Regulatory Toxicology and Pharmacology 70 (2014) 149–154 Contents lists available at ScienceDirect Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph Flatworm models in pharmacological research: The importance of compound stability testing Sofie Stalmans a,1, Maxime Willems b,1, Els Adriaens b, Jean-Paul Remon b, Matthias D’Hondt a, Bart De Spiegeleer a,⇑ a b Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460 (Second Floor), 9000 Ghent, Belgium Laboratory of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460 (Third Floor), 9000 Ghent, Belgium a r t i c l e i n f o Article history: Received 5 December 2013 Available online 3 July 2014 Keywords: Assay design Flatworms Quality practices Stability testing Stem cells Toxico-pharmacological test U(H)PLC a b s t r a c t Flatworms possess adult pluripotent stem cells, which make them extraordinary experimental model organisms to assess in vivo the undesirable effects of substances on stem cells. Currently, quality practices, implying evaluation of the stability of the test compound under the proposed experimental conditions, are uncommon in this research field. Nevertheless, performing a stability study during the rational design of in vivo assay protocols will result in more reliable assay results. To illustrate the influence of the stability of the test substance on the final experimental outcome, we performed a short-term International Conference on Harmonization (ICH)-based stability study of cyclophosphamide in the culture medium, to which a marine flatworm model Macrostomum lignano is exposed. Using a validated U(H)PLC method, it was demonstrated that the cyclophosphamide concentration in the culture medium at 20 °C is lowered to 80% of the initial concentration after 21 days. The multiwell plates, flatworms and diatoms, as well as light exposure, did not influence significantly the cyclophosphamide concentration in the medium. The results of the stability study have practical implications on the experimental set-up of the carcinogenicity assay like the frequency of medium renewal. This case study demonstrates the benefits of applying appropriate quality guidelines already during fundamental research increasing the credibility of the results. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction Flatworms are known for their amazing regenerative capacity. Even a fragment as small as 1/279th of the size of the original individual has the capacity to regenerate into an entire animal (Newmark and Sánchez Alvarado, 2002). The key to the regenerative prowess of these creatures are totipotent stem cells, called Abbreviations: CI, confidence interval; CP, cyclophosphamide; GLP, Good Laboratory Practices; HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatography; ICH, International Conference on Harmonization; l.c., label claim; LoD, limit of detection; LoQ, limit of quantification; PDA, photodiode array; Ph. Eur., European Pharmacopoeia; r.h., relative humidity; S/N, signal-to-noise ratio; U(H)PLC, ultra (high) performance liquid chromatography; WHO, World Health Organization. ⇑ Corresponding author. Fax: +32 9 264 81 93. E-mail addresses: Sofie.Stalmans@UGent.be (S. Stalmans),Maxime.Willems@ UGent.be (M. Willems), Els.Adriaens@UGent.be (E. Adriaens), JeanPaul.Remon@ UGent.be (J.-P. Remon), Matthias.Dhondt@UGent.be (M. D’Hondt), Bart.Despiegeleer@U Gent.be (B. De Spiegeleer). 1 Sofie Stalmans and Maxime Willems contributed equally to this work. http://dx.doi.org/10.1016/j.yrtph.2014.06.026 0273-2300/Ó 2014 Elsevier Inc. All rights reserved. neoblasts, distributed throughout the organism. Thus, neoblasts are the flatworm equivalent of somatic stem cells, making these remarkable creatures an excellent new model system for studying stem cell biology. In vivo studies of somatic stem cells are not easy in vertebrate models due to the fact that their experimental accessibility is relatively more challenging. Alternatively, nonmammalian model systems, such as flatworms, can be used to assess in vivo the effect of substances on stem cells. This has been done extensively in the past to test diverse pharmacological and carcinogenic compounds and recently their use has regained much interest (Alonso and Camargo, 2006, 2011; Best and Morita, 1982; Chan and Marchant, 2006; Demircan and Berezikov, 2013; Foster, 1963, 1969; González-Estévez et al., 2012a; Hall et al., 1986a,b; Isolani et al., 2012; Oviedo et al., 2008; Oviedo and Beane, 2009; Pagan et al., 2006, 2012, 2013; Plusquin et al., 2012a,b; Schaeffer et al., 1991; Schaeffer, 1993; Sánchez Alvarado, 2006; Simanov et al., 2012; Wu et al., 2012; Zhang et al., 2013). The power of these alternative animal models lies in their ease of culture and experimental accessibility. However, it is important to be aware that the specific test conditions, i.a. culture media, day/night regimes, 150 S. Stalmans et al. / Regulatory Toxicology and Pharmacology 70 (2014) 149–154 type of animal containers, feeding scheme and exposure time, can affect