Prediction of embryotoxic effects of valproic acid-derivatives with molecular in vitro methods

Therapy with the antiepileptic drug valproic acid (2-propyl-pentanoic acid, VPA) during early pregnancy can cause similar teratogenic effects (neural tube defects) in humans and mice. In this study a new molecular bioassay is presented using following endpoints: differentiation of F9 teratocarcinoma cells, altered cell morphology, induction of possible targed genes, and th e induction of viral RSV-promoter: The induction of a transiently transfect ed viral (RSV) promoter driven luciferase gene by VPA was used to screen a set of VPA-derivates. Structure-activity investigations showed; the longer the aliphatic side chain the more the induction of the RSV-reporter gene. T he specific induction was stereoselective. The teratogenic enantiomer S-4-y n-VPA (2-propyl-4-pentynoic acid) induced the RSV-driven reporter gene whil e the non teratogenic R-4-yn-VPA does not. Neptyl-4-yn-VPA was the most pot ent teratogen in vitro and in vivo. Non teratogenic VPA-derivatives like R- 4-yn-VPA and 2-en-VPA (2-propyl-2-penterzoic acid) were ineffective in this system. Thus, the teratogenic effect of VPA and VPA-derivatives in the mou se correlated with the specific induction of the viral RSV-promoter, contro lled reporter in F9-cells. Acid compounds such as fatty acids are known to interact with peroxisome proliferator-activated receptors (PPARs). To test structure-activity relationships by VPA or its derivatives we used CHO cell s stably expressing hybrid proteins of the ligand-binding domain of either of the PPARs. The teratogen VPA and the teratogenic derivatives of VPA acti vated the PPAR-delta construct in a very specific structure and stereoselec tive way which correlated well with the activities in the reporter gene ass ay (bioassay) and those in vivo. No such correlation was found with respect to activation of PPAR-alpha or PPAR-gamma. These structure-activity relati onships indicate that PPAR-delta may be a potential mediator of VPA-induced differentiation of F9 cells aid may possibly be involved in the mechanism of teratogenicity of VPA in vivo. Furthermore two bioassays were designed w ith clearly defined endpoints, amenable to automation and screening of grea t number of compounds. The lest system allows to replace animal experiments in the preclinical development of new antiepileptic drugs with reduced ter atogenic risk. Supported by BgVV-ZEBET (Berlin).