Abstract
Reaction phenotyping studies to identify specific enzymes involved in the metabolism of drug candidates are increasingly important in drug discovery efforts. Experimental approaches used for CYP reaction phenotyping include incubations with cDNA expressed CYP enzyme systems and incubations containing specific CYP enzyme inhibitors. Since both types of experiments present specific advantages as well as known drawbacks, these studies are generally viewed as complementary approaches. Although glucuronidation pathways are also known to present potential drug–drug interaction issues as well as challenges related to their polymorphic expression, reaction phenotyping approaches for glucuronidation are generally limited to cDNA expressed systems due to lack of availability of specific UGT inhibitors. This article presents a limited review of current approaches to reaction phenotyping studies used within the pharmaceutical industry.
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A. D. Rodrigues, and T. H. Rushmore. Cytochrome P450 pharmacogenetics in drug development: in vitro studies and clinical consequences. Curr. Drug Metab. 3:289–309 (2002).
K. Nakamura, F. Goto, W. A. Ray, C. B. McAllister, E. Jacqz, G. R. Wilkinson, and R. A. Branch. Interethnic differences in genetic polymorphism of debrisoquin and mephenytoin hydroxylation between Japanese and Caucasian populations. Clin. Pharmacol. Ther. 38:402–408 (1985).
M. Kimura, I. Ieiri, K. Mamiya, A. Urae, and S. Higuchi. Genetic polymorphism of cytochrome P450s, CYP2C19, and CYP2C9 in a Japanese population. Ther. Drug Monit. 20:243–247 (1998).
R. Jose, and A. Chandrasekaran. The pharmacogenetics of CYP2C9 and CYP2C19: ethnic variation and clinical significance. Curr. Clin. Pharm. 2(1):93–109 (2007).
E. Garcia-Martin, C. Martinez, J. M. Ladero, and J. A. G. Agundez. Interethnic and intraethnic variability of CYP2C8 and CYP2C9 polymorphisms in healthy individuals. Mol. Diagn. Ther. 10:29–40 (2006).
E. Tanaka. Clinically important pharmacokinetic drug–drug interactions: role of cytochrome P450 enzymes. J. Clin. Pharm. Ther. 23:403–416 (1998).
G. K. Dresser, J. D. Spence, and D. G. Bailey. Pharmacokinetic-pharmacodynamic consequences and clinical relevance of cytochrome P450 3A4 inhibition. Clin. Pharm. 38:41–57 (2000).
A. Hsu, G. R. Granneman, and J. Bertz. Ritonavir: Clinical pharmacokinetics and interactions with other anti-HIV agents. Clin. Pharm. 35:275–291 (1998).
A. Hsu, G. R. Granneman, G. Cao, L. Carothers, A. Japour, T. El-Shourbagy, S. Dennis, J. Berg, K. Erdman, J. M. Leonard, and E. Sun. Pharmacokinetic Interaction between ritonavir and indinavir in healthy volunteers. Antimicrob. Agents Chemother. 42:2784–2791 (1998).
S. B. Koukouritaki, P. Simpson, C. K. Yeung, A. E. Rettie, and R. N. Hines. Human hepatic flavin-containing monooxygenases 1 (FMO1) and 3 (FMO3) developmental expression. Pediatr. Res. 51:236–243 (2002).
S. Larsen-Su, and D. E. Williams. Dietary indole-3-carbinol inhibits FMO activity and the expression of flavin-containing monooxygenase form 1 in rat liver and intestine. Drug Metab. Dispos. 24:927–931 (1996).
C. Guillemette. Pharmacogenomics of human UDP-glucuronosyltransferase enzymes. Pharmacogenomics J. 3:136–158 (2003).
E. Levesque, R. Delage, M.-O. Benoit-Biancamano, P. Caron, O. Bernard, F. Couture, and C. Guillemette. The impact of UGT1A8, UGT1A9, and UGT2B7 genetic polymorphisms on the pharmacokinetic profile of mycophenolic acid after a single oral dose in healthy volunteers. Clin. Pharm. Ther. 81:392–400 (2007).
T. K. L. Kiang, M. H. H. Ensom, and T. K. H. Chang. UDP-glucuronosyltransferases and clinical drug–drug interactions. Pharmacol. Ther. 106:97–132 (2005).
A. D. Rodrigues. Integrated cytochrome P450 reaction phenotyping: attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes. Biochem. Pharmacol. 57:465–480 (1999).