the stability of the substance under investigation. To our knowledge, this aspect has never been mentioned in any past or recent reported studies. We use the flatworm model organism Macrostomum lignano (Ladurner et al., 2005, 2008; Mouton et al., 2009; Willems et al., 2014) for our toxicological studies. Being a small (about 1.5 mm) marine species, flatworms are cultured in plastic or glass (multiwell) dishes with artificial seawater media under a specific day/night light regime and temperature-controlled conditions. The effect of all aforementioned factors are usually not mentioned in the stability data sheet of toxic substances but, in a pharmaceutical context where regulatory GLP (Good Laboratory Practices) are requested, it is essential to rationally design high quality experiments to maximize the reliability of the outcomes (Verbeken et al., 2012; Vergote et al., 2008). Therefore, we initiated an analytical U(H)PLC stability study of cyclophosphamide (CP) under experimental conditions, which is used as a standard test compound for screening of genotoxicity and carcinogenicity using a flatworm model (Willems et al., 2014). The study design was based on the International Conference on Harmonization (ICH) guidelines (D’Hondt et al., 2012; European Medicines Agency, 1998, 2003). Moreover, to our knowledge, it is the first time an U(H)PLC method is described for evaluation of the stability of CP. In literature, both HPLC and HPTLC methods are available for testing the CP stability in different formulation types (Bouligand et al., 2005; Kennedy et al., 2010; Menard et al., 2003; Mittner et al., 1999). The aim of this study was to quantitatively evaluate the influence of experimental conditions on the CP concentration in the used medium, hence on the final conclusions of the biological experiment. Using this study, we want to demonstrate the benefits of including appropriate quality practices already during research phases of fundamental science and encourage researchers to apply appropriate quality assurance practices to ensure the credibility of their results. 2. Materials and methods 2.1. Materials Cyclophosphamide monohydrate (CAS No. 6055-19-2) was purchased from Sigma (Diegem, Belgium). Acetonitrile U(H)PLC grade (CAS No. 75-05-8) for the mobile phase was obtained at Fisher Chemical (Erembodegem, Belgium), while ultrapure water (18.2 MO  cm, CAS No. 7732-18-5) was produced using an Arium 611 VF water purification system (Sartorius, Vilvoorde, Belgium). HPLC glass vials with Teflon/polypropylene closures were purchased from Waters (Zellik, Belgium), polystyrene multiwell plates and covers from Novolab (Geraardsbergen, Belgium) and closable Quartz cuvets (10 mm) from Hellma GmbH & Co. (Müllheim, Germany). et al. (2005, 2008), Mouton et al. (2009) and Rieger et al. (1988). The worms were exposed to a specific compound (CP in our case) dissolved in 4 ml of culture medium at 20 °C in polystyrene multiwell (typically six well) plates under the same day/night regime as described above. To achieve ad libitum feeding during the exposure time, diatoms were inoculated and allowed to grow on the multiwell plates two weeks prior to the start of the experiment. Exposure time can vary from two days to three months. Fig. 1 shows the model organism and the exposure condition in one well of a multiwell plate during the carcinogenicity assay. In the considered in vivo carcinogenicity assay, the flatworms are exposed to a 28 lg/ml (100 lM) CP solution in culture medium during two weeks at 20 °C under a 14 h light/10 h dark cycle. 2.3. U(H)PLC-PDA method for determination of CP The Acquity H-class U(H)PLC apparatus consisted of a quaternary solvent manager, an automatic sample injection system, combined with a flow through needle, a column heater and an ultra-performance LC photodiode array (PDA) detector, equipped with Empower 2 software for data acquisition (all from Waters, Zellik, Belgium). A BEH C18 column (Waters, 2.1 mm  100 mm, 1.7 lm) was selected for isocratic separation using a mobile phase containing 75% ultrapure water and 25% acetonitrile. The flow rate was 0.3 ml/min and the total run time was 5 min. The injection volume was set at 2 ll. The BEH C18column was thermostated at 40 °C (±3 °C) and the sample compartment at 25 °C (±5 °C). PDA-detection was performed from 190 to 400 nm, with quantification at 195 nm. Ultrapure water was used as needle rinsing liquid, as CP is water-soluble. All samples were prepared by dissolving an appropriate quantity of CP in ultrapure water. The CP reference sample (100% label claim (l.c.)) has a concentration of 28 lg CP/ml or 100 lM CP. During method validation, the limit of quantification (LoQ, S/ N = 10 according to the European Pharmacopoeia (Ph. Eur.)) was calculated to be 0.42 lg/ml (1.50% l.c.), while the limit of detection (LoD, S/N = 3 (Ph. Eur.)) was 0.13 lg/ml (0.45% l.c.). Injection of an over-concentrated CP sample (5 mg/ml) did not result in peaks in the subsequently injected blank solution (i.e., <LoD), indicating there was no significant carry-over. The method was linear in the tested range from 3% l.c. to 200% l.c. (R2 = 0.9998; ANOVA F-value = 23378). During the repeatability evaluation of the U(H)PLC-method, the relative standard deviation of six successive injections of a 100% l.c. reference sample was 1.44%. The performed stress stability study in acidic, basic, hydrogen peroxide and elevated temperature conditions, resulted in a similar impurity profile as previously described (Dhakane and Ubale, 2013). These results indicate the selectivity for CP of the new U(H)PLC-method, allowing the stability-indicating use of the method. 