Intradermal, in skin, parenteral, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, trachea, in the intraperitoneal, tumor, perfusion, lavation, direct injection, and dosage forms for oral administration and preparation.As described in detail below, reactive compound can be used as medical gas by sucking or intubation is used, as the injection liquid agent by in the blood vessel, intravenous, intra-arterial, Intraventricular (intracerobroventicular), intraperitoneal, subcutaneous administration, use with liquid agent or gel or solid oral dosage form as the part.

And described amount can change according to type (genus of cell type, types of organization, biology and kind etc.) and/or its size (body weight, surface area etc.) of biological substance.Normally biology is big more, and dosage is just big more.Therefore, the effective dose that is used for mice usually will be lower than the effective dose that is used for rat, and the effective dose that is used for rat will be lower than the effective dose that is used for Canis familiaris L. usually, and the effective dose that is used for Canis familiaris L. will be lower than the effective dose that is used for the people usually.Hydrogen sulfide realizes that in human body the valid density of stagnating depends on dosage form and route of administration.For inhalation, in some embodiments, valid density is in the scope of sending 50ppm to 500ppm continuously.Use for intravenous, in some embodiments, valid density is in the scope of 0.5 to 50 milligram of every kg body weight of sending continuously.

Similarly, the length of time of application can change according to type (genus of cell type, types of organization, biology and kind etc.) and/or its size (body weight, surface area etc.) of biological substance, and will depend in part on dosage form and route of administration.In special embodiment, provide reactive compound approximately or at least 30 seconds, 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 24 hours or be longer than 24 hours.Reactive compound can single dose or multiple dose use the time quantum difference between the dosage of using.

In the situation of transplanting, the present invention can be before operation and or the operation back use so that host or graft material dormancy.In specific embodiment, surgical site injectable or perfusion contain the preparation of chalcogenide.Continue the described perfusion back of can performing the operation, as by allowing conduit be implanted in the surgery location place.

B. injectable compositions and preparation

The method for optimizing that is used to send oxygen antagonist of the present invention or other reactive compound is the perfusion and the dosage forms for oral administration of inhalation, intravenous injection, special section.Yet, pharmaceutical composition disclosed herein can be alternative as United States Patent (USP) 5,543,158, United States Patent (USP) 5,641,515 and United States Patent (USP) 5,399,363 (with every piece at this especially by being incorporated herein by reference in full) in description, through parenteral, Intradermal, intramuscular, through skin or or even intraperitoneal use.

The solution of reactive compound can suitably and in the blended water of surfactant (for example hyprolose) prepare.Dispersion also can and prepare in oil at glycerol, liquid macrogol and composition thereof.Under general storage and service condition, these preparations contain antiseptic in order to prevent microbial growth.The medicament forms that suitable injection is used comprises sterile aqueous solution or dispersion and is used for preparing the sterile powder of aseptic injectable solution or dispersion (United States Patent (USP) 5,466,468 is incorporated herein by reference by full text at this especially) temporarily.In all scenario, described form must be aseptic, and should be to flow to the degree of injecting easily.It must be stable under manufacturing and condition of storage, and must it is anticorrosion to prevent the contamination of microorganism such as antibacterial and fungus.Carrier can be to contain for example solvent or the disperse medium of water, ethanol, polyhydric alcohol (as glycerol, propylene glycol and liquid macrogol etc.) and suitable mixture and/or vegetable oil.Suitable flowability can for example be passed through with coating such as lecithin, passes through the required granularity of maintenance in dispersive situation, and by using surfactant to keep.Prevention to microbial action can be passed through multiple antibacterial and antifungal, and for example p-hydroxybenzoic acid esters, methaform, phenol, sorbic acid, thimerosal wait and realize.In many situations, will preferably include isotonic agent, for example sugar or sodium chloride.The prolongation of composition for injection absorbs can be by using the reagent of delayed absorption in said composition, for example aluminum monostearate and gelatin are realized.

For parenteral administration, for example, should suitably cushion this solution if necessary and at first give this liquid diluent isotonicity with enough saline or glucose with aqueous solution.These special aqueous solutions are particularly suitable in intravenous, intramuscular, subcutaneous, the tumor and intraperitoneal is used.In this connection, according to present disclosure, spendable sterile aqueous media will be that those skilled in the art are known.For example, a dosage can be dissolved in the isoosmotic NaCl solution of 1ml, and be added in the 1000ml h inf fluid or inject (referring to for example, " Remington ' s Pharmaceutical Sciences " the 15th edition, 1035-1038 and 1570-1580 page or leaf) in the infusion site of suggestion.Some variations of dosage will inevitably take place according to subject experimenter's situation.Under any circumstance, the people who is responsible for using will be identified for the suitable dose of individual subjects.In addition, use for the people, preparation should meet aseptic, pyrogenicity, Generally Recognized as safe and the purity rubric that requires as FDA biological product standards office (Office of Biologics standards).

Aseptic injection with solution by like this preparation: the reactive compound of requirement various other compositions of above enumerating with needs are mixed in the suitable solvent, afterwards filtration sterilization.Usually, dispersion is mixed in the sterile carrier by the active component with various sterilizations and is prepared, and described carrier contains basic disperse medium and required from above-named other those compositions.In the situation of the sterile powder that is used for preparing the sterile injectable solution agent, preferred manufacturing procedure is vacuum drying and Freeze Drying Technique, and described technology produces the powder that active substance adds any extra required composition from its aseptic in advance-filtering solution.

As used herein, " carrier " comprise any and all solvents, disperse medium, excipient, coating, diluent, antibacterial and antifungal, etc. blend reagent, buffer, carrier solution, suspension, colloid of delayed absorption etc.This medium and reagent are used for the purposes of pharmaceutically active substances and know in the art.Except with inconsistent any conventional media of active component or preparation, consider in their being used for the treatment of property compositionss.Auxiliary active component also can mix in the said composition.

Phrase " pharmaceutically acceptable " or " pharmacology is last acceptable " refer to be administered to molecular entity and the compositions that man-hour can not produce anaphylaxis or similar adverse effect.Containing protein is fully to understand in this area as the preparation of the Aquo-composition of active component.Usually, this based composition can be prepared into the injectable agent, as liquid solution agent or suspensoid; Also can be prepared into dissolving before being adapted at injecting or be suspended in solid form in the liquid.

C. intravenous preparation

In one embodiment, reactive compound of the present invention can be prepared and be used for parenteral administration (for example intravenous, intra-arterial).Reactive compound at room temperature is under the situation of gas therein, considers to contain the solution known and gas molecule of expecting concentration, and described gas molecule is dissolved in liquid or the solution that is used for parenteral administration.The preparation of active compounds solution can be by for example, allows gas contact (for example, bubbling or injection) with solution so that this gas molecule is dissolved in solution finishes.Those technical staff in this area will recognize, the amount that is dissolved in the gas in the solution will depend on many variablees, include, but is not limited to the chemical composition of dissolubility, liquid or the solution of gas in liquid or solution, its temperature, its pH, its ionic strength, and the concentration of gas and exposure level (for example, the speed of bubbling or injection and persistent period).Reactive compound can adopt the known method of those skilled in the art to determine in the concentration of liquid that is used for parenteral administration or solution.The stability of reactive compound in liquid or solution can be by after preparation or producing oxygen antagonist solution, behind different intervals, measure the concentration of dissolved oxygen antagonist and measure, wherein represent the loss or the chemical conversion of reactive compound with the minimizing of initial concentration comparison oxygen antagonist concentration.

In some embodiments, the solution that contains the chalcogenide chemical compound is dissolved in sterilized water or the saline (0.9% sodium chloride) by the chalcogenide with salt form and produces to produce pharmaceutically useful intravenous dosage form.Available buffer intravenous fluid dosage form reaches certain pH, with dissolubility that strengthens the chalcogenide chemical compound or the ionized state that influences the chalcogenide chemical compound.In the situation of hydrogen sulfide or Selenium hydride., any one of the known many salt forms of those skilled in the art can be satisfied the demand, and includes but not limited to sodium, calcium, barium, lithium or potassium.In another embodiment preferred, hydrogen sulfide or Selenium hydride. are dissolved in the sterile phosphate buffered saline, but and to regulate pH with hydrochloric acid be 7.0 to obtain the solution that intravenous or intra-arterial are administered to experimenter's concentration known.

Consider to be about the reactive compound saturated solution to pharmaceutical composition of the present invention in some embodiments.This solution can be any pharmaceutically acceptable preparation, and wherein great majority are known, for example Ringer's mixture.In specific embodiments, the concentration of reactive compound is approximately, at least about or about at the most 0.001,0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2.0,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4.0,4.1,4.2,4.3,4.4,4.5,4.6,4.7,4.8,4.9,5.0M or bigger, can come from any scope (under standard temperature and pressure (STP) (STP)) wherein.For H ₂S, for example, in some embodiments, concentration can be about 0.01 to about 0.5M (under STP).Consider that particularly above-mentioned concentration can be applicable to be arranged in independently or together the carbon monoxide and the carbon dioxide of solution.

In addition, be intravenous when using when using, consideration can be used following parameters.Flow velocity approximately, at least about or about at the most 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100gtts/ minute or μ gtts/ minute, maybe can come from any scope wherein.In some embodiments, the amount of solution illustrates by volume (concentration that depends on solution).The amount of time can be approximately, at least about or about at the most 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60 minute; 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 hour; 1,2,3,4,5,6,7 day; 1, in 2,3,4,5 weeks and/or 1,2,3,4,5,6,7,8,9,10,11,12 month, maybe can come from any scope wherein.

1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,441,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980,990,1000ml or liter, or the volume of any scope wherein can totally be used or use in independent a period of time.

In some embodiments, be used for parenteral administration reactive compound solution therein oxygen liquid or solution are being prepared with the liquid or the solution that have been removed before reactive compound contacts.Some oxygen antagonist, especially some chalcogenide chemical compound (for example hydrogen sulfide, Selenium hydride .) because they and oxygen carry out the ability of chemical reaction, causes their oxidation and chemical conversion but unsettled when having oxygen.Can use methods known in the art that oxygen is removed from liquid or solution, described method includes, but is not limited to apply negative pressure (vacuum degassing) in liquid or solution, with solution or liquid with cause oxygen in conjunction with or the reagent of " chelating " contact, effectively it is removed from solution.

In another embodiment, being used for the oxygen antagonist of parenteral administration or the solution of other reactive compound can store at airtight container.When oxygen being removed in advance with restriction or anti-block antagonist or other reactive compound oxidation from solution, this especially expects.In addition, in airtight container, store and to suppress oxygen antagonist gas or other reactive compound volatilizees from liquid or solution, make the constant density of dissolved oxygen antagonist be maintained.Airtight container is that those skilled in the art are known, and includes, but is not limited to contain " the intravenous bag " of air-locked structure material, or the sealed glass bottle.In order to prevent to be exposed to the air in the air-tightness storage capsule, can introduce in the container as nitrogen or argon with noble gas before sealing.

D. topical preparation and using method thereof

Method and composition of the present invention is used in the shallow-layer of skin and oral mucosa and induces stagnation, and the shallow-layer of described skin and oral mucosa includes, but is not limited to mouthful and hair follicle cell, capillary tube epithelial cell and the epithelial cell of tongue.Be used for the treatment of the normal cell in the radiotherapy of cancer and chemotherapy meeting damage hair follicle and the oral mucosa, cause not expecting side effect, alopecia and the oral mucositis of only debilitant cancer treatment respectively.In hair follicle cell that provides blood to hair follicle and/or vascular cell, induce stagnation can slow down, limit or prevent to follow radiotherapy and chemotherapeutical to the damage of hair follicle cell and the alopecia that causes, or other baldness, male pattern alopecia, female pattern alopecia, or hair is from other disappearance of its skin area that exists usually.In mouth epithelial cells and Interstitial cell, induce stagnation can slow down, limit or prevent to the cells injury of liner at mouth, esophagus and tongue, and the pain condition of the oral mucositis that causes.

In certain embodiments, local application reactive compound.This is by being mixed with Emulsion, gel, paste or collutory with this reactive compound, and said preparation is applied directly to the zone (for example, scalp, mouth, tongue, larynx) that need be exposed to reactive compound realizes.

Topical compositions of the present invention can be mixed with oil preparation, Emulsion, lotion, ointment etc. by selecting suitable carriers.Suitable carriers comprises that vegetable oil or mineral oil, white vaseline (paraffinum molle alba), a chain fatty or oil, Animal fat and high molecular weight alcohol are (greater than C ₁₂).Preferred carrier is that active component therein can dissolved those carriers.Also can comprise emulsifying agent, stabilizing agent, wetting agent and antioxidant, and the reagent of giving color or fragrance, if necessary.In addition, the transdermal penetration reinforcing agent can be used in these topical preparations.The example of this class reinforcing agent can be at United States Patent (USP) 3,989, finds in 816 and 4,444,762.

Emulsion is preferably with the mixture preparation of the Cera Flava and the water of mineral oil, self emulsifying, in this mixture fusion be dissolved in active component in the small quantity of oil (for example almond oil).The representative instance of this Emulsion is to contain 40 parts the water of having an appointment, the Emulsion of the almond oil of about 20 parts Cera Flava, about 40 parts mineral oil peace treaty portion.

Ointment can be by mixing the active ingredient solution in the vegetable oil (as almond oil) with gentle soft paraffin, and allow this mixture cool off and prepare.The representative instance of this ointment is by weight the ointment of the paraffinum molle alba that contains have an appointment 30% almond oil and about 70%.

Lotion can be by preparing in the alcohol (for example propylene glycol or Polyethylene Glycol) that active component is dissolved in the proper polymer amount easily.

But the possible pharmaceutical preparation that per rectum uses comprises, suppository for example, and its combination by one or more reactive compounds and suppository base is formed.Suitable suppository base for example is, natural or synthetic triglyceride or paraffin hydrocarbon.In addition, it also is possible using the gelatin rectal capsule of being made up of the combination of reactive compound and substrate.Possible host material for example comprises, liquid triglycerides, Polyethylene Glycol or paraffin hydrocarbon.

E. solid dosage forms

Pharmaceutical composition comprises that reactive compound wherein is captured or hidden solid dosage forms in reaching crystalline state, solid-state porous carrier framework.This solid dosage forms with gas storage ability is known in the art, and can be with pharmaceutically acceptable form production (for example, Yaghi etc., 2003).Peculiar advantage of this pharmaceutical composition and chalcogenide chemical compound (as, hydrogen sulfide, carbon monoxide, Selenium hydride .) relevant, the chalcogenide chemical compound has toxicity to some mammals when being in their free form under some concentration.In certain embodiments, described chemical compound can be prepared and be used for dosage forms for oral administration.

F. perfusion system

The perfusion system that is used for cell can be used for tissue or organ are exposed to the reactive compound of liquid or semi-solid form.Perfusion refers to that successive flow of solution passes cell mass or flow through from cell mass.This means and cultivate on the contrary with continuous-flow that cell is stranded in the culture, described continuous-flow cultivation washes out cell (for example chemostat) with isolation medium (withdrawn media).Perfusion makes can control culture environment (pH, pO better ₂, trophic level, reactive compound level etc.), and be the method for utilization that is used for the surface area of cell attachment in a kind of remarkable increase culture.

Exploitation perfusion technology wherein continues to the cells in vivo environment of cell with blood, lymph or other body fluid in order to simulation.Perfusion physiology nutritional solution not, the cells in culture experience is fed and hungry alternating phases, thereby has limited the abundant performance of their growths and metabolism potential.In the scope of the invention, perfusion system also can be used for to cell perfusion oxygen antagonist in order to induce stagnation.

Those skilled in the art are familiar with perfusion system, and exist many can the commercial perfusion system that obtains.Can adopt any one of these perfusion systems in the present invention.An example of perfusion system is to use the perfusion packed bed reactor (CelliGen of the bed matrix of non-woven fabrics ^TM, New Brunswick Scientific, Edison, NJ; Wang etc., 1992; Wang etc., 1993; Wang etc., 1994).In brief, this reactor comprises the improvement reactor that is used to cultivate anchorage-dependent cell and non-anchorage-dependent cell.Reactor is designed to have the packed bed of the device of the interior recirculation of providing.Preferably, the fibrous matrix carrier is placed basket in the reactor vessel.The top of this basket and bottom are all porose, allow culture medium can flow through basket.Specially designed impeller provides the culture medium recirculation nutrient to be provided and to remove refuse to guarantee homogeneous by the space that fibrous matrix occupies.This total cell mass of having guaranteed negligible quantity simultaneously is suspended in the culture medium.The combination of basket and recirculation also provides the oxygenate culture medium bubble-freely to flow through fibrous matrix.Fibrous matrix is the non-woven fabrics with 10 μ m to 100 μ m " hole " footpath, and very big inner capacities is provided, and pore capacities is equivalent to 1 to 20 times of individual cells capacity.

The perfusion packed bed reactor has several advantages.Have the fibrous matrix carrier, the protection cell is avoided from stirring and blistered mechanical stress.Culture medium flows through oxygen, pH and the nutrient that basket offers cell optimum adjustment level freely.Product can constantly shift out from culture medium, and the product of gathering is acellular and can produces that this can promote ensuing purification step in low-protein culture medium.This technology has detailed explanation in WO 94/17178 (on August 4th, 1994, Freedman etc.), therefore it be incorporated herein by reference it by full text.

Cellcube ^TM(Corning-Costar) module provides big styrene (styrenic) surface area for the fixing and growth of substrate attached cell.It is the aseptic disposable operative installations that integral body is sealed, and it has a series of parallel culture plate, and culture plate connects and the laminar flow space of the sealing that generation approaches between adjacent panels.

Cellcube ^TMModule has the relative entrance and exit that also helps the flow of adjusting culture medium in diagonal angle each other.In a couple of days of beginning in the growth course, usually that culture is saturated by being contained in intrasystem culture medium in initial inoculation back.Time quantum between initial inoculation and culture medium perfusion begin depends on the density and the cell growth rate of cell in the inoculum of inoculation.The measurement of nutrient concentrations is a good index of culture state in the circulation culture medium.After having determined program, might be necessary to monitor the nutrition composition of multiple different perfusion speed, to determine most economical and productive operating parameter.

Other can comprise by the commercial perfusion system that obtains, for example CellPerf_ (LaboratoriesMABIO International, Tourcoing, France) and Stovall Flow Cell (StovallLife Science, Inc., Greensboro, NC).

The selection of time of the production phase of culture and parameter depend on the type and the purposes of special cells system.Many cultures need be different from the required culture medium of culture growth stage and be used for producing.In traditional cultivation, need a plurality of washing steps probably from of the conversion of a stage to another stage.Yet one of advantage of perfusion system provides the ability of gentle conversion between a plurality of operational phases.Perfusion system also can promote from growth stage to passing through the inductive stagnation of oxygen antagonist (static) phase transition.Equally, perfusion system can promote by replacing the solution that contains the aerobic antagonist with physiology Nutrient medium for example, changes to growth stage from the lag phase.

G. conduit

In certain embodiments, conduit is used for reactive compound is offered biology.Particularly importantly use a kind of like this medicament to heart or vascular system.Usually, conduit is used for this purpose.Yaffe etc., 2004 have discussed conduit especially in about the living content that stagnates, though the use of conduit is exactly generally known before this publication.

H. gas sends

1. respiratory system

A kind of typical gas delivery system 100 illustrates in Fig. 9.Delivery system 100 is fit to send the gas (comprising activating agent) that can the suck respiratory system to the experimenter.Gas delivery system 100 comprises one or more gas sources 102.

In each gas source 102 each is connected to actuator 104 and effusion meter 106.Carburator 108, outlet controller 110, remover (scavenger) 112 that gas delivery system 100 also comprises activating agent source 107, chooses wantonly, and alarm/monitoring system 114.Delivery system 100 can comprise usually sends some element that uses in the instrument in anesthesia.For example, anesthesia is sent instrument and is comprised a high tension loop, low tension loop, breathing circuit and one usually and remove loop (scavenging circuit).As in Figure 10-11, describing, can provide one or more gas sources 102, carburator 108, control of export device 110, remover 112 and/or alarm/monitoring system 114 as having high pressure, low pressure, breathing and/or remove the part of the device in loop, and these elements can be similar to anesthesia send in the instrument normally used those.Anesthesia is sent instrument for example at United States Patent (USP) 4,034, describe in 753,4,266,573,4,442,856 and 5,568,910, therefore with the content of these patents by being incorporated herein by reference in full.

Gas source 102 can provide by compression gas tank; Yet, should be appreciated that gas source 102 can be the fluid supply that gas source maybe can change into gas.For example, carburator 108 can be used for making the liquified gas source vaporization.Actuator 104 comprises the valve of the air pressure that reduces each gas source 102.Depressed gas is subsequently by one of them effusion meter 106, and this flowmeter survey and control are from the flow of the gas of each respective gas sources 102.

Gas source 102 can be the carrier gas that is used for active agent delivery 107.Can select carrier gas in order to offer the environment of having been sent from an expectation of experimenter of the activating agent in source 107.For example, if activating agent is given the patient as the gas delivery that can suck, then carrier gas can comprise that oxygen, nitrous oxide or the air of q.s satisfy patient's needs.Can use other noble gas or active gases.

In some embodiments, one of gas source 102 comprises activating agent source 107.Activating agent from source 107 can be that perhaps this activating agent can be a gas source, for example Compressed Gas under high pressure by the liquefaction source of the gas of carburator 108 vaporizations.Activating agent can with one or more mixing the in the gas source 102.110 controls of outlet controller offer the amount of experimenter's admixture of gas.

Remover 112 is to offer the device or the system of experimenter's gas clean-up and/or ventilation.For example, if offer the patient from the activating agent in source 107 as the gas that can suck, then remover 112 can be used for removing the waste gas of the carbon dioxide of inhalant (for example activating agent), untapped oxygen and exhalation.

Alarm/monitoring system 114 is included in the one or more position monitoring gas flows in the delivery system 100 and/or the pick off of gas content.For example, the flow or the flow of monitoring oxygen comprise the enough oxygen for the patient to guarantee carrier gas in the time of can offering the patient as the gas that can suck at the activating agent from source 107.Alarm/monitoring system 114 also comprises a user interface, and this user interface is through setting in order to sound or visual alarm signal or the monitoring information to the user of delivery system 100, for example screen demonstration, light or acoustic alarm to be provided.Can set alarm/monitoring system 114 notifies user and/or information about gas level is provided when satisfying predetermined condition.

About Figure 10, system 100A comprises a high tension loop 116, low tension loop 118, breathing circuit 120 and removes loop 122.

High tension loop 116 comprises compressed gas source 102, and it is connected to control valve 104b, 104a.Control valve 104a control is from the amount of each gas source 102 effluent air, and can open control valve 104b in order to for example by providing the opening that leads to surrounding atmosphere to increase the pressure of gas.

Low tension loop 118 comprises effusion meter 106, activating agent source 107 and carburator 108.Admixture of gas from gas source 102 is provided by effusion meter 106, and its control is from the amount of every kind of gas of gas source 102.As illustrated in fig. 10, activating agent source 107 is a liquid.Vaporize by carburator and be added in the admixture of gas in activating agent source 107.

Breathing circuit 120 comprises outlet controller 110, two check valves 124,126 and absorbers 128.Remove loop 122 and comprise valve 112a, reservoir 112b and outlet 112c.The gas that experimenter 130 receives from the admixture of gas of outlet controller 110 and generation passes through to remove loop 122 ventilations.More specifically special, 110 controls of outlet controller are delivered to the amount of experimenter 130 admixture of gas by check valve 124.Breath flows through check valve 126 and arrives valve 112a and arrive reservoir 112b.Excess air is discharged by the outlet 112c of remover 112.Some gases can recirculatioies and are flow through absorber 128 and enter in the breathing circuit 120.Absorber 128 can be the carboloy dioxide canister that is used for reducing the carbon dioxide of breath.In this structure, but the oxygen of exhalation and/or activating agent recirculation and re-use.

One or more pick off S can add at the diverse location among the 100A of system.Gas among pick off S perception and/or the monitoring system 100A.For example, if one of them gas source 102 is an oxygen, then one of them pick off S can be through setting and placing the appropriate location to be used for the oxygen of monitoring system 100A, so that the patient accepts the oxygen sensor of appropriate amount oxygen.Pick off S and alarm/monitoring system 114 communication (see figure 9)s.If that do not expect or dangerous gas level appears in the system 100, then but the user of alarm/monitoring system 114 warning system 100A is so that can take adequate measures, and for example increase offers experimenter 130 oxygen level or allows experimenter 130 break away from delivery system 100A.

About Figure 11, shown the 100B of system, wherein activating agent source 107 is connected to two control valve 104b, 104a.If activating agent source 107 is liquefaction sources of the gas, then provide optional carburator 108 to make the vaporization of liquefaction source of the gas.If activating agent source 107 is gas (as gases at high pressure), can omit carburator 108 so.Activating agent from source 107 mixes with other gas source 102 with the amount of being controlled by effusion meter 106 in low tension loop 118.Low tension loop 118 comprises gas reservoir 109, and it is contained in admixture of gas any admixture of gas that overflows when flowing to breathing circuit 120.Should be appreciated that activating agent source 107 and/or any gas source 102 can be used as the liquefaction source of the gas and offer carburator.Element at system 100B illustrated in fig. 11 is identical with those elements about Figure 10 of above describing basically, and will can not describe in addition.

Can adopt system 100,100A, 100B to implement, in Figure 12, illustrate according to the method for embodiment of the present invention.The mixture (202 parts) of one or more gas sources that suck is provided.The gas source that can suck can be as obtaining from gas source 102 that relevant Fig. 9-11 describes.The activating agent of scheduled volume is added in the admixture of gas (204 parts), and the activating agent source 107 among for example relevant Fig. 9-11 is shown.Admixture of gas is administered to experimenter 120 (306 parts).Breath is for example come ventilation and/or recirculation (208 parts) by remover 112.Describe though the method for Figure 12 is system 100,100A, the 100B with respect to Fig. 9-11, should be appreciated that any suitable system or device can be used for finishing the step among Figure 12.

