Alprazolam is a triazolobenzodiazepine which is most commonly used in the short-term management of anxiety disorders, often in combination with antipsychotics. The four human members of the CYP3A subfamily are mainly responsible for its metabolism, which yields the main metabolites 4-hydroxyalprazolam and α-hydroxyalprazolam. We performed a comparison of alprazolam metabolism by all four CYP3A enzymes upon recombinant expression in the fission yeast Schizosaccharomyces pombe. CYP3A4 and CYP3A5 show the highest 4-hydroxyalprazolam production rates, while CYP3A5 alone is the major producer of α-hydroxyalprazolam. For both metabolites, CYP3A7 and CYP3A43 show lower activities. Computational simulations rationalize the difference in preferred oxidation sites observed between the exemplary enzymes CYP3A5 and CYP3A43. Investigations of the alprazolam metabolites formed by three previously described CYP3A43 mutants (L293P, T409R, and P340A) unexpectedly revealed that they produce 4-hydroxy-, but not α-hydroxyalprazolam. Instead, they all also make a different metabolite, which is 5-N-O alprazolam. With respect to 4-hydroxyalprazolam, the mutants showed fourfold (T409R) to sixfold (L293P and P340A) higher production rates compared to the wild-type (CYP3A43.1). In the case of 5-N-O alprazolam, the production rates were similar for the three mutants, while no formation of this metabolite was found in the wild-type incubation.
The detection of a putative 18‐methyl‐19‐nortestosterone metabolite in a forensic bodybuilder's urine sample collected as part of a criminal proceeding has triggered a follow‐up investigation. Four different dietary supplements in the possession of the suspect were examined with regard to possible precursor steroids. This led to the detection of the declared ingredient methoxydienone, which was confirmed by both, GC–MSMS and LC‐HRMSMS. As neither 18‐methyl‐testosterone, nor 18‐methyl‐19‐nortestosterone were detectable in the supplements, the possibility that the metabolite originates from methoxydienone was investigated. For this purpose, the metabolic fate of methoxydienone was studied in vitro using human HepG2 cells and in vivo by a single oral administration. While the 18‐methyl‐19‐nortestosterone metabolite was not generated by HepG2 cells incubated with methoxydienone, it was observed in the urine samples collected at 2, 6, 10 and 24 h after methoxydienone administration. Moreover, the potential binding of methoxydienone as ligand to the human androgen receptor was modelled in silico in comparison with 18‐methylnandrolone, for which androgen receptor activation had been shown in an in vitro approach before. In conclusion, we could ascribe the presence of the 18‐methyl‐19‐nortestosterone metabolite in a forensic urine sample to originate from methoxydienone present in dietary supplements. Methoxydienone was observed to slowly degrade by demethylation of the methoxy substituent in liquid solutions. While no compound‐specific intermediates were identified that allowed differentiation from other 18‐methyl steroids, the 18‐methyl‐19‐nortestosterone metabolite proved to be a suitable marker for reliable detection in doping analysis.
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