2.4. In use stability test of CP 2.2. Carcinogenicity assay We developed an in vivo flatworm carcinogenicity bioassay, in which stem cell proliferation is used as an endpoint to assess the carcinogenic potential of compounds. A detailed description of this bioassay can be found in Willems et al. (2014) and is briefly summarized hereafter in order to understand which parameters were chosen for stability testing. During the in vivo flatworm carcinogenicity assay, M. lignano cultures were grown in a medium consisting of artificial seawater enriched with a f/2 marine water solution (Guillard, 1975), under a 14 h day/10 h dark regime and fed ad libitum with the diatom Nitzschia curvilineata (PAE culture collection; UGent http:// www.pae.ugent.be/collection.htm), as described by Ladurner In order to represent the production variability of the culture medium, three different batches of the medium, each containing 28 lg CP/ml or 100 lM CP, were used. For the kinetic stability testing, the three batches, as well as a placebo sample, i.e., no CP in culture medium, were divided over different HPLC glass vials and stored in ICH conform storage rooms (Weiss Technik Belgium, Liedekerke, Belgium) at following conditions: 5 °C (50% relative humidity (r.h.)), 25 °C (60% r.h.) and 40 °C (75% r.h.), all protected from light. All the samples were analyzed using the previously described U(H)PLC-method at day T0, T1, T2, T5, T8, T10, T11 and T12. The freshly prepared reference sample consisted of a 28 lg/ ml (100 lM) aqueous CP solution and was also used to test system suitability prior to analysis by evaluation of the plate number S. Stalmans et al. / Regulatory Toxicology and Pharmacology 70 (2014) 149–154 151 Fig. 1. Left: Photograph of the flatworm model organism M. lignano. ey, eyes; mo, mouth; te, testes; ov, ovaria; tp, tail plate; g, gut. The gut is brown due to ingestion of diatoms. Scale bar 150 lm. Right: Schematic representation of one polystyrene well of a multiwell plate set-up for the carcinogenicity assay. Culture medium containing the test compound is pseudo-coloured blue, diatoms at the bottom of the well brown and worms are represented as black lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) (N P 17  103) and peak asymmetry (As 6 2). Typical chromatograms of T12 samples are given in Fig. 2. To evaluate the influence of the polystyrene multiwell plates (instead of a glass recipient), each batch was also stored in multiwell plates at the three different storage conditions (multiwell samples) and CP concentrations were analyzed at day T12. To test whether CP interacts with the biological components of the carcinogenicity test, three batches of an experimental sample, i.e., culture medium with flatworms and diatoms stored in a multiwell plate, were stored at 5 °C (50% r.h.) and 25 °C (60% r.h.) (diatom samples). The concentration of CP in these diatom samples was determined at day T0 and T12. During light stability testing of batch 1 and 2, the CP concentration was evaluated by incubation of quartz cuvettes filled with culture medium containing 28 lg/ml CP under visible (VIS, six cool white fluorescent lamps TDL 18 W/33) and ultraviolet (UV, six fluorescent lamps F18W/BLB-T8) light. Storage temperature and humidity were 25 °C and 60% r.h., respectively. As a control, cuvettes were wrapped in aluminium foil to represent light-untreated samples. The CP concentration of the light-(un)treated samples was determined at day T3 for the UV-treated samples and at day T8 for the VIS-treated samples. The aforementioned light-treated conditions complied with ICH requirements for stress degradation (European Medicines Agency, 1998). Fig. 2. Typical chromatograms of kinetic stability samples (CP in culture medium contained in glass HPLC vials) stored at three different conditions and analyzed at day T12. ⁄ Represents peaks also present in the placebo sample analyzed at day T12. 152 S. Stalmans et al. / Regulatory Toxicology and Pharmacology 70 (2014) 149–154 Therefore, we initiated a stability study on cyclophosphamide, one of our standard test compounds, focusing on the aforementioned parameters, i.e., temperature, container and experimental medium, as well as light. The results will be discussed within the framework of the experimental set-up of our carcinogenicity assay. 