Review
The pivotal role of hepatocytes in drug discovery

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Abstract

This review promotes the value of isolated hepatocytes in modern Drug Discovery programmes and outlines how increased understanding, particularly in the area of in vitroin vivo extrapolation (IVIVE), has led to more widespread use. The importance of in vitro metabolic intrinsic clearance data for predicting in vivo clearance has been acknowledged for several years and the greater utility of hepatocytes, compared with hepatic microsomes and liver slices, for this application is discussed. The application of hepatocytes in predicting drug–drug interactions (DDIs) resulting from reversible and irreversible (time-dependent) inhibition is relatively novel but affords the potential to study both phase I and phase II processes together with any impact of drug efflux and/or uptake (cellular accumulation). Progress in this area is reviewed along with current opinions on the comparative use of primary hepatocytes and higher throughput reporter gene-based systems for studying cytochrome P450 (CYP) induction. The appreciation of the role of transporter proteins in drug disposition continues to evolve. The study of hepatic uptake using isolated hepatocytes and the interplay between drug transport and metabolism with respect to both clearance and DDIs and subsequent IVIVE is also considered.

Introduction

The liver is the body's largest internal organ, comprising approximately 2% of the total body mass. It is made up of many different cell types with distinct functions; the parenchymal cells (hepatocytes) constitute 65% of total liver cells, occupy over 80% of total liver volume [1] and are the primary site of metabolic drug transformation in the body. The majority of drugs are eliminated from the body predominantly by hepatic metabolism involving CYP-dependent oxidation (phase I metabolism) and most clinical DDIs occur via inhibition or induction of CYP enzymes.

Twenty years ago it was suggested that 80% of candidate drugs failed in development because of sub-optimal ADME [2]. Major liabilities such as metabolic instability and DDI potential have resulted in automated screening programs within drug discovery designed to optimise these parameters using in vitro tools [3]. As a result of this, in vitro drug metabolism and kinetic data have been used with increasing refinement to predict in vivo clearance and DDIs and this focus may account for purported improvements in late stage attrition statistics [4], [5]. For the in vitro study of drug metabolism in the liver, the most widely used tools are isolated hepatocytes, hepatic microsomes and heterologously expressed recombinant human CYPs. These have been incorporated into automated drug discovery methodologies in order to generate the data required for new molecular entity (NME) optimisation [6], [7], [8], [9], [10], [11], [12].

Availability of good quality, fresh liver tissue has limited human hepatocyte experiments but the progress in cryopreservation over the last decade has seen an explosion in the use of human hepatocytes within the pharmaceutical industry and academia allowing hepatocytes to become an ‘off-the-shelf’ reagent [13]. This review details the current and potential uses of hepatocytes in drug discovery from the perspective of the authors’ laboratory.

Section snippets

General use of isolated hepatocytes in drug discovery

In vivo hepatic metabolic clearance can be estimated successfully from the turnover or intrinsic clearance (CLint) derived from in vitro tissue preparations, such as hepatocytes and hepatic microsomes [14], [15]. Many laboratories use hepatic microsomes rather than hepatocytes to determine CLint[16], [17] since they have traditionally been viewed as a more flexible system with which to study oxidative biotransformations in terms of ease of preparation from many species, long-term storage and

Use of hepatocytes in the prediction of metabolic clearance

In the early 1990s in vitro systems (isolated hepatocytes, hepatic microsomes and liver slices) came into widespread use in the pharmaceutical industry. A general strategy for predicting in vivo clearance from in vitro data was presented by Houston [26] which illustrated how existing mathematical models for describing hepatic clearance (CLH), incorporating terms for liver blood flow (Q), CLint and blood binding (fub) [27], [28], [29], [30], could be adopted to facilitate IVIVE with CLint being

The use of hepatocyes to assess drug uptake

To date, there are two main pathways responsible for the uptake of endogenous and xenobiotic substrates into hepatocytes. The sodium-dependent route is catalyzed exclusively at the basolateral membrane by the sodium-taurocholate cotransporting polypeptide (NTCP). NTCP is primarily responsible for the uptake of conjugated bile salts and sulphated steroids but has a more limited role in the uptake of drugs [24]. The organic anion transporting polypeptides (OATPs) are a superfamily of enzymes that

Use of hepatocytes in the assessment of drug–drug interactions

Drug discovery departments in the pharmaceutical industry employ a battery of in vitro screens to assess the potential of new chemical entities to cause pharmacokinetic based DDIs via inhibition and induction of drug metabolising enzymes. Although metabolic DDIs have been reported for several enzyme families, it is recognised that inhibition and induction of CYP dependent metabolism are the most prevalent source of DDIs, which may lead to serious clinical consequences [89], [90].

