Inactivation of Human Cytochrome P450 Enzymes and Drug–Drug Interactions

  • R. Scott Obach
  • Odette A. Fahmi
  • Robert L. Walsky


Inactivation of human P450 enzymes represents an important mechanism of drug–drug interactions (DDIs). Inactivators are distinct from other inhibitors in that the affected enzyme is responsible for bioactivating an otherwise inert drug into an intermediate that can irreversibly damage the enzyme, and recovery of activity in vivo requires the biosynthesis of new enzyme. Whether a new drug will be a mechanism-based inactivator depends on the identity of chemical substituents present in the substrate and their metabolism by the P450 enzyme. Experimental approaches used to define new drugs as possible time-dependent inhibitors and mechanism-based inactivators are described. Finally, it has been demonstrated that inactivation kinetic parameters generated in vitro can be used to predict DDI. The methods used to do this are described along with existing uncertainties in the input parameters needed for accurate predictions.


Human Liver Microsome Heme Iron Reversible Inhibition Ethinyl Estradiol IC25 Concentration 
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  1. Baer BR, Wienkers LC and Rock DA (2007) Time-dependent inactivation of P450 3A4 by raloxifene: identification of Cys239 as the site of apoprotein alkylation. Chem Res Toxicol 20:954–964.CrossRefPubMedGoogle Scholar
  2. Bertelsen KM, Venkatakrishnan K, Von Moltke LL, Obach RS and Greenblatt DJ (2003) Apparent mechanism-based inhibition of human CYP2D6 in vitro by paroxetine: comparison with fluoxetine and quinidine. Drug Metab Dispos 31:289–293.CrossRefPubMedGoogle Scholar
  3. Correia M and Ortiz de Montellano PR (2005) Inhibition of cytochrome P450 enzymes, in Cytochrome P450 Structure, Mechanism, and Biochemistry (Ortiz de Montellano PR ed) pp 247–322, Kluwer Academic/Plenum Publishers, New York.Google Scholar
  4. Delaforge M, Jaouen M, and Mansuy D (1984) The cytochrome P-450 metabolite complex derived from troleandomycin: properties in vitro and stability in vivo. Chem-Biol Int. 51:371–376.Google Scholar
  5. De Matteis F (1974) Covalent binding of sulfur to microsomes and loss of cytochrome P-450 during the oxidative desulfuration of several chemicals. Mol. Pharmacol. 10:849–854.Google Scholar
  6. Einolf HJ (2007) Comparison of different approaches to predict metabolic drug–drug interactions. Xenobiotica 37:1257–1294.PubMedGoogle Scholar
  7. Faber MS and Fuhr U (2004) Time response of cytochrome P450 1A2 activity on cessation of heavy smoking. Clin. Pharmacol. Ther. 76:178–184.Google Scholar
  8. Fahmi OA, Maurer TS, Kish M, Cardenas E, Boldt S and Nettleton D (2008) A combined model for predicting CYP3A4 clinical net drug–drug interaction based on CYP3A4 inhibition, inactivation, and induction determined in vitro. Drug Metab Dispos 36:1698–1708.CrossRefPubMedGoogle Scholar
  9. Franklin MR (1974) Complexes of metabolites of amphetamines with hepatic cytochrome P-450. Xenobiotica 4:133–142.Google Scholar
  10. Franklin MR (1991) Cytochrome P450 metabolic intermediate complexes from macrolide antibiotics and related compounds. Meth. Enzymol. 206:559–573.Google Scholar
  11. Galetin A, Burt H, Gibbons L and Houston JB (2006) Prediction of time-dependent CYP3A4 drug–drug interactions: impact of enzyme degradation, parallel elimination pathways, and intestinal inhibition. Drug Metab Dispos 34:166–175.CrossRefPubMedGoogle Scholar
  12. Ghanbari F, Rowland-Yeo K, Bloomer JC, Clarke SE, Lennard MS, Tucker GT and Rostami-Hodjegan A (2006) A critical evaluation of the experimental design of studies of mechanism based enzyme inhibition, with implications for in vitro-in vivo extrapolation. Curr Drug Metab 7:315–334.CrossRefPubMedGoogle Scholar
