Dissertation: In vivo and In vitro Metabolic Studies of Anabolic Steroids

The knowledge of drug metabolism is fundamental for scientific fields where a comprehensive understanding of steroid metabolism is of high relevance, including but not only limited to anti-doping studies, endocrinology, forensic toxicology, and safety assessment in drug development. This work concentrates on the metabolism of anabolic androgenic steroids (AAS) in both in vivo and in vitro models, with a specific focus on the AAS compounds testosterone (T), metandienone (MD), methyltestosterone (MT), clostebol (CLT), dehydrochloromethyltestosterone (DHCMT), and methylclostebol (CLMT) due to their structural similarities. It introduces new alternative models for studying AAS metabolism and provides valuable knowledge on metabolic properties based on chemical structural characteristics, offering the possibility for the enhancement of AAS detection. Firstly, in order to have a deep understanding of the A-ring reduction in MD, isolated enzyme assays (AKR1C2, AK1C3, ALR1C4, and AKR1D1) were performed. The results obtained substantiate the sequence of A-ring reduction in MD, as previously suggested in the literature [93, 136, 137], i.e., the 4,5-double bond is reduced first, then the 3 oxo group, and finally the 1,2-double bond. Moreover, it appears that AKR1C2, AKR1C3, and AKR1C4 exhibited varying stereoselectivity in catalyzing the 3-oxo reduction in 5α- or 5β-DHMD. AKR1C2 and AKR1C4 showed both 3α- and 3β-HSD activities, whereas AKR1C3 functioned as 3α-HSD only. The sequence of A-ring reduction provided valuable insights for the metabolic pathway analysis for subsequent in vitro studies in human skin cells. The metabolism of T, MT, CLT, and CLMT by keratinocytes and fibroblasts derived from human foreskins produced metabolites with partially or fully reduced A-ring. However, no metabolites of MD or DHCMT could be detected. The metabolite profiles suggest that 3α-HSD, 3β-HSD, and 5α-reductase activities play important roles in steroid metabolism by human keratinocytes and fibroblasts, whereas 17β HSD activity is weak. The stereochemistry of fully reduced metabolites (i.e., 3α,4α,5α-THCLT, 3β,4α,5α-THCLT, 3α,4α,5α-THCLMT, and 3β,4α,5α-THCLMT) of CLT and CLMT was newly identified and confirmed in this study. Differences in the chemical structures of compounds appear to affect A-ring reduction order and cellular metabolic capacities, especially the chlorine group at position 4. Keratinocytes appear to have a higher metabolic capability for compounds containing a chlorine substituent in position 4, whereas the opposite is true in fibroblasts. The medaka embryo model was used for the first time as an alternative model for in vivo studies of AAS. There were four metabolites detected after incubation of medaka embryos with MD, including 6βOH-MD and 5β-DHMD as well as tentatively assigned 18OH-MD and 16OH-MD. These metabolites have also been reported in previous human administration studies [136, 177, 180]. In comparison to the in vitro models, the medaka embryo model allows for simultaneous analysis of biotransformation and potential toxicity. Given the similar outcomes with human administration studies, medaka embryos may serve as an alternative model to identify potential metabolites for human biotransformation of doping-relevant compounds. Investigations on the metabolism of [13C3]-T and MT in the medaka embryo model show that the main metabolic reactions for both two substrates include hydrogenation of the 4,5-double bond and reduction of the 3-oxo function. Additionally, the oxidation of 17β-hydroxy group in [13C3]-T was also observed. The metabolites observed indicate the activities of steroid 5α-reductases, steroid 5β-reductases, 3α-HSD, 3β-HSD, and 17β-HSD in medaka embryos. 3β,5α- and 3α,5β-isomers were detected as the main fully reduced A-ring metabolites in both incubations. However, this study only showed preliminary results, further studies are necessary. In conclusion, appropriate in vivo and in vitro models were evaluated and used for metabolic studies of AAS. The findings presented in this thesis hold significant implications not only for doping control analysis, but also for other scientific areas where a comprehensive understanding of steroid metabolism is highly relevant, such as endocrinology, forensic toxicology, and safety assessment in drug development.