Summary

Over the last 10–15 years, the use of hepatocytes for studying the routes and rates of drug metabolism has become relatively commonplace. The increase in popularity has been fuelled by the ability to optimise CYP-related biotransformation in Drug Discovery programmes, a desire to minimise affinity at the hERG channel (resulting in an increase in acidic phase II substrates) and the need to address phase II metabolism and hepatic transporter-related events.

In contrast, the application of

References (121)

  • M.G. Ismair et al.

    Hepatic uptake of cholecystokinin octapeptide by organic anion-transporting polypeptides OATP4 and OATP8 of rat and human liver

    Gastroenterology

    (2001)
  • Y. Cui et al.

    Hepatic uptake of bilirubin and its conjugates by the human organic anion transporter SLC1A6

    J. Biol. Chem.

    (2001)
  • M. Sasaki et al.

    Transcellular transport of organic anions across a double-transfected Madin-Darby canine kidney II cell monolayer expressing both human organic anion-transporting polypeptide (OATP2/SLC21A6) and multidrug resistance-associated protein 2 (MRP2/ABCC2)

    J. Biol. Chem.

    (2002)
  • K.J. Spears et al.

    Directional trans-epithelial transport of organic anions in orcine LLC-PK1 cells that co-express human OATP1B1 (OATP-C) and MRP2

    Biochem. Pharmacol.

    (2005)
  • Y. Shitara et al.

    Function of uptake transporters for taurocholate and estradiol-17β-d-glucuronide in cryopreserved human hepatocytes

    Drug Metab. Pharmacokin.

    (2003)
  • C.L. Crespi

    Xenobiotic-metabolizing human cells as tools for pharmacological and toxicological research

    Adv. Drug Res.

    (1995)
  • R.G. Tirona et al.

    Polymorphisms in OATP-C. Identification of multiple allelic variants associated with altered transport activity among European and African-Americans

    J. Biol. Chem.

    (2001)
  • C. Michalski et al.

    A naturally occurring mutation in the SLC21A6 gene causing impaired membrane localization of the hepatocyte uptake transporter

    J. Biol. Chem.

    (2002)
  • F.B. Oleson et al.

    An evaluation of the P450 inhibition and induction potential of daptomycin in primary human hepatocytes

    Chem. Biol. Interact.

    (2004)
  • E.R. Weibel et al.

    Correlated morphometric and biochemical studies on the liver cell. I. Morphometric model, stereologic methods, and normal morphometric data for rat liver

    J. Cell Biol.

    (1969)
  • R.A. Prentis et al.

    Pharmaceutical innovation by the seven UK-owned pharmaceutical companies (1964–1985)

    Br. J. Clin. Pharmacol.

    (1988)
  • H. Van de Waterbeemd et al.

    ADMET in silico modelling: towards prediction paradise?

    Nat. Rev. Drug Discov.

    (2003)
  • R. Weaver et al.

    Cytochrome P450 inhibition using recombinant proteins and mass spectrometry/multiple reaction monitoring technology in a cassette incubation

    Drug Metab. Dispos.

    (2003)
  • D.F. McGinnity et al.

    Prediction of CYP2C9 mediated drug–drug interactions: a comparison using data from recombinant enzymes and human hepatocytes

    Drug Metab. Dispos.

    (2005)
  • A. Atkinson et al.

    Automated assessment of time-dependent inhibition of human cytochrome P450 enzymes using liquid chromatography-tandem mass spectrometry analysis

    Drug Metab. Dispos.

    (2005)
  • H.-K. Lim et al.

    Automated screening with confirmation of mechanism-based inactivation of CYP3A4, CYP2C9, CYP2C19, CYP2D6, and CYP1A2 in pooled human liver microsomes

    Drug Metab. Dispos.

