Human hepatocytes are a physiologically relevant tool useful in evaluating liver-related pharmacokinetics, including non-cytochrome P-450 (CYP) metabolism, due to their broad spectrum of metabolic enzyme activity. To verify the usefulness of human hepatocytes in evaluating non-CYP metabolism for drug discovery, we compared intrinsic clearance values (CL_int) in freshly isolated and cryopreserved hepatocytes using 14 compounds primarily metabolized by non-CYP enzymes, including UDP-glucuronosyltransferase, carbonyl/aldo-keto reductase, aldehyde oxidase, flavin-containing monooxygenase, and monoamineoxidase. Cryopreservation resulted in a >20% reduction (maximum: 50%) in CL_int in 7/14 compounds (statistically significant for 5 compounds) on comparing CL_int values in freshly isolated and cryopreserved hepatocytes from the same donors (n = 4). However, the number of compounds with >20% CL_int reduction decreased to 3 on comparing average of CL_int values including un-matched donors (dolasetron: −27%, naltorexone: −32%, and phthalazine: −48%; statistically significant for phthalazine, n = 6–11). These findings suggest that fresh hepatocytes are useful in evaluating intact non-CYP enzyme activities. However, we must note that the reduction in CL_int by cryopreservation could be rendered negligible if high-activity lots are selected for assay. We therefore recommend using cryopreserved hepatocytes for large-scale screening for non-CYP metabolism in drug discovery research considering the advantages in usability with cryopreserved hepatocytes.

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