CommentaryFactors confounding the successful extrapolation of in vitro CYP3A inhibition information to the in vivo condition
Introduction
The issue of metabolism-based drug interactions continues to draw attention from the medical community, regulatory agencies, and the pharmaceutical industry. Over the past 5 years several prominent drugs have been withdrawn from the market due to serious adverse events associated with drug–drug interactions (e.g. Posicor (mibefradil); Hismanal (astemizole)). In this light, drug–drug interactions are not only a medical problem for clinicians and patients, but also represent a profound economic loss for the sponsoring pharmaceutical companies. As a means of avoiding disastrous in vivo drug interactions, the FDA requires identification of the specific metabolic pathways from which potential inhibition or induction interactions may be inferred and, most recently, the effect of the new drug on hepatic P450 metabolism (Guidance for Industry, 1997). As a consequence, many pharmaceutical companies employ in vitro drug–drug assays early in drug discovery to predict potential interactions of new drug candidates in an attempt to minimize untoward characteristics associated with novel compounds and therefore underwrite the health and safety of patients.
The cytochrome P450s are a super-family of drug metabolizing enzymes found principally in liver and intestine and are responsible for the oxidative metabolism of a wide range of xenobiotic and endobiotic compounds (Guengerich, 2001). In humans P450 3A4 (CYP3A4) is the most abundant P450 enzyme and is responsible for the biotransformation of the majority of drugs currently on the market (Bertz and Granneman, 1997). As a consequence, there are several well-documented clinical drug interactions involving compounds that are principally metabolized by CYP3A4 (Thummel and Wilkinson, 1998).
Comment is provided here to discuss some of the limitations of extrapolating drug–drug interaction data derived from in vitro experiments to the in vivo situation. Emphasis is placed on the confounding features associated with in vitro CYP inhibition studies in particular with CYP3A4, and the possible constraints these findings place on the ability to extrapolate in vitro data in order to predict in vivo inhibitory drug interaction potential.
Section snippets
In vitro approaches to predict drug interactions in vivo
Historically, most drug–drug interaction studies were conducted relatively late in development (typically Phase II or III clinical trials focusing primarily on the therapeutic indices of candidate drug and the likelihood of concurrent medications). Unfortunately, the assessment of interaction potential at such a late stage was not very practical for if clinical studies revealed that a drug candidate caused serious drug interactions, it was probably too late to terminate the development of the
Factors which impact accurate Ki determinations
There are numerous experimental factors that may affect the accuracy of Ki estimation. For many drug inhibition studies, inaccuracy in the Ki estimation is a reflection of inappropriate incubation conditions. There exist a variety of conditions that confound one or more key assumptions that form the basis of this type of analysis. For instance, complications arise when the concentration of substrate and/or inhibitor in solution is significantly reduced by nonspecific binding to components of
CYP3A4-mediated drug metabolism
The active site of CYP3A4 is generally considered to be spacious as evidenced by its ability to oxidize a wide range of structurally diverse molecules. Moreover, CYP3A4 may also exhibit atypical kinetic profiles including positive co-operativity (Ueng et al., 1997) and substrate inhibition (Wang et al., 2000). In deference to the large CYP3A4 active site and the atypical kinetics associated with certain CYP3A4 mediated oxidations, it has been hypothesized that two substrates may physically
Conclusion
As the end result, the various anomalies associated with CYP3A4 inhibition kinetics suggest that in vitro conditions and resulting substrate-velocity curves may not be consistent with the Michaelis–Menten kinetic model and thus, may not be appropriate in predicting drug interactions in vivo. Moreover, the observed atypical kinetics is not restricted to CYP3A4, since nonMichaelis–Menten kinetics has been reported for other P450s enzymes (Hutzler et al., 2001, Lin et al., 2001). From a pragmatic
References (39)
- et al.
