Fishing for teratogens: a consortium effort for a harmonized zebrafish developmental toxicology assay.

A consortium of biopharmaceutical companies previously developed an optimized Zebrafish developmental toxicity assay (ZEDTA) where chorionated embryos were exposed to non-proprietary test compounds from 5 to 6 h post fertilization and assessed for morphological integrity at 5 days post fertilization. With the original 20 test compounds, this achieved an overall predictive value for teratogenicity of 88% of mammalian in vivo outcome [Gustafson, A. L., Stedman, D. B., Ball, J., Hillegass, J. M., Flood, A., Zhang, C. X., Panzica-Kelly, J., Cao, J., Coburn, A., Enright, B. P., et al. (2012). Interlaboratory assessment of a harmonized Zebrafish developmental toxicology assay-Progress report on phase I. Reprod. Toxicol. 33, 155-164]. In the second phase of this project, 38 proprietary pharmaceutical compounds from four consortium members were evaluated in two laboratories using the optimized method using either pond-derived or cultivated-strain wild-type Zebrafish embryos at concentrations up to 100µM. Embryo uptake of all compounds was assessed using liquid chromatography-tandem mass spectrometry. Twenty eight of 38 compounds had a confirmed embryo uptake of >5%, and with these compounds the ZEDTA achieved an overall predictive value of 82% and 65% at the two respective laboratories. When low-uptake compounds (≤ 5%) were retested with logarithmic concentrations up to 1000µM, the overall predictivity across all 38 compounds was 79% and 62% respectively, with the first laboratory achieving 74% sensitivity (teratogen detection) and 82% specificity (non-teratogen detection) and the second laboratory achieving 63% sensitivity (teratogen detection) and 62% specificity (non-teratogen detection). Subsequent data analyses showed that technical differences rather than strain differences were the primary contributor to interlaboratory differences in predictivity. Based on these results, the ZEDTA harmonized methodology is currently being used for compound assessment at lead optimization stage of development by 4/5 of the consortium companies.

Improving the odds of success in drug discovery: choosing the best compounds for in vivo toxicology studies.

A set of molecules that advanced into exploratory animal toxicology studies (two species) was examined to determine what properties contributed to success in these safety studies. Compounds were rigorously evaluated across numerous safety end points and classified as "pass" if a suitable in vivo therapeutic index (TI) was achieved for advancement into regulatory toxicology studies. The most predictive end point contributing to compound survival was a predicted human efficacious concentration (Ceff) of ≤250 nM (total drug) and ≤40 nM (free drug). This trend held across a wide range of CNS modes of action, encompassing targets such as enzymes, G-protein-coupled receptors, ion channels, and transporters.

Toxicity assessments of nonsteroidal anti-inflammatory drugs in isolated mitochondria, rat hepatocytes, and zebrafish show good concordance across chemical classes.

To reduce costly late-stage compound attrition, there has been an increased focus on assessing compounds in in vitro assays that predict attributes of human safety liabilities, before preclinical in vivo studies are done. Relevant questions when choosing a panel of assays for predicting toxicity are (a) whether there is general concordance in the data among the assays, and (b) whether, in a retrospective analysis, the rank order of toxicity of compounds in the assays correlates with the known safety profile of the drugs in humans. The aim of our study was to answer these questions using nonsteroidal anti-inflammatory drugs (NSAIDs) as a test set since NSAIDs are generally associated with gastrointestinal injury, hepatotoxicity, and/or cardiovascular risk, with mitochondrial impairment and endoplasmic reticulum stress being possible contributing factors. Eleven NSAIDs, flufenamic acid, tolfenamic acid, mefenamic acid, diclofenac, meloxicam, sudoxicam, piroxicam, diflunisal, acetylsalicylic acid, nimesulide, and sulindac (and its two metabolites, sulindac sulfide and sulindac sulfone), were tested for their effects on (a) the respiration of rat liver mitochondria, (b) a panel of mechanistic endpoints in rat hepatocytes, and (c) the viability and organ morphology of zebrafish. We show good concordance for distinguishing among/between NSAID chemical classes in the observations among the three approaches. Furthermore, the assays were complementary and able to correctly identify "toxic" and "non-toxic" drugs in accordance with their human safety profile, with emphasis on hepatic and gastrointestinal safety. We recommend implementing our multi-assay approach in the drug discovery process to reduce compound attrition.

