The CYP3A4 pitfall in cytochrome P450 identification: when human liver microsomes and recombinant enzymes misidentify the main drug-metabolising enzymes

In recognition of the potential adverse clinical effects pharmacokinetic drug-drug interactions (DDIs) can pose, regulatory authorities provide detailed guidance on multi-step in vitro methods to characterise a new drug-entity’s risk for DDIs. Identification of the main enzyme(s) responsible for metabolism is a key first step and, for drug-metabolising cytochrome P450s (CYPs); human hepatocytes (HH), liver microsomes (HLM) and recombinant enzymes are considered complementary in vitro models. However, HH may exhibit lower intrinsic CYP3A4 activities relative to HLM that have been difficult to explain. Here, using savolitinib, a cMET inhibitor, we will show that differences in CYP3A4 activities between in vitro models can manifest as misidentification of CYP3A4 as the main metabolising enzyme by HLM and recombinant enzymes while HH, consistent with clinical data, correctly identify CYP1A2. Further examples supported by clinical data confirm a systemic misidentification of CYP3A4 by HLM and recombinant enzymes leading to intriguing insights into a possible underlying mechanism. Altogether, the data supports a new approach to CYP phenotyping that utilises HH as the main model with complete exclusion of HLM. Recombinant enzymes may be considered as a refining tool that, as exemplified by savolitinib, serves to confirm the misidentification because recombinant CYP3A4 does not form the major in vivo savolitinib-metabolite despite displaying over ten-fold higher savolitinib intrinsic clearance relative to other CYPs.

1. Facilitate re-evaluation of historical data generated predominantly in human liver microsomes and recombinant enzymes for inaccurate CYP phenotyping. 

2. Enable regulators to start using this information when evaluating/recommending additional work for DDI sections of new drug applications. 

3. Appreciate that the use of HH as the main CYP reaction phenotyping tool relies heavily on the use of appropriate probes and selective inhibitors whose drawbacks due to overlap with other CYPs should be well-understood. 

 4. Gain an appreciation of the challenges inhibitor/probe non-selectivity pose, thereby ensuring appropriate controls which will likely be compound-specific are included. 

Published with Communications Biology.