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Cryopreserved human hepatocytes as alternative in vitro model for cytochrome P450 induction studies

  • Martha GarciaEmail author
  • Joseph Rager
  • Qing Wang
  • Robert Strab
  • Ismael J. Hidalgo
  • Albert Owen
  • Jibin Li

Summary

Induction of cytochrome P450 (CYP) by drugs is one of major concerns for drug-drug interactions. Thus, the assessment of CYP induction by novel compounds is a vital component in the drug discovery and development processes. Primary human hepatocytes are the preferred in vitro model for predicting CYP induction in vivo. However, their use is hampered by the erratic supply of human tissue and donor-to-donor variability. Although cryopreserved hepatocytes have been recommended for short-term applications in suspension, their use in studies on induction of enzyme activity has been limited because of poor attachment and response to enzyme inducers. In this study, we report culture conditions that allowed the attachment of cryopreserved human hepatocytes and responsiveness to CYP inducers. We evaluated the inducibility of CYP1A1/2 and CYP3A4 enzymes in cryopreserved hepatocytes from three human donors. Cryopreserved human hepatocytes were cultured in serum-free medium for 4 d. They exhibited normal morphology and measurable viability as evaluated by the reduction of tetrazolium salts (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) by cellular dehydrogenases. Treatment with β-naphthoflavone (10 μM) for 3 d increased ethoxyresorufin-O-deethylase activity (CYP1A1/2) by 6- to 11-fold over untreated cultures and increased CYP1A2 messenger ribonucleic acid (mRNA) expression by three- to eightfold. Similarly, treatment of cryopreserved human hepatocytes with rifampicin (25 μM) for 3 d increased testosterone 6β-hydroxylase activity (CYP3A4) by five- to eightfold over untreated cultures and increased CYP3A4 mRNA expression by four- to eightfold. The results suggest that cryopreserved human hepatocytes can be a suitable in vitro model for evaluating xenobiotics as inducers of CYP1A1/2 and CYP3A4 enzymes.

