nComparative 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.

Keywords: Apicomplexan parasites; Bumped kinase inhibitors; CDPK1; Embryo development; Interference; Pregnancy; Zebrafish.