A. Parkinson. An overview of current cytochrome P450 technology for assessing the safety and efficacy of new materials. Toxicol. Pathol. 24:48–57 (1996).
A. D. Rodrigues. Use of in vitro human metabolism studies in drug development. An industrial perspective. Biochem. Pharmacol. 48:2147–2156 (1994).
C. Emoto, S. Murase, and K. Iwasaki. Approach to the prediction of the contribution of major cytochrome P450 enzymes to drug metabolism in the early drug-discovery stage. Xenobiotica. 36:671–683 (2006).
J. B. Houston. Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance. Biochem. Pharm. 47:1469–1479 (1994).
T. Iwatsubo, N. Hirota, T. Ooie, H. Suzuki, N. Simada, K. Chiba, T. Ishizaki, G. E. Green, C. A. Tyson, and Y. Sugiyama. Prediction of in vivo drug metabolism in the human liver from in vitro metabolism data. Pharmacol. Ther. 73:147–171 (1997).
R. S. Obach, J. G. Baxter, T. E. Liston, B. M. Silber, B. C. Jones, R. MacIntyre, D. J. Rance, and P. Wastal. The prediction of human pharmacokinetic parameters from preclinical and in vitro metabolism data. J. Pharmacol. Exp. Ther. 283:46–58 (1997).
N. J. Proctor, G. T. Tucker, and A. Rostami-Hodjegan. Predicting drug clearance from recombinantly expressed CYPs: intersystem extrapolation factors. Xenobiotica. 34:151–178 (2004).
S.-I. Ikushiro, E. Yoshikazu, K. Yoshihisa, S. Yamada, and T. Sakaki. Monospecific antipeptide antibodies against human hepatic UDP-glucuronosyltransferase 1A subfamily (UGT1A) isoforms. Drug Metab. Pharmacokinet. 21:70–74 (2006).
J. H. Lin, and B. K. Wong. Complexities of glucuronidation affecting in vitro–in vivo extrapolation. Curr. Drug Metab. 3:623–646 (2002).
M. H. Court, S. X. Duan, L. L. Von Moltke, D. J. Greenblatt, C. J. Patten, J. O. Miners, and P. I. MacKenzie. Interindividual variability in acetaminophen glucuronidation by human liver microsomes: identification of relevant acetaminophen UDP-glucuronosyltransferase isoforms. J. Pharmacol. Exp. Ther. 299:998–1006 (2001).
K. R. Yeo, A. Rostami-Hodjegan, and G. T. Tucker. Abundance of cytochromes P450 in human liver: a meta-analysis. Br. J. Clin. Pharmacol. 57:687–688 (2004).
S. Krishnaswamy, S. X. Duan, L. L. Von Moltke, D. J. Greenblatt, and M. H. Court. Validation of serotonin (5-hydrosytryptamine) as an in vitro substrate probe for human UDP-glucuronosyltransferase (UGT) 1A6. Drug Metab. Disp. 31:133–139 (2003).
M. B. Fisher, M. Vandenbranden, K. Findlay, B. Burchell, K. E. Thummel, S. D. Hall, and S. A. Wrighton. Tissue distribution and interindividual variation in human UDP-glucuronosyltransferase activity: relationship between UGT1A1 promoter genotype and variability in a liver bank. Pharmacogenetics. 10:727–739 (2000).
C. P. Strassburg, S. Kneip, J. Topp, P. Obermayer-Straub, A. Barutt, R. H. Turkey, and M. P. Manns. Polymorphic gene regulation and interindividual variation of UDP-glucuronosyltransferase activity in human small intestine. J. Biol. Chem. 46a:36164–36171 (2000).
R. Fulceri, G. Banhegyi, A. Gamberucci, R. Giunti, J. Mandl, and A. Benedetti. Evidence for the intraluminal positioning of p-nitrol UDP-glucuronosyltransferase activity in rat liver microsomes. Arch. Biochem. Biophys. 309:43–46 (1994).
M. B. Fisher, K. Campanale, B. L. Ackermann, M. Banderbranden, and S. A. Wrighton. In vitro glucuronidation using human liver microsomes and the pore-forming peptide alamethicin. Drug Metab. Disp. 25:560–566 (2000).
J. M. Little, P. A. Lehman, S. Nowell, V. Samokyszyn, and A. Radominska. Glucuronidation of all-trans-retinoic acid and 5,6-epoxy-all-trans-retinoic acid. Drug Metab. Disp. 25:5–11 (1997).
