Received for publication June 27, 2006.
Revised November 2, 2006.
Accepted for publication November 17, 2006.

Evaluation of Cryopreserved Human Hepatocytes as an Alternative In Vitro System to Microsomes for the Prediction of Metabolic Clearance

Abstract

Human liver microsomes have typically resulted in marked under-prediction of in vivo human intrinsic clearance (CL_int), therefore the utility of cryopreserved hepatocytes as an alternative in vitro system has become an important issue. In this study, 10 compounds (tolbutamide, diclofenac, S-warfarin, S-mephenytoin, dextromethorphan, bufuralol, quinidine, nifedipine, testosterone and terfenadine) were selected as substrate probes for CYP2C9, 2C19, 2D6 and 3A4, and the kinetics of metabolite formation (n=14 pathways) were investigated in three individual lots of cryopreserved hepatocytes, and in a pool of human liver microsomes. For the majority of the compounds, lower unbound K_M or S₅₀ values were observed in hepatocytes compared to microsomes, on average by 50% over a 200-fold range (0.5-140µM). Expressed on an equivalent liver weight basis, a good correlation between microsomal and hepatocyte V_max values was observed for most pathways over 5-orders of magnitude (0.16-216 nmoles/min/g liver). Unbound hepatocyte CL_int (CL_int,u) when scaled to the whole liver, (range 0.38-4000 ml/min/kg) were on average 2.5-fold higher than microsomal CL_int,u values, with the exception of tolbutamide and diclofenac where lower hepatocellular CL_int,u values were observed. Hepatocyte predicted CL_int values were compared with human in vivo CL_int values and in order to supplement our data, in vitro data from cryopreserved hepatocytes were collated from four other published sources. These data show that for 41 drugs, there is on average a 4.2-fold under-prediction of the in vivo CL_int using cryopreserved hepatocytes, representing a significant reduction in prediction bias compared to human microsomes.

Key words: drug clearance, hepatocytes, in vitro-in vivo prediction, in vitro-in vivo scaling, liver microsomes

This article has been cited by other articles:

				X. Wei, G. Dai, Z. Liu, H. Cheng, Z. Xie, R. Klisovic, G. Marcucci, and K. K. Chan Enzyme Kinetics of GTI-2040, a Phosphorothioate Oligonucleotide Targeting Ribonucleotide Reductase Drug Metab. Dispos., November 1, 2008; 36(11): 2227 - 2233. [Abstract] [Full Text] [PDF]

				J. B. Dennison, M. A. Mohutsky, R. J. Barbuch, S. A. Wrighton, and S. D. Hall Apparent High CYP3A5 Expression Is Required for Significant Metabolism of Vincristine by Human Cryopreserved Hepatocytes J. Pharmacol. Exp. Ther., October 1, 2008; 327(1): 248 - 257. [Abstract] [Full Text] [PDF]

				P. J. Kilford, M. Gertz, J. B. Houston, and A. Galetin Hepatocellular Binding of Drugs: Correction for Unbound Fraction in Hepatocyte Incubations Using Microsomal Binding or Drug Lipophilicity Data Drug Metab. Dispos., July 1, 2008; 36(7): 1194 - 1197. [Abstract] [Full Text] [PDF]

				A. J. Parker and J. B. Houston Rate-Limiting Steps in Hepatic Drug Clearance: Comparison of Hepatocellular Uptake and Metabolism with Microsomal Metabolism of Saquinavir, Nelfinavir, and Ritonavir Drug Metab. Dispos., July 1, 2008; 36(7): 1375 - 1384. [Abstract] [Full Text] [PDF]

				A. Rowland, D. J. Elliot, K. M. Knights, P. I. Mackenzie, and J. O. Miners The "Albumin Effect" and in Vitro-in Vivo Extrapolation: Sequestration of Long-Chain Unsaturated Fatty Acids Enhances Phenytoin Hydroxylation by Human Liver Microsomal and Recombinant Cytochrome P450 2C9 Drug Metab. Dispos., May 1, 2008; 36(5): 870 - 877. [Abstract] [Full Text] [PDF]

				Cheng Li, Tongtong Liu, Xiaoming Cui, A. S. Uss, and K.-C. Cheng Development of In Vitro Pharmacokinetic Screens Using Caco-2, Human Hepatocyte, and Caco-2/Human Hepatocyte Hybrid Systems for the Prediction of Oral Bioavailability in Humans J Biomol Screen, December 1, 2007; 12(8): 1084 - 1091. [Abstract] [PDF]

				H. S. Brown, A. Chadwick, and J. B. Houston Use of Isolated Hepatocyte Preparations for Cytochrome P450 Inhibition Studies: Comparison with Microsomes for Ki Determination Drug Metab. Dispos., November 1, 2007; 35(11): 2119 - 2126. [Abstract] [Full Text] [PDF]

				B. Karanam, M. Madeira, S. Bradley, L. Wenning, R. Desai, E. Soli, D. Schenk, A. Jones, B. Dean, G. Doss, et al. Absorption, Metabolism, and Excretion of [14C]MK-0524, a Prostaglandin D2 Receptor Antagonist, in Humans Drug Metab. Dispos., July 1, 2007; 35(7): 1196 - 1202. [Abstract] [Full Text] [PDF]