2. decompression delivery system

The embodiment of gas delivery system 300 illustrates with respect to Figure 13.Gas delivery system 300 is placed on the experimenter 302.Gas delivery system 300 is particularly suitable for sending the tissue that activating agent in the admixture of gas is given experimenter 302, for example wound tissue.

System 300 comprises a pressure-reducing chamber 304, and it has the veil 306 of the processing region that covers experimenter 302.This pressure-reducing chamber 304 is connected to vacuum pump 310 by pump discharge 310a.Pressure-reducing chamber 304 comprises air inlet 308a and outlet 308b, and it is connected to activating agent source 307 successively.Controller 320 is connected to activating agent source 307 and vacuum pump 310.Pressure-reducing chamber and vacuum pump system be at United States Patent (USP) 5,645, discuss in 081 and 5,636,643, therefore with its content by being incorporated herein by reference in full.

Be provided with zone that pressure-reducing chamber 304 surrounds experimenter 302 with fluid-tight is provided or the air-tightness cover, realize should the zone with 307 pairs in decompression or negative pressure and activating agent source processing.Pressure chamber 304 can be used the covering (not shown), and for example flexible, polymer flake adhesive, fluid impermeable is attached on the experimenter 302.This covering can have a viscosity backing, and it is used for living skin around the edges cover that is subject to processing the zone, and is used to provide usually the sealing of bubble-tight or fluid-tight and is used for fixing chamber 304 in suitable position.

Veil 306 is positioned on experimenter 302 the processing region.For example, if experimenter 302 processing region comprises wound, then this veil 306 can be positioned on the wound in order to prevent its undue growth.The size of scalable veil 306 and structure are with suitable individual processing region, and its available multiple porous material forms.This material should be fully porous so that oxygen and any other gas, and for example the gas from activating agent source 307 can arrive processing region.For example, veil 306 can be out the form of chamber foam of polymers, and as polyurethane foam plastics, it is fully porous so that gas can flow to processing region and/or handle the zone outflow certainly.But the foam plastics that used thickness is different with hardness, if but the patient in therapeutic process, must lie on this device, then the comfortable use sponge material for the patient may be ideal.This foam plastics also can punch to strengthen the weight of air-flow and mitigation system 300.Veil 306 can cut into suitable shape and size being adapted in the processing region, or alternately, veil 306 can enough come greatly and skin on every side overlapping.

Vacuum pump 310 provides a suction to come from the pressure-reducing chamber 304.Activating agent source 307 provides a certain amount of activating agent for pressure-reducing chamber 304.Controller 320 is controlled the amount of the vacuum that puts on pressure-reducing chamber 304 and the amount of controlling the activating agent of supply chambers 304 by activating agent source 307 by vacuum pump 310.

Should be appreciated that controller 320 can abundant constant mode, circularly or adopt multiple fluctuation or pattern or their combination in any to apply vacuum and/and activating agent.In some embodiments, activating agent provides by activating agent source 307, and the effect of pumping of alternative vacuum with vacuum pump 310 provides.That is to say that controller 320 alternately activates vacuum pump 310 when 307 inactivations of activating agent source, when vacuum pump 310 inactivations, activate activating agent source 307 then.Pressure in the pressure-reducing chamber 304 allows fluctuation.In other embodiments, substantially invariable pressure is kept by vacuum pump 310, and activating agent source 307 provides the activating agent of abundant constant basis to the chamber in reduced pressure atmosphere 304.In some embodiments, substantially invariable pressure is kept by vacuum pump 310, and the amount of activating agent changes with periodic manner.In other embodiments, the pressure in the pressure-reducing chamber 304 fluctuates by vacuum pump 310, and the amount of the activating agent that is provided by source 307 also fluctuates.The fluctuation of pressure in vacuum pump 310 and the chamber 304 that produces, perhaps the fluctuation of the amount of the activating agent that is provided by source 307 can be periodic or acyclic.

The method according to embodiment of the present invention that available system 300 carries out is illustrated in Figure 14.Chamber 304 is positioned on experimenter 302 the processing region (402 parts).Pressure in the chamber 304 reduces by vacuum pump 310 (404 parts).The activating agent from activating agent source 307 of scheduled volume is applied to chamber (406 parts).Though the method for Figure 14 is to describe with respect to the system among Figure 12 300, should be appreciated that, any suitable system or device all can be used for implementing the step among Figure 14.For example, outlet 308b can omit, and activating agent can be supplied to chamber 304 by single air inlet 308a.Other gas also can be for example be added in the chamber 304 with single air inlet or an air inlet and an outlet, as illustrating with respect to activating agent source 307 and air inlet 308a and outlet 308b.In some embodiments, vacuum pump 310 is connected to the other collection container that is used between pump 310 and the chamber 304 to collect from the exudate of handling the zone, for example, as United States Patent (USP) 5,636, describes in 643.

In some embodiments, negative-pressure gas delivery system 500 as illustrating among Figure 22 A, is contained in an active oxygen antagonist source in the container 502, and container 502 is connected to cap rock 504 by conduit 508 through air inlet 506.This cap rock forms the envelope of sealing with respect to tissue site 510 (may be wound site).In some embodiments, this cap rock has the outlet 512 that is communicated with negative pressure source 514 by conduit 516.In some embodiments, dedicated waste tanks 518, it can be dismountable dedicated waste tanks, and is communicated with between outlet and the negative pressure source.In some embodiments, loop outlet 520 is communicated with container 502 by conduit 522.In some embodiments, as showing among Figure 22 B, carburator 524 is inserted in the connection thing between container 502 and the overcover 504.

Conduit can be flexible, and can be suitably for the material flexible pipe of similar plastics.Negative pressure source 514, it can be suitably for vacuum pump, carries out fluid communication by conduit 516 and outlet 512 in some embodiments, is used to promote fluid to discharge, as known in the art.In some embodiments, dedicated waste tanks 518 is positioned under the vacuum, collects the fluid of discharge by fluid communication.Preferably, with a filter (not shown), it can be the hydrophobic film filter, inserts between dedicated waste tanks and the negative pressure source, is used for preventing to suck the fluidic pollution of discharge in the dedicated waste tanks.In some embodiments, cap rock 504 contains elastomeric material, so its pressure that can adapt in the intermittent operation process of negative pressure source on the tissue site zone changes.In some embodiments, the edges cover of cap rock has pressure-sensitive adhesive, and it can be an acrylic adhesive, is used for cap rock is sealed in tissue site.

Can be used for handling zones of different as the negative-pressure gas delivery system 300 and 500 that illustrates among Figure 12 and Figure 22 A-B and be used for the treatment of, and be particularly useful for handling wound.The wound that can adopt system 300 to handle comprises infected open wound, decubital ulcer, the otch that splits, segment thickness burn, and the multiple injury region that has connected flap or graft.Treatment to wound can be carried out like this: with the treatment position that is fixed in of gas delivery system such as previous demonstration and description, the pressure of keeping continuous decrease basically in the pressure-reducing chamber 304 or periodically reducing, and to continue basically or periodic mode provides activating agent to reach the improvement situation of expectation up to wound to chamber 304.The state of the improvement situation of selecting can comprise form the granulation tissue that is enough to be used in adhering to flap or graft, the infected by microbes that reduces the wound, prevention or reverse that burn penetrates, closure, flap or the graft of wound are integrated with following injured tissues, the healing fully of wound or be suitable for wound or syndromic other improvement of wound or the healing stage of given type.Especially when use combines the gas delivery system of veil on wound or in the wound, but gas delivery system periodic variation in therapeutic process for example changes with 48 hours interval.This method can be put into practice with the negative pressure or the decompression of 0.01 to 0.99 barometric pressure range, and perhaps this method can be put into practice with the negative pressure or the decompression of scope between 0.5 to 0.8 atmospheric pressure.Being used for using the time cycle of this method on wound can be at least 12 hours, but can for example extend to 1 day or a couple of days.Do not exist the use of this method to surpass its then no longer valid upper limit; This method can increase closed speed when wound is closed really.The gratifying treatment of polytype wound can be equivalent to force down about decompression of 2 to 7Hg than atmosphere by utilization and finish.

With intermittently or periodic mode provide decompression to gas delivery system, for example describe in as mentioned, be used in treatment wound when having activating agent.Intermittently or periodically provide decompression can realize by manually or automatically controlling vacuum system to gas delivery system.Period ratio in this intermittent reduced pressure treatment, promptly " work " time can hang down by 1: 10 or up to 10: 1 with the ratio of " not working " time.Typical ratio is about 1: 1, and finish at its interval that provides and do not provide with 5 minutes the decompression that hockets usually.

Suitable vacuum system comprises can provide at least 0.1 pound of suction force to wound, or 3 pounds suction force at the most, or any suction pump of 14 pounds of suction forces at the most.This pump can be the common suction pump that any suitable medical purpose of required suction force can be provided.The size of pump and the middle conduit that is connected of decompressor is provided the ability control of the required suction force level of operation by this pump.The conduit of 1/4 inch diameter can be suitable.

Embodiment of the present invention also comprise the method for handling damaged tissues, and it is included in time of selection and uses negative pressure to wound with apply the step of activating agent with the value of selecting (it is enough to reduce the bacterial density of wound).Open wound almost always is subjected to harmful germ contamination.Generally speaking, every gram tissue 10 ⁵The bacterial density of individual bacterium living beings is thought infected.It is generally acknowledged that at this infection level the tissue of transplanting will can not be attached on the wound.These antibacterials must reply or kill by some externalist methodologies by wound host's the natural immunity before wound closure.Use negative pressure and activating agent can reduce wound to wound bacterial density.It is believed that this effect may be because the blood flow of the incompatible or wound area of antibacterial and subnormal ambient increases and the combination that is exposed to activating agent, because blood has brought cell and enzyme to destroy antibacterial to it.Method according to embodiment of the present invention can be used for the bacterial density of wound is reduced by at least half.In some embodiments, it can be used for bacterial density being reduced by at least 1,000 times or at least 1,000,000 times.

Embodiment of the present invention also comprise the method for handling burn, and this method comprises with predetermined negative pressure with in the step that is enough to suppress to apply in the time that the through thickness burn forms negative pressure and the activating agent burn to certain zone.Segment thickness burn is to have the surface layer of dead tissue and the burn of following stagnant layer, is subjected to abundant infection usually, and it will be transformed into the through thickness burn in 24-48 hour like this, i.e. the burn that all is damaged of all epidermal structures wherein.Applying negative pressure and a certain amount of activating agent to wound can protect from infection and become fully serious and epidermal structure below causing destroys.The size that pressure is used, pattern and persistent period can change with individual wound.

Embodiment of the present invention also comprise and are used to strengthen the method that living tissue is attached to wound, this method comprises following step: at first connect living tissue to wound to form wound-organize complex, apply wound to the zone of the negative pressure of size of selection or decompression and a certain amount of activating agent-organize complex then, the amount of described negative pressure or decompression and activating agent is enough to promote epithelium and subcutaneous tissue to migrate to this complex, allows negative pressure and be exposed to the cycle that activating agent keeps being enough to promoting the selection of wound closure.It is the commonsense method that can take various ways that living tissue is attached to wound.For example, a kind of ordinary skill is to utilize " flap ", promptly wherein will separate but remain on the fourth face from three of the skin histology of wound adjacent domain to connect, and then it is moved to the technology on the wound.The another kind of technology of often using is open skin transplantation, and wherein skin separates fully with another skin surface and is transplanted on the wound.Apply negative pressure and activating agent to wound-graft complex and reduced the bacterial density in this complex and increased blood flow, thereby improved adhering to of transplanted tissue to wound.

I. Other Instruments

In certain embodiments of the invention, replenishing the method that is used for the treatment of the patient that will suffer or suffer wound of the present invention expects with the ability of external control patient's core temperature.In this, patient's core temperature can be handled by invasive or Noninvasive approach in conjunction with method of the present invention.The invasive method that is used to operate core temperature comprises, for example, utilizes heart-lung pump to heat or cools off patient's blood, thereby raise or reduce patient's core temperature.The Noninvasive approach of handling core temperature comprises system and the instrument that advances or migrate out patient body heat passage.

J. other delivery apparatus or instrument

In some embodiments, consideration method or compositions will be referred to specific delivery apparatus or instrument.Can be with any device that is used to send or use in any method of this argumentation, include but not limited to that those devices of discussing finish here.

For local application reactive compound of the present invention, reactive compound of the present invention can be mixed with solution, gel, ointment, Emulsion, suspensoid etc., as known in the art.Whole body can comprise with preparation and being designed for by injection or infusion, in for example subcutaneous, intravenous, intramuscular, the sheath or those preparations of using of peritoneal injection, and those preparations that are designed for transdermal, saturating mucosa, per os or use through lung.

For dosage forms for oral administration, reactive compound of the present invention will be formulated into the tablet, pill, lozenge, capsule, fluid agent, gel, syrup, the what that are used for by being treated patient's oral uptake and starch agent, suspensoid etc., or the liquid preparation of per os, for example suspending agent, elixir and solution.

For oral administration, form such as the tablet that compositions can be taked to prepare in a usual manner, lozenge.Send in other the mucosa and can pass through suppository or intranasal delivery.

For being applied directly to pulmonary by suction, chemical compound of the present invention can be delivered to pulmonary easily by multiple different device.For example,

Metered-dose inhaler (MDI): the metered-dose inhaler (" MDI ") that use can be contained the gas tank of suitable low boiling propellant (as dichlorodifluoromethane, Arcton 11, dichlorotetra-fluoroethane, carbon dioxide or other suitable gas) is used for directly sending chemical compound of the present invention to pulmonary.The MDI device can obtain from many suppliers, 3M Corporation (for example network address 3m.com/us/healthcare/manufacturers/dds/pdf/idd_valve_can ister_brochure.pdf-) for example, Nasacort (for example network address products.sanofi-aventis.us/Nasacort_HFA/nasacort_HFA.htm l-63k-) from A Fansi (Aventis), Bao Er Yin Haimu (Boehringer Ingelheim), (for example network address boehringer-ingelheim.com/corporate/home/download/r_and_d 2003.pdf), Aerobid (for example network address frx.com/products/aerobid.aspx) from Fu Site laboratory (Forest Laboratories), Ge Lansu-welcon (Glaxo-Wellcome) (for example on the network address www.gsk.com/research/newmedicines/newmedicines pharma.html) and the clever Bao Er (Schering Plough) of elder generation, (network address www.schering-plough.com/schering_plough/pc/allergy_respi ratory.jsp).

Diskus (DPI): the DPI device usually adopt a kind of mechanism for example gas gush out and in container, form the spray of dry powder, it can be sucked by the patient subsequently.The DPI device also is well known in the art and can buys from many sale persons, comprise, for example from the Foradil aerolizer of Schering Corp (Schering Corporation), (for example, www.spfiles.com/piforadil.pdf), from the Advair Diskus of Ge Lansu-welcon (for example, www.us.gsk.com/products/assets/us_advair.pdf-).Most popular variant is multiple dose DPI (" MDDPI ") system, and it makes transmissibility more than one therapeutic dose.The MDDPI device can obtain from a plurality of companies, for example (for example from the Plumicort Turbuhaler of A Sijielikang (AstraZeneca), www.twistclickinhale.com/), the plain welcon of Ge Lan (for example, www.us.gsk.com/products/assets/us_advair.pdf-) and earlier clever Bao Er, for example, www.schering-plough.com/schering_plough/pc/allergy_respi ratory.jsp).Further consider to change this class device, or be used for single dose at any other device of this argumentation and use.

Electrofluid Mechanics (Electrohydrodynamic) is aerosol delivery (EHD): the EHD aerosol device utilizes electric energy to make liquid medicine solution or suspensoid aerosolization (referring to for example Noakes etc., U.S. Patent No. 4,765,539, Coffee, U.S. Patent No. 4,962,885, Coffee, the PCT application, WO 94/12285, Coffee, PCT application, WO 94/14543, Coffee, the PCT application, WO 95/26234, Coffee, PCT application, WO 95/26235, Coffee, the PCT application, WO 95/32807).The comparable existing pulmonary delivery technology of EHD aerosol device more effectively delivering drugs to pulmonary

Nebulizer: nebulizer is by utilizing, and for example ultrasonic energy forms the fine particle that can suck easily, produces aerosol from liquid pharmaceutical formulation.The example of nebulizer comprises that the device that is provided by Sheffield/Systemic Pulmonary Delivery Ltd. is (referring to Armer etc., U.S. Patent No. 5,954,047; Van der Linden etc., U.S. Patent No. 5,950,619; Van der Linden etc., U.S. Patent No. 5,970,974), the suction nebulizer scheme that provides by A Fansi (Intal nebulizer solution) (for example, www.fda.gov/medwatch/SAFETY/2004/feb_PI/Intal_Nebulizer_ PI.pdf).

In order directly to use gas to pulmonary by sucking, can adopt the multiple delivering method that is used to send oxygen that can obtain from the market at present.For example, can adopt resuscitator, for example medical bag (referring to, United States Patent(USP) Nos. 5,988,162 and 4,790,327).Medical bag is made up of the squeeze bag of the flexibility that is connected to face shield, and it is made by the doctor and is used for air is introduced in victim's lung.

Portable, portable drug delivery device can produce the propellant that the patient be fit to suffer from respiratory tract disease sucks by nebulizer.In addition, this delivery apparatus provides a kind of means, and wherein the dosage of inhalant can and change as required by doctor or doctor's remote monitoring.Referring to U.S. Patent No. 7,013,894.Sending of chemical compound of the present invention can be given the people by being used to send make-up gas, finishes in conjunction with the method for monitoring people's ventilation, and the both does not use the face shield (for example in U.S. Patent No. 6,938, in 619 description) of sealing to finish.In the respiratory therapy process, use the pneumatic oxygen save set that is used for effectively distributing oxygen or other gas, had only the initial part of patient respiratory to contain aerobic or other treatment gas (referring to U.S. Patent No. 6,484,721) like this.The gas delivery device that use starts when the patient begins to suck.After tail gas stream is during initial suction regularly, be delivered to the patient, be delivered to the patient to prevent gas pulses.By this way, gas only is delivered to the patient during the beginning part that sucks, and prevents that gas delivery from only can fill the air flue of patient pulmonary.By effectively utilizing oxygen, the oxygen of the cylindrical bottle of using when being mobile the patient will last much longer, and can littler and easier transporting.Send gas by pneumatic type and give the patient, without battery or electronic equipment.

All devices described here can have gas extraction system and come the combination or the chemical compound of the present invention that neutralizes.

The using and to realize by contain medicine device or the plaster that are fixed on the patient skin of The compounds of this invention through skin.Plaster allows to be contained in medicinal compound in the plaster and absorbs by skin layer and enter in patient's the blood flow.This class plaster can commercial obtain, as from the Nicoderm CQ plaster (www.nicodermcq.com/NicodermCQ.aspx) of GlaxoSmithkline and for example from the Ortho Evra (www.ortho-mcneilpharmaceutical.com/healthinfo/womensheal th/products/orthoevra.html) of Ortho-McNeil Pharmaceuticals.Transdermal drug delivery has reduced to use relevant pain with medicine injection and intravenous drug, and the infection risk relevant with these technology.Transdermal drug delivery has also been avoided the gastrointestinal tract metabolism of drug administration, has reduced the elimination of liver to medicine, and the lasting release of drug administration is provided.Transdermal drug delivery is also owing to the lasting release of relatively easy property of using and medicine has strengthened the compliance of patient to drug regimen.

Other of plaster changes form and comprises ultrasonic plaster, and it makes with material design and can transmit the ultrasonic plaster that passes through, and realized being stored in sending of medicine in the plaster, and can be used in combination (seeing U.S. Patent No. 6,908,448) with the ultrasonic medicinal delivering method.Plaster (U.S. Patent No. 6,958,154) in the bottle comprises a kind of fluid composition, spray in some embodiments for example, and it can be used as fluid administration to the surface, but subsequent drying and form cladding element, for example plaster on host's surface.So the cladding element that forms has noncohesive outer surface covering and help plaster below and adheres to tacky surfaces on the substrate.

Another kind of drug delivery system comprises one or more spheric semiconductor aggregations (aggregation) and promotes to be stored in the release of the medicine in the storage.First aggregation is used for perception and memory, and second aggregation is used for the control aspect, for example is used for pumping and distributes medicine.This system can be for a long time and the tele-control system communication, or operate independently with local power, be used for based on the patient demand, under system's control, regularly discharge, or send delivering drugs according to measurement markers.Referring to U.S. Patent No. 6,464,687.

Pump and infusion device: infusion pump or filling apparatus (perfusor) are infused into fluid, medicine or nutrient in patient's the blood circulation.Infusion pump can very reliable and inexpensive manner application of fluid.For example, they per hour can use few injection to 0.1mL (concerning instiling very little), per minute injection, the multiple disposable dosage injection that the patient requires, up to maximum quantity hourly (for example in the analgesia of patient's control), or its capacity is with the fluid of one day time variation.Dissimilar infusion devices has had description in the patent application below United States Patent and Trademark Office accepts.These include but not limited to U.S. Patent No. 7,029,455, U.S. Patent No. 6,805,693, U.S. Patent No. 6,800,096, U.S. Patent No. 6,764, and 472, U.S. Patent No. 6,742,992, U.S. Patent No. 6,589,229, U.S. Patent No. 6,626, and 329, U.S. Patent No. 6,355,019, U.S. Patent No. 6,328,712, U.S. Patent No. 6,213, and 738, U.S. Patent No. 6,213,723, U.S. Patent No. 6,195,887, U.S. Patent No. 6,123, and 524 and U.S. Patent No. 7,022,107.In addition, can infusion pump also be from Baxter International company (www.baxter.com/products/medication_management/infusion_p umps/), Alaris Medical Systems (www.alarismed.com/products/infusion.shtml) and from the B BraunMedical (www.bbraunusa.com/index.cfm of company? uuid=001AA837D0B759A1E34666434FF604ED) obtain.

The disposable dosage of oxygen/gas is sent (bolus delivery) device: thisly be used to send gas and can obtain for chronic obstructive pulmonary disease (COPD) patient's device from Tyco Healthcare (www.tycohealth-ece.com/files/d0004/ty_zt7ph2.pdf).It also can be used for sending chemical compound of the present invention.Said apparatus is that cost is effective, lightweight, inconspicuous and portable.

Plaster (U.S. Patent No. 6,958,154) in the bottle comprises a kind of fluid composition, spray in some embodiments for example, and it can be used as fluid and is applied to the surface, but subsequent drying and form cladding element, for example plaster on host's surface.So the cladding element that forms has noncohesive outer surface covering and help plaster below and adheres to tacky surfaces on the substrate.

Implantable drug delivery system: another kind of drug delivery system comprises one or more spheric semiconductor aggregations and promotes to be stored in the release of the medicine in the storage.First aggregation is used for perception and memory, and second aggregation is used for the control aspect, for example is used for pumping and distributes medicine.This system can be for a long time and the tele-control system communication, or operate independently with local power, be used for based on the patient demand, under system's control, regularly discharge, or send delivering drugs according to measurement markers.Referring to U.S. Patent No. 6,464,687.

Each patent quoted in this part, discussed and the content of network address at this by being incorporated herein by reference.

VIII. therapeutic alliance

Compounds and methods for of the present invention can be used for many treatments and diagnostic application field.In order to increase effectiveness, be ideal with effective other medicament in those diseases of treatment and disease (second treatment) combination with these components with component of the present invention (for example oxygen antagonist or other reactive compound) treatment.For example, treatment apoplexy (anti-stroke treatment) generally includes anti-platelet agents (aspirin, clopidogrel, dipyridamole, Ticlopidine), anticoagulant (heparin, warfarin) or thrombolytic agent (tissue plasminogen activator).

Can adopt different combinations, for example reactive compound such as H ₂S be " A " and second the treatment be " B ".

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/AA/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/AA/B/A/A A/A/B/A

If using oxygen antagonist of the present invention and/or other reactive compound will toxic, then consider the toxicity that oxygen antagonist (or other reactive compound) is treated according to the general approach that is used to use those second special treatments for biological substance.Expection will be according to the need repetitive therapy cycle.Also consider, can be with the therapy of multiple standards, and surgical intervention and described therapy use in conjunction.

IX. embodiment

Comprise that the following example is in order to explanation the preferred embodiments of the invention.Those skilled in the art should be appreciated that, disclosed technology representative is well played a role in the present invention's practice by the technology that the present inventor finds in ensuing embodiment, and therefore can think and constituted the optimal way that is used for its practice.Yet those skilled in the art should be appreciated that according to present disclosure, can carry out many changes and it still obtains similar or similar result and can not deviate from the spirit and scope of the present invention in disclosed particular embodiment.

Embodiment 1:

The preservation of nematicide in carbon monoxide

Atmosphere contains 210, the oxygen of 000ppm.Be exposed to low-level oxygen, or cause people's primary cellular defect and death in the hypoxia.In nematicide (Caenorhabditis elegans), the oxygen concentration between 100ppm and 1000ppm also is fatal.By strictly studying the reaction of nematicide to a series of oxygen tension, finding to be lower than 10ppm is can be not fatal with the oxygen concentration that is higher than 5000ppm.With in the 10ppm oxygen of nitrogen balance, nematicide enters reversible stagnant living state, wherein can be observed all to stop (Padilla etc., 2002) aspect all of survival condition under optical microscope.In 5000ppm (using nitrogen balance) and higher oxygen concentration, nematicide normally makes progress by their biocycle.Avoid having tested carbon monoxide in the medicine of hypoxia infringement searching protection nematicide.