    (2005)
  • D.F. McGinnity et al.

    Automated definition of the enzymology of drug oxidation by the major human drug metabolizing cytochrome P450s

    Drug Metab. Dispos.

    (2000)
  • J.B. Houston et al.

    Prediction of hepatic clearance from microsomes, hepatocytes and liver slices

    Drug Metab. Rev.

    (1997)
  • K. Grime et al.

    The impact of in vitro binding on in vitroin vivo extrapolations, projections of metabolic clearance and clinical drug–drug interactions

    Curr. Drug Metab.

    (2006)
  • R.S. Obach et al.

    The prediction of human pharmacokinetic parameters from preclinical and in vitro metabolism data

    J. Pharm. Exp. Ther.

    (1997)
  • S.E. Clarke et al.

    Utility of metabolic stability screening: comparison of in vitro and in vivo clearance

    Xenobiotica

    (2001)
  • A. Ayrton et al.

    Role of transport proteins in drug absorption, distribution and excretion

    Xenobiotica

    (2001)
  • S.J. Griffin et al.

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

    Drug Metab. Dispos.

    (2004)
  • G.T. Tucker et al.

    Optimizing drug development: strategies to assess drug metabolism/transporter interaction potential-towards a consensus

    Clin. Pharmacol. Ther.

    (2001)
  • N. Mizuno et al.

    Impact of drug transporter studies on drug discovery and drug development

    Pharmacol. Rev.

    (2003)
  • P. Olinga et al.

    Characterization of the uptake of rocuronium and digoxin in human hepatocytes: carrier specificity and comparison with in vivo data

    J. Pharamcol. Exp. Ther.

    (1998)
  • R.J. Riley et al.

    The influence of DMPK as an integrated partner in modern drug discovery

    Curr. Drug Metab.

    (2002)
  • J.B. Houston

    Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance

    Biochem. Pharmacol.

    (1994)
  • M. Roland et al.

    Clearance concepts in pharmacokinetics

    J. Pharmacokinet. Biopharm.

    (1973)
  • G.R. Wilkinson et al.

    Commentary: a physiological approach to hepatic drug clearance

    Clin. Pharmacol. Ther.

    (1975)
  • K.S. Pang et al.

    Hepatic clearance of drugs. I. Theoretical considerations of a “well-stirred” model and a “parallel tube” model. Influence of hepatic blood flow, plasma and blood cell binding, and the hepatocelluar enzyme activity on hepatic drug clearance

    J. Pharmacokinet. Biopharm.

    (1977)
  • M.S. Roberts et al.

    Hepatic elimination-dispersion model

    J. Pharm. Sci.

    (1985)
  • K. Ito et al.

    Comparison of the use of liver models for predicting drug clearance using in vitro kinetic data from hepatic microsomes and isolated hepatocytes

    Pharm. Res.

    (2004)
  • T. Iwatsubo et al.

    Prediction of in vivo drug disposition from in vivo data based on physiological pharmacokinetics

    Biopharm. Drug Dispos.

    (1996)
  • K. Ito et al.

    Quantitative prediction of in vivo drug clearance and drug interactions from in vitro data on metabolism, together with binding and transport

    Annu. Rev. Pharmacol. Toxicol.

    (1998)
  • R. Niro et al.

    Application of a convective-dispersion model to predict in vivo hepatic clearance from in vitro measurement utilizing cryopreserved human hepatocyes

    Curr. Drug Metab.

    (2003)
  • H.C. Rawden et al.

    Microsomal prediction on in vivo clearance and associated interindividual variability of six benzodiazepines in humans

    Xenobiotica

    (2005)
  • J.H. Lin

    Applications and limitations of interspecies scaling and in vitro extrapolation in pharmacokinetics

    Drug Metab. Dispos.

    (1998)
  • T. Iwatsubo et al.

    Prediction of in vivo drug metabolism in the human liver from in vitro metabolism data

    Pharmacol. Ther.

    (1997)
  • D.J. Carlile et al.

    Microsomal prediction of in vivo clearance of CYP2C9 substrates in humans

    Br. J. Clin. Pharmacol.

    (1999)
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