Comparative assessment of the effects of bumped kinase inhibitors on early zebrafish embryo development and pregnancy in mice

Bumped kinase inhibitors (BKIs) are effective against a variety of apicomplexan parasites. Fifteen BKIs with promising in vitro efficacy against Neospora caninum tachyzoites, low cytotoxicity in mammalian cells, and no toxic effects in non-pregnant BALB/c mice, were assessed in pregnant mice. Drugs were emulsified in corn oil and applied by gavage for 5 days. Five BKIs did not affect pregnancy, 5 BKIs exhibited 15-35% of neonatal mortality, and 5 compounds caused strong effects (infertility, abortion, stillbirth and pup mortality). Additionally, the impact of these compounds on zebrafish ( Danio rerio ) embryo development was assessed by exposing freshly fertilized eggs to 0.2-50μM of BKIs and microscopical monitoring of embryo development in a blinded manner during 4 days. We propose an algorithm that includes quantification of malformations and embryo deaths, and established a scoring system that allows to calculate an impact score (S i ) that indicates at which concentrations BKIs visibly affect zebrafish embryo development. Comparison of the two models showed that for 9 compounds no clear correlation between S i and pregnancy outcome was visible. However, those 3 BKIs affecting zebrafish embryos only at high concentrations (40μM or higher) did not impair mouse pregnancy at all, and those 3 compounds that inhibited zebrafish embryo development already at 0.2μM showed detrimental effects in the pregnancy model. Thus, the zebrafish embryo development test has a limited predictive value to foresee pregnancy outcome in BKI-treated mice. We conclude, that maternal health-related factors such as cardiovascular, pharmacokinetic and/or bioavailability properties also contribute to BKI-pregnancy effects.