K. He, S. J. Ludtke, W. T. Heller, and H. W. Huang. Mechanism of alamethicin insertion into lipid bilayers. Biophys. J. 71:2669–2679 (1996).
E. Lett, W. Kriszt, V. de Sandro, G. Ducrotoy, and L. Richert. Optimal detergent activation of rat liver microsomal UDP-glucuronosyltransferases toward morphine and 1-naphthol: contribution to induction and latency studies. Biochem. Pharmacol. 43:1649–1653 (1992).
C. B. Trapnell, R. W. Klecker, C. Jamis-Dow, and J. M. Colling. Glucuronidation of 3¢-azido-3¢-deoxythymidine (zidovudine) by human liver microsomes—relevance to clinical pharmacokinetic interactions with atovaquone, fluconazole, methadone and valproic acid. Antimicrob. Agents Chemother. 42:1592–1596 (1998).
H. V. Gelboin, and K. Krausz. Monoclonal Antibodies and Multifunctional Cytochrome P450: Drug Metabolism as Paradigm. J. Clin. Pharmacol. 46:353–372 (2006).
K. W. Krausz, I. Goldfarb, J. T. Buters, T. J. Yang, F. J. Gonzalez, and H. V. Gelboin. Monoclonal antibodies specific and inhibitory to human cytochromes P450 2C8, 2C9, and 2C19. Drug Metab. Dispos. 29:1410–1423 (2001).
D. Sesardic, A. R. Boobis, B. P. Murray, S. Murray, J. Segura, R. de la Torre, and D. S. Davies. Furafylline is a potent and selective inhibitor of cytochrome P4501A2 in man. Br. J. Clin. Pharm. 29:651–663 (1990).
S. E. Clarke, A. D. Ayrton, and R. J. Chenery. Characterization of the inhibition of P4501A2 by furafylline. Xenobiotica. 24:517–526 (1994).
M. Bourrie, V. Meunier, Y. Berger, and G. Fabre. Cytochrome P450 isoform inhibitors as a tool for the investigation of metabolic reactions catalyzed by human liver microsomes. J. Pharmacol. Exp. Ther. 277:321–332 (1996).
A. Mancy, S. Dijols, S. Poli, F. P. Guengerich, and D. Mansuy. Interaction of sulfaphenazole derivatives with human liver cytochromes P4502C: Molecular origin of the specific inhibitory effects of sulfaphenazole on CYP2C9 and consequences for the substrate binding site topology of CYP2C9. Biochemistry. 35:16205–16212 (1996).
H. Suzuki, M. B. Kneller, R. L. Haining, W. F. Trager, and A. E. Rettie. (+)-N-3-benzyl-nirvanol and (−)-N-3-benzylphenobarbital: New potent and selective in vitro inhibitors of CYP2C19. Drug Metab. Dispos. 30:235–239 (2002).
R. L. Walsky, and R. S. Obach. Verification of the selectivity of (+)N-3-benzylnirvanol as a CYP2C19 inhibitor. Drug Metab. Dispos. 31:343 (2003).
D. J. Newton, R. W. Wang, and A. Y. Lu. Cytochrome P450 inhibitors. Evaluation of specificities in the in vitro metabolism of therapeutic agents by human liver microsomes. Drug Metab. Dispos. 23:154–158 (1995).
A. D. Rodrigues, and E. M. Roberts. The in vitro interaction of dexmedetomidine with human liver microsomal cytochrome P4502D6 (CYP2D6). Drug Metab. Dispos. 25:651–655 (1997).
H. Yamazaki, and T. Shimada. Comparative studies of in vitro inhibition of cytochrome P450 3A4-dependent testosterone 6beta-hydroxylation by roxithromycin and its metabolites, troleandomycin, and erythromycin. Drug Metab. Dispos. 26:1053–1057 (1998).
F. Marre, G. de Sousa, A. M. Orloff, and R. Rahmani. In vitro interaction between cyclosporin A and macrolide antibiotics. Br. J. Clin. Pharm. 35:447–448 (1993).
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Harper, T.W., Brassil, P.J. Reaction Phenotyping: Current Industry Efforts to Identify Enzymes Responsible for Metabolizing Drug Candidates. AAPS J 10, 200–207 (2008). https://doi.org/10.1208/s12248-008-9019-6
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DOI: https://doi.org/10.1208/s12248-008-9019-6