Reproductive toxicity assessment of sunitinib, a multitargeted receptor tyrosine kinase inhibitor, in male and female rats.

BACKGROUND: Sunitinib (SUTENT, Pfizer Inc., New York, NY) is a multitargeted inhibitor of selected receptor tyrosine kinases, which produces an antiproliferative and antiangiogenic effect by blocking pathways fundamental to tumor growth and survival. We investigated the effects of sunitinib on male and female fertility and early embryonic development in the rat. METHODS: In the female fertility and early embryonic development phase, untreated males were paired with treated females dosed at 0 (control), 0.5, 1.5, and 5 mg/kg/day from 14 days premating, through mating, to gestation day 7. In the male fertility phase, the same males were then treated 58 days at doses of 0 (control), 1, 3, and 10 mg/kg/day, mated with untreated females, with continued daily dosing for a total of 74 days. RESULTS: There was no systemic toxicity- or treatment-related effects on fertility in female rats. Females exposed at 5 mg/kg/day had an increase in the number of early resorptions with associated decrease in viable embryos. In the males, body weight and food consumption were decreased at 10 mg/kg/day compared to the controls. Male reproductive capacity, as assessed by copulation, fertility, and conception indices, was not impacted at any dose level. Sperm morphology, concentration, and motility were also unaffected by treatment. CONCLUSIONS: There were no effects on male reproduction. An increase in corpora lutea and an increase in early resorptions with associated reduction in viable embryos was noted in the females dosed 5 mg/kg/day. Sunitinib at doses up to 1.5 and 10 mg/kg/day had no effects on female and male reproduction, respectively.

Endless possibilities: stem cells and the vision for toxicology testing in the 21st century.

The National Research Council's (NRC) toxicity testing vision lays out a bold future for our field. It depends heavily on computational algorithms based on the latest knowledge of cellular biochemistry and protein interaction pathways, exposing human cells to novel compounds in vitro, and being able to understand the changes seen. At the same time, significant strides are being made in our understanding of the control, production, and "behavior" of stem cells. While stem cells offer seemingly limitless possibilities for regenerative medicine, they have already delivered new assays to predict embryo-fetal developmental toxicity in vitro. In addition to providing a model of cells undergoing differentiation and proliferation, stem cells will play a major role by giving rise to many of the differentiated cell types on which this new vision depends. These will not be pure populations of single cell types but mixtures of cells much more representative of tissues in vitro. Moving from cells alone in a culture dish toward the more physiological condition of multiple cell types being able to interact to maintain homeostasis in the face of a disequilibrating force (like a toxic exposure) will lead us toward more useful and correct predictions of in vivo toxicities. Despite the seemingly insurmountable hurdles, persistence and creativity are on our side. We expect that a long series of successive iterations of predictive models will eventually yield a working process that approximates the NRC's vision and delivers on the promise of faster evaluation of chemicals with reduced animal use.

Assessment of the Embryonic Stem Cell Test and application and use in the pharmaceutical industry.