In order to obtain specific atmospheric condition, used time array apparatus: the tip has for example glass syringe pipe of LUER-LOK of locking device (locking device), the big opening of syringe tube produces airtight sealing with the steel (custom-machined steel) and the sealing of rubber accessory of customization, be locked to this syringe tube on the air inlet of environmental chamber by locking device, described environmental chamber has an air inlet and a gas outlet, and each mouth is equipped with for example LUER-LOK accessory of locking device.(ByrneSpecialty Gas, Seattle discharge to come humidification and offer environmental chamber by the Drexel bottle (500ml Kimex) that is full of DDW in WA) from compressed tanks by at first allowing gas with the gas determined.Drexel bottle is connected with environmental chamber through a gas flowmeter.Gas flowmeter is used for providing between whole 24 hour incubation period 70cc/ minute flow of adjusting to pass through the environmental chamber.

Whether inductive in order to detect, reversible stagnation can realize in Caenorhabditis elegans, collect 2-cell Caenorhabditis elegans embryo, L3 larva or adult nematicide and with its at room temperature be exposed in the effective 100%CO environment, 100%N ₂In the environment, contain in the environment of the equilibrated 500ppm oxygen of useful carbon monoxide, or be exposed in the environment of 100,500 or the 1000ppm oxygen that contain useful nitrogen balance.Nematicide is observed art (being also referred to as the Nomarski optics) video picture with differential-interference contrast microscope.Images acquired is also used NIH image and AdobePhotoshop 5.5 analyses.Embryo is about 50 μ m.

The result of these tests shows that 100% carbon monoxide is nonlethal and has induced reversible stagnant life.Nematicide can not survive in in the 500ppm oxygen of nitrogen balance,, has entered stagnant life and is survived with those nematicides of the equilibrated 500ppm oxygen treatments applied of carbon monoxide.Vide infra:

Embodiment 2:

The preservation of application on human skin in carbon monoxide

Carbon monoxide is to be very deleterious to the people, because it is bonded to hemoglobin with the oxygen competition consumingly, hemoglobin is to distribute the main molecules of oxygen to tissue.The nematicide of hemoglobin-free to carbon monoxide have resistance and even also prevent hypoxia infringement by this medicine, this fact has been pointed out such probability: carbon monoxide will be therein exist in people's tissue in the situation of blood, for example, prevent the hypoxia infringement in the surgical area of tissue grafts or depletion of blood.In order to check this hypothesis, used application on human skin.

Obtain three blocks of people's foreskins and be used for this purpose.Prepuce tissues is stored in the keratinocyte growth medium (KGM) that contains insulin, EGF (0.1ng/ml), hydrocortisone (0.5mg/ml) and Niu Chuiti extract (about 50 micrograms/ml protein).With foreskin rinsing in PBS, and remove the excess fats tissue.Every foreskin sample is divided into 2 pieces such as grade.With every container that places separately, described container is equipped with has 24mg/ml Bacillus polymyxa Neutral proteinase II (from Bacillus Polymyxa EC 3.4.24.4:Roche Diagnostics Corp., Indianapolis, PBS solution IN).A container (the foreskin piece in the PBS that contains Bacillus polymyxa Neutral proteinase II is housed) is placed a humidity indoor of fume hood.Another container (having second half foreskin piece in the PBS that contains Bacillus polymyxa Neutral proteinase II) is placed in the environmental chamber of the moistening 100%CO of being full of of identical fume hood.These two samples were all at room temperature kept 24 hours.The method that is used for setting up definite atmospheric condition and embodiment 1 use those are consistent.

In being exposed to normal oxygen (normoxia) or 100%CO after 24 hours, according to Boyce etc. (1983; 1985; Each is here by being incorporated herein by reference in full) method described isolates keratinocyte from foreskin.In brief, will move on to from the epidermis of every foreskin sample in the fresh vessel that contain PBS.Epidermis is at room temperature hatched 5 minutes chopping and homogenize before in 3ml 0.05% trypsin, 1mM EDTA, so that basal cell separates with epidermis.After hatching, and adding 6ml 400 μ g/ml (microgram/soybean trypsin inhibitor ml), the BSA of 1mg/ml, and sample is centrifugal with 900RPM.Discard the supernatant of each sample and with sample deposit seed resuspending in the KGM of 10ml.Each sample branch is gone in the flat board of two 10cm, and each flat board contains the HEPES pH 7.3 (N-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid) of 5ml KGM and 100 μ l.Flat board was hatched 5 days in 37 ℃ the calorstat that is full of 95% room air, 5% carbon dioxide.

Adopt inverted phase contrast microscope macroscopy cell.All three keratinocyte groups that are exposed in the normal oxygen show very little growth or not growth.All three keratinocyte groups that are exposed among the 100%CO show remarkable growth.Quantize quantitatively (by the colony formation judgement) of two keratinocyte number alive in three blocks of foreskins.See Fig. 1.

It is quantitative that table 1-colony forms

Foreskin	Atmosphere	The colony sum
Foreskin	Atmosphere	The colony sum	1	100％CO	542 colonies (majority in them is very big)
1	Normal oxygen	2 colonies (all little)	1	100％CO	542 colonies (majority in them is very big)
1	Normal oxygen	2 colonies (all little)	2	100％CO	780 colonies (majority in them is very big)
2	Normal oxygen	0 colony	2	100％CO	780 colonies (majority in them is very big)

Embodiment 3:

With the further preservation test of nematicide

The information overlap that following embodiment contains and expanded disclosed information among the embodiment 1.

A. material and method

Environmental chamber and deviceHypoxia test is used by the customization atmosphere chamber (Van Voorhies etc., 2000) of W.Van Voorhies design and is undertaken.This chamber is a 30mL glass syringe (Fisher#14-825-10B) that the steel stopper of customization is installed, and described steel plug lining has two synthetic rubber (viton) o type ring to guarantee sealing.This steel plug drills through, and externally has a steel on the face and lure lock (lure lock), so that can connect the flexible pipe that delivers Compressed Gas.The admixture of gas of determining is delivered to this chamber from compressed tanks with constant compression force and flow velocity, gas is at first by rotometer (Aalborg, flowtube 032-41ST) or mass flow control device (Sierra Instruments#810) with the monitoring flow velocity, the 500ml Drexel bottle (Fisher#K28220-5001) by 250ml water is housed is so that gas hydrate then.Adopt 1/4 " OD nylon (Cole-Parmer#P-06489-06) or FEP (Cole-Parmer#A-06450-05) pipe, and between pipe and the actuator and the connection between pipe and the rotometer realize with pyrite John-Guest-type joint (Byrne Gas).All other connect with miniflow fast connecting joint (Cole-Parmer#A-06363-57, #A-06363-52) or the joint of luring of standard (Cole-Parmer#A-06359-37 #A-06359-17) finishes.

The viability of nematicide under hypoxia.Bristol strain N2 is continued to maintain under 20 ℃, note guaranteeing that this colony can not die of hunger.The adult Caenorhabditis elegans of selection logarithmic (log) phase is put into a sterilized water that contains 100 μ g/ml ampicillins, 15 μ g/ml tetracyclines and 200 μ g/ml streptomycins on the glass plate.Adult minced with razor blade and with a thin mouthful of pipette (mouthpipet) selection 2-cell stage.30-60 2-cell stage is transferred in the small-sized glass boat (customization is to be fit to atmosphere chamber, Avalon GlassWorks, Seattle WA) of filling 1% agarose among the 3ml M9.Subsequently this glass boat is placed moist chamber 2 hours so that embryo's maturation places environmental chamber then.With environmental chamber at room temperature to continue the pure N of perfusion in 70cc/ minute ₂(grade is 4.5), 100ppm O ₂/ N ₂, 500ppm O ₂/ N ₂, 1000ppm O ₂/ N ₂Or 5000ppm O ₂/ N ₂24 hours.After exposure, the agar caked sugar that will contain the embryo cuts out from boat, and allows the embryo face up to place on the medium sized NGM flat board with escherichia coli (OP50) inoculation.After exposure, embryo's assessment was hatched 24 hours, and the L1 ' s of hatching is transferred to the surface of NGM flat board, and enter the manhood subsequently.Inexplicable animal is removed from overall.(Seattle WA) provides all gas by Byrne Gas.Guarantee pure N ₂Contain the impurity that is less than 10ppm, and prove all O ₂/ N ₂Mixture is that ± 2% oxygen content (for example, proves the O of 100ppm ₂/ N ₂Contain the O between 98ppm and the 102ppm ₂).1,000,000/umber and kPa transform and are based on next 1,000,000 parts=101kPa of 1 atmospheric pressure.

Nematicide is based on the viability in the atmosphere of carbon monoxide.From Bristol N2 and 30-60 the embryo of hif-2 (ia04) strain results who continues as described above to keep.With environmental chamber at room temperature to continue perfusion pure CO (CP level) or 500ppm O in 70cc/ minute ₂/ CO 24 hours.In order to reach 2500ppm O ₂/ CO or 2500ppm O ₂/ N ₂, with 5000ppm O ₂/ N ₂With 1: 1 ratio and pure CO or with pure N ₂Mix, come the precise monitoring flow with two mass flow control devices (SierraInstruments 810).In whole 24 hours process-exposed, every kind of gas was passed into three-way valve (Cole-Parmer#A-30600-23) with 50cc/ minute, then with the mixture that obtains by Drexel bottle and enter in the environmental chamber.(Seattle WA) provides all gas by ByrneGas.Proof 500ppm O ₂/ CO mixture is ± 2% oxygen content and contains 7000ppm N ₂To guarantee in the use of compressed tanks the being O of unanimity ₂/ CO ratio.

Cytobiology is analyzed.In order to be determined at, to allow the 2-cell stage be exposed to the hypoxia of multiple degree as described above, and take pictures immediately, or take pictures after 12 hours recovery stages in the chamber of humidity based on the growth advance degree (table 2) in the atmosphere of nitrogen.Whether in atmosphere, stop in order to measure the embryo,, and take pictures immediately or be placed on 100% carbon monoxide or 0.05kPa O 2-cell stage in room air ripe 2 hours based on carbon monoxide ₂Among/the CO 24 hours and after exposure, take pictures immediately.In all scenario, by the embryo being placed under the coverslip on the 1% thin agarose pad and on Zeiss axioscope, observing and carry out the DIC microscopy.Obtain photo with RS Image and Adobe Photoshop software then.

B. result

Reported in the past that HIF-1 was at slight hypoxia (0.5kPa O ₂(Padilla etc., 2002) and 1kPa O ₂(Jiang etc., 2001)) in Caenorhabditis elegans in required, and known stagnant life is at anoxia (＞0.001kPa O ₂) in be possible (Padilla etc., 2002).In order accurately to determine every kind of active scope of these reactions, measure wild type Caenorhabditis elegans embryo's viability after 24 hours being exposed to multiple oxygen tension between slight hypoxia and the anoxia.Be exposed to anoxybiotic embryo and entered stagnant life as former report, thereby and in exposure, survive, have high viability.At 0.5kPa O ₂In the embryo in whole process-exposed, keep life, and also survived, have high viability.Yet, be exposed to oxygen tension (the 0.1kPa O of the intermediate range between slight hypoxia and the anoxia ₂To 0.01kPa O ₂) the embryo be survived (Fig. 2) surprisingly.

The embryo is not hatching in being exposed to the hypoxia process of this medium range, shows that they successfully do not finish the reaction of HIF-1 mediation.In order to determine whether they occur stagnating, and have detected whether the embryo has stopped embryogenesis in process-exposed in this intermediate range.Embryo in the lethal oxygen tension does not stop embryogenesis, and the amount of oxygen increase increases relevant (table 2) with embryo's growth progress degree.Again behind the oxygenate, the unusual L1s of many conducts stops among those embryos that these embryo's great majority have failed to hatch and hatch.These data show, the hypoxia of this intermediate range be a kind of uniqueness stress, wherein the neither enough height of oxygen level promote the vigor that continues also enough not hang down to induce to stagnate to give birth to.

Based on these discoveries, suppose that then it will provide the hypoxia of this lethal range of protection antagonism if carbon monoxide (the bonded competitive inhibitor of a kind of oxygen) can induce to stagnate gives birth to when having low levels of oxygen.In order to check this probability, at first measured the viability of Caenorhabditis elegans embryo in the carbon monoxide of multiple concentration.Although high-caliber carbon monoxide can have toxic action in some systems, it is found that the Caenorhabditis elegans embryo tolerates the carbon monoxide tension force of wide scope significantly.In fact, the Caenorhabditis elegans embryo can withstand and continue to be exposed among the 101kPa CO (100%CO) 24 hours and also have high viability (81.5% survival is to the manhood, Fig. 3.) it should be noted that in 101kPa CO the embryo does not make progress by embryogenesis, shows that they have entered stagnant life in process-exposed.Be exposed to the equilibrated 0.05kPa O of carbon monoxide in order to check carbon monoxide whether can when having the lethal oxygen tension, protect the embryo, to have measured ₂In embryo's viability.Use N with being exposed to ₂Equilibrated 0.05kPa O ₂In embryo's (wherein great majority are not survived) relatively, these embryos return to the manhood (Fig. 3) with 96.2% viability.And, be similar to the embryo who handles with 101kPa CO, with the equilibrated 0.05kPa O of carbon monoxide ₂In the embryo stopped embryogenesis, show that they have entered stagnant life.Therefore, carbon monoxide can be protected from the hypoxia infringement from birth by inducing to stagnate when having the lethal oxygen tension.

For further research can be used for solving the embryo's (hif-1 (ia04) strain) who lacks the HIF-1 function whether the protection of avoiding the hypoxia infringement also is possible at slight hypoxia by the oxygen tension scope of excessive carbon monoxide protection.At the 0.1kPa O that detects with nitrogen balance ₂And 1kPaO ₂Between multiple oxygen tension after, find that the greatest requirements to HIF-1 is the 0.25kPa O of infra ground usefulness nitrogen balance ₂In.Under this atmosphere, the normal progress of wild type embryo has been finished growth and has been demonstrated high viability, but hif-1 (ia04) embryo fails to finish embryogenesis and shows 100% fatality rate (table 3).Therefore, checked carbon monoxide whether can protect 0.25kPa O ₂In hif-1 (ia04) embryo.Using the equilibrated 0.25kPa O of carbon monoxide ₂In, wild type and hif-1 (ia04) embryo entered stagnant life and in this exposure with high viability survive (being respectively 78.7% and 84.0% survives the manhood) (table 3).Therefore, induce by carbon monoxide and stagnate to give birth to up to 0.25kPa O ₂Oxygen tension the time be possible, and even when lacking the HIF-1 function carbon monoxide also can protect the slight hypoxia of antagonism.

Grow the quantitative of progress in the table 2-hypoxia

Atmosphere

Embryo's percentage ratio in the scope

The scope of embryogenesis (the number of minutes behind the 2-cell stage)

N

＞0.001kPa O ₂/N ₂	100％±0.0	20-40 minute	35
＞0.001kPa O ₂/N ₂	100％±0.0	20-40 minute	35	0.01kPa?O ₂/N ₂	92.9％±6.0	40-80 minute	115
0.05kPa?O ₂/N ₂	97.7％±2.0	100-140 minute	108	0.01kPa?O ₂/N ₂	92.9％±6.0	40-80 minute	115
0.05kPa?O ₂/N ₂	97.7％±2.0	100-140 minute	108	0.1kPa?O ₂/N ₂	91.4％±1.3	300-340 minute	60

Wild type 2-cell stage is placed the hypoxia 24 hours of multiple degree, and their development are finished the degree marking of embryogenesis.Be exposed to and cause making progress the increase of finishing embryogenesis in the atmosphere of the oxygen that contains recruitment.Measured the embryo's who in given 20-40 minute scope, stops in the embryogenesis percent.Data are results of 3 independent trialss.

Table 3-carbon monoxide protection hif-1 embryo resists slight hypoxia

	0.25kPa?O ₂/N ₂	n	0.25kPa?O ₂/CO	N
	0.25kPa?O ₂/N ₂	n	0.25kPa?O ₂/CO	N	N2	94.2％±1.2	49	18.7％±21.9	109
hif-1(ia04)	0.0％±0.0	68	83.9％±13.8	108	N2	94.2％±1.2	49	18.7％±21.9	109

In wild type and hif-1 (ia04) embryo, at the 0.25kPa O that is exposed to 24 hours ₂/ N ₂Or 0.25kPa O ₂Detected the viability of the phase that reaches an adult age behind/the CO.All data points all are the results of at least 3 independent trialss and inexplicable anthelmintic are removed from overall.

Nematicide responds hypothermic viability.

The viability of nematicide also is a temperature sensitivity, is being exposed to (4 ℃ of low temperature; Figure 15) colony's 100% death after 24 hours.Yet, if entering anoxic conditions (＜10ppm oxygen) by balance before temperature reduces, nematicide induced stagnation in following 1 hour, the nematicide of vast scale is being exposed to 4 ℃ survive after following 24 hours (Figure 15).In this experiment, nematicide keeps stagnating during hypothermia, and keeps stagnating 1 hour after they return to room temperature.Oxygen free condition (pure N ₂), upgrowth situation and viability measurement be described below.

Embodiment 4:

The reduction of core temperature and breathing in the mice

A. material and method

The implantation of telemetering equipment.The standard method that provides according to manufacturer, give female C57BL/6J mice (Jackson Laboratories-Bar Harbor, Maine) implant telemetering equipment (PDT-4000HR E-Mitter-MiniMitter Inc.-Bend, OR).Allow mice recover several weeks so that body temperature and heart rate signal are stable.The core temperature of mice, heart rate and motion are continued to monitor by telemetering equipment and with VitalView software (being provided by MiniMitter) record.(Onset Computer Corp.-Pocasset MA) monitor, and analyzes with BoxCar software (being provided by Onset Computer Corp.) by data with HOBO for ambient temperature.

Mice is exposed under the atmosphere of being regulated.(a) that every mice be exposed to 1L/ minute contains 500ppm H ₂(Byrne Specialty Gas-Seattle Washington) mixes (use from Aalborg-Orangeburg, 3 channel gas of NewYork distribute (proportioner) meter) and reaches the H that final concentration is 80ppm the equilibrated nitrogen of S with room air ₂S and 17% O ₂Atmosphere, or (b) nitrogen to mix with room air and reach final concentration be 17% O ₂Atmosphere.H ₂S and O ₂Measurement (Thermo Gas Tech-Newark California) carries out with the portable gas monitor of Innova GasTech GT series.

Before in being exposed to the atmosphere of being regulated He regulated, testing and in this process, mice placed comprise a glass cage and (have drinking water, do not have food) plenum chamber in, described glass cage is equipped with (the Vernon Hills from Cole-Parmer, the input pipe and the outlet tube of FEP pipe Illinois) are used for importing and discharging atmosphere.(Sigma-St.Louis Missouri.) seals this cage with lid with Dow Corning silicone vacuum grease.Gas from each cage enters chemical hood by the discharge pipe discharge.In order to ensure this system is bubble-tight, the portable monitor of GasTech GT is used for detecting leaks.

Respirometry.In some experiments, the consumption of oxygen is by using the PA-10a O that uses according to manufacturers instruction ₂Analyzer (Sable Systems) is measured.Similarly, the carbon dioxide of animal generation adopts the LI-7000 CO that uses according to manufacturers instruction ₂/ H ₂O analyzer (Li-Cor company) monitoring.These equipment placements are placed point-blank with environmental chamber, like this their pick test from gas inlet pipe and outlet tube.

The adjusting of ambient temperature.(Sheldon Manufacturing company-Cornelius, Oregon) middle stable breeding is mice attemperation and photoperiod (8AM turns on light, and 8PM turns off the light) by the calorstat that carries out illumination round the clock at Shel Lab low temperature with mice.Mice is exposed in the atmosphere of being regulated as described above.When mice is exposed in the atmosphere of being regulated, the calorstat internal temperature is reduced to desired temperatures, for example to 10 ℃ or 15 ℃.Maintain mice in the atmosphere of being regulated and be in following 6 hours of the temperature of reduction.Atmosphere in the plenum chamber is replaced to room air, and allow mice get back to normal room temperature (22 ℃) and allow its recovery.

B. result

Base-line data.In order to measure the reaction of mice to the hydrogen sulfide of sublethal dose, the present inventor at first by one-period interocclusal record have the data of the mice of wireless set to determine the baseline of core temperature, heart rate and motion from four implantation in the calorstat, described calorstat keeps at ambient temperature and is full of room air.Base-line data proves that mice has circadian rhythm, the night of activity peak after lamp just extinguishes, and the early morning before lamp is just bright.Core temperature between their active stages high 37 ℃ to changing low 33.5 ℃ between their craticular stages.Heart rate changes to the 250bpm between their craticular stages from the 750bpm between their active stages (per minute is beaten).Heart rate may be associated with core temperature (the higher then heart rate of temperature is higher).Equally, total motor activity is not long ago the highest in night and dawn.

Mice is exposed to the atmosphere of being regulated under the room temperature.The first time test that mice is exposed to hydrogen sulfide comprises and at first mice is placed calorstat to remain in the plenum chamber under 27 ℃ 1 hour.After 1 hour, this chamber is full of the as above general 80ppm that describes, and the temperature of calorstat is reduced to 18 ℃ in experimentation.Though do not detect the change immediately of heart rate and total motor activity, observe core temperature and significantly reduce.Allow experiment carry out 90 minutes, during this period of time, core temperature is reduced to low 5 degree of any one minimum temperature of four mices of writing down in the baseline study of 28.6 ℃-ratio description in the above.Between the convalescent period after this chamber is full of room air, the present inventor notices that animal is motionless relatively (catching easily) at first; Yet in 60 minutes, it has returned to core temperature and the activity of normal range.Second mice is exposed in the identical testing program; Yet this time the charge operation of 80ppm is 3 hours.During this period of time, the present inventor notices that heart rate is reduced to 250bpm from 600bpm significantly, and total motor activity is shown as does not almost have activity, and core temperature drops to 18.6 ℃.

Respiratory variations follows core temperature to reduce.Mice is exposed to 80ppm H ₂S also causes metabolic rate to reduce, and metabolic rate is measured by measuring oxygen consumption and carbon dioxide generating.For example, the mice of having measured core temperature and carbon dioxide generating simultaneously proves that the rapid minimizing of carbon dioxide generating reduces (Fig. 4 A) prior to the core temperature of animal.Carbon dioxide generating reduces about 3 times and is being exposed to H ₂Determined a new baseline behind the S in about 5 minutes.

Table 4 has shown from measuring the O that is exposed to the mice under the room air simultaneously ₂And CO ₂The result of experiment of concentration, the CO in the described room air ₂Be cleaned (therefore contrast is 0 value), contain or do not contain H ₂S (80ppm).This measurement was carried out during 15 minutes, and mice is in the environmental chamber of 0.5L sealing, and the atmosphere flow velocity is 500cc/ minute.The consumption of oxygen is by obtaining with the oxygen concentration that deducts in the contrast when mice does not exist when mice exists.Equally, the generation of carbon dioxide is by obtaining with the gas concentration lwevel that deducts in the contrast when mice does not exist when mice exists.RQ represents respiratory quotient, and the ratio of the oxygen of carbon dioxide that equals to produce and generation.This result proves, has H ₂Oxygen consumption reduces 2-3 doubly under the situation of S, and the generation of carbon dioxide also reduces 3-4 doubly.The variation of respiratory quotient has reflected in existence or has not had H ₂Under the situation of S, oxygen consumption of mice and carbon dioxide generating different.

Table 4-H ₂S exposes the breathing that has suppressed mice

The existence of mice	H ₂The existence of S	[O ₂] ppm	[CO ₂] ppm	RQ
The existence of mice	H ₂The existence of S	[O ₂] ppm	[CO ₂] ppm	RQ	-	-	207,000	0
+	-	203,600	2800		-	-	207,000	0
+	-	203,600	2800			Consume, produce	3,400	2800	0.82
-	+	166,200	0			Consume, produce	3,400	2800	0.82
-	+	166,200	0		+	+	164,900	750
	Consume, produce	1300	750	0.58	+	+	164,900	750

The different parameters of stagnating (oxygen consumption reduces, carbon dioxide generating reduces or activeness reduces) can be by diversified algoscopy and technology assessment.For example, H has been used in measurement ₂What induce stagnation in the mice of S may the easiest method be by observing their breathing.Really, this has comprised all three parameters, because it has represented the oxygen consumption, carbon dioxide generating and the activeness that reduce.Normal mouse in the room air under the standard conditions will be breathed 200 times by per minute approximately.If used the H of 80ppm ₂S gives mice, and core temperature is reduced to 15 ℃, then breathes and reduces the order of magnitude between about 1-10 the breathing of per minute at least.In fact, observe mice under these conditions and breathe no more being longer than in period of 1 hour, the stagnation that shows deep level is accessible.Therefore, this has represented Cellular respiration (that is, oxygen consumption and carbon dioxide generating) at least about 1-20 minimizing doubly.

Mice is exposed in the atmosphere of being regulated under the ambient temperature that reduces.In order to begin to determine that hydrogen sulfide reduces the limit of the active ability of mice, the present inventor has carried out several tests, uses the mice of no telemetering equipment in the test, and the mice of carrying telemetering equipment by exposure obtains data afterwards.First test is to allow the mice of no telemetering equipment accept 80ppm H under the temperature inside the box of 10 ℃ reduction ₂The atmosphere of being regulated of S is described in material and method basically as mentioned, except before being exposed to gas and reducing ambient temperature with mice in placing under 27 ℃ the plenum chamber 1 hour.The performance in this is handled of the mice of no telemetering equipment is good, and reactivates in about 90 minutes after shifting out from plenum chamber.The mice that telemetering equipment is arranged of accepting the same terms also shows well, and shows that the minimizing core temperature is to about 12.5 ℃.The present inventor can not accurately measure this temperature, because this electronic equipment is not worked in the time of 15.3 ℃.Therefore, temperature is reduced to 12.5 ℃ and is based on the descending slope before not working and stays in the estimated value of indoor time the electronic equipment back animal of not working.

