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<p>NADPH-P450 oxidoreductase (POR) is the accepted redox-partner for drug-metabolising cytochrome P450s (CYPs) over NADH-cytochrome <i>b</i><sub><i>5</i></sub> reductase (Cyt<i>b</i><sub><i>5</i></sub>R). Accordingly, POR-centric NADPH-supplemented human liver microsomes (HLM) and recombinant CYP-POR systems complement human hepatocytes (HH) as drug-metabolism models. However, HH may exhibit inexplicably lower CYP activities relative to NADPH-HLM, particularly for CYP3A4-substrates. Here, we show the phenomenon can manifest as disparate CYP phenotyping in which NADPH-HLM and recombinant CYP-POR systems incorrectly identify CYP3A4 while HH accurately assign the main-metabolising CYP exemplified by CYP1A2 for savolitinib. Mechanistically, we serendipitously discover that HH CYP3A4-mediated midazolam-metabolism is increased by gefitinib and find that this can only be recapitulated in non-canonical Cyt<i>b</i><sub><i>5</i></sub>R-dependent NADH-HLM and recombinant CYP3A4-Cyt<i>b</i><sub><i>5</i></sub>R. We conclude that Cyt<i>b</i><sub><i>5</i></sub>R is important for HH-CYP3A4 and show HH-consistent CYP3A4-activities in NADH-HLM. Imaging NADH/NADPH in hepatocytes shows equivalent concentrations suggesting CYP redox-partnerships are cofactor-independent and likely influenced by protein-protein interactions as mimicking the dense-intracellular protein using albumin recapitulates HH savolitinib-metabolism in HLM.</p>

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A mismatch in enzyme-redox partnerships underlies divergent cytochrome P450 activities between human hepatocytes and microsomes

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月6日

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  • Tashinga E. Bapiro,
  • Scott Martin,
  • Thomas S. Blacker,
  • Stephen D. Wilkinson,
  • Alexandra L. Orton,
  • Niresh Hariparsad,
  • Rhys D. O. Jones,
  • Michael R. Duchen,
  • Stephanie Harlfinger

摘要

NADPH-P450 oxidoreductase (POR) is the accepted redox-partner for drug-metabolising cytochrome P450s (CYPs) over NADH-cytochrome b5 reductase (Cytb5R). Accordingly, POR-centric NADPH-supplemented human liver microsomes (HLM) and recombinant CYP-POR systems complement human hepatocytes (HH) as drug-metabolism models. However, HH may exhibit inexplicably lower CYP activities relative to NADPH-HLM, particularly for CYP3A4-substrates. Here, we show the phenomenon can manifest as disparate CYP phenotyping in which NADPH-HLM and recombinant CYP-POR systems incorrectly identify CYP3A4 while HH accurately assign the main-metabolising CYP exemplified by CYP1A2 for savolitinib. Mechanistically, we serendipitously discover that HH CYP3A4-mediated midazolam-metabolism is increased by gefitinib and find that this can only be recapitulated in non-canonical Cytb5R-dependent NADH-HLM and recombinant CYP3A4-Cytb5R. We conclude that Cytb5R is important for HH-CYP3A4 and show HH-consistent CYP3A4-activities in NADH-HLM. Imaging NADH/NADPH in hepatocytes shows equivalent concentrations suggesting CYP redox-partnerships are cofactor-independent and likely influenced by protein-protein interactions as mimicking the dense-intracellular protein using albumin recapitulates HH savolitinib-metabolism in HLM.