Comparison of fresh and cryopreserved rat hepatocyte suspensions for the prediction of in vitro intrinsic clearance.

Freshly isolated hepatocytes are currently regarded as the most superior in vitro model for use in prediction studies, in particular to provide estimates of in vivo intrinsic clearance (CL(int)). However, due to their loss of viability within 4 h and a decrease in cytochrome P450-dependent metabolism upon culture, newer cellular models are being developed. Cryopreserved hepatocytes have several potential advantages, but to date evaluation of the utility of this model for estimating in vitro CL(int) has been limited to the substrate depletion approach. We have incubated eight compounds with suspensions of freshly isolated and cryopreserved rat hepatocytes and obtained in vitro CL(int) via metabolite formation kinetics (for 14 pathways). A substantial range of in vitro CL(int) values (0.1-98 microl/min/10(6)cells) was obtained in both models, and the freshly isolated suspension data were in good agreement with the literature. Cryopreserved suspensions were able to give a comparable estimation (within 2-fold) of in vitro CL(int) to fresh cells for six pathways, namely tolbutamide, three diazepam metabolites, propranolol, and 7-hydroxylation of warfarin. A higher estimation of in vitro CL(int) was obtained for the three other metabolites of warfarin due to a decrease in the K(M) values. Lower estimations of in vitro CL(int) were observed for four compounds (six pathways), and this was particularly pronounced (4-16%) for pathways showing atypical Michaelis-Menten kinetic profiles (dextromethorphan, nordiazepam) but less so (25-45%) for pathways showing biphasic Michaelis-Menten kinetics (7-ethoxycoumarin and phenytoin).

[1]  J. Houston,et al.  Drug metabolizing capacity in vitro and in vivo--II. Correlations between hepatic microsomal monooxygenase markers in phenobarbital-induced rats. , 1990, Biochemical pharmacology.

[2]  J B Houston,et al.  Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance. , 1994, Biochemical pharmacology.

[3]  D. Friend,et al.  HIGH-YIELD PREPARATION OF ISOLATED RAT LIVER PARENCHYMAL CELLS , 1969, The Journal of cell biology.

[4]  Yuichi Sugiyama,et al.  Utility of hepatocytes in predicting drug metabolism: comparison of hepatic intrinsic clearance in rats and humans in vivo and in vitro. , 2003, Drug metabolism and disposition: the biological fate of chemicals.

[5]  F. Oesch,et al.  Drug metabolizing capacity of cryopreserved human, rat, and mouse liver parenchymal cells in suspension. , 1999, Drug metabolism and disposition: the biological fate of chemicals.

[6]  J B Houston,et al.  Prediction of hepatic clearance from microsomes, hepatocytes, and liver slices. , 1997, Drug metabolism reviews.

[7]  D. Jack.  Handbook of Clinical Pharmacokinetic Data , 1992, Palgrave Macmillan UK.

[8]  Masato Chiba,et al.  Prediction of hepatic clearance and availability by cryopreserved human hepatocytes: an application of serum incubation method. , 2002, Drug metabolism and disposition: the biological fate of chemicals.

[9]  Yau Yi Lau,et al.  Development of a novel in vitro model to predict hepatic clearance using fresh, cryopreserved, and sandwich-cultured hepatocytes. , 2002, Drug metabolism and disposition: the biological fate of chemicals.

[10]  H. Thijssen,et al.  Comparison of the rat liver microsomal metabolism of the enantiomers of warfarin and 4'-nitrowarfarin (acenocoumarol). , 1991, Xenobiotica; the fate of foreign compounds in biological systems.

[11]  A. Yacobi,et al.  Protein binding of warfarin enantiomers in serum of humans and rats , 1977, Journal of Pharmacokinetics and Biopharmaceutics.

[12]  P Skett,et al.  Problems in using isolated and cultured hepatocytes for xenobiotic metabolism/metabolism-based toxicity testing-Solutions? , 1994, Toxicology in vitro : an international journal published in association with BIBRA.

[13]  J. Houston,et al.  Prediction of in vivo disposition from in vitro systems: clearance of phenytoin and tolbutamide using rat hepatic microsomal and hepatocyte data. , 1995, The Journal of pharmacology and experimental therapeutics.

[14]  G. J. Conard,et al.  Binding of 5,5-diphenylhydantoin and its major metabolite to human and rat plasma proteins. , 1971, Journal of pharmaceutical sciences.

[15]  A. Li,et al.  Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential. , 1999, Chemico-biological interactions.

[16]  J. Houston,et al.  Sigmoidal kinetics of CYP3A substrates: an approach for scaling dextromethorphan metabolism in hepatic microsomes and isolated hepatocytes to predict in vivo clearance in rat. , 1999, The Journal of pharmacology and experimental therapeutics.

[17]  J. Villeneuve,et al.  Propranolol disposition in the rat: variation in hepatic extraction with unbound drug fraction. , 1992, Journal of pharmaceutical sciences.

[18]  J. Houston,et al.  In vivo clearance of ethoxycoumarin and its prediction from In vitro systems. Use Of drug depletion and metabolite formation methods in hepatic microsomes and isolated hepatocytes. , 1998, Drug metabolism and disposition: the biological fate of chemicals.

[19]  R. Branch,et al.  Intact hepatocyte theory of impaired drug metabolism in experimental cirrhosis in the rat. , 1979, Gastroenterology.

[20]  B. Burchell,et al.  In vitro analysis of human drug glucuronidation and prediction of in vivo metabolic clearance. , 2002, The Journal of pharmacology and experimental therapeutics.

[21]  J. Houston,et al.  Kinetics of diazepam metabolism in rat hepatic microsomes and hepatocytes and their use in predicting in vivo hepatic clearance. , 1995, Xenobiotica; the fate of foreign compounds in biological systems.

[22]  K H Antonin,et al.  Pharmacokinetics and plasma binding of diazepam in man, dog, rabbit, guinea pig and rat. , 1976, The Journal of pharmacology and experimental therapeutics.

[23]  J B Houston,et al.  In vitro-in vivo scaling of CYP kinetic data not consistent with the classical Michaelis-Menten model. , 2000, Drug metabolism and disposition: the biological fate of chemicals.