Changes in Alprazolam Metabolism by CYP3A43 Mutants

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 alpha-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 alpha-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 alpha-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.

Automated summary

Alprazolam, a commonly used drug for managing anxiety disorders, is metabolized by CYP3A enzymes. This study compared the metabolism of alprazolam by all four human CYP3A enzymes and found that CYP3A4 and CYP3A5 have the highest production rates of 4-hydroxyalprazolam, while CYP3A5 alone is the major producer of alpha-hydroxyalprazolam. Computational simulations explained the difference in preferred oxidation sites between CYP3A5 and CYP3A43. Unexpectedly, three CYP3A43 mutants produced a different metabolite, 5-N-O alprazolam. The mutants also showed higher production rates of 4-hydroxyalprazolam compared to the wild-type, while the wild-type did not produce 5-N-O alprazolam.