Because the restriction of instrument, the present inventor has the atmosphere of being regulated that contains the 80ppm hydrogen sulfide of having an appointment, or is having in the plenum chamber of room air next basically as described above, detects every of 4 mice with telemetering equipment 6 hours.The temperature of calorstat (be exposed in the atmosphere of being regulated, or mice being exposed to the 0 o'clock time of room air) when test is initial is reduced to constant 15 ℃.When 6 hours cycles ended up, middle as mentioned generality was described, and mice is returned to the atmosphere of room air and 22 ℃ ambient temperature.Core temperature obviously descend (Fig. 4 B) in depending on all 4 mices that utilize 80ppm hydrogen sulfide.Also there is the obvious reduction of heart rate relevant and total motor activity with the body temperature reduction.Mice was supported for 4 weeks, and the behavior of animal does not significantly change.

Embodiment 5

About reducing the Mus research of radiation damage

A. scientific basic principle

Though radiation damage model aspect can and be estimated in cell culture, the ability that detects trial drug influence damage and agglutination need comprise affected all response systems.At present, the unique channel that realizes it is in intact animal.The present inventor proposes mice is used for this research as only model.Selected the C57BL/6 mice to be used for research, because the mice of this strain is easily to radiation pulmonary lesion sensitivity, the radiation level of this strain tolerance is definite, and the present inventor has shown H recently ₂S has reduced the core temperature of this mouse species.

According to this conceptual design the test of two unanimities.Each test will be studied H ₂The inductive hypothermia of S-is to the effectiveness of the development of radiation induced pulmonary lesion.Every group of 10 mices will be exposed to four experiment condition (H ₂The irradiation of S/17.5Gy chest, H ₂S/ does not have chest irradiation, no H ₂The irradiation of S/17.5Gy chest does not perhaps have H ₂One of S/ does not have the chest irradiation), continued for 13 weeks then.Every group of 12 animal will expose similarly and continue for 26 weeks (n of increase compensates for the mortality rate that occurs in the course of disease later stage to increase required.)

For these tests, variance analysis (ANOVA) will be with the statistical model that acts on data analysis.4 groups of complete intersection and randomized two factor ANOVA (accept H ₂S or do not accept H ₂The exposure mice of S or unirradiated mice) and two intervals (13 or 26 week) will be used in the temporary variation of the hydroxyprolin levels of analyzing bronchoalveolar lavage inflammatory cell number and total protein concentration and lung.Suppose 80% power of test, 5% significance and two-tailed test, 5 survival mice of the combination of each damage group, intervention group and time point will allow detected difference between the cell mean more than or equal to 1.7 times of potential group of internal standard difference.Expection group internal standard difference equals about 25%.Therefore, the change of the 35-50% inflammatory cell number of control value or pulmonary's collagen content should be recognizable in these trials.

H ₂The exposure of S and chest irradiation will be carried out in the SLU AHR in the linear accelerator installation.The acquisition of bronchoalveolar lavage and lung will be carried out in AHR mice necropsy chamber when necropsy.The measurement of the hydroxyproline content of bronchoalveolar lavage cell counting and protein concentration and lung will be carried out in another laboratory (D3-255).Wild gene type C57BL/6 mice will be accepted the chest irradiation of 17.5Gy.Mice will be anaesthetized with avertin (Avertin) intraperitoneal, be placed in the independent cloth matter mice restraint device (restrains), and by linear accelerator with 3Gy/ minute close rate with the 8.5Gy irradiation by through calibrating only two side regions of targeting chest (total chest dosage is 17.5Gy).

B. scheme

Anesthesia.Wild gene type C57BL/6 mice will be anaesthetized because of using isoflurane in the trachea.Depth of anesthesia will be by the breathing rate monitoring to the reaction of tactual stimulation.The peritoneal injection of avertin (0.4-0.7ml/ mice i.p.) will be used to anaesthetize the animal that is used for the chest irradiating step.Depth of anesthesia will be by breathing rate with to the reaction monitoring of tactual stimulation.

Be exposed to hydrogen sulfide.Mice is placed the lucite plenum chamber (IR1606) that similarly seals with the plenum chamber that before was used for mice.This chamber will have two mouths (input port and delivery outlet).To contain the equilibrated H of useful room air ₂The gas of S (80ppm) feeds with the speed of 1 liter of per minute that this is indoor.Employing have extend to the room from the output vent the indoor ventilation system of flexible pipe of exhaust ventilation mouth with gas from indoor discharge.

Using of hazardous agents.Use linear accelerator, irradiation mice so that the accumulated dose of 17.5 gray(Gy)s is in mice is in plenum chamber.This exposure dose will be induced subacute pulmonary lesion in mice, it makes progress into fibre modification.Mice can not be radioactive, otherwise staff or other animal are damaged.Because this irradiation does not need special monitoring, inhibition or processing.

Predetermined euthanasia.After chest irradiation about 13 and 26 weeks of back, allow animal pass through deep anaesthesia (adopting avertin 0.4-0.7ml i.p.) euthanasia, puncture blood-letting by postcava afterwards.Implement bronchoalveolar lavage and be used to measure inflammatory cell number, differential counting and eluate protein concentration.Shift out lung and esophageal tissue and be used for Histological evaluation and collagen content analysis.

Dying animal.The chest irradiation is relevant with the limited mortality rate of mice, and mice 22 weeks after irradiation 50% are dead in that 10 weeks after the irradiation are dead for 15% mice.Researcher will be monitored the ill effect (originally every day 2-3 time is stable up to their performances, begins progress up to disease once a day then, will return to this time point inventor and observe repeatedly every day) of animal every day.If the weight of animals alleviates, no longer ornamenting, show serious RD, and/or activity clumsy or that obviously reduce, then with it with the excessive euthanasia of carrying out of avertin.When putting into practice, will be used for the histology to the animal enforcement bronchoalveolar lavage and the tissue collecting of these irregular euthanasia.

Chest irradiation should be able to produce pulmonary lesion, itself be not pain but itself can show as that breathing rate increases, slight loss of appetite, slightly lose weight and/or no longer ornamenting (the 10th week).Researcher and animal facility staff will monitor this ill effect of animal every day.If animal seems not ingest, then can provide softish food and fluid support.Be in the pain if perceive animal, then use butorphanol (0.2mg/kg i.p.) or buprenorphine (1.0mg/kg bid s.q.) pain relieving as required.If animal shows misery and the measure of taking stopgap measures property does not cause improving, then make its euthanasia immediately.Collection lung and esophageal tissue are used for histopathology evaluation and collagen content analysis when predetermined necropsy.

Postradiation management.Relay the risk minimization that any pathogen is given the remainder of described facility in order to make; and in order to protect these animals in a way during non-responsiveness, will carry out (before in vitro any other animal) at first every day and will in the bio-safety chamber, carry out all management work of these animals at them.In order to make the risk minimization of external infection, mice will be used the cage and the bed accessory of autoclave sterilization.And they will be fed through shining the standard rodent food of pathogen kill.

Wild gene type C57BL/6 mice will be accepted the chest irradiation of 17.5Gy.Mice will be used avertin anesthesia by intraperitoneal, place independently cloth matter mice restraint device and shift-in and the similar lucite plenum chamber (IR1606) that seals of the plenum chamber that before was used for mice.This chamber will have two mouths (input port and delivery outlet).To contain the equilibrated H of useful room air ₂The gas of S (80ppm) feeds with the speed of 1 liter of per minute that this is indoor.Employing have extend to the room from the output vent the indoor ventilation system of flexible pipe of exhaust ventilation mouth with gas from indoor discharge.In case enter plenum chamber, by linear accelerator with 3Gy/ minute dose rates with 8.5Gy irradiation mice by through calibrating only two side regions of targeting chest (total chest radiological dose is 17.5Gy).After finishing chest irradiation, animal is put back in little isolation cage that they are monitored up to recovering from narcotism.

Predetermined necropsy.The 13rd week will be carried out necropsy to a treated animal after irradiation, with the inflammatory stage of assessment damage.To carry out euthanasia to second treated animal in the 26th week, with the fibre modification stage of assessment damage.Animal is used avertin anesthesia, then blood-letting.With lung with 1000 μ l PBS lavations and with eluate remain on be used on ice total cell count and the classification cell counting.Gather right lung then and be used for the hydroxyproline content analysis, and with left lung NBF by trachea perfusion 10% under 25-30cm pressure.Esophagus, trachea, left lung and heart immersed among 10% the NBF and deliver to FHCRC histology shared resource laboratory (FHCRC) and be used for processing and pathological evaluation.

Chest irradiation should be able to produce pulmonary lesion, itself be not pain but itself can show as that breathing rate increases, slight loss of appetite, slightly lose weight and/or no longer ornamenting (the 10th week).Researcher and animal facility staff will monitor this ill effect of animal every day.If animal seems not ingest, then can provide softish food and fluid support.Be in the pain if perceive animal, then use butorphanol (0.2mg/kg i.p.) or buprenorphine (1.0mg/kg bid s.q.) pain relieving as required.If animal shows misery and the measure of taking stopgap measures property does not cause improving, then pass through CO immediately ₂Suffocate and make its euthanasia.

Main problem may be esophagitis (causing the picked-up of food and water to reduce) and respiratory insufficiency (reducing the oxygen picked-up).The present inventor will check every day these animals 2-3 time up to be sure of they be stable and performance good, at this moment the present inventor can reduce and checks that frequency to once a day, begins progress up to disease, returns at this time point and checks repeatedly every day.Supportive care will provide in many ways.If the animal animal can not ingest or drink water well (show as and lose weight and the grooming problem), the present inventor will provide softish food and fluid fill-in (Ru Suanlingeshi solution on probation, the 1-2ml/ mice is adopted little vent needle (＞20G) subcutaneous injection, every day 1-2 time).Be in the pain if perceive animal, then use butorphanol (0.2mg/kg i.p.) or buprenorphine (1.0mg/kg bid s.q.) pain relieving as required.If animal shows misery and the measure of taking stopgap measures property does not cause improving, then pass through CO immediately ₂Suffocate and make its euthanasia.If animal experiences remarkable pain or misery when chest shines, will pass through CO ₂Suffocate and make animal euthanasia.

The 3rd experiment is basically as described above, allows the mice of telemetering equipment suffer 80ppm H under the indoor temperature of 10.5 ℃ reduction ₂The atmosphere of being regulated of S.In this experimentation, the mice and of detecting by an unaided eye by its activity of web camera record, and note down the remote measurement measurement result as described above.Mice is exposed to 80ppm H ₂In the atmosphere of being regulated of S, and indoor temperature is reduced to constant 10.5 ℃.When about 6 hours cycle ends up, come to the indoor heat that applies by indoor temperature being set at 25 ℃.The H that allows mice regulated ₂Intensification is between 17 ℃ and 18 ℃ up to the core temperature of mice in the S atmosphere, behind this time point, the atmosphere of being regulated is replaced to room air.The core temperature of mice obviously is reduced to 10.5 ℃ in the atmosphere of being regulated, and follows total motor activity obviously to reduce.Breathing rate is reduced to can not detected speed about 1 hour 15 minutes by perusal.After heating up in the chamber, when reaching 14 ℃, the core temperature of mice observes weak breathing.In the temperature rise period, when core temperature is increased between 17 ℃ and 18 ℃, and mice shows and breathes and when movable, the atmosphere of being regulated is replaced to room air.Normal movable obvious fully when core temperature returns to 25 ℃ with breathing.Compare with undressed animal, mice does not demonstrate the significant change in the behavior.

Embodiment 6:

Cell and mammal research

A. Canis animals research

Canis animals research will be carried out with Canis familiaris L., and described Canis familiaris L. underwent operative has been implanted telemetering equipment to monitor their core temperature.To when existing or lack the hydrogen sulfide of sublethal dose, zoologize 10 hours.During this period, will continue to monitor their vital sign by telemetry.Ambient temperature also will be reduced to 15 ℃ 30 minutes, whether have any influence in order to determine this core temperature to animal.

This program will be with 2 groups of Canis familiaris L.s, and 2 every group (4 altogether) carry out.Because the cost of telemetering equipment, the present inventor will carry out these tests continuously.If show that from first group result hypothesis is wrong, then will repeat this research with two Canis familiaris L.s of second group.If do not support hypothesis from second group result, then will abandon this project.

Toxicologic study shows, works as H ₂When the S level is higher than people's the OSHA limit (10ppm), proved in the past, rat and mice in 90 days 6 hours every days, be exposed to the H of 80ppm in 5 days weekly ₂Show among the S and do not observe detrimental effect.When this is included in the processing end intestinal, lung, the heart, liver, kidney or other organ are carried out macroscopy and histopathological examination.According to present inventor's knowledge, there is not the relevant information that Canis familiaris L. is exposed in the hydrogen sulfide to utilize.

Use H ₂The key issue of S operation is not surpass to disclose about rodent being exposed to the described dosage of other people (80ppm) of the research of not finding illeffects in the hydrogen sulfide.In the gas science, exist considerable experience to utilize, and the present inventor can send the gas of prescribed dose to mice.Taked many preventive measures can not come to harm in order to guarantee animal and research worker.These preventive measures comprise and continue to monitor admixture of gas, and alarm is set to the OSHA limit and sensitivity is 1ppm, and can mix to specifications and send gas and can not leak into or go out the multiple device of system.

The timeline of this scheme provides in table 5.

The timeline of table 5-test

My god	Movable	Details
My god	Movable	Details	-1	Before the operation	To implement the CBC/ chemistry; Canis familiaris L. is with fasting in the afternoon, but permission is freely drunk water.
0	Operation	(preemptive) analgesia before operation sticks the fentanyl transdermal plaster to be used for emptying noon before that day.Securement head conduit before the surgical operation; Acepromazine, buprenorphine, glycopyrronium bromide carry out premedicate; Use ketamine: the feasible energy of diazepam or Propofol induction intubate; Keep anesthesia by isoflurane and oxygen.Allow Canis familiaris L. dorsal part recumbency and prune abdominal part/operation and prepare and drape.The anesthetis percentage ratio of monitoring pulse, breathing rate, end-tidal carbon dioxide, suction, SpO ₂Per 15 minutes or implement more continually and record.To carry out the fluid support in operation process and after the operation.In case Canis familiaris L. is stable and carried out suitable preparation for this program, will implement abdominal part median line laparotomy, begin to extend 5-10cm to umbilical part and towards afterbody from afterbody.An aseptic emitter is put in the peritoneal cavity.Check that placement location can move freely to guarantee emitter; Impulse force (momentum) will be replaced, and the closure of peritoneal cavity will be carried out with 3 layers.Canis familiaris L., can be stood thermoregulation and use the chest recumbency up to it is removed pipe monitored.Monitor cutting part, abdominal part (by palpation and ultrasonic, if indicated words), appetite, temperature (postoperative initial 3-5 days), weight and the activity of Canis familiaris L. every day.	-1	Before the operation

7	Determine baseline	This date is flexible.Have only this step to carry out through approval.Four animals will place on the receiver apparatus (this do not relate to animal is shifted out and will carry out at AHR from their cage), and determine the baseline of the vital sign of all these four animals.
7	Determine baseline		8	Be exposed to H ₂S	Animal will be transferred in the room that will measure, there they will be put into the cage that food and water are arranged of the atmosphere with inclosure.After determining baseline, two in four animals is the H of 80ppm with acceptor concentration ₂S.After exposing 10 hours, atmosphere will return to indoor air temperature and animal will be put back in their cage.Be exposed to H ₂S will repeat weekly once, to begin determining whether the arbitrary data collection is repeatably.

B. people's platelet

Use the oxidative phosphorylation inhibitor to can be used for making people's this idea of being benefited in order to check, the present inventor induces the life state that stagnates to be exposed to oxygen to protect them to avoid lethal in people's tissue.In pilot plant test, the present inventor places application on human skin 100% CO environment.Present inventors have observed that 100 times of those Skin Cells in the survival ratio room air of the Skin Cell after 24 hours in CO.These results are very infusive: they have proved that the oxidative phosphorylation inhibitor may be in people's tissue effectively.

Another has been organized evidence and has induced the life that stagnates to hematoblastic protective effect.With the platelet of a unit in two.Be stored under the standard storage condition the first half, the standard storage condition comprises constant joltily preserve at room temperature (22-25 ℃) of platelet.Second half is placed the anaerobic environment (＜10ppm oxygen) of having removed oxygen with standard method.The 0th day, the 5th day and the 8th day these two groups of platelet of comparison.In one group of 5 different vitro detection, those that preserve under the platelet of preserving under oxygen free condition performance and the standard conditions are the same good or better, and described performance comprises centralize ability, morphocytology, annexin-V dyeing (Phosphatidylserine turn on the adventitia as natural death of cerebral cells labelling) in early days etc.This shows that control metabolic activity, particularly oxidative phosphorylation can be finished by removing deoxidation, and cellular function has protective effect in stagnation process over a long time.

The sulfuration Hydrogen Energy is the same as with CO cytochrome C oxidase, and stops oxidative phosphorylation when needed.It is so effective in hindering oxidative phosphorylation, to such an extent as to if the people has inhaled in the atmosphere that contains 0.1% hydrogen sulfide without a break, they should not inhale another mouthful.Otherwise, their incident of " knocking down " of can falling over immediately-in industrial background, be commonly referred to.As if this also is reversible, because if move on to rapidly in the fresh air (and fall be without damage), these individualities can be recovered and live on sometimes and do not had neurological problem.This is a kind of such material, and it is common in our world not only, in fact, even produces in we self cell, and is the effective reversible oxidative phosphorylation inhibitor of not realizing that oxygen is sent.

C. Mus research

Use H ₂S induces the state of similar hibernation.According to definition, Homoiotherm is kept core temperature and is higher than ambient temperature 10-30 ℃.Accomplish this point for these animals, they must produce heat from the energy that produces by oxidative phosphorylation.Combined enzyme agent terminal in the oxidative phosphorylation is a cytochrome c oxidase.Because hydrogen sulfide suppresses this complex (Petersen, 1977; Khan etc., 1990), present inventor's prediction is exposed to hydrogen sulfide with Homoiotherm and will prevents that this class animal from keeping its core temperature and being higher than ambient temperature.

In order to check this hypothesis, the present inventor will continue to monitor core temperature and the level of activation of Homoiotherm (mice).The telemetering equipment of implanting in the mouse peritoneum can be finished this two kinds of things, and has the advantage (Briese, 1998) that can not introduce deviation owing to handling mice to reading.In addition, they can be exposed to hydrogen sulfide gas process medium-long range monitoring mice.Before showed, and continued to reach 10 weeks for exposure, the hydrogen sulfide dosage of 80/1000000ths parts (ppm) is to being that harmless (CIIT 1983 for mice; Hays, 1972).Therefore, for these tests, the present inventor adopts the hydrogen sulfide dosage of 80ppm to check our hypothesis.The atmosphere that generation contains 80ppm hydrogen sulfide is not minor matter.Along with the time goes over, when having oxygen, hydrogen sulfide will be oxidized to sulfate.For this reason, can continue mice is exposed in the atmosphere that contains 80ppm hydrogen sulfide in order to make the present inventor, the present inventor constantly mixes room air with one jar of hydrogen sulfide with the 500ppm of nitrogen balance.

The sign of core temperature control

Mice is exposed to 80ppm H ₂S is reduced to its core temperature to be higher than ambient temperature about 2 degrees centigrade (Fig. 5).This effect is highly repeatably, follows similar pattern (Fig. 5) because be exposed to the average core temperature of 7 mices of 6 hours of 80ppm hydrogen sulfide.In 13 ℃ ambient temperature, the minimum average core temperature of these 7 mices is 15 ℃.When atmosphere was converted into the atmosphere that only contains room air, all these mices successfully recovered behind rewarming.In contrast, the present inventor replaces hydrogen sulfide with nitrogen and finds that core temperature does not have essence to reduce.

Though these mices are normal (although core temperature and breathing rate temporarily reduce) to the eye, the present inventor has carried out one group of behavior test and has got rid of such probability: be exposed to that hydrogen sulfide gas, core temperature sharply descend, breathing rate reduces, or the combination of these effects has caused neurological damage.All tests are implemented mice before being exposed to hydrogen sulfide and afterwards.These behavior tests are selected from the SHIRPA method (Rogers etc., 1997) by Mouse Models for Human Diseaseconsortium development.After being exposed to gas, not existing in mice can detected behavior difference.In view of the above, the present inventor concludes, and the state that enters similar hibernation is not deleterious.

H ₂S The initial optimization of dosage.The influence of 80ppm hydrogen sulfide to the mice core temperature described in top test.In order to determine to be enough to lose thermotaxic concentration of hydrogen sulfide, the present inventor is exposed to a series of concentration of hydrogen sulfide (20ppm, 40ppm, 60ppm and 80ppm) (Fig. 6) with mice.Though the hydrogen sulfide of 20ppm and 40ppm is enough to cause the core temperature of mice to descend, this is less with the viewed suppression ratio of hydrogen sulfide of using 60ppm and 80ppm.According to this test, the present inventor reaches a conclusion: the concentration of the hydrogen sulfide that offers mice is directly depended in the forfeiture of calorigenic action.This preliminary study of the pharmacokinetics of dosage range and hydrogen sulfide has been emphasized to need more fully to analyze.

The preliminary of low core temperature boundary determined.The present inventor is also interesting to set up the understanding more fully of tolerance of the time length that core temperature scope and mice are allowed under this state.Above-mentioned test shows that the present inventor can repeatedly reduce the core temperature of mice when needed to 13-15 ℃.In addition, as if mice can tolerate this processing a few hours.Adopt identical scheme, when reducing ambient temperature, the present inventor successfully makes the core temperature of mice reach 10.7 ℃ (Fig. 7).Further trial is reduced to core temperature lower, and the longer time will carry out in the future.Although be preliminary, these results prove, the core temperature that exists the mouse biological of remarkable scope to allow, and this scope can be by losing thermoregulation and probe into because of being exposed in the hydrogen sulfide.

EndogenousH ₂S The adjusting of level.Well-known mammalian cell endogenous ground produces hydrogen sulfide (Wang 2002).Because this chemical substance dynamically produces in cell, the foundation level of understanding under different condition is extremely important, because this can influence the pharmacokinetics of the hydrogen sulfide of external application significantly.For this importance of the research that solves us, the present inventor has begun to detect the endogenous hydrogen sulfide levels in the mice.The extraction alkanisation technology of present inventor's employing and gas chromatography and the associating of quality specific detection is come quantitative hydrogen sulfide (Hyspler etc., 2002).Use this method, the present inventor has observed the hydrogen sulfide levels in the undisturbed mice.Fig. 8 A shows the hydrogen sulfide that has significant quantity in this mice body.In addition, as if hydrogen sulfide levels depends on the ambient temperature of mice.Particularly, when mice is in cold environment, they reduced endogenous sulfide level and, be positioned at warm following time of ambient temperature mice, they have increased endogenous sulfide level.In view of the above, the present inventor reaches a conclusion: mice response environment temperature and the sulfide level of regulating them.

The variable effect of endogenous levelH ₂S Effectiveness.Because ambient temperature changes the endogenous level of sulfide in the mice body, present inventor's supposition, in case be exposed to external hydrogen sulfide, ambient temperature may influence the change of core temperature.Allow mice adapt to low temperature～12 ℃, produced persistent maintenance level, the present inventor observes this maintenance level (Fig. 8 B) after core temperature begins to descend.Therefore, the core body cooling that causes of this effect that shows that this adaptation to cold makes mice more tolerate hydrogen sulfide gas.Yet, allow mice before being exposed to gas, adapt to warm pining for property (thermoneutral) temperature and eliminated this maintenance level.In fact, the room temperature mice when being exposed to hydrogen sulfide than the mice that seasons oneself to cold rapider the cooling (Fig. 8 B) of Duoing.These data show that the endogenous level of hydrogen sulfide has a direct impact the effectiveness of external hydrogen sulfide in the mice body.

H ₂S The protection mice avoids the hypoxia influence.Normal room air contains 21% the oxygen of having an appointment.In the preliminary test of research stagnation to the protective effect of hypoxia, the mice that is exposed to 80ppm hydrogen sulfide survives in 11 minutes 5.2% oxygen, and after 3 weeks, mice still shows well in mouse model.Previously disclosed work shows, 90% expose by this way and do not have these animals (C57Bl) of hydrogen sulfide not survive (Zhang etc., 2004).This test comprises the mice pre-equilibration in 80ppm H ₂Among the S 3 hours, then as the oxygen tension of describing in the top experiment in the chamber that makes reduce.Use identical flow velocity (being to be 500cc/mL in the 0.5L chamber) as described above.Those people that are familiar with this field determine well, if one group of mice is exposed in 4% the oxygen, and then 100% will be dead in 15 minutes.Yet, wherein be reduced in 4% process and used H in oxygen tension ₂The mice of S still maintains vigor under these hypoxia conditions, even lasting time expand (up to 1 hour).As if mice is not subjected to these condition effect after recovery, and test the time has vigor and can normal reaction after 24 hours.This test is different with top experiment, and difference is that mice still remains on H when exposed hypoxia finishes ₂Return to normal level (21%O up to oxygen tension among the S ₂).

Embodiment 7

Other zooscopy

A. protect to avoid the unfavorable conditions influence

Carry out the ability of this test in order to survive under the condition that detects mice its common meeting death therein in ' similar hibernation ' state.Unfavorable conditions is a hypoxia, and it is had documents claim mice (C57BL6/J is male) can survive at most 20 minutes (Zhang etc., 2004) in 5% oxygen.

As showing in the table 6, test comprises the H that mice is exposed to 80ppm (unless otherwise indicated) ₂Time shown in continuing among the S reduces the oxygen tension in the chamber afterwards, yet still at H ₂Under the S.Record is exposed to the time (following explanation) of hypoxia and measures the viability of mice.

