Estimation of Fraction Metabolized by Cytochrome P450 Enzymes Using Long-Term Cocultured Human Hepatocytes

Estimation of the fraction of a drug metabolized by individual hepatic CYP enzymes relative to hepatic metabolism (fm,CYP) or total clearance h as been challenging for low turnover compounds due to insufficient resolution of the intrinsic clearance (CLint) measurement in vitro and difficulties in quantifying the formation of low abundance metabolites. To overcome this gap, inhibition of drug depletion or selective metabolite formation for 7 marker CYP substrates was investigated using chemical inhibitors and a micro-patterned hepatocyte coculture system (HepatoPac). The use of 3 μM itraconazole was successfully validated for estimation of fm,CYP3A4 by demonstration of fm values within a 2-fold of in vivo estimates for 10 out of 13 CYP3A4 substrates in a reference set of marketed drugs. Other CYP3A4 inhibitors (ketoconazole and posaconazole) were not optimal for estimation of fm,CYP3A4 for low turnover compounds due to their high CLint. The current study also demonstrated that selective inhibition sufficient for fm calculation was achieved by inhibitors of CYP1A2 (20 μM furafylline), CYP2C8 (40 μM montelukast), CYP2C9 (40 μM sulfaphenazole), CYP2C19 [3 μM (-)N-3-benzyl-phenobarbital], and CYP2D6 (5 μM quinidine). Good estimation of fm,CYP2B6 was not possible in this study due to the poor selectivity of the tested inhibitor (20 μM ticlopidine). The approach verified in this study can result in an improved fm estimation that is aligned with the regulatory agencies' guidance and can support a victim drug-drug interaction risk assessment strategy for low clearance discovery and development drug candidates. SIGNIFICANCE STATEMENT: Successful qualification of a chemical inhibition assay for estimation of fraction metabolized requires chemical inhibitors that retain sufficient unbound concentrations over time in the incubates. The current cocultured hepatocyte assay enabled estimation of fraction metabolized, especially by CYP3A4, during the drug discovery phase where metabolite quantification methods may not be available. The method enables the assessment of pharmacokinetic variability and victim drug-drug interaction risks due to enzyme polymorphism or inhibition/induction with more confidence, especially for low clearance drug candidates.