3.1. Kinetic stability testing Fig. 3. Kinetic stability of cyclophosphamide in the culture medium, stored in glass HPLC vials, visualized as the percentage label claim of CP (here batch 1) in function of time and storage condition. Table 1 Percentage label claim at day T12 of CP in the culture medium stored in glass HPLC vials at 5 °C (50% r.h.), 25 °C (60% r.h.) and 40 °C (75% r.h.). Batch 1 2 3 Percentage label claim of CP at T12 (%) 5 °C (50% r.h.) 25 °C (60% r.h.) 40 °C (75% r.h.) 94.0 91.7 99.9 75.4 73.0 79.8 11.9 11.0 11.9 2.5. Data-analysis The stability of CP in the culture medium was assessed by comparing the percentage of the label claim (28 lg CP/ml) remaining at each time interval. To determine a statistically significant degradation, the slope and its 95% confidence interval (CI) was calculated for the percentage label claim versus time curve. If zero was not included in the 95% CI around the slope, a statistically significant degradation was demonstrated. When significant degradation was observed, the degradation constant k was calculated from linear regression data, assuming first-order degradation kinetics which was found to be the best fitting model for the experimental data (highest R2 compared to zero-order and second-order model). The frequency factor A and activation energy Ea of the degradation reaction were derived from the Arrhenius equation. 3. Results and discussion The model flatworm M. lignano is currently being used in carcinogenicity assays. When performing such assays, the assessment of compound stability over the test period is of paramount importance. Factors such as temperature, light and medium play a key role in that respect. In addition, these factors vary depending on the model organism specific culture and experimental conditions. The percentage label claim of CP was calculated for the three different batches at the three different storage conditions. As an example, the percentage label claim for batch 1 in function of time and storage condition is visualized in Fig. 3. The curves of batches 2 and 3 show the same trend. Fig. 3 and Table 1 indicate that samples stored at 25 °C and 40 °C showed a clear degradation profile of CP in the culture medium. Approximately 75% of CP is recovered in the culture medium when stored at 25 °C (60% r.h.) and only about 11% when stored at 40 °C (75% r.h.) (Table 1). In contrast, CP did not show a statistically significant degradation in the culture medium at 5 °C during the test period of 12 days as zero is not included in the 95% confidence interval of the slope of the degradation curve for the three batches. As our carcinogenicity assay is currently performed at 20 °C for different time periods varying from two days to three months, our data were extrapolated to estimate the incubation time until 80% of the original CP concentration is present in the medium. Therefore, the degradation rate constant (k), using a first-order model, was calculated for the three batches at 25 °C and 40 °C. The observed k-values of the three batches were not significantly different, based on their 95% confidence intervals, indicating that the extent of degradation of CP is similar for the three batches (Table 2). Using these k-values, the activation energy (Ea) and frequency factor (A) were calculated applying the Arrhenius equation, which are summarized in Table 2. Using these kinetic data, it was estimated that it takes on average 21 days before the CP concentration is lowered to 80% of the initial concentration when stored at 20 °C. The above data thus provide information on the stability and the stability robustness (i.e., batch consistency) of the test compound in the culture medium, which is also important from a practical point of view, i.e., time scheme for renewal of the culture medium in order to maintain a reliable test. Applied on the carcinogenicity test, the media should be renewed minimally every three weeks as at least 80% CP must be present in the medium. This 80% CP was the stability specification under limit currently used in our laboratory for this type of basic research studies. The results of the multiwell samples show for all batches and all storage conditions a higher percentage label claim than the kinetic samples. It appears that our stability results of the multiwell samples are confounded by evaporation of the medium, probably because the cover of the multiwell plate is not gas tight in contrast to the cap of a HPLC vial used to store the kinetic samples. This experiment indicated that not only degradation of the test compound needs to be considered, but also other phenomena like evaporation loss of the solvent during the test set-up, resulting in higher concentration of CP in the medium. Therefore, renewal of Table 2 Overview of the degradation constant k of the three batches of CP in culture medium stored in glass HPLC vials at 25 °C and 40 °C and the calculated activation energy Ea and frequency factor A based on the Arrhenius equation (95% confidence interval is indicated between brackets). 