Cortisol metabolism in vitro—III. Inhibition of microsomal 6 beta-hydroxylase and cytosolic 4-ene-reductase
J. Steroid Biochem. Mol. Biol.
(1993) - et al.
High rates of substrate hydroxylation by human cytochrome P450 3A4 in reconstituted membranous vesicles: influence of membrane charge
Biochem Biophys Res. Commun.
(1996) - et al.
Involvement of cytochrome P450 3A4 in N-dealkylation of buprenorphine in human liver microsomes
Life Sci.
(1997) - et al.
Covalent alteration of the CYP3A4 active site: evidence for multiple substrate binding domains
Arch. Biochem. Biophys.
(2001) - et al.
Lack of electron transfer from cytochrome b5 in stimulation of catalytic activities of cytochrome P450 3A4. Characterization of a reconstituted cytochrome P450 3A4/NADPH-cytochrome P450 reductase system and studies with apo-cytochrome b5
J. Biol. Chem.
(1996) - et al.
Allosteric behavior in cytochrome p450-dependent in vitro drug–drug interactions: a prospective based on conformational dynamics
Chem. Res. Toxicol.
(2001) - et al.
Use of in vitro and in vivo data to estimate the likelihood of metabolic pharmacokinetic interactions
Clin. Pharmacokinet.
(1997) - et al.
Warfarin-fluconazole. II. A metabolically based drug interaction: in vivo studies
Drug Metab. Dispos.
(1996) - et al.
Biotransformation of tirilazad in human: 2. Effect of ketoconazole on tirilazad clearance and oral bioavailability
J. Pharmacol. Exp. Ther.
(1996) - et al.
Effect of inhibitor depletion on inhibitory potency: tight binding inhibition of CYP3A by clotrimazole
Drug Metab. Dispos.
(1999)
Metabolism of artelinic acid to dihydroqinqhaosu by human liver cytochrome P4503A
Xenobiotica
Metabolism of beta-arteether to dihydroqinghaosu by human liver microsomes and recombinant cytochrome P450
Drug Metab. Dispos.
Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity
Chem. Res. Toxicol.
Analysis of human cytochrome P450 3A4 cooperativity: Construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics
Proc. Natl. Acad. Sci. USA
The mechanism of the interaction between amiodarone and warfarin in humans
Clin. Pharmacol. Ther.
Elucidation of distinct ligand binding sites for cytochrome P450 3A4
Biochemistry
In vitro–in vivo scaling of CYP kinetic data not consistent with the classical Michaelis–Menten model
Drug Metab. Dispos.
Dapsone activation of CYP2C9-mediated metabolism: evidence for activation of multiple substrates and a two-site model
Drug Metab. Dispos.
Cited by (16)
Evaluation of fluorescence- and mass spectrometry-based CYP inhibition assays for use in drug discovery
2008, Journal of Biomolecular ScreeningDrug-Associated Disease: Cytochrome P450 Interactions
2006, Critical Care ClinicsCatalytic turnover of pyrene by CYP3A4: Evidence that cytochrome b <inf>5</inf> directly induces positive cooperativity
2005, Archives of Biochemistry and BiophysicsCitation Excerpt :The practice of extrapolating in vitro P450 data to predict human clearance and metabolic inhibitory potency is a fundamental activity of the drug discovery process. However, environmental factors such as the ionic strength of the buffer, pH, lipids, and divalent cations can affect P450 function [8,17,37,38] and may confound drug metabolism predictions. Biological factors such as reductase [39,40], P450 [25], and cytochrome b5[24,41] may also alter P450 structure/function.
Substrate inhibition kinetics in drug metabolism reactions
2011, Drug Metabolism ReviewsThe biochemistry of drug metabolism - An introduction: Part 7. Intra-individual factors affecting drug metabolism
2009, Chemistry and BiodiversityEvaluation of 3-O-methylfluorescein as a selective fluorometric substrate for CYP2C19 in human liver microsomes
2007, Drug Metabolism and Disposition