The mice short time is exposed to H ₂Protect mice to avoid hypoxia among the S (being at least 80ppm) unsuccessfully, but at least one mice arranged at H ₂Only just can survive under 50 minutes exposed hypoxia after 8 minutes among the S.In addition, observe and be exposed to 90ppm H ₂Among the S only 10 minutes mice under 5% oxygen condition, can survive the longer time, although its at last can be dead.

Mice is exposed to 80ppm H ₂The longer time avoids hypoxia to the protection mice among the S intensive effect up to 1 hour.

Table 6

Ambient temperature	Before being exposed to hypoxia at H ₂Time among the S	Oxygen %	Time in hypoxia	The result
Ambient temperature	Before being exposed to hypoxia at H ₂Time among the S	Oxygen %	Time in hypoxia	The result	20℃	5 hours	5.20％	11 minutes	Survival
20℃	5.5 hour	5.00％	25 minutes	Survival	20℃	5 hours	5.20％	11 minutes	Survival
20℃	5.5 hour	5.00％	25 minutes	Survival	20℃	5 hours	5.00％	60 minutes	Survival
20℃	5 hours	4％	28 minutes	Survival	20℃	5 hours	5.00％	60 minutes	Survival
20℃	5 hours	4％	28 minutes	Survival	24℃	No H ₂S	5％	14 minutes	Dead
24℃	Take place simultaneously	5.10％	10 minutes	Dead	24℃	No H ₂S	5％	14 minutes	Dead
24℃	Take place simultaneously	5.10％	10 minutes	Dead	24℃	8 minutes	5％	20 minutes	Dead
24℃	8 minutes	4.00％	8 minutes	Dead	24℃	8 minutes	5％	20 minutes	Dead
24℃	8 minutes	4.00％	8 minutes	Dead	24℃	8 minutes	4.50％	23 minutes	Dead
30℃	8 minutes	4.50％	6 minutes	Dead	24℃	8 minutes	4.50％	23 minutes	Dead
30℃	8 minutes	4.50％	6 minutes	Dead	24℃	10 minutes (90ppm)	5％	56 minutes	Dead
24℃	8 minutes	5.00％	50 minutes	Survival	24℃	10 minutes (90ppm)	5％	56 minutes	Dead

B. strengthen the anoxia toleration

1. background

Studied carbon dioxide (CO ₂) and hydrogen sulfide (H ₂S) strengthen the purposes that complicated metazoa fruit bat (Drosophila melanogaster) survives under anoxia.These tests show, these materials, especially H ₂S can increase the anoxia toleration of adult fruit bat.

The Caenorhabditis elegans embryo stagnates from birth at anoxia (＜10ppm O by entering ₂) survive down, and growth can be at 0.5% O ₂In carry out.Yet, the lethal oxygen concentration (0.01-0.1%O of 10 times of scopes of existence ₂).In addition, stop the oxygen utilization can prevent embryo's hypoxia infringement with carbon monoxide.Therefore, if there is no enough oxygen is utilized by effective biological activity, then is more preferably not have (or use) any oxygen.

In more complicated metazoa, the cell oxygen concentration not necessarily oxygen level with environment is identical.In Caenorhabditis elegans, oxygen is given tissue by transdermal delivery.Yet, in higher organism, exist in conjunction with oxygen so that the protein that transportation oxygen is extremely organized, for example hemoglobin.Therefore, when the oxygen level of environment descends, in cell, may there be residual oxygen.

Most of biologies can not survive and be exposed to environmental hypoxia.A kind of probability is that the residual oxygen on the cellular level is virose, is equivalent to the oxygen scope of observed lethal in the Caenorhabditis elegans embryo.In this case, can not utilize if residual oxygen is removed or becomes, then the survival under the anoxia should be able to increase.CO ₂Promote O ₂From hemoglobin, discharge and H ₂S is a kind of effective oxidative phosphorylation inhibitor.

2. material and method

Basic experimental program.The adult fly is introduced in the pipe of being made by glass (Balsh pipe) that 35mL has the air-tightness rubber closure.This is usually by finishing like this: use CO ₂With fly anesthesia, the fruit bat group moved in the bottle that food is arranged recovered at least 2 hours, then they are shifted in the Balsh pipe.In order to exchange the gaseous environment in the Balsh pipe, 2 No. 18 pins are inserted in the rubber closure, and allow gas be blown in the entry needle with 100mL/ minute.In order to prevent drying, gas before by the Balsh pipe by making it to steep humidification by the 10mL ascites.Before on-test with the water in the bubbler with gas balance at least 20 minutes.

For " stopping-flowing (stopped-flow) " test, gas exchange carried out 60 minutes before the sealing test tube.For " slow-stream (low-flow) " test, air-flow continues in whole experiment.CO ₂From indoor source (100%), and anaerobic environment is passed through with 100% nitrogen (N ₂) blow out room air and set up.With atmosphere from CO ₂Convert N to ₂The time, note preventing from room air is introduced in this system.

After anoxic treatment, oxygen is introduced in the Balsh pipe once more by charging into room air 20 minutes.Remove rubber stopper then and a food bottle is inverted in Balsh pipe top with Parafilm.If fruit bat reactivates it is evaluated as alive.After finishing, anoxic treatment viability is estimated at least 18 hours the time.After 2 weeks,, think that then fly can educate if contain larva and/or pupa in the food bottle.

3. result

Before being exposed to anoxia, use CO ₂Handle.If the adult fly is at first used CO ₂Pretreatment, then they show higher anoxia survival rate.Stopping-after anoxia exposes 19 hours in the flow test, use CO ₂The adult fruit bat of

pretreatment

30 or 90 minutes shows 54% or 28% survival respectively.Being exposed to anoxia without CO ₂Pretreatment or be exposed to CO ₂Do not observe survivor in the anoxybiotic contrast and next be exposed to.In addition, if fruit bat also is exposed to CO at once after anoxia exposes 20 minutes ₂, then use CO ₂Pretreatment does not have fly to survive to expose in anoxia.

Short time is exposed to CO ₂In be enough to strengthen anoxybiotic survival.In stopping of exposing of 22 hours anoxias-flow test, if before converting nitrogen atmosphere to, use CO ₂0.5-5 minute, the ratio of Cun Huo fly the highest (Figure 16) then.Therefore, for experiment subsequently, standard scheme is to use CO before being exposed to anoxia ₂Handled 10 minutes.In the slow-stream test of this scheme of use, 6% adult fruit bat survives in 20 hours anoxia exposure, and this survival needs CO ₂Pretreatment.

Test shows, at CO ₂Handle and set up N ₂Prevent to introduce again O between the environment ₂Be important.When the water of the bubbler that is used for humid gas is blowing out CO ₂Before without N ₂During balance, do not have fruit bat to survive in these trials and expose, no matter N in 13 hours anoxia ₂Be with 10,50 or introduced in 100mL/ minute.Under these conditions, CO ₂Atmosphere N ₂/ O ₂The mixture purge comes out, wherein N ₂/ O ₂Mixture is by O ₂Be dissolved in the water and produce.

Carry out a series of slow-the stream test in order to determine with without pretreated comparison, CO ₂The time that the anoxia that exposure is carried out exposes, every kind of condition detects (Figure 17) in duplicate.In these data, tendency is CO ₂Pretreatment causes bigger survival.An important explanation is, these tests have departed from standard scheme, and difference is that fruit bat is used CO ₂Anesthesia also is transferred in the Balsh pipe, and at beginning CO ₂Only allow to recover 10-20 minute (except testing 2 the 18th hour time point) before the processing.

Implemented several other tests, it is not for using CO ₂Whether pretreatment is the favourable information that provides.For example, in a test, have 10 minutes CO ₂Do not observe the survivor in anoxia after 17,22 and 24 hours in the slow-stream test of pretreatment stage.Yet in other test, many fruit bats survived after 17 hours.This can illustrate that other factors has influenced the result in some case, for example the change of the age of adult, circadian rhythm or room temperature.Another relatively stop-test of mobility program and slow-stream scheme in, do not have fly to survive and expose in 17 or 19.5 hours anoxia; Yet in this case, mould contamination may promote the death of fruit bat.

Before exposing, anoxia usesH ₂S Handle.In pretreating scheme, comprise H ₂S has strengthened the ability that the adult fly survives more significantly under anoxia.In the test of those that in a series of Figure 17 of being similar to, show, add 50ppm H ₂S to CO ₂(H in the pretreatment ₂S/CO ₂) increased the ratio (Figure 18) of the fruit bat that in processing, survives.These flies look like healthy, and have produced the offspring after exposure.Yet, in similarly testing, at H ₂S/CO ₂In do not have fly to survive in 18,20,25 or 30 hours anoxia after 10 minutes.The reason of this species diversity is unclear.With H ₂The favourable influence that S handles conforms to, after 15 hours anoxias expose, 50% use H ₂The pretreated fruit bat of S survives, and is not exposed to H ₂The contrast fruit bat of S does not recover.In this test, fruit bat is used CO ₂Handled 10 minutes, and used H then ₂S/CO ₂Handled 10 minutes, and used N subsequently ₂/ H ₂S handled 10 minutes, used N at last ₂Handle the persistent period that continues slow-stream test.

H ₂The dependent anoxia survival of S-increase does not require CO ₂Handle.25% cause anoxia before 18.5 hours with the 50ppm H in the room air ₂The fruit bat that S handles is survived.If before setting up anaerobic environment, increase and be exposed to H ₂S/CO ₂10 minutes, then the ratio of Cun Huo fruit bat was unaffected.Therein fruit bat before anoxia exposes only with CO handle in the examination according to the facts, have only 11% fruit bat to recover.

Use H ₂As if the time of S be important to increasing the anoxia survival.If H ₂S (20 hours) in whole anoxia process-exposed exists, and does not then have fruit bat to restore, no matter H ₂S is at CO ₂Whether exist in the preprocessing process.Yet, if 50ppm is H ₂S is present in CO ₂In the pretreatment, and when setting up anaerobic environment, remove then 35% fruit bat survival subsequently.In parallel test, using CO ₂(no H ₂S) pretreatment after 10 minutes 6% the anoxybiotic fruit bat of being exposed to survive.

Preliminary test with larva and embryo.After handling with CO and the CO that contains HS, the anoxybiotic survival of increase is also observed in embryo and larva.Be exposed to anoxia after 24 hours, 7 pupas are formed by one group of 0-19 hour big embryo.Yet, using CO ₂Pretreatment is observed 20 pupas in 10 minutes the paired pond.Similarly, be exposed to 24.5 hours larvas in the anoxia only at them through CO ₂Or H ₂S/CO ₂Just can when oxygenate again, reactivate during pretreatment.0-24 hour big embryo survives in 18.5 hours anoxia exposes and grows to the adult stage, no matter uses CO ₂Or H ₂S/CO ₂Whether pretreatment.

Cold treatment in the anoxia process.Reduce ambient temperature and can prolong the time span that the adult fruit bat can survive and expose in anoxia.At room temperature, stop-not having in the mobility program fruit bat and surviving in 15.5 hours anoxia exposes, but keeping anoxybiotic those fruit bats to be recovered simultaneously under 4 ℃ of 20% the maintenances.Similarly, the anoxia that is converted under 4 ℃ also moves to the not survival of the following 16.5 hours fruit bat of room temperature subsequently.Yet, 16.5 hours and even after 40 hours, in whole process-exposed, remain on 4 ℃ fruit bat and recovered and can educate.Before setting up anaerobic environment, use CO ₂Pretreatment does not have significant difference in these trials.

Embodiment 8:

The decline of core temperature

In rat and mice, studies show that and utilize H ₂S and CO ₂, can reduce the metabolism output, show as core temperature and reduce.Figure 20 A-B shows, ought at first apply H in the time 0 ₂S or CO ₂The time, the core temperature of animal begins to descend.After 6 hours, when removing H ₂S or CO ₂The time, it is normal that body temperature begins to recover.Obviously bigger mammal needs more H ₂S influences metabolism.

Embodiment 9 portentous:

Gas matrix

In order to measure the concentration of every kind of composition gas in the mixed atmosphere that the customization of the maximum capacity of metabolism flexibility in the control mammal is provided, can implement following test.Gas comprises oxygen (O ₂), nitrogen (N ₂), carbon dioxide (CO ₂), hydrogen sulfide (H ₂S) and helium (He).At H ₂When S may reduce oxygen demand in the mitochondrion, CO ₂Can further reduce oxygen demand.In addition, the reduction of having found core temperature is essential to reducing metabolism.Therefore, helium can provide the cooling means of simple and Noninvasive with its high thermal capacity.In addition, with 100% O ₂, 20.95% oxygen of normal oxygen can be maintained at other one-tenth wherein and be grouped in 79.05% any admixture of gas that is less than total amount.And last, nitrogen is used for this mixture to 100% of balance.

These tests have been described a kind of progressive method and have been come at first mice then rat, subsequently single and combine detection gas in Canis familiaris L..The target of this gas matrix is basis of studying with a plurality of variablees with logical order thereon of development.This experimental design is described in Figure 21.

One of feature of this gas matrix is: it shows that the test general only can just carry out when finishing in test (connecting by arrow) in front.Bulk testing will can not implemented in any animal model when at first not optimizing composition gas.In addition, gas or admixture of gas will not use in rat when at first not optimizing dosage in mice, and will not use in Canis familiaris L. before at first not optimizing in rat.Therefore, it shown with pure gas to multiple gases mixture (read on the portion left side on earth from top the right) and from the mice to the rat to the experiment progress of Canis familiaris L. (read on the right from the top left to the bottom).Mice will always at first be used to measure the concentration that provides the optimally-controlled composition gas of metabolism flexibility.In case with mouse assay the most effective dose of gas, identical test will be carried out in rat.Simultaneously, will be with a kind of gas or admixture of gas under the mouse assay.In case concentration is determined in rat, will check this gas in Canis familiaris L..Following table provides slightly different mode to observe gas matrix and confirmed test order:

In proper order	Mice	Rat	Canis familiaris L.
In proper order	Mice	Rat	Canis familiaris L.	1	H ₂S+CO ₂	CO ₂
2	He/O ₂	H ₂S+CO ₂	CO ₂	1	H ₂S+CO ₂	CO ₂
2	He/O ₂	H ₂S+CO ₂	CO ₂	3	H ₂S+CO ₂+He	He/O ₂	H ₂S+CO ₂
4		H ₂S+CO ₂+He	He/O ₂	3	H ₂S+CO ₂+He	He/O ₂	H ₂S+CO ₂
4		H ₂S+CO ₂+He	He/O ₂	5			H ₂S+CO ₂+He

Program 1. carbon dioxide (CO ₂)

Mice: discover 15% CO ₂Control to the metabolism flexibility is provided in mice.Yet in view of we mix the limitation of ability, we can not check higher concentration.This idea is not correct, and we can check the CO up to 80% ₂Concentration.Therefore, we will be from 15% CO ₂Beginning also increases to 40% with 5% increment, increases to 80% with 10% increment then.Animal will expose 6 hours.Mice will expose with the glass chamber of 375ml, will provide water and pre-mixed gas atmosphere to flow into wherein with the speed of 500 milliliters of per minutes to animal therein.These glass chambers will be contained in the calorstat so that the may command ambient temperature.This table provides our height-concentration C O ₂The framework of test, but may need other experiment to understand effect better.Mice can be used in a plurality of experiments, but we will can be than also using an animal continually once in a week.In addition, animal can be used as they self contrast in test subsequently.

％CO ₂	15	20	25	30	35	40	50	60	70	79
％CO ₂	15	20	25	30	35	40	50	60	70	79	％O ₂	21	21	21	21	21	21	21	21	21	21
％N ₂	64	59	54	49	44	39	29	19	9	0	％O ₂	21	21	21	21	21	21	21	21	21	21

To monitor metabolism (O ₂Consume and core temperature) and movable.CO 15% ₂Following, tidal volume increases but breathing rate remains unchanged.Mice does not show " asthma ".Increase CO ₂Concentration should strengthen anaesthetic effect.

These tests will be carried out in calorstat, so that we can reduce ambient temperature to 10 ℃ subsequently in order to detect the relation between core temperature and the metabolism output.

Rat: the experiment in the rat will be with 3% CO ₂With 21% O ₂Equilibrated nitrogen environment begins.It is normal that this is considered to blood carbonic acid, because it is CO ₂Exhalation concentration.Since 3%, we will increase to 15% with 2% increment.This increase can be carried out in one baseline test, and we increase CO in described baseline test ₂Concentration is observed metabolic change up to us.Metabolism will be by measuring O ₂Consume and the core temperature monitoring.Single rat in test can be used for measuring this minimum CO ₂Dosage.In test subsequently, wherein will detect 6 hours CO ₂The effect that exposes, single rat will be used for only a kind of CO ₂Dosage (that is, level will can not change in process of the test).Rat can be used for a plurality of experiments; They use weekly once no more than.Will be with 2, the 800ml glass container exposes animal, will provide water and pre-mixed gas to flow into wherein with the speed of 3 liters of per minutes in the glass container.This table has shown the initial CO that utilizes rat ₂The structure of test, but need other test fully to study effect.

％CO ₂	3	5	7	9	11	13	15
％CO ₂	3	5	7	9	11	13	15	％O ₂	21	21	21	21	21	21	21
％N ₂	76	74	72	70	68	66	64	％O ₂	21	21	21	21	21	21	21

From 15% to 80%, test will be as the progress of implementing with mice (15%-40% is 5% increment, and 40%-80% is 10% increment).To monitor metabolism and behavior.In case be identified for the CO of rat ₂Effective dose, whether as use H will reduce ambient temperature with the research metabolism ₂The core temperature reduction of passing through that S carries out further reduces.These tests will be carried out in calorstat, and this calorstat will provide cooling and heat when off-test in on-test and experimentation.

Finish the CO of rat ₂After the research, will know whether rat needs than the more CO of mice ₂(to H ₂S is correct), or identical, or CO still less ₂Be used to reduce their metabolism.Understand this crucial friction speed growth (allometric) trend and will be the effective CO in the Canis familiaris L. ₂Dosage provides better supposition.

To name a person for a particular job be O in the termination of these programs in mice and the rat ₂Consume minimizing 99% or CO ₂Produce and reduce 99%.

Canis familiaris L.: be used for Canis familiaris L. experimental design will with identical (the adopting 10 liters/minute flow velocity) that be used for rat and mice.Test will be with 3% CO ₂Beginning also increases up to observing physiological reaction.With anaesthetic mask Canis familiaris L. is exposed to mist, these Canis familiaris L.s adapt to this anaesthetic mask in advance before exposure.To monitor O ₂Consume and core temperature.Identical one or two Canis familiaris L.s can be used for these tests.

Program 2. hydrogen sulfide and carbon dioxide (H ₂S+CO ₂)

By mixing H ₂S and CO ₂, we will seek the cooperative effect of these two kinds of gases.That is H, ₂S and CO ₂Can make together and be used for reducing metabolism with the single the same degree of depth of gas ground of planting of higher concentration.In initial test, use mice, will use the H of 20ppm ₂CO is gone in S and titration ₂Reach 15% (or other concentration of in program 1, determining).An animal can be used for changing CO ₂Concentration and keep H simultaneously ₂S is constant.Secondly, we will use 40ppmH ₂S also adds CO ₂Reach 15%.The 3rd, we will use 80ppm H ₂S also allows CO ₂Reach 15%.In these tests each can be used an animal.To monitor O ₂Consumption, core temperature, and behavior.The test of listing in table is research H ₂S+CO ₂The effect of mixture provides the foundation, and will need other to test to understand effect.

Test	O ₂％	H ₂S?ppm	CO ₂％
Test	O ₂％	H ₂S?ppm	CO ₂％	1	21	20	5-10-15
2	21	40	5-10-15	1	21	20	5-10-15
2	21	40	5-10-15	3	21	80	5-10-15

In case measured effective mixture, whether can play synergist with low core temperature and be used for further reducing metabolic rate with reducing admixture of gas that ambient temperature optimizes in order to research.In these temperature dependency tests, the concentration of the gas in the mixture will not change.Again, an animal can be used for a plurality of experiments, weekly every no more than test procedure of animal.

Use the test of rat and Canis familiaris L. will adopt identical method to implement.The CO that is used for rat and Canis familiaris L. ₂Concentration will be optimized in program 1, but suppose that they will be between 5% and 15%.For Canis familiaris L., high H ₂S concentration is undetermined greater than 400ppm but still.

Program 3 helium (He)

Helium is a kind of effective dissipation of heat device; Its conduction of heat is bigger 6 times than nitrogen.It has comprised many mammals uses among rat, Canis familiaris L. and the people to promote cooling.It is nontoxic, cheap, and is easy to handle.Expectation is the same with other gas with helium, is used for 80%/20% mixture (He-O with oxygen ₂) in, be used for by the Repiration increased thermal conductivity.Propose 5 pilot studys and analyzed He-O ₂Influence to metabolism and behavior.The 80%-20% mixture of widely used standard will be adopted.The mixture that also detection is contained 60%He is in order to reflect the He-O of the minimum that we will use better in scheme 5 ₂Mixture, we have mixed H in scheme 5 ₂S, CO ₂And He.

Test	Animal	Temperature	He-O ₂-N ₂
Test	Animal	Temperature	He-O ₂-N ₂	1	Mice	23℃	80-20-0
2	Mice	10℃	80-20-0	1	Mice	23℃	80-20-0
2	Mice	10℃	80-20-0	3	Mice	23℃	60-20-20
4	Mice	10℃	60-20-20	3	Mice	23℃	60-20-20
4	Mice	10℃	60-20-20	5	Rat	23℃	80-20-0
6	Rat	10℃	80-20-0	5	Rat	23℃	80-20-0
6	Rat	10℃	80-20-0	7	Rat	23℃	60-20-20
8	Rat	10℃	60-20-20	7	Rat	23℃	60-20-20
8	Rat	10℃	60-20-20	9	Canis familiaris L.	23℃	80-20-0
10	Canis familiaris L.	23℃	60-20-20	9	Canis familiaris L.	23℃	80-20-0

To monitor oxygen consumption, carbon dioxide generating, core body peaceable conduct in order to understanding He-O ₂The same effect that whether can induce other gas to have.

Program 4: oxygen (O ₂)

The minimizing oxygen concentration can reduce core temperature and be shown with Cavia porcellus in 1936 by Gellhorn and Janus.Expectation then in rat, and reappears these tests at first in mice at last in Canis familiaris L., the O that reduces in order to research ₂Whether concentration reduces metabolism.

Mice: proposed mice wherein and will be exposed to the O that is low to moderate 6% minimizing ₂The experiment of concentration.Test will be with 5% increment progress.To measure O ₂Consumption, CO ₂Output, core temperature, and behavior.If observe the extremely convulsions behavior of hypoxia of indication, then with O ₂Return to 21%.Following table provides this O ₂The summary of test.Open-assembly time is to carry out 6 hours in pre-mixed gas inflow control room (water is arranged) wherein.Because have the evidence of hypoxic preconditioning, four independent animals are used for this four experiments.

Test	％O ₂	％N ₂
Test	％O ₂	％N ₂	1	21	79
2	16	84	1	21	79
2	16	84	3	11	89
4	6	94	3	11	89

In case determined O ₂Relation between dividing potential drop and the metabolism may importantly repeat this experiment in cold environment.These tests will be implemented as the test of front fully, and just the temperature of calorstat will be reduced to 10 ℃.

Rat: expectation adopts rat to implement the identical experiment of before having implemented with mice.If observe the extremely convulsions behavior of hypoxia of expression, with O ₂Concentration returns to 21%.

Canis familiaris L.:, then expect to implement identical a series of tests with Canis familiaris L. if in mice and/or rat, observe positive correlation between oxygen tension minimizing and the metabolic rate minimizing.

Program 5: hydrogen sulfide, carbon dioxide, helium and oxygen (H ₂S+CO ₂+ He+O ₂)

These tests are targets of this gas matrix; With determine to provide to the strongest (robust) of metabolism and reversible control, with the O of the ambient temperature combination of optimizing ₂, CO ₂, H ₂The mixture of S and He.Therefore, as the basis, the mixture of measuring these four kinds of gases provides optimally-controlled mixture to find to the metabolism flexibility with single concentration of planting gas of determining in the program in front.O ₂, CO ₂And H ₂S will relative to each other change, and helium will be used for this mixture of balance simultaneously.Below in the mouse test of Xian Shiing, at O ₂And H ₂S keeps the constant CO that changes simultaneously ₂To change helium in order to keep constant flow.We will adopt the identical metabolic determination method that comprises oxygen consumption and core temperature.An animal can be used for a plurality of experiments.Mice will expose 6 hours.The lower ambient temperature of employing repeats these tests then, how much can influence metabolism in order to understand the core temperature that adopts this admixture of gas to reduce.

Mice:

	O ₂	?H ₂S	CO ₂	He
	O ₂	?H ₂S	CO ₂	He	Test	Concentration %	Concentration ppm	Concentration %	Balance
1	21	?20	5-10-15		Test	Concentration %	Concentration ppm	Concentration %	Balance
1	21	?20	5-10-15		2	21	?40	5-10-15
3	21	?80	5-10-15		2	21	?40	5-10-15
3	21	?80	5-10-15		4	16	?20	5-10-15
5	16	?40	5-10-15		4	16	?20	5-10-15
5	16	?40	5-10-15		6	16	?80	5-10-15
7	11	?20	5-10-15		6	16	?80	5-10-15
7	11	?20	5-10-15		8	11	?40	5-10-15
9	11	?80	5-10-15		8	11	?40	5-10-15
9	11	?80	5-10-15		10	9	?20	5-10-15
11	9	?40	5-10-15		10	9	?20	5-10-15
11	9	?40	5-10-15		12	9	?80	5-10-15
13	6	?20	5-10-15		12	9	?80	5-10-15
13	6	?20	5-10-15		14	6	?40	5-10-15
15	6	?80	5-10-15		14	6	?40	5-10-15

Rat: after having understood the optimal gas mixture that is used for mice what is, those the same experiments that will carry out and implement with mice with rat are except H ₂S concentration be 100,200 and 300ppm beyond.To handle rat 6 hours.