1 Batch k (day ) (25 °C) 1 2 3 0.022 [0.018; 0.025] 0.022 [0.019; 0.026] 0.022 [0.019; 0.026] k (day 1 ) (40 °C) 0.180 [0.174; 0.186] 0.181 [0.174; 0.188] 0.182 [0.177; 0.188] Ea (kJ mol 1 ) 108.6 [107.4; 109.8] A (day 1 ) 2.3  1017 [1.5  1017; 3.7  1017] 153 S. Stalmans et al. / Regulatory Toxicology and Pharmacology 70 (2014) 149–154 Table 3 Percentage label claim of the UV- and VIS-treated and untreated samples of CP in culture medium stored in closed cuvettes at 25 °C (60% r.h.). Batch 1 2 a Percentage label claim of CP (%) Time (day) UV-treated UV-untreated Time (day) VIS-treated VIS-untreateda T0 T3 T0 T3 97.4 107.2 94.8 101.2 97.4 106.6 94.8 101.0 T0 T8 T0 T8 97.4 95.9 94.8 90.9 97.4 95.5 – – For batch 2 no results are available for the VIS-untreated sample. the medium during the carcinogenicity assay is not only dependent on the stability of the test compound, but also on the evaporation of the medium during the experimental period. Moreover, this study indicated also no biologically significant adsorption of the test compound to the test materials, an often overlooked phenomenon (Pezeshki et al., 2009). The diatom samples were also stored in multiwell plates, thus evaporation loss is also observed for these results. The worms and diatoms do not induce a significant loss of CP, i.e., more than 20%, in the culture medium, which justifies the ad libitum feeding regime, which is required during long time exposure protocols. Starvation of the worms would lead to a decrease in stem cell division, which would interfere with the endpoint of the assay (González-Estévez et al., 2012b; Nimeth et al., 2004). Although the presence of worms and diatoms had no biologically significant effect on the CP concentration, uptake of CP by the worms cannot be excluded by this study. 3.2. Light stability testing As already mentioned, M. lignano is reared under a 14 h light/ 10 h dark regime to ensure diatom growth. Experiments occur under the same regime. Photoreactivity of drugs is compound-specific and may take place through different mechanisms (Moore, 2004). This implies that testing photostability is of great importance to assure reliable carcinogenicity assay results if the medium is exposed to light during the test period. Analysis of the light stability samples indicated that CP is not significantly degraded during exposure to both ultraviolet and visible light (Table 3). Consequently, the light/dark regime will not influence the concentration of CP during the carcinogenicity assay. While the influence of compound purity on biological tests was previously demonstrated by our research group (Verbeken et al., 2012; Vergote et al., 2008), this study illustrates the importance of the evaluation of test compound stability in the used medium under the assay conditions prior to fixing the assay protocol (Fig. 4). Regulatory quality practices are not obliged during the research stage of fundamental science. Nevertheless, the World Health Organization (WHO) provides quality guidelines in fundamental biomedical research and demonstrates that quality assurance is required for obtaining credibility of the resulting data, which possibly can be used for further research for detection, prevention and treatment of diseases (World Health Organization, 2006). Moreover, once our in vivo carcinogenicity test would be implemented in a pharmaceutical context, regulatory guidelines and GLP would require to demonstrate the test compound stability (OECD, 2005; World Health Organization, 2009). Therefore, we encourage researchers working in fundamental medical research to already take appropriate quality guidelines into account during their research activities. 4. Conclusion In this study, in use stability testing during the rational design of flatworm in vivo carcinogenicity screening assays was introduced in the biological research field, where this procedure is not common. Using the example of cyclophosphamide, it was demonstrated that in function of time, storage condition and used container, the concentration of test compound altered in a significant manner. The results of this stability study are used to optimize the experimental set-up, e.g., frequency of renewal of the medium, justification of the application of a light/dark regime, storage temperature and used container. Stability testing of the test substance prior to implementation of the screening assay fits in the application of appropriate quality practices and will result in more reliable test outcomes. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This research was funded by a Ph.D. grant to S.S. of the Special Research Fund (BOF) of Ghent University (No. 01D38811) and by an OZM grant to M.W. (No. 100631) and a Ph.D. grant to M.D. (No. 101529), both by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT). We gratefully thank Nadia Lemeire for her technical assistance during the early stage of the method development. References Fig. 4. 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