A practical and direct comparison of intrinsic metabolic clearance of several non-CYP enzyme substrates in freshly isolated and cryopreserved hepatocytes

Drug Metab Pharmacokinet. 2012;27(2):181-91. doi: 10.2133/dmpk.dmpk-11-rg-097. Epub 2011 Oct 25.


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.

Publication types

  • Comparative Study

MeSH terms

  • Alcohol Oxidoreductases / metabolism
  • Aldehyde Oxidase / metabolism
  • Aldehyde Reductase
  • Aldo-Keto Reductases
  • Cell Survival / physiology
  • Cryopreservation* / methods
  • Drug Evaluation, Preclinical / methods
  • Enzymes / metabolism*
  • Hepatocytes / enzymology*
  • Humans
  • Metabolic Clearance Rate / physiology
  • Pharmaceutical Preparations / chemistry
  • Pharmaceutical Preparations / metabolism*
  • Substrate Specificity / physiology


  • Enzymes
  • Pharmaceutical Preparations
  • Alcohol Oxidoreductases
  • Aldo-Keto Reductases
  • Aldehyde Reductase
  • Aldehyde Oxidase