BACKGROUND: The European Centre for the Validation of Alternative Methods (ECVAM) designed the Embryonic Stem Cell Test (EST) as a tool for classifying developmentally toxic compounds. An in vitro tool to assess developmental toxicity would be of great value to the pharmaceutical industry to help with toxicity-associated attrition. METHODS: ECVAM's EST protocol was used, but employing a different mouse embryonic stem cell (ESC) line and an alternative differentiation medium. A subset of the compounds used to validate the EST assay along with a number of in-house pharmaceutical compounds plus marketed pharmaceutical compounds were used to assess the EST performance with receptor-mediated compounds. RESULTS: Our results with ECVAM compounds mirrored ECVAM's. Compounds that were developmentally toxic in vivo were classified by the EST as moderate risk. Overall, the accuracy was 75% with the current set of data and the predictivity of low-, moderate-, and high-risk compounds was 90, 71, and 60% while the precision was 59, 86, and 100%, respectively. Interestingly, a number of the non-developmentally toxic compounds had values for the 3T3 IC(50) values, which were lower than the ESC IC(50) and ID(50), a situation not taken into account by ECVAM when designing the EST algorithm. CONCLUSIONS: The assay as currently constructed has a significant false-positive rate (approximately 40%), but a very low false-negative rate (approximately 7%). Additional moderate- and high-risk compounds need to be assessed to increase confidence, accuracy, and understanding in the EST's predictivity.

Embryonic stem cells in predictive cardiotoxicity: laser capture microscopy enables assay development.

Embryonic stem (ES) cells offer unprecedented opportunities for in vitro drug discovery and safety assessment of compounds. Cardiomyocytes derived from ES cells enable development of predictive cardiotoxicity models to increase the safety of novel drugs. Heterogeneity of differentiated ES cells limits the development of reliable in vitro models for compound screening. We report an innovative and robust approach to isolate ES-derived cardiomyocytes using laser microdissection and pressure catapulting (LMPC). LMPC cells were readily applied onto 96-well format in vitro pharmacology assays. The expression of developmental and functional cardiac markers, Nkx 2.5, MLC2V, GATA-4, Connexin 43, Connexin 45, Serca-2a, cardiac alpha actin, and phospholamban, among others, was confirmed in LMPC ES-derived cardiomyocytes. Functional assays exhibited cardiac-like response to increased extracellular calcium (5.4 mM extracellular Ca2+) and L-type calcium channel antagonist (1 microM nifedipine). In conclusion, laser microdissection and pressure catapulting is a robust technology to isolate homogeneous ES-derived cell types from heterogeneous populations applicable to assay development.

Rat embryos express transcripts for cyclooxygenase-1 and carbonic anhydrase-4, but not for cyclooxygenase-2, during organogenesis.

BACKGROUND: Acetylsalicylic acid (ASA) is a rat teratogen, and exposures on gestational days (GDs) 9 and 10 induce diaphragm, cardiac, and midline defects. ASA inhibits members of the cyclooxygenase (COX) family and potentially members of the carbonic anhydrase (CA) family. The objective of this study was to determine whether the mRNA developmental expression pattern for any COX or CA isoform was consistent with a model in which ASA teratogenicity is mediated through direct interaction with one of these enzymes within embryos or within the adjacent ectoplacental cone (EPC) or yolk sac. METHODS: Staged embryos, over a range (GD 9.5-12) that included ASA-sensitive and ASA-insensitive stages of organogenesis, were assayed for COX and CA mRNA levels by three techniques: microarrays; in situ hybridization quantitated by a micro-imager; and quantitative reverse transcription polymerase chain reaction. ASA- and vehicle-treated embryos also were compared to determine whether inhibition led to upregulated COX or CA mRNA expression. RESULTS: COX-2 mRNA was undetectable in embryos throughout organogenesis by any assay (although it was abundant in EPC). In contrast, COX-1 mRNA was moderately abundant in embryos throughout organogenesis. One CA isoform, CA-4, demonstrated developmentally regulated embryonic mRNA expression that coincided with ASA sensitivity ASA exposure failed to induce upregulation of any of these mRNAs. CONCLUSIONS: Although ASA may affect the embryo indirectly through interaction with COX-2 within EPC, failure to detect embryonic COX-2 mRNA argues against COX-2 functioning as a direct mediator of ASA teratogenic activity in induction of cardiac, diaphragm, and midline defects. Correlation of COX-1 and CA-4 expression with ASA sensitivity suggested that embryonic COX-1 and possibly CA4 are much more likely candidates for mediators of ASA developmental toxicity.