Canis familiaris L.: the test of use Canis familiaris L. will be consistent with those tests of using mice and rat.Concentration will start from 300ppm and forward the concentration that will measure to.Animal can be used for a plurality of experiments but is once no more than weekly.Mice and rat will be handled 6 hours, and Canis familiaris L. will be handled 2 hours.

Predictive embodiment 10:

The selection of philtrum hydrogen sulfide dosage

Can be by any one of multiple dosage form and route of administration, include but not limited to suck gas form or intravenous is used hydrogen sulfide solution, use hydrogen sulfide and induce stagnation to the animal or human.Described and be used for determining being enough to inducing the dosage form of hydrogen sulfide of stagnation and the method for route of administration at the whole biology that needs are stagnated.Test organism (for example rat, Canis familiaris L., pig, monkey) is exposed to the hydrogen sulfide of using as disposable dosage intermittence or seriality that increases concentration, and the monitoring physiological status includes but not limited to core temperature, oxygen consumption, carbon dioxide generating, heart rate, blood pressure, breathing rate, blood pH, motion and wakefulness when a plurality of time points shift out blood sample (0.5mL).The concentration of hydrogen sulfide that will be present in the blood plasma that is derived from experimental animal blood is measured with methods known in the art, includes, but are not limited to that X derives, the Y extraction, and use gas chromatography and mass spectroscopy quantitatively.

At the dependency of realizing stagnation in varying degrees, determined to be enough in experimental animal, to induce the effective dose of the hydrogen sulfide of stagnation in the steady state blood plasma level of the hydrogen sulfide that in experimental animal, produces and the experimental animal by special dosage regimen.Be used for inducing the effective dose of stagnation to induce therein under the condition of stagnation by definite the people that needs are stagnated, acquisition is measured with dosage, route of administration and the dosage regimen of the hydrogen sulfide of the stable plasma concentration of the identical hydrogen sulfide that obtains in experimental animal in the people.Hydrogen sulfide realizes that in human body the valid density of stagnating depends on dosage form and route of administration.For inhalation, in some embodiments, valid density is in the scope of sending 50ppm to 500ppm continuously.Use for intravenous, in some embodiments, valid density is in the scope of sending 0.5 to 50 milligram of every kg body weight continuously.

Scope in every kind of situation is characterized by the stagnation degree with the increase of the dosage acquisition of the hydrogen sulfide that increases.Be enough to cause outside hospital, to suffer asystole and heartbeat recovery and unconscious people's core temperature continues when recovering, 12-24 hour reduces the dosage of 3-5 degree centigrade to 32-34 degree centigrade hydrogen sulfide, expectation has significant survival advantage with respect to the people of the kind who is not exposed to hydrogen sulfide, as describing among the Bernard etc. 2002.

Embodiment 11:

The Study on pretreatment of animal

What show among the embodiment 7 studies have shown that, uses H ₂S in advance and the male C57Bl/6 mice of processing that continues can strengthen the ability that they are survived under the hypoxia condition of the oxygen of 5% oxygen or 4%.

In order to be determined at the hypoxia condition private H that places an order ₂The S pretreatment (does not continue to be exposed to H to the influence of viability in the hypoxia process ₂S), mice is being exposed to 5%O ₂(5%), 4%O ₂(4%), 1 hour 5% O ₂After be 4% O ₂The O of (4%+1 hour 5%) or 1 hour 5% ₂After be 3% O ₂(3%+1 hour 5%) is exposed under 30 minutes the room air (without pretreatment) before, and the room air that perhaps is exposed to 10 minutes is exposed to the 20 minutes 150ppm hydrogen sulfide (pretreatment) in the room air after down, and measures their time-to-live.Test stopped in the time of 60 minutes, and the animal that will still live is put back in their cage.As showing among Figure 23, at the 150ppm H that is exposed in advance in the room air ₂All mices in 20 minutes the treated animal of S all survive in 5%O subsequently ₂Exposure, and all control animals that only are exposed to room air all are being exposed to 5%O ₂Dead in 15 minutes.Therefore, mice is exposed to H in advance ₂S has set up feasible can the survival for a long time and be the physiological status of lethal hypoxia under other situation in this mice.At H ₂Observed protective effect is considerably beyond the known protective effect of whole body hypoxic preconditioning of report in the literature in the pretreated mice of S, in described document at 5%O ₂In viability only prolonged 2 times (Zhang etc., 2004).Though in Figure 23, do not show some H ₂The pretreated mice of S can be at 5%O ₂Middle survival was longer than 4 hours, and can recover and not significant motion or behavioral deficiency.

In order to determine H ₂The S pretreatment whether strengthen in addition the viability of lower oxygen tension, mice is exposed to lower O ₂Concentration.As showing H among Figure 24 ₂The S pretreatment has strengthened widely at the O that has 5% ₂Situation under survival.Comparatively speaking, there is 4%O ₂The time, H ₂The S pretreatment has increased survival only lessly.Yet, if with H ₂The pretreated mice of S is exposed to the O that gradually reduces ₂Level, they at first are exposed to 5% O then through pretreatment like this ₂1 hour, be exposed to 4% O subsequently ₂Or 3% O ₂, their time-to-live be enhanced to they at H ₂Be exposed to 5% O after the S pretreatment ₂The time observed identical level (Figure 28).Therefore, be exposed to H in advance ₂S has set up a kind of wherein mice can survive that (21% normal oxygen is reduced to 3% O in the minimizing step by step that surpasses 80% oxygen tension ₂) physiological status.And, in some tests, at H ₂Reduce oxygen tension after the S pretreatment step by step and show, mice can be in being low to moderate 2.5% oxygen tension survival 1 hour.

These data and those proofs of describing in embodiment 7 are exposed to H ₂S has pharmacological effect, wherein is greatly improved for the survival rate in the lethal hypoxia in other cases.About this point, H ₂The pharmacological effect of S depends on and is exposed to H ₂The dosage level of S and persistent period, those skilled in the art can change to obtain the parameter of best viability to the lethal hypoxia.It will be apparent to one skilled in the art that and also can change route of administration (for example, sucking parenteral administration) comes to obtain the lethal hypoxia tolerance of expectation in mammal effect.In addition, described pharmacological effect can be at H ₂Observe in the time of during S exposes and is confined to pretreatment or expands to hypoxia.Equally, can change and be exposed to H ₂The selection of time that S begins with respect to the lethal hypoxia is used so that the viability maximization that improves.The hypothesis that these data fit are such: make that by the oxygen demand minimizing that causes with reactive compound (as the oxygen antagonist) pretreatment can survive in other cases is the oxygen supply minimizing of lethal to animal.

Pass through H in order to be characterized in ₂The metabolization that exists in the environment of enhanced viability to the lethal hypoxia that S handle to produce is being exposed to H ₂H in the S process and then ₂S handles the O that stops and be exposed to 5% subsequently ₂Process in, measure the CO of mice ₂Produce.CO ₂The variation that produces shows in Figure 25.Measurement is exposed to room air 30 minutes (without pretreatment) or room air is exposed to 150ppm H after 10 minutes ₂Be converted to 5%O in the mice of S 20 minutes (pretreatment) ₂Or 4%O ₂The time CO ₂The variation that produces.In addition, measured at the O that progressively transits to 5% ₂1 hour, be converted to 4% O afterwards ₂The time CO ₂The variation that produces.The result of these tests provides in Figure 29.

CO ₂Be created in H ₂Reduce about 2 to 3 times in the pretreated initial 5-10 of S minute, shown the 150ppm H in using room air ₂During the S pretreatment 20 minutes, in mice, induced stagnation.Yet, the O of animal ₂Consumption and core temperature are at H ₂Significantly do not change in the S preprocessing process (data not shown), show be exposed to H ₂In the S process, set up feasible physiological status rather than the stagnation that the viability of lethal hypoxia is improved in the mice.The size that this state can be characterized by the metabolism minimizing in the biomaterial is lower than the size that is defined as stagnation.In order to stagnate with reactive compound, biological substance be necessary must transition by oxygen consumption and CO in the biological substance wherein ₂Produce to reduce and be lower than 2 times the state of hypometabolism step by step.This wherein metabolism or Cellular respiration are described to " pre--as to stagnate " state by the continuous state that reactive compound is reduced to the degree that is lower than twice.In Figure 25, show, at H ₂The S pretreatment stops and induces behind the lethal hypoxia CO ₂The continuous monitoring that produces proves CO ₂Produce and reduce about 50 times, illustrate to stagnate in being exposed to lethal hypoxia process, to obtain.In being exposed to lethal hypoxia process, O in the mice that follows ₂The minimizing that consumes and the strong attenuation of activeness are further supported such observed result: obtain subsequently to stagnate in being exposed to lethal hypoxia process.

Measured with at H ₂After the S pretreatment or without being converted to 5%O after the pretreatment ₂Or 4%O ₂The relevant CO of hypoxia condition ₂The variation that produces.As in Figure 28, showing, there be not H ₂Be exposed to 5% O during the S pretreatment ₂Mice or have H ₂Be exposed to 4% O under the pretreated situation of S ₂Mice show CO ₂Produce a large amount of the minimizing.Comparatively speaking, with at H ₂The pretreated new baseline values of S relatively is exposed to 5%O subsequently ₂Perhaps at 5%O ₂Be exposed to 4%O afterwards ₂Through H ₂The pretreated mice of S is at CO ₂The generation aspect does not demonstrate any obvious change.These results proved that metabolic activity reduces and death between dependency, described death with do not having H ₂Be exposed to 5%O during the S pretreatment ₂, H is perhaps arranged ₂Be exposed to 4%O during the S pretreatment ₂Relevant.In addition, these digital proofs are at H ₂Be exposed to 5% O after the S pretreatment ₂, or from 5% O ₂Gradually reducing is 4% O ₂Do not cause the extra minimizing of metabolic activity.In order to sum up these results, at the CO that when normal oxygen changes the lethal hypoxia into, takes place ₂H is being used in the minimizing of emitting ₂In the pretreated mice of S is slow.Change the lethal hypoxia into from normal oxygen and cause CO ₂Emit and reduce 40%, but use H ₂The S pretreatment, itself causes CO ₂Emit minimizing 50-60% and reach new, a lower baseline, prevented CO when being transformed into the lethal hypoxia ₂Any further minimizing of emitting.H is used in these digital proofs separately ₂The S pretreatment has prevented the extra minimizing of the metabolic activity relevant with changing the lethal hypoxia into usually, thereby has improved the survival under hypoxia condition.In addition, these data have been supported such model, and wherein biological substance being exposed in advance reactive compound is enough to strengthen viability and/or reduces injury from damage or disease infringement.

Embodiment 12:

Selenium hydride. is at the core temperature of the low mice of the lowering of concentration that reduces

Reported in the literature in the past, greater than the H of 1ppm ₂Se is fatal to animal.Test is carried out according to material and the method described among the embodiment 4, except using even comparing H ₂The H of S lower concentration ₂Se.The H that uses ₂Se has the initial concentration from 20ppm in the nitrogen that comes carrying shield, then with its with room air dilution into about 10/1000000000ths parts or 100 parts (ppb).Then animal is exposed to this mixture.

Two mices are exposed to 100ppb H ₂Se is less than 10 minutes.Figure 26 has shown by breathing in the mice and has shown that core temperature reduces and metabolic activity reduces 3 times.

H ₂The concentration of Se even further be reduced to 10ppb.Be exposed to 10ppb H ₂The mice of Se has also experienced the minimizing (Figure 27) of core temperature and breathing.

In addition, use H based on being used for assessment ₂Reversible experiment of S (Blackstone etc., 2005, therefore with it by being incorporated herein by reference), H ₂The effect of Se shows as completely reversibility.

Embodiment 13:

Hydrogen sulfide protection antagonism lethal is hemorrhage

What show among the embodiment 7 and 11 studies have shown that, with hydrogen sulfide (H ₂S) handle mice and strengthened the ability that they are survived under the hypoxia condition of the oxygen of 5% oxygen or 4%.In order to determine H ₂Whether the S processing also can be used for reducing mortality rate and/or the tissue injury relevant with relevant more clinically ischemic hypoxia acute injury model, uses H in the hemorrhage process of in check lethal ₂S handles rat, and described lethal is hemorrhage have been reduced the oxygen that offers tissue and caused death (Blackstone etc., 2005).In this research, use H ₂The rat that S handles survives and loses blood and recovery fully in lethal.

Rat is used H in check lethal hemorrhage (losing blood 60%) process ₂S handles.At the operation implantation catheter with after recovering, blood was shifted out in conscious animal body in 40 minutes.Will with blended a small amount of (300ppm) H of room air ₂S after the beginning blood-letting 20 minutes (promptly in 30% back of losing blood) is administered to the animal that is subject to processing.When blood-letting finishes, animal is put back into and do not have H ₂In the room air of S.Finish back 3 hours in blood-letting, the animal intravenous of survival is used the Ru Suanlingeshi solution of effusive blood volume.

Great majority (6/7) H ₂The rat that S handles survives in shock stage and recoveries (table 7) fully hemorrhage and 3 hours.The rat of these survivals does not have one to show behavior or functional defect after recovery.A H ₂The rat that S handles is in hemorrhage end death in back 174 minutes.All undressed animals are dead in back 82 minutes of hemorrhage end; The mean survival time of undressed animal is 35+/-26 minute.Adopt two tail Fischer (Fishers) accurate T-check, the p value is 0.0047.

In initial 20 minutes hemorrhage (losing blood before 30%), rat has increased the oxygen carrying capacity that breathing rate and tidal volume reduce owing to losing blood in order to compensation.The increase of this ventilation has caused the carbon dioxide generating (V that breathes _CO2) minimizing (table 7).Losing blood after 60% H ₂What S handled all shows V with undressed animal _CO2Reduce.Arterial blood lactic acid increases and pCO ₂, bicarbonate ([HCO ₃ ^-]), pH and base excess reduce (table 7).Therefore hemorrhagely cause having the compensatory metabolic acidosis of respiratory.Yet, at H ₂In the rat that S handles, these variations are less in size, the reduction of expression metabolic acidosis.In addition, at H ₂In the animal that S handles, V _CO2After hemorrhage, do not continue to reduce.In undressed animal, V _CO2Stably reduce and cease breathing up to animal.H ₂S uses demonstration and has prevented that shock reaction is developed to death.

Table 7. usesH ₂S The survival and the physiology of the hemorrhage model of rat

	H ₂S handles	Undressed
	H ₂S handles	Undressed	Survival
Return to the time of non-survivor death fully	85.7％(6/7) 174(1/7)	0％(0/7) 35+/-26	Survival
Return to the time of non-survivor death fully	85.7％(6/7) 174(1/7)	0％(0/7) 35+/-26	Ml/kg/ minute CO ₂Produce (VCO ₂)：
The hemorrhage end in hemorrhage centre before hemorrhage	25+/-4 20+/-2 16+/-2	26+/-6 21+/-3 11+/-3	Ml/kg/ minute CO ₂Produce (VCO ₂)：

Hemorrhage back 15 minutes	17+/-3	7+/-5
Hemorrhage back 15 minutes	17+/-3	7+/-5	The blood CO2 content (pCO2) of mmHg
Hemorrhage end before hemorrhage	45+/-6 35+/-6	44+/-3 21+/-3	The blood CO2 content (pCO2) of mmHg
Hemorrhage end before hemorrhage	45+/-6 35+/-6	44+/-3 21+/-3	The blood bicarbonate radical content ([HCO3-]) of mmol/L
Hemorrhage end before hemorrhage	32+/-3 21+/-3	30+/-1 12+/-3	The blood bicarbonate radical content ([HCO3-]) of mmol/L
Hemorrhage end before hemorrhage	32+/-3 21+/-3	30+/-1 12+/-3	Blood pH
Hemorrhage end before hemorrhage	7.46+/-0.03 7.41+/-0.02	7.45+/-0.02 7.35+/-0.06	Blood pH
Hemorrhage end before hemorrhage	7.46+/-0.03 7.41+/-0.02	7.45+/-0.02 7.35+/-0.06	The BEb of mmol/L
Hemorrhage end before hemorrhage	8+/-2 -5+/-4	6+/-1 -14+/-3	The BEb of mmol/L
Hemorrhage end before hemorrhage	8+/-2 -5+/-4	6+/-1 -14+/-3	The blood lactic acid of mmol/L
Hemorrhage end before hemorrhage	1.4+/-0.5 6.6+/-1	1.2+/-0.2 11+/-3	The blood lactic acid of mmol/L

Embodiment 14:

Be exposed to the benefit of hydrogen sulfide a middle or short term in hemorrhage process

Male this pula-Dao Lai Shi rat of heavy 275-350 gram each test the last week available from Charles River Laboratories and allow its adaptation.Testing the same day, operative catheter is implanted in right femoral artery and the vein.Conduit is drawn at facies posterior scapulae.Give rat administration of buprenorphine and allow its recovery after operation.

Anticoagulation medicine heparin (80-100 unit) is used with disposable dosage intravenous, in order to the coagulation ability that reduces blood and strengthen hemorrhage.After using heparin, the unrestricted rat of consciousness is placed 2.75 liters of crystallizing dish that have glass cover individually.Conduit, temperature probe and gas sampler are passed through in the central boring of lid.Temperature keeps roughly constant temperature (27+/-2 ℃).

Hemorrhage model definition was to shift out total body inner blood of 60% in the process of bleeding at 40 minutes.Blood shifts out with peristaltic pump.In order determine to constitute the amount of total body inner blood of 60%, rat is weighed and the following Equation for Calculating of blood volume: (0.06X body weight)+0.77 (Lee etc., (1985)).

Allow processed group accept to be exposed to the room air (experimental animal) that contains hydrogen sulfide, or contain the room air (control animal) of nitrogen, described air is used with the speed of 3 liters of per minutes by caloic stream controller (SierraInstruments).

With hydrogen sulfide (H ₂S) (20,000ppm uses nitrogen balance) (Byrne SpecialtyGas) dilution advances in the room air to reach the 2000ppm concentration that is used to handle.Blood is shifted out via femoral catheter with the speed that calculates.Blood when shifting out, it is weighed.After 20 minutes (or blood-letting in 40 minutes 50% o'clock), experimental animal is exposed in the room air that contains 2000ppm hydrogen sulfide.This is exposed to when animal shows asphyxia and dystonia and stops.Be exposed to hydrogen sulfide (H ₂S) length average time was generally between 1 and 2 minute.Indoor maximum H ₂S concentration be estimated as 1000 and 1500ppm between.When observing asphyxia and dystonia, allow animals received be exposed in the room air.Experimental animal recovers breathing pattern clocklike in 20 to 30 seconds after exposure.Control animal with the speed blood-letting the same with experimental animal, but is not accepted to use hydrogen sulfide treatment.Control animal does not show asphyxia or dystonia in process of the test.

Metabolic rate is passed through measure CO ₂Produce and measure (Licor Li7000).Collect temperature and CO ₂Data (ADI PowerLab).Measure arterial blood values (I-Stat bichromatic analyser).After blood-letting, animal was placed cage 3 hours and observation.When finishing in 3 hours, arbitrarily provide lactated Ringer solution to the rat that survives.Animal for not surviving ceases breathing and CO animal ₂When stopping, generation announces the death time.After recovery, rat is transferred in the clean cage with food and water, and about 16 hours of 30 ℃ of following stable breedings.Conduit is shifted out in operation, and allows animal to recover a few hours down in 30 ℃ before transfer is got back in the colony.Behavior and function test are selected from the battery of tests of describing in the SHIRPA scheme (Rogers etc., 1997).

In these trials, in hemorrhage process through hydrogen sulfide (H ₂S) animal 8 of Chu Liing merely hit 7 (88%) survive in hemorrhage.Two control animals of accept handling were 3 hours viewing duration death.

Embodiment 15:

Additional result from embodiment 2

As discussing the preservation that people's foreskin is used for assessing cell and is organized in carbon monoxide among the top embodiment 2.8 blocks of people's foreskins (embodiment 2 has reported 3) have altogether finally been assessed.Great-hearted keratinocyte number is assessed (table 8 and Figure 30) with trypan blue.This shows the number that carbon monoxide exposes has increased living cells.

Table 8. adopts trypan blue (tb) staining to detect from being exposed to room air (RA)

Or the viability of isolating keratinocyte in 24 hours the foreskin of CO.

RA		CO
RA		CO				(tb-) that lives	Dead (tb+)	Live Mark	Live (tb-)	Dead (tb+)	Live Mark
SUM						(tb-) that lives	Dead (tb+)	Live Mark	Live (tb-)	Dead (tb+)	Live Mark
SUM	0 1 0 0 7 0 1 0 9	3 1 0 0 34 1 2 1 42	0.00 0.50 0.17 0.00 0.33 0.00 0.18	10 4 10 5 49 24 3 1 106	1 4 3 1 42 6 3 1 61	0.91 0.50 0.77 0.83 0.54 0.80 0.50 0.50 0.63

Untreated CO

The cell 51 167 that # recovers

The ratio 0.18 0.63 that lives

T check 0.000883884

Embodiment 16:

Low-level long-termH ₂S Exposing increases viability

Use method and the instrument described among the embodiment 1, the Caenorhabditis elegans nematicide is exposed to low-level H ₂S (＜100ppm).The nematicide that is adapted to this processing demonstrates the life-span of increase and to the resistance of heat stress, yet, do not exist as inducing and stagnate the recognizable minimizing of metabolic activity that has.

In nematicide, can not carry out aerobic metabolism (by oxygen concentration around reducing or adding CO) and cause inducing stagnant give birth to or stagnate (referring to embodiment 1).Yet stagnant life can not be by being exposed to them in the room air＜H of 100ppm ₂S induces.Greater than 100ppm dosage the time, H ₂S can cause the sizable fatality rate of nematode population that exposes.Be enjoyably, even most of therein anthelmintic is by H ₂In the situation that S kills, those of survival are acted normally and not injured by this material.At about 50ppm H ₂The anthelmintic that grows among the S is finished embryogenesis, be developed to sexual maturity and with do not having H ₂The born of the same parents identical speed of raising in the environment of S is produced the offspring.Comparatively speaking, the oxygen concentration of metabolic rate reduction therein (is lower than 3.5%O ₂) in, all these processes have all slowed down.In addition, at H ₂The anthelmintic of raising among the S has produced the offspring of the quantity identical with the contrast in the room air, shows the illeffects that does not exist from these conditions.These data show, H ₂S does not reduce Caenorhabditis elegans metabolic activity under these conditions.

At H ₂The contrast of the age-matched of nematicide ratio in independent room air of growing among the S is anti-heat stress more.In this algoscopy, will be at 50ppm H ₂The anthelmintic of raising among the S is at H ₂Be exposed to high temperature among the S, and the anthelmintic that will raise is exposed to room air in room air.Therefore, H ₂S-inductive to stress resistance and metabolic activity reduce uncorrelated.Yet, this to heat-stress resistance need nematicide to adapt to H ₂The S environment.In room air, raise and at H ₂Be exposed among the S heat-stress anthelmintic they expose more quickly death in room air such as fruit.At H ₂Anthelmintic of raising among the S and the anthelmintic that in room air, is exposed to heat stress than the contrast of in room air, raising survive better in the scope, to H ₂The adaptation of S continues.In addition, adapt to nontoxic low concentration H ₂The anthelmintic that the anthelmintic of S (for example 50ppm) can be resisted not adapting to is the H of the higher concentration of lethal ₂S.

These data and following data consistent: the of short duration H that is exposed to ₂The fruit bat of S subsequently can than undressed contrast survive better in anoxia (referring to, embodiment 7 and embodiment 11).Be protected from heat stress (in anthelmintic) and oxygen deprivation stress (in fruit bat) shows H ₂S can strengthen the viability under the multiple disadvantageous or stress state that may run into clinically.

Adapt to 50ppm H ₂The anthelmintic of S and isogenic undressed comparing life-span (Figure 32) with increase.This is consistent with following idea: they be in usually more tolerance with old and feeble relevant multiple stress state under.In fact, at H ₂The old anthelmintic ratio of growing among the S is without H ₂Those anthelmintics at the similar age that S handles seem more strong and healthy.In addition, relatively from the anthelmintic of the every kind of groups that is in middle of life the time, this also be correct (that is, room air contrast and H ₂The anthelmintic that S-handles is not to be complementary at the age in time, but wherein 50% of each colony this point of dying in heaven is complementary).Therefore, be adapted to H ₂The S chronic primary cellular defect relevant in the Caenorhabditis elegans that can slow down with aging.

Embodiment 17:

The enforcement of gas matrix test

Adopt the gaseous environment that changes in mice, rat and Canis familiaris L., to assess the metabolism flexibility.Three parameters are used to define metabolic this minimizing, comprise the carbon dioxide generating of measuring by respirometry, the change of oxygen consumption, and as the change of the core temperature measured with telemetry.In test, animal is placed closed chamber with a gas input port and a gas outlet with mice and rat.For Canis familiaris L., face shield is placed the snout of animal, this face shield is connected with two flexible pipes (input port and delivery outlet).The gas flow rate that is used for every kind of animal: mice-per minute 500cc, 2 liters of rat-per minutes, and Canis familiaris L. is 40 liters of per minutes.Every kind of atmosphere is by diluting indoor air and making up from Compressed Gas, unless indicate in addition.For rat and mouse test, ambient temperature is 7-10 ℃ in being exposed to the test gas process.For Canis familiaris L., ambient temperature is room temperature (22 ℃).

Table 9: make up the metabolism flexibility that is used for detecting mice, rat and Canis familiaris L.

The description of gaseous environment.

