Abstract
The liver is the major drug-metabolizing organ which is responsible for the clearance of approximately 60% of drugs available on the market through cytochrome P450 (CYP)-mediated metabolism. Therefore, hepatic clearance is undeniably one of the most critical pharmacokinetic parameters. In the early stages of drug discovery, the in vitro hepatic metabolic stability assay carries great importance as it provides a time- and cost-effective route for measuring the metabolic clearance in a high-throughput fashion and predicts in vivo hepatic clearance through in vitro-in vivo extrapolation. This valuable information permits effective and critical decision-making before the in vivo studies take place. The hepatic metabolic stability assay measures the disappearance of a test compound over time in the presence of an in vitro system, for example, liver microsomes, S9, recombinant CYP450 enzymes, hepatocyte suspensions, and hepatocyte cocultures. Hepatocyte suspensions are extensively used among various available in vitro systems, as it is a more representative and integrated system with a full complement of metabolic enzymes and retained cellular compartments. Compound percent remaining is quantified by liquid chromatography tandem mass spectrometry (LC/MS/MS) with an individually tuned, condition optimized, signal quality high-throughput balanced method. Half-life is calculated from the natural logarithm of the compound remaining in percent over time. Intrinsic clearance is calculated from the half-life combined with known scaling factors. Intrinsic clearance, together with blood protein binding (fub) and liver blood flow (QH), are used in the prediction of in vivo hepatic clearance. Predicted in vivo clearance facilitates a better understanding of in vitro-in vivo correlations and guides the best use of in vitro pharmacokinetics data. This chapter offers a detailed high-throughput in vitro method to assess metabolic clearance of test compounds using hepatocyte suspensions.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Iyanagi T (2007) Molecular mechanism of Phase I and Phase II drug-metabolizing enzymes: implications for detoxification. Int Rev Cytol 260:35–112
Kadlubar S, Kadlubar FF (2010) Enzymatic basis of Phase I and Phase II drug metabolism. In: Pang KS, Rodrigues AD, Peter RM (eds) Enzyme- and transporter-based drug-drug interactions: progress and future challenges. Springer, New York, New York, NY, pp 3–25
Benet LZ, Zia-Amirhosseini P (1995) Basic principles of pharmacokinetics. Toxicol Pathol 23(2):115–123
Choi GW, Lee YB, Cho HY (2019) Interpretation of non-clinical data for prediction of human pharmacokinetic parameters: in vitro-in vivo extrapolation and allometric scaling. Pharmaceutics 11(4):168
Gillette JR (1971) Factors affecting drug metabolism. Ann N Y Acad Sci 179:43–66
Rowland M, Benet LZ, Graham GG (1973) Clearance concepts in pharmacokinetics. J Pharmacokinet Biopharm 1(2):123–136
Wilkinson GR, Shand DG (1975) Commentary: a physiological approach to hepatic drug clearance. Clin Pharmacol Ther 18(4):377–390
Asha S, Vidyavathi M (2010) Role of human liver microsomes in in vitro metabolism of drugs-a review. Appl Biochem Biotechnol 160(6):1699–1722
Fonsi M, Orsale MV, Monteagudo E (2008) High-throughput microsomal stability assay for screening new chemical entities in drug discovery. J Biomol Screen 13(9):862–869
Richardson SJ, Bai A, Kulkarni AA, Moghaddam MF (2016) Efficiency in drug discovery: liver S9 fraction assay as a screen for metabolic stability. Drug Metab Lett 10(2):83–90
Chalbot S, Morfin R (2205) Human liver S9 fractions: metabolism of dehydroepiandrosterone, epiandrosterone, and related 7-hydroxylated derivatives. Drug Metab Dispos 33(4):563–569
Galetin A, Brown C, Hallifax D, Ito K, Houston JB (2004) Utility of recombinant enzyme kinetics in prediction of human clearance: impact of variability, CYP3A5, and CYP2C19 on CYP3A4 probe substrates. Drug Metab Dispos 32(12):1411–1420
Stringer RA, Strain-Damerell C, Nicklin P, Houston JB (2009) Evaluation of recombinant cytochrome P450 enzymes as an in vitro system for metabolic clearance predictions. Drug Metab Dispos 37(5):1025–1034
Sudo M, Nishihara M, Takahashi J, Asahi S (2017) Long-term stability of cryopreserved human hepatocytes: evaluation of Phase I and II drug-metabolizing enzyme activities and CYP3A4/5 induction for more than a decade. Drug Metab Dispos 45(7):734–736
Bischof I, Köster J, Segner H, Schlechtriem C (2016) Hepatocytes as in vitro test system to investigate metabolite patterns of pesticides in farmed rainbow trout and common carp: comparison between in vivo and in vitro and across species. Comp Biochem Physiol C Toxicol Pharmacol 187:62–73
Smith CM, Nolan CK, Edwards MA, Hatfield JB, Stewart TW, Ferguson SS et al (2012) A comprehensive evaluation of metabolic activity and intrinsic clearance in suspensions and monolayer cultures of cryopreserved primary human hepatocytes. J Pharm Sci 101(10):3989–4002
Burton RD, Hieronymus T, Chamem T, Heim D, Anderson S, Zhu X et al (2018) Assessment of the biotransformation of low-turnover drugs in the HmicroREL human hepatocyte coculture model. Drug Metab Dispos 46(11):1617–1625
Chan TS, Yu H, Moore A, Khetani SR, Tweedie D (2019) Meeting the challenge of predicting hepatic clearance of compounds slowly metabolized by cytochrome P450 using a novel hepatocyte model, HepatoPac. Drug Metab Dispos 47(1):58–66
Pelkonen O, Turpeinen M (2019) In vitro–in vivo extrapolation of hepatic clearance: Biological tools, scaling factors, model assumptions and correct concentrations. Xenobiotica 37(10–11):1066–1068
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Cai, J., Shalan, H. (2021). Assessment of Cytochrome P450 Metabolic Clearance Using Hepatocyte Suspension. In: Yan, Z., Caldwell, G.W. (eds) Cytochrome P450. Methods in Pharmacology and Toxicology. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1542-3_15
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1542-3_15
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1541-6
Online ISBN: 978-1-0716-1542-3
eBook Packages: Springer Protocols