Complex Metabolism of the Novel Neurosteroid, Ganaxolone, in Humans: A Unique Challenge for Metabolites in Safety Testing Assessment

The human pharmacokinetics, metabolism, and excretion of [14C]-ganaxolone (GNX) were characterized in healthy male subjects (n 5 8) following a single 300-mg (150 lCi) oral dose. GNX exhibited a short half-life of 4 hours in plasma, whereas total radioactivity had a half-life of 413 hours indicating extensive metabolism to long-lived metabo-lites. Identification of the major GNX circulating metabolites required extensive isolation and purification for liquid chromatography-tandem mass spectrometry analysis, together with in vitro studies, NMR spec-troscopy, and synthetic chemistry support. This revealed that the ma-jor routes of GNX metabolism involved hydroxylation at the 16a-hydroxy position, stereoselective reduction of the 20-ketone to afford the corresponding 20a-hydroxysterol, and sulfation of the 3a-hydroxy group. This latter reaction yielded an unstable tertiary sulfate, which eliminated the elements of H2SO4 to introduce a double bond in the A ring. A combination of these pathways, together with oxidation of the 3b-methyl substituent to a carboxylic acid and sulfation at the 20a po-sition, led to the major circulating metabolites in plasma, termed M2 and M17. These studies, which led to the complete or partial identifica-tion of no less than 59 metabolites of GNX, demonstrated the high com-plexity of the metabolic fate of this drug in humans and demonstrated that the major circulating products in plasma can result from multiple sequential processes that may not be easily replicated in animals or with animal or human in vitro systems. SIGNIFICANCE STATEMENT Studies on the metabolism of [14C]-ganaxolone in humans revealed a complex array of products that circulated in plasma, the two major components of which were formed via an unexpected multi-step path-way. Complete structural characterization of these (disproportionate) human metabolites required extensive in vitro studies, along with contemporary mass spectrometry, NMR spectroscopy, and synthetic chemistry efforts, which served to underscore the limitations of tradi-tional animal studies in predicting major circulating metabolites in man.