	Mice	Rat	Canis familiaris L.
	Mice	Rat	Canis familiaris L.	Hydrogen sulfide Selenium hydride. hydrogen phosphide carbon dioxide H ₂S+CO ₂?CO ₂+ low O ₂?CO ₂+ low O ₂+He?+77％	Have 0.01% to have 0.0001% nothing 0.016% to have 15% to have 0.01%+15% to have 15%+8% that 15%+8%+77% is arranged	Have 0.03% to have 0.003% N/D to have 15% N/D to have 15%+8% that 15%+8%+77% is arranged	Not having 0.85% N/D N/D does not have 9% N/D N/D 9%+15% is arranged

Table 9 has shown the amount of every kind of gas, and its percentage ratio as room air atmosphere provides, unless otherwise indicated.In 6 hours processing procedures, as judging that by carbon dioxide generating or oxygen consumption metabolic rate is described by " having " greater than 5 times inhibition sign; " nothing " (these values reduce or be lower than 5 times minimizing describe like this); " N/D " expression test is not carried out.Temperature descends in 30 minutes process-exposed about 1.5 ℃ in Canis familiaris L. test (carbon dioxide+low oxygen+helium).Such temperature reduces to be thought significantly, because Canis familiaris L. is not observe such temperature in the recording process of baseline widely of 12kg and the animal in room air to descend.

The animal that is exposed to the atmosphere of multiple structure demonstrates the metabolism flexibility, and described metabolism flexibility proves (Figure 33-40) by the change near the core temperature (CBT) of ambient temperature.Figure 33 has proved to be exposed to and has contained 15%CO ₂, 8%O ₂With the rat of the atmosphere of 77%He with under conditions of similarity, be exposed to 300ppm H ₂The metabolism that the rat of S relatively has acceleration suppresses.Figure 34 proof is being exposed to 1.2ppm H ₂CBT significantly reduces in the mice of Se.The rat that is exposed to room air under 10 ℃ ambient temperature does not show the remarkable reduction (Figure 35) of CBT.Under 7 ℃, be exposed to 80%He, 20%O ₂Rat do not show that CBT significantly reduces (Figure 36) yet.Under 7 ℃ ambient temperature, be exposed to 15%CO ₂, 20%O ₂, 65%He the rat of atmosphere show that CBT significantly reduces (Figure 37).Equally, Figure 38 is presented under 7 ℃ and is exposed to 15%CO ₂, 8%O ₂, 77%He the rat of atmosphere in CBT significantly reduce.The remarkable reduction of CBT also is being exposed to 9%CO ₂, 20%O ₂, 71%He the Canis familiaris L. of atmosphere in obtain proof (Figure 39).The amplitude of this reduction is lower, may be because this animal large-size and to the restriction of thermal diffusion.Be exposed to the CO of variable concentrations ₂Canis familiaris L. in observe similar reduction.

Embodiment 18:

The screening of chemical compound

Implement screening compound and identified the test compound that can cause the reversible reduction of subcutaneous temperature in the mice.They provide and are protected from the lethal hypoxia (with 21% O to have detected the test compound identified subsequently ₂The typical environment of equilibrated nitrogen environment compares, the O 4% ₂The time measure) ability.Whole screening sequence comprises 3 steps:

1) first step (1 °) screening will cause that to measure the subcutaneous temperature of test mice can survey the minimum effective dose of the test compound of reduction;

2) screening of second step (2 °) to be measuring the reversibility that temperature reduces, as by having normal behaviour in back 24 hours in processing and at 24 hours or still less return to the test mice definition of normal subcutaneous temperature in the time; And

3) screening of the 3rd step (3 °) is to compare with undressed contrast experimenter under identical hypoxia condition, and the evaluation test mice survives in lethal hypoxia (4%O ₂) ability.

The mice of using in these researchs is the male C57BL/6 mice (Taconic) of big, jugular vein intubate (JVC) of 5-6 week, its back is implanted with subcutaneous RFID temperature sensor (IPTT-300, Bio Medic Data Systems, Inc. (BMDS)) and allow it to recover at least 24 hours.With 1 or Luer-Lok syringe (Becton Dickinson) and the infusion pump (Harvard Apparatus) of 5ml, come to the mice administration by inlying catheter infusion test compound.From the DAS-6008 data acquisition module of BMDS subcutaneous temperature by transponder (transponder) record mice, and with the electrical form of this data input computer and draw figure with respect to the time.

The first step (1 °) screening:

For first step screening, the transfusion of test compound is with the prepared at concentrations of the concentration that is considered to largest optimization.With NaOH or HCl with pH regulator to 6-8, with sodium chloride permeability is adjusted to 250-350mOsm, and the accumulated dose of the test compound of using (mg) can not surpass 400% of its disclosed mg/kg LD50 in mice divided by test experimenter's body weight (kg).

Mice is placed container at the bottom of the high glass with opaque wall, and by the jugular vein infusion.Test compound step-by-step program infusion, infusion rates increased (table 10) at 2 hours in the process.

The infusion step-by-step program of table 10. test compound

Time (minute)	Infusion rates (mul/min)	The microlitre number of infusion	The microlitre number of total infusion
Time (minute)	Infusion rates (mul/min)	The microlitre number of infusion	The microlitre number of total infusion	0-20 20-40 40-60 60-80 80-100 100-120	0.8 1.6 3.2 6.3 12.7 25.4	15.875 31.75 63.5 127 254 508	15.875 47.625 111.125 238.125 492.125 1000.125

In infusion process, read the subcutaneous temperature of mice in every 3-5 minute, and any change of record mice behavior.First step results of screening has shown whether test compound has subcutaneous temperature to 33 ℃ or the lower ability of reducing, and shown and reduced the required effective dose of subcutaneous temperature that described required effective dose is that the infusion rates of test compound is measured when observing for the first time stable temperature and reduce.

The screening of second step (2 °):

The subcutaneous temperature that causes mice is reduced to 33 ℃ or lower test compound and detects in the screening of second step.Go on foot in the screening 50% of effective infusion rates of mensuration speed during mice is screened with the first step, infusion test compound 60 minutes second.In infusion process, by measuring the subcutaneous temperature of monitoring mice in every 3-5 minute.If subcutaneous temperature does not reduce in initial 60 minutes, then infusion rates is doubled and continued infusion other 60 minutes.When the subcutaneous temperature of mice is reduced to 33 ℃ or when lower, stop infusion at once, and assess the recovery of mice by the behavior of measuring subcutaneous temperature and observing mice.Observed and write down temperature and the behavior of mice in back 24 hours in processing.Whether second step results of screening confirmed test chemical compound has caused the reversible reduction of subcutaneous temperature and has not had lethality.

The 3rd step/lethal hypoxia (3 °) screening: in the screening of the 3rd step, with the speed infusion test compound of mice to determine in the screening of second step.The subcutaneous temperature of measuring mice in every 3-5 minute is reduced to 33 ℃ up to it, as in the screening of second step.Stop infusion and mice is transferred to the low (4%O of oxygen content immediately with control mice ₂) indoor, described control mice infusion carrier (saline solution, 148mM, permeability=300) or unprocessed.The glass chamber that this is airtight is with the air and the nitrogen perfusion of continuous-flow, to reach the 4%O of expectation ₂Hypoxic atmosphere.If mice has survived in hypoxic atmosphere 60 minutes, it is shifted back under the room air, and by writing down subcutaneous temperature and monitoring its recovery 24 hours by behavior observation.

Control mice is dead in 6-15 minute usually.

Mice with sodium sulfide (effective dose is 0.79mmol/kg), sulfo-Feldalat NM (effective dose is 4.61mmol/kg) or sodium rhodanate (effective dose is 4.67mmol/kg) infusion survives in being exposed to the lethal hypoxia 60 minutes.Mice with mercaptamine (effective dose 7.58mmol/kg) infusion has survived in the lethal hypoxia 45 minutes; Mice with mercaptamine-S-sodium ascorbyl phosphate infusion has survived in the lethal hypoxia 31 minutes; And the mice with tetrahydric thiapyran-4-alcohol infusion has survived in the lethal hypoxia 15 minutes.The survival rate of these survival rates and control mice is compared, and described control mice is dead in 6-15 minute under low-oxygen environment usually.

Comparatively speaking, in first step screening, be defined as having some other test compounds that reduce body temperature ability antagonism lethal hypoxia that do not watch for animals.Thiacetic acid., selenourea and D2EHDTPA S-(2-((3-aminopropyl) amino) ethyl) ester has all reduced body temperature, but does not have to improve the survival under hypoxia.2-sulfydryl-ethanol, sulfydryl second and 2-sulfydryl ether have all reduced body temperature, but are virose under the dosage that effectively reduces temperature.Do not reduce subcutaneous temperature under thiourea, dimethyl sulphide, sodium selenide, methyl-sulfinic acid sodium, the maximum dose level that the N-acetyl group-the L-cysteine provides in this research.Got rid of dimethyl sulfoxide, be considered to be used for medicinal purpose because of effective dose (10%DMSO) Tai Gao.

These are determined, and the screening sequence of development can be successfully used to identify the chemical compound that can protect the animal that suffers the lethal hypoxia.In addition, the result of this research shows, compounds identified, and will can be used for protecting the patient to avoid damage and ischemia injury that hypoxia causes with other chemical compound that this program is identified.

Embodiment 19:

Be exposed to reactive compound every day

The last adaptation of physiology hydrogen sulfide (H ₂S) ability of long-term disposal and time of meeting the needs of are detected in mice.Adaptation is defined as, and when animal is exposed to 80ppm hydrogen sulfide in the room air and carries out long-term disposal, does not have the reduction (greater than 4 ℃) of displaing core body temperature.Long-term disposal is defined as in six weeks 4 days weekly, is exposed to the 80ppm hydrogen sulfide in the room air 4 hours every days.

The mice of using in these researchs is big male C57BL/6 mice or a male C129 mice of 5-6 week.The body cavity of telemetering equipment being implanted mice before test is in order to the record core temperature.

At the plexiglas box with chamber that is used for every mice separately, being exposed to flow velocity is the hydrogen sulfide of 10 liters of per minutes with mice (handling 8) at every turn.The carbon dioxide generating and the oxygen consumption that detect the animal in the 500cc glass bowl have the flow velocity that per minute 0-5 rises.

Between one-period, mice has adapted to the hydrogen sulfide exposure on an average.Adapt to be defined as when animal and be exposed to 80ppm hydrogen sulfide in the room air in the time of 4 hours, do not have the reduction (greater than 4 ℃) of displaing core temperature.The mice that does not have the displaing core temperature to reduce is considered to have the physiological adaptability to hydrogen sulfide.The mice that has produced the usefulness hydrogen sulfide treatment that adapts to has shown that than carbon dioxide generating (vCO2) oxygen consumption (vO2) increases (Figure 42) when comparing with undressed control mice.Adapt to H ₂The mice of S shows lower respiratory quotient (RQ ratio), and respiratory quotient is defined as the carbon dioxide of the ratio of vCO2/vO2 or generation and the ratio (Figure 43) of the oxygen of consumption.

Embodiment 20:

To thalassemic application

Based on the current results in other model system of here introducing, expection has the erythrocyte of the animal of hematology's obstacle (thalassemia), when handling them with sulfide, will have the ability of the opposing oxidative damage of increase, cause the erythrocyte survival that prolongs.To carry out following test confirms can protect with the processing of reactive compound and suffers from thalassemic animal and avoid oxidative damage.

In first campaign, suffer from thalassemic animal and will treat in the activation thing by long term exposure.After the initial trial of determining baseline, will be according to the scheme begin treatment of summing up below.If erythropoiesis or erythrocyte survival are improved, may observe effect in week as far back as 1-2, because the erythrocytic half-life of thalassemia is estimated as 4-7 days.We will observe smear and obtain reticulocyte count when two weeks, and begin to study widely after other two weeks.If detect erythrocytic improvement the (identifying by the reticulocyte count and the blood smear that improve) in mice, this research will continue the stable state in observing the metrics that is used for mensal research.This research has the final terminal point in 1 year of expectation.In the time of 1 year, will finish red cell survival study, and will put to death animal.If do not observe improvement, exposure will continue up to other 1 year, will finish survival research at that time and put to death animal.

Scheme 1:

1) animal will carry out initial trial.

2) after preliminary research in 1 week, captive animal similarly, and:

A) be exposed to 80ppm H ₂S 8 hours/day;

B) do not expose; Perhaps

C) has the water of 0.25% dimethyl sulphide (DMSO), and allow arbitrarily to drink that (the estimated value hint mice from previous research will consume 5-10cc/ days/mice, has 2.5-25 microgram/sky DMSO content, use the average weight of every mice 18g, consumption is estimated as 700-1,400ug/kg/ days).

In the second series test, will measure intrauterine (in utero) effect of handling with reactive compound according to the scheme of summing up below.

Scheme 2:

1) became pregnant back 3 days, accept processing during conceived female Mus (Plugged dam) one of will be organized below:

A) be exposed to 80ppm H ₂S 8 hours/day;

B) do not expose; Perhaps

C) have the water of 0.25% dimethyl sulphide (DMSO), and allow arbitrarily to drink and (will consume 5-10cc/ days/mice from previous research estimated value hint mice, and have 2.5-25 microgram/sky DMSO content.Use the average weight of every mice 18g, consume and be estimated as 700-1,400ug/kg/ days).

2) will allow conceived female Mus natural production, and after production, identify the genotype of cub soon and its execution is used for detail analysis.

Will be in the whole process of these tests the monitoring test animal, point out as following.

Monitoring:

1) preliminary research:

1. reticulocyte count;

2. blood smear;

3. the computer tomography of spleen and skeleton (CT);

4.O ₂Consume and CO ₂Produce;

5. body weight;

The sulfide metabolite and

7. hematocrit (60 μ l total blood volume).

2) when two weeks: reticulocyte count and blood smear (5 μ l blood).

3) mensal research:

1. reticulocyte count;

2. the computer tomography of spleen and skeleton;

3.O ₂Consume and CO ₂Produce;

4. body weight; With

5. sulfide metabolite (blood of extraction is less than 30 μ l).

4) putting to death previous month: red cell survival study.

5) execution and detailed analyzed in vitro.

Embodiment 21:

Application for sicklemia

In order to check this hypothesis, will use the mouse model of sicklemia (SCD), strain makes it no longer express mice Hba and Hbb through transformation in this mouse model, but expressing human HBA and HBB (Patsy, etc., 1997).It simulates gene, hematology and the histopathology feature of finding in suffering from the people of sicklemia, comprise erythrocyte, anemia and many organopathies Neo-Confucianism of irreversible sickling.Significantly the meniscocyte mice of percentage ratio can not survive to the manhood.

Use this mouse model, will detect the effectiveness of plurality of reagents with the chemical compound antagonism SCD that contains sulfide.Exposure will be acute with chronic, and animal will expose at birth or in uterus.Surviving to being born or surviving to the manhood for the cub of intrauterine exposure will be to measure a terminal point of rendeing a service.The phenotype effect will be estimated by reticulocyte count, hematocrit and erythrocyte (RBC) half-life measurement (compare with wild type, it lacks 20 times usually for SCD).

Embodiment 21:

The cyanide exposure test

This embodiment shows, when mice was exposed to 80ppm cyanide in the room air, they reduced their core temperature gradually to about 34 ℃.

A target of the flexible research of these metabolism is to identify can reduce oxygen consumption and watch for animals to avoid the chemical compound of hypoxia injury.Before, proved hydrogen sulfide (H ₂S), a kind of inhibitor of effective oxygen consumption can reduce metabolism and protection mice and rat and avoid hypoxia injury.Blausure (German) (HCN) is similar to H in many aspects ₂S, and we want whether understand it with our algoscopy of metabolism output can be used to regulate metabolism.Picture H ₂It is synthetic and found it in many biosystems (comprising the people) that S, HCN are widely used in industrial chemistry.Do not know whether HCN only is whether the by-product of carbon-nitrogen metabolism or its have special biologic activity.Picture H ₂S, HCN are considered to by for example oxidase and dehydrogenase reaction work with the protein that contains transition metal.HCN not with the composition kickback of hemoglobin.

In the people, NIOSH IDLH (life threatening and healthy) immediately value is 50ppm.OSHA PEL (admissible exposure limit) TWA (8 hours time weighted averages) is 10ppm.LC50 for rat is 143ppm 60 minutes.

In order to measure HCN, mice is exposed to the HCN (beginning to be 1ppm) that increases concentration to metabolic influence.Measure or assessment oxygen (O ₂) consume, carbon dioxide (CO ₂) produce, core temperature (BCT) and behavior.The concentration of HCN seems demonstrate painful sign up to observing to metabolic influence or animal with the increment increase of 10ppm.To be defined as above-described any measured value in the variation that is less than in 10 minutes 10% to metabolic influence.

Find that when mice at room temperature is exposed to the cyanide of the 80ppm in the room air they reduce their core temperature gradually to about 34 ℃.This is different from the being seen reduction of use 80ppm hydrogen sulfide, and core temperature is reduced to about 28 ℃ when using 80ppm hydrogen sulfide.And, and be exposed to hydrogen sulfide (about 2 hours) relatively, the recovery of core temperature very slow (about 14 hours) in being exposed to the mice of cyanide.

Checked such hypothesis: in cyanide, recovering (judging by core temperature) slowly can rescue by the of short duration hydrogen sulfide that is exposed to 80ppm.This is based on such idea: a kind of conservative enzyme is that rhodanese (and other similar enzyme) may use hydrogen sulfide and cyanide to produce relative hypotoxic material Hydrogen thiocyanate.Shown that rhodanese can use cyanide and thiosulfate to produce Hydrogen thiocyanate.(Chen 1933) in addition, it is the nursing standard that the U.S. is used for the treatment of cyanide poisoning that intravenous is used thiosulfate.Discovery recovers the time of core temperature by reducing with the of short duration processing of hydrogen sulfide after being exposed to cyanide.These results suggest, hydrogen sulfide expose and can be used to treat the situation that patient wherein suffers cyanide poisoning.

*************

According to present disclosure, can prepare and carry out all and not need undue experimentation in this open and claimed compositions and method.Though the compositions and methods of the invention are to describe according to embodiment preferred, concerning those technical staff of the present invention, be apparent that, can change described compositions and method, and in the order of the step of method described here or step, change and do not deviate from notion of the present invention, spirit and scope.More particularly, the alternative material described here of all relevant some material on chemistry and the physiology can be used in, identical or similar result will be obtained simultaneously being apparent that.Significantly all should similarly replace and modify and were considered in spirit of the present invention, scope and notion by incidental claim definition to those skilled in the art.

List of references

By reference especially will following list of references (on they provide those the exemplary process of listing at this or the additional degree of other details) be incorporated herein.

United States Patent (USP) 3,777,507

United States Patent (USP) 3,881,990

United States Patent (USP) 3,989,816

United States Patent (USP) 3,995,444

United States Patent (USP) 4,034,753

United States Patent (USP) 4,186,565

United States Patent (USP) 4,266,573

United States Patent (USP) 4,292,817

United States Patent (USP) 4,442,856

United States Patent (USP) 4,444,762

United States Patent (USP) 4,447,415

United States Patent (USP) 4,473,637

United States Patent (USP) 4,502,295

United States Patent (USP) 4,559,258

United States Patent (USP) 4,723,974

United States Patent (USP) 4,745,759

United States Patent (USP) 4,798,824

United States Patent (USP) 4,828,976

United States Patent (USP) 4,938,961

United States Patent (USP) 4,951,482

United States Patent (USP) 5,066,578

United States Patent (USP) 5,157,930

United States Patent (USP) 5,217,860

United States Patent (USP) 5,231,025

United States Patent (USP) 5,285,657

United States Patent (USP) 5,326,706

United States Patent (USP) 5,370,989

United States Patent (USP) 5,395,314

United States Patent (USP) 5,399,363

United States Patent (USP) 5,405,742

United States Patent (USP) 5,434,045

United States Patent (USP) 5,466,468

United States Patent (USP) 5,470,738

United States Patent (USP) 5,476,763

United States Patent (USP) 5,543,158

United States Patent (USP) 5,552,267

United States Patent (USP) 5,568,910

United States Patent (USP) 5,569,579

United States Patent (USP) 5,580,781

United States Patent (USP) 5,599,659

United States Patent (USP) 5,636,643

United States Patent (USP) 5,641,515

United States Patent (USP) 5,645,081

United States Patent (USP) 5,693,462

United States Patent (USP) 5,699,793

United States Patent (USP) 5,719,174

United States Patent (USP) 5,736,397

United States Patent (USP) 5,739,169

United States Patent (USP) 5,752,929

United States Patent (USP) 5,801,005

United States Patent (USP) 5,830,880

United States Patent (USP) 5,846,945

United States Patent (USP) 5,912,019

United States Patent (USP) 5,952,168

United States Patent (USP) 6,013,256

United States Patent (USP) 6,046,046

United States Patent (USP) 6,054,261

United States Patent (USP) 6,057,148

United States Patent (USP) 6,100,082

United States Patent (USP) 6,187,529

United States Patent (USP) 6,365,338

United States Patent (USP) 6,490,880

United States Patent (USP) 6,492,103

United States Patent (USP) 6,524,785

United States Patent (USP) 6,552,083

United States Patent (USP) 6,602,277

United States Patent (USP) 6,790,603

U.S. Patent application 10/971,575,

U.S. Patent application 10/971,576

U.S. Patent application 10/972,063

U.S. Provisional Application 60/513,458

U.S. Provisional Application 60/548,150

U.S. Provisional Application 60/557,942

Alam，Antioxid?Redox?Signal..4(4)：559-62，2002.

Amersi etc., Hepatology, 35 (4): 815-823,2002.

Austin-Ward and Villaseca, Revista Medica de Chile, 126 (7): 838-845,1998.

Barton?&?Ollis，Oxford，UK，Jones(Ed.)，Pergamon?Press，3：373-487，1979.

Baskin and Wang, Tetrahedron Lett., 43:8479-8483,2002.

Baskin etc., Org.Lett., 4:4423,2002.

Beauchamp etc., Crit.Rev.Toxicol.13,25,1984.

Beck etc., Proc.Soc.Exp.Biol.Med.86,823,1954.

Behringer etc., Crit.Care Med., 31 (5): 1523-1531,2003.

Bellamy etc., Crit.Care Med., 24 (2Suppl): S24-47,1996.

Bernard etc., J.Thorac.Cardiovasc.Surg.90:235-242,1985.

Bernard etc., N.Engl.J.Med., 346 (8): 557-563,2002.

Blackstone etc., Science, 308:518,2005.

Boyce and Ham, J.Invest.Dermatol., 81:335-405,1983.

Boyce and Ham, J.Tissue Culture Methods, 9:83-93,1985.

Briese，Neurosci.Biobehav.Rev.，22(3)：427-436，1998.

Brizel，Seminars?Radiation?Oncol.，8(4Supp1)：17-20，1998.

Brouard etc., J.Biol.Chem., 277 (20): 17950-17961,2002.

Bukowski etc., Clinical Cancer Res., 4 (10): 2337-2347,1998.

Burns?&?Murphey，Arch.Biochem.Biophys.，339：33-39，1997.1997

Burns etc., Arch.Biochem.Bipophys, 10:60-68,1995.

Cairns etc., J.Am.Chem.Soc., 74:3982,1952.

Chapter?IV；Chapter?VI；Chapter?VII；Chapter?VIII；ChapterIX?of?Klayman，D.L.；Gunther，W.H.H.Eds，Wiley?Interscience，New?York，1973.

Chasteen and Bentley, Chem.Rev., 103 (1): 1-25,2003.

Christodoulides etc., Microbiology, 144 (Pt 11): 3027-3037,1998.

CIIT(Chemical?Industry?Institute?of?Toxicology)，In：90?dayvapor?inhalation?toxicity?study?of?hydrogen?sulfide，Toxigenics，420-0710，1983.

Clive etc., J.Org.Chem., 47:1641,1982.

Cloarec?&?Charette，Org.Lett.，6：4731，2004.

Cohen etc., Ann.Thorac.Surg., 67 (5): 1489-1491,1999.

Curran，Seminars?Radiation?Oncol.，8(4Supp1)：2-4，1998.

Davidson etc., J.Immunother., 21 (5): 389-398,1998.

Davis(1994)

Demuynck and Vialle, Bulletin de la Siociete Chimique de France, 4:1213-1218,1967

Demuynck etc., Bulletin de la Societe Chimique de France, 3366-3367,1966.

Demuynck etc., Bulletin de la Societe Chimique de France, 8:2748-2754,1967.

Dhanasekaran etc., J.Biol.Chem., 279:37575-37587,2004.

Dillman，Cancer?Biother.Radiopharm.，14(1)：5-10，1999.

Dittmer and Hoey, In:The Chemistry of Sulphinic Acids, Esters, and Their Derivatives, Wiley:Chichester, U.K., 239-273,1990.

Neurotoxicol.Teratol. such as Dorman, 22 (1): 71-84,2000.

Dulak etc., Antioxid.Redox Signal, 4 (2): 229-240,2002.

Duus，.In?Comprehensive?Organic?Chemistry：The?Synthesis?andReactions?of?Organic?Compounds，1 ^st?Ed.，1994.

Eto etc., Biochem.Biphys.Res.Commun., 293:1483-1488,2002.

Ganther，Carcinogenesis?20(9)：1657-66(1999)

Gilbert etc., LANCET, 355:375-376,2000.

Gladysz etc., J.Org.Chem., 43:1204,1987.

Glass，Phosph.Sulfur?Silicon?Rel.Elem.，136，137，138：159-174，1998.

Gorman etc., Toxicology, 187 (1): 25-38,2003.

Guillemin etc., Cell, 89 (1): 9-12,1997.

Hanibuchi etc., Intl.J.Cancer, 78 (4): 480-45,1998.

Hannan etc., JAMA, 290 (6): 773-780,2003.

Harris，J.Org.Chem.，25：225，1960.

Harris，J.Org.Chem.，30：2190，1965.

Hays，In：Studies?of?the?Effects?of?Atmospheric?Hydrogen?Sulfidein?Animals，thesis?dissertation，University?of?Missouri-Columbia，1972.

