A practical and direct comparison of intrinsic metabolic clearance of several non-CYP enzyme substrates in freshly isolated and cryopreserved hepatocytes.
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.
Astellas Research Institute of America LLC. takako-furukawa@jp.astellas.com
Takafumi
T
Akabane
Nicolas
N
Gerst
Yoichi
Y
Naritomi
Jeffrey N
JN
Masters
Kouichi
K
Tamura
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D042931:Aldehyde Oxidase / Q000378:metabolism; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D002470:Cell Survival / Q000502:physiology; D015925:Cryopreservation / Q000379:methods; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004798:Enzymes / Q000378:metabolism*; D022781:Hepatocytes / Q000201:enzymology*; D006801:Humans; D008657:Metabolic Clearance Rate / Q000502:physiology; D004364:Pharmaceutical Preparations / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity / Q000502:physiology
D004798:Enzymes; D004364:Pharmaceutical Preparations; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; D042931:Aldehyde Oxidase