Key words

CYP1A1/2 CYP3A4 drug-drug interactions drug-screening model 

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References

  1. Bapiro, T.; Anderson, T. B.; Otter, C.; Hasler, J. A.; Masimirembwa, C. M. Cytochrome P450 1A1/2 induction by antiparasitic drugs: dose-dependent increase in ethoxyresorufin O-deethylase activity and mRNA caused by quinine, primaquine and albendazole in HepG2 cells. Eur. J. Clin. Pharmacol. 58:537–542; 2002.PubMedCrossRefGoogle Scholar
  2. Bjornsson, T. D.; Callaghan, J. T.; Einolf, H. J., et al. The conduct of in vitro and in vivo drug-drug interaction studies: a pharmaceutical research and manufacturers of America (PhRMA) perspective. Drug Metab. Dispos. 31:815–832; 2003.PubMedCrossRefGoogle Scholar
  3. Chr.'t Hoen, P. A.; Commandeur, J. N. M.; Vermeulen, N. P. E.; Van Berkel, T. J. C.; Bijsterbosch, M. K. Selective induction of cytochrome P450 3A1 by dexamethasone in cultured rat hepatocytes. Biochem. Pharmacol. 60:1509–1518; 2000.CrossRefGoogle Scholar
  4. de Sousa, G.; Langouët, S.; Nicolas, F.; Lorenzon, G.; Placidi, M.; Rahmani, R.; Guillouzo, A. Increase of cytochrome P-4501A and glutathione transferase transcripts in cultured hepatocytes from dogs, monkeys, and humans after cryopreservation. Cell Biol. Toxicol. 12:351–358; 1996.PubMedCrossRefGoogle Scholar
  5. Donato, M. T.; Castell, J. V. Strategies and molecular probes to investigate the role of cytochrome P450 in drug metabolism. Clin. Pharmacokinet. 42:154–178; 2003.CrossRefGoogle Scholar
  6. Donato, M. T.; Gómez-Lechón, M. J.; Castell, J. V. A microassay for measuring cytochrome P4501A1 and P450IIB1 activities in intact human and rat hepatocytes cultured on 96-well plates. Anal. Biochem. 213:29–33; 1993.PubMedCrossRefGoogle Scholar
  7. Gómez-Lechón, M. J.; Donato, T.; Jover, R.; Rodriguez, C.; Ponsoda, X.; Glaise, D.; Castell, J. V.; Guguen-Guillouzo, C. Expression and induction of a large set of drug-metabolizing enzymes by the highly differentiated human hepatoma cell line BC2. Eur. J. Biochem. 268:1448–1459; 2001.PubMedCrossRefGoogle Scholar
  8. Hengstler, J. G.; Utesch, D.; Steinberg, P., et al. Cryopreserved primary hepatocytes as a constantly available in vitro model for the evaluation of human and animal drug metabolism and enzyme induction. Drug Metab. Rev. 32:81–118; 2000.PubMedCrossRefGoogle Scholar
  9. Hewitt, N. J.; Büring, K.-U.; Dasenbrock, J.; Haunschild, J.; Ladstetter, B.; Utesch, D. Studies comparing in vivo:in vitro metabolism of three pharmaceutical compounds in rat, dog, monkey and human using cryopreserved hepatocytes, microsomes and collagen gel immobilized hepatocyte cultures. Drug Metab. Dispos. 29:1042–1050; 2001.PubMedGoogle Scholar
  10. Li, A. P.; Lu, C.; Brent, J. A.; Pham, C.; Fackett, A.; Ruegg, C. E.; Silber, P. M. Cryopreserved human hepatocytes: characterization of drugmetabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential. Chem. Biol. Interact. 121:17–35; 1999.PubMedCrossRefGoogle Scholar
  11. Madan, A.; Dehaan, R.; Mudra, D.; Carroll, K.; LeCluyse, E.; Parkinson, A. Effect of cryopreservation on cytochrome P-450 enzyme induction in cultured rat hepatocytes. Drug Metab. Dispos. 27:327–335; 1999.PubMedGoogle Scholar
  12. Madan, A.; Graham, R. A.; Carroll, K. M., et al. Effects of prototypical microsomal enzyme inducers on cytochrome P450 expression in cultured human hepatocytes. Drug Metab. Dispos. 31:421–431; 2003.PubMedCrossRefGoogle Scholar
  13. Marshall, N. J.; Goodwin, C. J.; Holt, S. J. A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function. Growth Regul. 5:69–84; 1995.PubMedGoogle Scholar
  14. Raucy, J. L. Regulation of CYP3A4 expression in human hepatocytes by pharmaceuticals and natural products. Drug Metab. Dispos. 31:533–539; 2003.PubMedCrossRefGoogle Scholar
  15. Rodriguez-Antona, C.; Donato, M. T.; Boobis, A.; Edwards, R. J.; Watts, P. S.; Castell, J. V.; Gómez-Lechón, M.-J.. Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells. Xenobiotica 32:505–520; 2002.PubMedCrossRefGoogle Scholar
  16. Shibata, Y.; Takahashi, H.; Chiba, M.; Ishii, Y. Prediction of hepatic clearance and availability by cryopreserved human hepatocytes: an application of serum incubation method. Drug Metab. Dispos. 30:892–896; 2002.PubMedCrossRefGoogle Scholar
  17. Silva, J. M.; Day, S. H.; Nicoll-Griffith, D. A. Induction of cytochrome-P450 in cryopreserved rat and human hepatocytes. Chem. Biol. Interact. 121:49–63; 1999.PubMedCrossRefGoogle Scholar
  18. Silva, J. M.; Morin, P. E.; Day, S. H.; Kennedy, B. P.; Payette, P.; Rushmore, T.; Yerger, J. A.; Nicoll-Griffith, D. A. Refinement of an in vitro cell model for cytochrome P450 induction. Drug Metab. Dispos. 26:490–496; 1998.PubMedGoogle Scholar

Copyright information

© Society for In Vitro Biology 2003

Authors and Affiliations

  • Martha Garcia
    • 1
    Email author
  • Joseph Rager
    • 1
  • Qing Wang
    • 1
  • Robert Strab
    • 1
  • Ismael J. Hidalgo
    • 1
  • Albert Owen
    • 1
  • Jibin Li
    • 1
  1. 1.Absorption SystemsLP, Oaklands Corporate CenterExton

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