Hellstrand etc., Acta Oncologica, 37 (4): 347-353,1998.

Higuchi and Fukamachi, Folia Pharmacologica Japonica, 73 (3): 307-319,1977.

Hobert etc., Organometallics, 20:1370,2001.

Hochachka etc., Comp.Biochem.Physiol.B Biochem.Mol.Biol., 130 (4): 435-459,2001.

Hochachka etc., Proc.Natl.Acad.Sci.USA, 93 (18): 9493-94938,1996.

Hui and Hashimoto, Infection Immun., 66 (11): 5329-5336,1998.

Hwang?&?Greenberg，Biochemistry，38：14248，1999.

Hyspler etc., J.Chromatography, 770:255-259,2002.

Innicenti etc., Bioorg.Med.Chem.Lett.14,5769 (2004).

Jiang etc., Am.J.Physiol.Cell Physiol., 280:1140-1150,2001.

Ju etc., J.Neuropathol.Exp.Neurol., 59 (3): 241-50,2000.

Kamoun，Amino?Acids?26，243，2004.

Kelso etc., J.Biol.Chem., 276:4588-4596,2001.

Khan etc., Toxicol.Applied Pharmacol., 103:482-490,1990.

Kilburn and Warshaw, Toxicology Indust.Health, 11 (2): 185-197,1995.

Kilburn，Environ.Health，54(3)：150，1999

Kilburn，Environ.Res.，81(2)：92-99，1999.

Knapp and Darout, Org.Lett, 7:203,2005.

Kontou etc., J.Agricultureal and Food Chem., 52:1212,2004.

Kuroda etc., Transplantation, 46 (3): 457-460,1988.

Lai etc., Biochemistry, 40:4904-4910,2001.

Langer etc., Biochemistr, y33:14034,1994.

Langer etc., Biochemistry, 33:10867,1997.

Ledingham etc., Circulation, 82 (2): IV351-358,1990.

Ledingham etc., J.Thorac.Cardiobasc.Surg., 93:240-246,1987.

J.Nuc.Med.26:72 such as Lee, 1985.

Liu etc., J.Org.Chem., 67:9267,2002.

Lundgren-Eriksson etc., Anticancer Res.2001Sep-Oct; 21 (5): 3269-74

Mehlhorn etc., Cardiovasc Surg., 9 (5): 482-486,2001.

Menasche etc., Eur.J.Cardio.Thorax.Surg., 8:207-213,1994.

Michaels etc., Circulation, 106 (23): e187-190,2002.

Mugesh etc., Chem.Rev., 101:2125,2001.

Murai and Kato, In:Organoselenium Chemistry:ModernDevelopments in Organic Synthesis, Wirth (Ed.), Springer, NY, Vo.28,2000.

Murai, etc., J.Org.Chem., 66:8101,2001.

Netherton?&?Fu，Org.Lett.，3：4295，2001.

Noguchi etc., Biochemistry, 42:11642,2003.

Nogueira etc., Chem.Rev., 104:6255,2004.

Nystul etc., Science, 302 (5647): 1038-1041,2003.

O ' Sullivan etc., J.Am.Chem.Soc., 126:2194,2004.

Olojo etc., J.Phys.Chem.A, 108:1018,2004.

Otterbein etc., Am.J.Physiol.Lung Cell Mol.Physiol., 279 (6): L1029-L1037,2000.

Otterbein etc., Trends Immunol., 24 (8): 449-455,2003.

Padilla etc., Molec.Biology of the Cell, 13:1473-1483,2002.

Padilla etc., Proc.Natl.Acad.Sci.USA, 98 (13): 7331-7335., 2001.

Partlo etc., Neurotoxicology, 22 (2): 177-189,2001.

PCT?Appln.WO?94/17178

Petersen，Biochemica?et?Biophysica?Acta，460：299-307，1977.

Pietras etc., Oncogene, 17 (17): 2235-2249,1998.

Punch etc., 2001

Qin etc., Proc.Natl.Acad.Sci.USA, 95 (24): 14411-14416,1998.

Quirante etc., j.Org.Chem., 67:2323,2002.

Rager etc., NC Med.J., 65 (1): 18-25,2004.

J.Med.Chem. such as Reigan, 48:392,2005.

Remington ' s Pharmaceutical Sciences, 15 ^ThEd., 1035-1038 page or leaf and 1570-1580 page or leaf, Mack Publishing Company, Easton, PA, 1980.

Rogers etc., Mamm.Genome, 8:711-713,1997.

Ryter and Otterbein, BioEssays, 26:270-280,2004.

Seburg?abd?Squires，Intl.J.Mass?Spectrometry?Ion?Proc.，167/168：541，1997.

Semenza，Cell，98(3)：281-284，1999.

Semenza，Trends?Mol.Med.，7(8)：345-350，2001.

Shaw(1996)

Shawali etc., J.Org.Chem., 61:4055,2001.

Shen etc., J.Agric.Food Chem., 50:2644,2002.

Smith etc., Eur.J.Biochem., 263:709-716,1999.

Soledad etc., Org.Lett., 3:1213,2001.

Steudel，Chem.Rev.，102：3905，2002.

Struve etc., Neurotoxicology, 22 (3): 375-385,2001.

Sundarrajan etc., Macromolecules, 35:3331,2002.

Supuran etc., Med.Res.Rev., 23 (2): 146-189,2003.

Sweeney，In：A?Survy?of?Compounds?from?the?AntiradiationDrug?Development?Program?of?the?U.S.Army?Medical?Research?andDevelopment?Command.Walter?Reed?Army?Institute?of?Research，Washington?D.C.，1979.

Teodoro and OFarrell, EMBO J., 22 (3): 580-587,2003.

The Hypothermia After Cardiac Arrest Study Group etc., 2002.

Tisherman，Crit.Care?Med.，32(2)：S46-S50，2004.

Van Voorhies etc., J.Exp.Biol., 203 (Pt 16): 2467-2478,2000.

Wang etc., 1992

Wang etc., 1993

Wang etc., 1994

Wang，FASEB?J.，16(13)：1792-1798，2002.

Yaffe etc., Crit.Care Med., 32 (2): S51-55,2004.

Yaghi etc., Nature, 423 (6941): 705-714,2003.

Yang etc., J.Agric.Food Chem., 52:7051,2004.

Yoshikawa etc., J.Biochem. (Tokyo), 71:859-872,1972.

Zhang etc., J.Appl.Physiol.96 (1): 392-397,2004.

Zhang etc., J.Org.Chem.63:5314,1998.

Ziegler，Ann.Rev.Biochem.54，305，1985.

Claims

1. be used to strengthen the method for the viability of biological substance, this method comprises at least a reactive compound that effective dose is provided to described biological substance.

2. the process of claim 1 wherein that described reactive compound is the oxygen antagonist.

3. the process of claim 1 wherein described material is exposed to reactive compound.

4. the process of claim 1 wherein that described reactive compound is a chalcogenide.

5. the process of claim 1 wherein that described reactive compound has the chemical constitution of formula I or formula IV.

6. the method for claim 5 wherein is exposed to described material the precursor of the chemical constitution of formula I or formula IV.

7. the process of claim 1 wherein the combination of reactive compound is provided to described material.

8. the process of claim 1 wherein in described material damage, seizure of disease or progress or hemorrhage before, during or afterwards, described reactive compound is provided for described material.

9. the method for claim 8, wherein said damage comprises hemorrhage.

10. the method for claim 9, wherein said damage is from the physical resource of outside.

11. the method for claim 8 is wherein providing described reactive compound before the damage or before the outbreak of disease or progress.

12. the method for claim 8, the metabolism of wherein said damage or disease and described material or the reduction of temperature are relevant.

13. the method for claim 12, wherein said damage is a surgical operation.

14. the method for claim 11 is wherein in damage or the outbreak of disease or between progressive stage or described reactive compound is not provided afterwards.

14.2. the method for claim wherein provides described reactive compound between the progressive stage of disease.

14.3. the method for claim 14.2, wherein said disease are thalassemia, drepanocytosis or cystic fibrosis.

15. the method for claim 11 provides described reactive compound wherein for described material, the amount of this chemical compound and the described persistent period that provides are enough to reduce CO ₂Emit at least 25%.

16. the method for claim 11 provides described reactive compound wherein for described material, the amount of this chemical compound and the described persistent period that provides can not cause that this material enters stagnation.

17. the method for claim 11; described reactive compound is provided wherein for described material; the amount of this chemical compound and the described persistent period that provides protect this material to avoid infringement or dead, and wherein said infringement or death are by damaging or the outbreak or the progress of disease cause.

18. the method for claim 11 provides described reactive compound wherein for described material, the amount of this chemical compound and the described persistent period that provides are enough to increase this material enters stagnation after the outbreak of damage or disease or progress speed.

19. the method for claim 11 provides described reactive compound wherein for described material, the amount of this chemical compound and the described persistent period that provides are enough to prevent CO after the outbreak of damage or disease or progress ₂The further reduction of emitting.

20. the method for claim 11 provides described reactive compound wherein for described material, the amount of this chemical compound and the described persistent period that provides are enough to cause that this material enters stagnation.

21. the process of claim 1 wherein under hypoxia or the anoxia condition or before being exposed to hypoxia or anoxia condition, described reactive compound be provided for described material.

22. the method for claim 21, wherein when no described reactive compound, described hypoxia or anoxia condition will damage described material.

22.4. the method for claim 22 wherein during being exposed to hypoxia or anoxia condition, is not exposed to described reactive compound with described material.

23. the method for claim 21 provides the described reactive compound of gas, semisolid liquid, liquid or solid form wherein for described material.

24. the method for claim 23 provides the described reactive compound of at least a gas form wherein for described material.

25. the method for claim 23 provides the described reactive compound of at least a semisolid liquid form wherein for described material.

26. the method for claim 23 provides the described reactive compound of at least a liquid form wherein for described material.

27. the method for claim 26 is wherein bubbled described reactive compound in described liquid.

28. the method for claim 26 wherein is dissolved in described reactive compound in the described liquid.

29. the process of claim 1 wherein at least two kinds of reactive compounds are provided successively.

30. the process of claim 1 wherein at least two kinds of oxygen antagonisies are provided together.

31. the process of claim 1 wherein that described material is exposed to one of described oxygen antagonist continues about 30 seconds to 30 days a period of time.

32. the method for claim 7, wherein said combination comprises the oxygen antagonist, and this oxygen antagonist has and is selected from following group at least a chemical formula:

A) has the chemical compound of formula I;

B) has the chemical compound of formula II;

C) has the chemical compound of formula III;

D) has the chemical compound of formula IV;

Or its salt or precursor.

33. the method for claim 32, wherein said combination comprise more than a kind of from the same group reactive compound mutually.

34. the method for claim 32, wherein at least a reactive compound are from group a).

35. the method for claim 32, wherein at least a reactive compound are chalcogenide or chalcogenide salt.

36. the method for claim 35, wherein at least a reactive compound comprises sulfur.

37. the method for claim 35, wherein at least a reactive compound comprises selenium.

38. the method for claim 35, wherein at least a reactive compound is a chalcogenide salt.

39. the method for claim 38, wherein said chalcogenide salt is selected from by Na ₂S, NaHS, K ₂S, KHS, Rb ₂S, Cs ₂S, (NH ₄) ₂S, (NH ₄) group formed of HS, BeS, MgS, CaS, SrS and BaS.

40. the method for claim 33, wherein said combination comprise more than a kind of from group reactive compound a).

41. the method for claim 40, the combination of wherein said oxygen antagonist comprises CO ₂

42. the method for claim 32, wherein at least a reactive compound is from group b).

43. the method for claim 42, wherein said reactive compound have the chemical formula of formula II (a), formula II (b) or formula II (c).

44. the method for claim 32, wherein at least a reactive compound is from group c).

45. the method for claim 44, wherein said reactive compound have the chemical formula of formula III (a), formula III (b), formula III (c), formula III (d), formula III (e), formula III (f), formula III (g) or formula III (h).

46. the process of claim 1 wherein by suction, injection, conduit insertion, dipping, lavation, perfusion, topical application, absorption, absorption or dosage forms for oral administration, described reactive compound be provided for described biological substance.

47. the method for claim 1, wherein pass through intravenous, Intradermal, intra-arterial, intraperitoneal; in the pathological changes; intracranial; intraarticular; in the prostate; in the pleura; in the trachea; intranasal; in the sheath; in the vitreous body; intravaginal; internal rectum; surface; in the tumor; intramuscular; intraperitoneal; ophthalmic; subcutaneous; under the conjunctiva; in the capsule; through mucous membrane; in the pericardium; in the umbilicus; ophthalmic; per os; surface; part; by sucking; by injection; pass through infusion; pass through continuous infusion; pass through regional perfusion; be applied to described biological substance via conduit or via lavation provides described reactive compound for described biological substance.

48. the method for claim 1, this method comprise described biological substance is exposed in check temperature and/or pressure environment.

49. the method for claim 48 wherein is exposed in check temperature environment with described biological substance.

50. the method for claim 49, wherein said biological substance reach non--physiological core temperature.

51. the method for claim 50 wherein is exposed to described biological substance and is lower than about 20 ℃ in check temperature environment.

52. the method for claim 49 wherein before one or more reactive compounds are provided and/or simultaneously, is exposed in check temperature environment with described biological substance.

53. the process of claim 1 wherein that one or more reactive compounds comprise can be with one or more reactive compound targeting to mitochondrial cationic structural.

54. the process of claim 1 wherein that described biological substance will be by preservation.

55. the method for claim 54, wherein said biological substance comprises platelet.

56. the method for claim 54, wherein said biological substance is transplanted.

57. the process of claim 1 wherein that described biological substance has the risk of ischemia reperfusion injury.

58. the method for claim 57 has wherein been induced cardioplegia in described biological substance.

59. the process of claim 1 wherein that described biological substance is the biology that the hemorrhagic shock risk is arranged.

60. the method for claim 1, this method further comprises the biological substance that evaluation need be handled.

61. the method for claim 1, this method further comprise the toxicity from described reactive compound of monitoring described biological substance.

62. the form with pharmaceutical composition that the process of claim 1 wherein offers described biological substance with described reactive compound.

63. be used to strengthen the method for the viability of biological substance, this method comprises to described material provides effective amount of i) combination of reactive compound or reactive compound and ii) hypoxia condition.

64. be used to strengthen the method for the viability of biological substance, this method comprises the compositions that effective dose is provided to described material, said composition has one or more chemical compounds with formula II or its salt or precursor.

65. be used to strengthen the method for the viability of biological substance, this method comprises the compositions that effective dose is provided to described material, said composition has one or more chemical compounds with formula III or its salt or precursor.

66. be used to strengthen the method for the viability of biological substance, this method comprises the compositions that effective dose is provided to described material, said composition has one or more chemical compounds with formula IV or its salt or precursor.

67. be used to strengthen the method for the viability of biological substance, this method comprises the chemical compound with formula I, formula II, formula III and/or formula IV or its salt or the prodrug of using effective dose to described material.

68. be used to prevent or reduce under unfavorable conditions method to the infringement of biological substance, this method comprises the reactive compound that effective dose is provided to described biological substance, wherein prevents or has reduced infringement.

69. be used to produce the method for the original seed of biological preservation, this method comprises the one or more chemical compounds that described biology are exposed to following group of being selected from of effective dose:

A) has the chemical compound of formula I;

B) has the chemical compound of formula II;

C) has the chemical compound of formula III;

D) has the chemical compound of formula IV;

Or its salt or prodrug.

70. be used for reversibly suppressing the metabolic method of biology, this method comprises the reactive compound that effective dose is provided to described biological substance, this reactive compound is not a rotenone.

71. the method for claim 69, this method further comprise described biology is exposed to hypoxia condition.

72. the method for claim 69, wherein said biology are fly class, Fish, batrachia or its embryo.

73. be used for the method at the biology induced hypnotic, this method comprises the reactive compound that effective dose is provided to described biology, wherein this effective dose is less than the amount that can induce stagnation in this biology.

74. be used to anaesthetize the method for biological substance, this method comprises the reactive compound that effective dose is provided to described material, wherein this effective dose is less than the amount that can induce stagnation in this biology.

75. the protection biological substance is avoided damaging, the outbreak of disease or progress or dead method; this method is included in before the outbreak or progress or death of described damage, disease; the reactive compound of effective dose is provided for described material, wherein this effective dose is less than the amount that can induce stagnation in this biological substance.

76. the method for claim 75, wherein said biological substance are just hemorrhage.

77. prevent bleed till death method of biology, this method comprises to described biology of bleeding provides the reactive compound of effective dose to prevent death.

78. the method for claim 77, wherein said biology enters hemorrhagic shock.

79. be used for inducing at biological substance the method for stagnation, this method comprises:

A) identify the biological substance that wherein expectation is stagnated; And

B) provide the reactive compound of at least a effective dose of effective dose to come the stagnation of biological substance in the inductor for this biological substance.

80. the method for claim 79 provides the different activities combination of compounds of effective dose wherein for described biological substance.

81. the pharmaceutical composition in the pharmaceutically acceptable preparation, it comprises at least a mixture that contains reactive compound.

82. the pharmaceutical composition of claim 81, wherein said reactive compound are the oxygen antagonisies.

83. the pharmaceutical composition of claim 82, wherein said oxygen antagonist are direct oxygen antagonisies.

84. the pharmaceutical composition of claim 82, wherein said oxygen antagonist is H ₂S.

85. the pharmaceutical composition of claim 84, this pharmaceutical composition comprises the H of effective dose ₂S when being administered to the patient, provides the C of 10 μ M to 10mM _MaxOr Cpss.

86. the pharmaceutical composition of claim 81, wherein said reactive compound have structure or its salt or the precursor of formula I, formula II, formula III, formula IV.

87. the pharmaceutical composition of claim 81, wherein said reactive compound is a chalcogenide.

88. the pharmaceutical composition of claim 72, wherein said chalcogenide is H ₂S or its salt or precursor.

89. the pharmaceutical composition of claim 66, wherein said compositions is a liquid.

90. a manufacture, it comprises packaging material and is included in reactive compound in these packaging material, and wherein said packaging material comprise and show that described reactive compound can be used for the label that biological substance is in vivo induced stagnation.

91. the manufacture of claim 90, it comprises pharmaceutically acceptable diluent.

92. the manufacture of claim 91, wherein said reactive compound provides in the container of first sealing, and described pharmaceutically acceptable diluent provides in the container of second sealing.

93. the manufacture of claim 92, it further comprises the description that is used to mix described reactive compound and described diluent.

94. the manufacture of claim 91, wherein said reactive compound is induced stagnation by the biological substance that reconstruct is used in vivo.

95. the manufacture of claim 90, it further comprises buffer agent.

96. the manufacture of claim 90, wherein said reactive compound have chemical constitution or its salt or the prodrug of formula I, formula II, formula III, formula IV.

97. the manufacture of claim 90, wherein said reactive compound are the oxygen antagonisies.

98. the manufacture of claim 90, wherein said reactive compound is a chalcogenide.

99. the manufacture of claim 98, wherein said chalcogenide is H ₂S or its salt or precursor.

100. the manufacture of claim 90, wherein said label show that described oxygen antagonist can be used for inducing stagnation the patient of this processing of needs.

101. a manufacture, it comprises following material packaging together: reactive compound, about using the description of this reactive compound, and it comprises: differentiate that (a) needs stagnate the in-vivo tissue of processing; (b) use the described reactive compound of effective dose for the interior biological substance of described body.

102. a manufacture, it comprises medical gas and the label that contains reactive compound, and this label comprises about details of inducing stagnation in biological substance or usage and uses.

103. be used for reactive compound is delivered to the test kit that needs are stagnated the tissue site that handles, comprise:

Be suitable for forming covering at the sealed envelope thing of tissue site;

The container that contains the aerobic antagonist; And

Import in described wound covering thing;

The wherein said container that contains reactive compound is communicated with described import.

104. the test kit of claim 103, it further comprises the outlet that is arranged in described covering, wherein should outlet be communicated with negative pressure source.

105. the test kit of claim 104 is wherein settled described outlet, makes it to be communicated with the negative pressure source fluid.

106. the test kit of claim 105, it further comprises flexible conduit, and this conduit is communicated with described outlet and negative pressure source.

107. the test kit of claim 106, it further comprises a jar, and fluid is communicated with between this jar and described outlet and the negative pressure source.

108. the test kit of claim 107, wherein said jar is dismountable jar.

109. the test kit of claim 106, the described container that wherein contains reactive compound is communicated with described inlet gas.

110. the test kit of claim 104, wherein said container contains gaseous active compound.

111. the test kit of claim 106, wherein said container contains the liquid gas reactive compound.

112. the test kit of claim 111, it further comprises gasifier, and this gasifier is communicated with the described container that contains reactive compound with between entering the mouth.

113. the test kit of claim 106, it comprises Returning outlet, and this outlet is communicated with the container that contains described reactive compound.

114. the test kit of claim 103, wherein said reactive compound are carbon monoxide, carbon dioxide or hydrogen sulfide.

115. the test kit of claim 103, wherein said tissue comprises the wound site.

116. the test kit of claim 103, wherein said covering comprises elastomeric material.

117. the test kit of claim 116, it further comprises the contact adhesive that covers described blanket edge.

118. the test kit of claim 103, wherein said negative pressure source is a vacuum pump.

119. be used for the oxygen antagonist is delivered to the method for biological substance, this method comprises the test kit that uses claim 103.

120. be used to screen the method for candidate's reactive compound, this method comprises:

A) zebrafish embryo is exposed to material;

B) the described embryo's of measurement heart rate;

Heart rate when embryo's heart rate is with this material not in the time of c) will having described material relatively, wherein the reduction of heart rate identifies that this material is candidate's reactive compound.

121. the method for claim 120 is wherein measured described embryo's heart rate by the number of times that calculates heart beating.

122. the method for claim 120 wherein when measuring heart rate, is observed described zebrafish embryo under anatomic microscope.

123. the method for claim 120, it further comprises

D) mice is exposed to described candidate's reactive compound and measure in the following parameter one or more:

I) core temperature;

Ii) oxygen consumption;

Iii) motoricity; Or

Iv) carbon dioxide generating.

124. the method for claim 120, wherein said chemical compound has the structure of formula I or formula IV.

125. be used to screen the method for candidate's reactive compound, this method comprises:

A) nematicide is exposed to material;

B) one or more in the following Cellular respiration factor of mensuration:

I) core temperature;

Ii) oxygen consumption;

Iii) motoricity; Or

Iv) carbon dioxide generating;

Cellular respiration factor when the Cellular respiration factor of nematicide is with this material not in the time of c) will having described material relatively, the minimizing of wherein said feature identifies that this material is candidate's reactive compound.

126. the method for claim 125 is wherein measured motoricity.

127. further comprising, the method for claim 125, this method identify described material.

128. the method for claim 124, wherein said chemical compound has the structure of formula I or formula IV.

129. the protection mammal avoids suffering the method for the primary cellular defect that caused by surgical operation, this method is included in provides for described mammal before the surgical operation to be enough to induce this mammal to enter the hydrogen sulfide of the amount of pre-stagnation.

130. the method for claim 129, wherein said surgical operation are selected from the surgical operation or the emergency surgery operation of elective surgery, plan.

131. the method for claim 129, wherein intravenous is used hydrogen sulfide.

132. the method for claim 129 is wherein used hydrogen sulfide by suction.

133. the method for claim 129, wherein said surgical operation is a cardiopulmonary surgery.

134. the protection mammal avoids suffering the method for the primary cellular defect that caused by disease or unfavorable medical condition, this method is included in provides for described mammal before described seizure of disease or the progress to be enough to induce this mammal to enter the hydrogen sulfide of the amount of pre-stagnation.

135. the method for claim 134, wherein said disease or disadvantageous medical condition are selected from: hemorrhagic shock, myocardial infarction, acute coronary syndrome, asystole, neonate hypoxia/ischemia, ischemic damage and reperfusion damage, unstable angina pectoris, postangioplasty, aneurysm, wound and lose blood.

136. be used for inducing apneic method the experimenter, this method comprises the hydrogen sulfide of using effective dose to described experimenter.

137. the method for claim 136, wherein said experimenter is bleeding or the risk of bleeding is being arranged.

138. further comprising from described experimenter, the method for claim 137, this method obtain blood sample.

139. the method for claim 138, this method comprise that further the hydrogen sulfide of estimating described blood sample exposes.

140. the method for claim 136, this method further comprises the experimenter that evaluation need be handled.

140.3. the method for claim 136 is provided wherein for described patient and surpasses 3, the hydrogen sulfide of 000ppm.

140.4. the method for claim 136 provides hydrogen sulfide to continue about 5 minutes or shorter time wherein for described patient.

140.5. the method for claim 140.4 is provided wherein for described patient and surpasses 3, the hydrogen sulfide of 000ppm continues about 5 minutes or shorter time.

140.6. the method for claim 140.5 provides hydrogen sulfide to continue about 3 minutes or shorter time wherein for described patient.

141. be used for the treatment of the method for hemorrhagic shock among the patient, this method comprises the hydrogen sulfide that effective dose is provided.

142. the method for claim 141 wherein uses aerosol apparatus hydrogen sulfide to be provided for described patient.

143. the method for claim 141 is provided wherein for described patient and surpasses 3, the hydrogen sulfide of 000ppm.

144. the method for claim 141 provides hydrogen sulfide to continue about 5 minutes or shorter time wherein for described patient.

145. the method for claim 144 is provided wherein for described patient and surpasses 3, the hydrogen sulfide of 000ppm continues about 5 minutes or shorter time.

146. the method for claim 145 provides hydrogen sulfide to continue about 3 minutes or shorter time wherein for described patient.

147. the method for claim 141 provides hydrogen sulfide wherein for continuously described patient.

148. the method for claim 141 provides the hydrogen sulfide of single dose wherein for described patient.

149. the method for claim 141 provides the hydrogen sulfide of multiple dose wherein for described patient.