Dissertation: Metabolism of androstane derivatives with focus on hydroxylation reactions

The most common type of performance enhancing drugs (PED) are anabolic androgenic steroids (AAS). These substances are derived from the male sex hormones testosterone and 5-dihydrotestosterone and are supposed to promote mainly anabolic effects. Cytochrome P450 (CYP) enzymes play an important role in the biosynthesis of steroid hormones. In Phase I metabolism of endogenous as well as exogenous steroids their part is also clearly relevant. Drug metabolizing CYP enzymes are seemingly as important as their steroidogenic counterpart. The most prominent reaction catalyzed by CYP enzymes is the insertion of a hydroxy group. The designer steroid 1-androstenedione (1-AED) and the endogenous prohormone androstenedione (AED) have been investigated regarding the formation of hydroxylated Phase I metabolites. Therefore, chemical synthesis of reference substances, in vitro biotransformation with CYP enzymes of interest (CYP19A1, CYP3A4 and human liver microsomes) and investigation of in vivo formation after administration of the precursors were conducted. The formation of 19‑hydroxylted 1-AED by CYP19A1 was verified with synthesized reference material. In vitro generation of this metabolite could not be confirmed and remains doubtful. The 2-hydroxylation of endogenous AED was shown in vivo (post administration) by comparison with a synthesized reference. Several other hydroxylated metabolites of AED and 1-AED and closely related steroids were postulated based on accurate mass spectrometric data and structure proposals are made. Future work could focus on structure confirmation of the proposed metabolites reference material. This could be achieved by chemical synthesis supported by biocatalytic hydroxylation.