P123 MetMax™ Pooled Donor Human Hepatocytes: A Novel In Vitro System for the Evaluation of Hepatic Drug Metabolism

David Ho , In Vitro ADMET Laboratories Inc., Malden, MA
Nick Ring , In Vitro ADMET Laboratories Inc., Malden, MA
Kirsten Amaral , In Vitro ADMET Laboratories Inc., Malden, MA
Albert P. Li , In Vitro ADMET Laboratories Inc., Columbia, MD
Cryopreserved human hepatocytes are used routinely for in vitro drug metabolism studies. The use of cryopreserved human hepatocytes requires thawing of the cells, recovery of the cells by centrifugation, quantification of cell numbers and viability, and dilution to the optimal cell concentration with an appropriate culture medium before initiation of the study. We described here a novel hepatocyte experimental system, MetMax™ pooled donor human hepatocytes (patent pending), which can be used directly after thawing by adding to equal volume of metabolism substrates for the evaluation of in vitro hepatic metabolism. Furthermore, MetMax™ hepatocytes can be stored at -80 deg. C in lieu of liquid nitrogen which is required for cryopreserved human hepatocytes. In this study, we compared MetMax™ and cryopreserved human hepatocytes in the key xenobiotic drug metabolizing enzyme activities. Human hepatocytes from ten donors (5 males and 5 females) were thawed, combined (pooled), and re-cryopreserved using routine procedures as “normal” or as MetMax™ pooled donor human hepatocytes. Both types of pooled donor hepatocytes were evaluated for the following drug metabolizing enzyme activities: phenacetin hydroxylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), bupropion hydroxylation (CYP2B6), paclitaxel 6α-Hydroxylation (CYP2C8), diclofenac 4-hydroxylation (CYP2C9), S-mephenytoin 4-hydroxylation (CYP2C19), dextromethorphan hydroxylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), midazolam 1’-hydroxylation (CYP3A4), testosterone 6β-hydroxylation (CYP3A4), 7-hydroxycoumarin glucuronidation (UGT) and sulfation (SULT), benzydamine N-oxidation (FMO), and kynuramine 4-hydroxylation (MAO). Time-dependent increases in metabolite formation was observed for both types of hepatocytes upon to the longest incubation time of 4 hrs. The specific activities calculated from the 30 min. incubation duration for the “normal” and MetMax™ human hepatocytes for each of the pathways were, respectively, CYP1A2: 58.5 (normal), 105.5 (MetMax™); CYP2A6: 102.2, 180.7; CYP2B6: 29.4, 42.9; CYP2C8: 141.1, 184.6; CYP2C9: 97.6, 182.4; CYP2C19: 19.7, 11.2; CYP2D6: 17.3, 14.8, CYP2E1: 47.2, 62.4; CYP3A-midazolam: 15.7, 23.6; CYP3A4-testosterone: 204.0, 323.8; UGT: 537.0, 1459.2; SULT: 20.0, 40.7; FMO: 761.3, 1286; and MAO: 1893, 1690. We observed that the MetMax™ hepatocytes yielded significantly lower inter-experimental variations than the “normal” hepatocytes. The results suggest that the MetMax™ hepatocytes represent an in vitro experimental system similar to cryopreserved hepatocytes for the evaluation of hepatic drug metabolism. The simplicity of the application of MetMax™ hepatocytes (storage in -80 deg. C; thaw-and-use without centrifugation and cell counting) and the robust drug metabolizing enzyme activities represent the attractive features of this novel experimental system, with potential applications include hepatic metabolic stability screening, metabolite profiling, metabolic pathway identification, and enzyme inhibition studies.