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Preparative‐Scale Production of Testosterone Metabolites by Human Liver Cytochrome P450 Enzyme 3A4
Author(s) -
Fessner Nico D.,
Srdič Matic,
Weber Hansjörg,
Schmid Christian,
Schönauer David,
Schwaneberg Ulrich,
Glieder Anton
Publication year - 2020
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.202000251
Subject(s) - chemistry , pichia pastoris , cytochrome p450 , metabolite , biotransformation , cyp3a4 , enzyme , biochemistry , drug metabolism , isozyme , chromatography , recombinant dna , gene
Just like the drugs themselves, their metabolites have to be evaluated to succeed in a drug development and approval process. It is therefore essential to be able to predict drug metabolism and to synthesise sufficient metabolite quantities for further pharmacological testing. This study evaluates the possibility of using in vitro biotransformations to solve both these challenges in the case of testosterone as a representative component for steroids. The application of cells of Pichia pastoris with expressed membrane‐associated human liver cytochrome P450 enzyme (P450) 3A4 in two cycles of a preparative‐scale bioreactor experiment enabled the isolation of the common metabolites 6β‐hydroxytestosterone and 6β‐hydroxyandrostenedione on a 100 mg scale. Side‐product formation caused by enzymes intrinsic to P. pastoris was reduced. In addition more polar testosterone metabolites formed by a P450 3A4‐catalysed bioconversion, than the known mono‐hydroxylated ones, are reported and 6‐dehydro‐15β‐hydroxytestosterone as well as the di‐hydroxylated steroids 6β,16β‐dihydroxytestosterone, 6β,17β‐dihydroxy‐4‐androstene‐3,16‐dione and 6β,12β‐dihydroxyandrostenedione were isolated and verified by NMR analysis. Their respective biological significance remains to be investigated. Whole‐cell P450 catalysts expressed in P. pastoris qualify as a tool for the preparative‐scale synthesis of human metabolites. Biotransformation processes in combination with standard chemical procedures allow the isolation and characterisation even of minor drug metabolite products.