  13. Gorski JC, Jones DR, Haehner-Daniels BD, Hamman MA, O‘Mara EM, Jr. and Hall SD (1998) The contribution of intestinal and hepatic CYP3A to the interaction between midazolam and clarithromycin. Clin Pharmacol Ther 64:133–143.CrossRefPubMedGoogle Scholar
  14. Grime KH, Bird J, Ferguson D and Riley RJ (2009) Mechanism-based inhibition of cytochrome P450 enzymes: an evaluation of early decision making in vitro approaches and drug–drug interaction prediction methods. Eur J Pharm Sci 36:175–191.CrossRefPubMedGoogle Scholar
  15. Grimm SW, Einolf HJ, Hall SD, et al., (2009). The conduct of in vitro studies to address time-dependent inhibition of drug-metabolizing enzymes: a perspective of the Pharmaceutical Research and Manufacturers of America. Drug Metab. Dispos. 37:1355–1370.Google Scholar
  16. Hallifax D, Galetin A and Houston JB (2008) Prediction of metabolic clearance using fresh human hepatocytes: comparison with cryopreserved hepatocytes and hepatic microsomes for five benzodiazepines. Xenobiotica 38:353–367.CrossRefPubMedGoogle Scholar
  17. Halpert J. (1981) Covalent modification of lysine during the suicide inactivation of rat liver cytochrome P-450 by chloramphenicol. Biochem. Pharmacol. 30:875–881.Google Scholar
  18. Hollenberg PF, Kent UM and Bumpus NN (2008) Mechanism-based inactivation of human cytochromes p450s: experimental characterization, reactive intermediates, and clinical implications. Chem Res Toxicol 21:189–205.CrossRefPubMedGoogle Scholar
  19. Jones DR, Gorski JC, Hamman MA, Mayhew BS, Rider S and Hall SD (1999) Diltiazem inhibition of cytochrome P-450 3A activity is due to metabolite intermediate complex formation. J Pharmacol Exp Ther 290:1116–1125.PubMedGoogle Scholar
  20. Kalgutkar AS, Obach RS and Maurer TS (2007) Mechanism-based inactivation of cytochrome P450 enzymes: chemical mechanisms, structure-activity relationships and relationship to clinical drug–drug interactions and idiosyncratic adverse drug reactions. Curr Drug Metab 8:407–447.CrossRefPubMedGoogle Scholar
  21. Kalgutkar AS, Vaz AD, Lame ME, Henne KR, Soglia J, Zhao SX, Abramov YA, Lombardo F, Collin C, Hendsch ZS and Hop CE (2005) Bioactivation of the nontricyclic antidepressant nefazodone to a reactive quinone-imine species in human liver microsomes and recombinant cytochrome P450 3A4. Drug Metab Dispos 33:243–253.CrossRefPubMedGoogle Scholar
  22. Kanamitsu SI, Ito K, Okuda H, Ogura K, Watabe T, Muro K and Sugiyama Y (2000) Prediction of in vivo drug–drug interactions based on mechanism-based inhibition from in vitro data: inhibition of 5-fluorouracil metabolism by (E)-5-(2-Bromovinyl)uracil. Drug Metab Dispos 28:467–474.PubMedGoogle Scholar
  23. Kartha JS and Yost GS (2008) Mechanism-based inactivation of lung-selective cytochrome P450 CYP2F enzymes. Drug Metab Dispos 36:155–162.CrossRefPubMedGoogle Scholar
  24. Kassahun K, Skordos K, McIntosh I, Slaughter D, Doss GA, Baillie TA and Yost GS (2005) Zafirlukast metabolism by cytochrome P450 3A4 produces an electrophilic alpha,beta-unsaturated iminium species that results in the selective mechanism-based inactivation of the enzyme. Chem Res Toxicol 18:1427–1437.CrossRefPubMedGoogle Scholar
  25. Khan KK, He YQ, Domanski TL and Halpert JR (2002) Midazolam oxidation by cytochrome P450 3A4 and active-site mutants: an evaluation of multiple binding sites and of the metabolic pathway that leads to enzyme inactivation. Mol Pharmacol 61:495–506.CrossRefPubMedGoogle Scholar
  26. Khojasteh-Bakht SC, Koenigs LL, Peter RM, Trager WF and Nelson SD (1998) (R)-(+)-Menthofuran is a potent, mechanism-based inactivator of human liver cytochrome P450 2A6. Drug Metab Dispos 26:701–704.PubMedGoogle Scholar
  27. Koenigs LL, Peter RM, Thompson SJ, Rettie AE and Trager WF (1997) Mechanism-based inactivation of human liver cytochrome P450 2A6 by 8-methoxypsoralen. Drug Metab Dispos 25:1407–1415.PubMedGoogle Scholar
  28. Kunze KL, Mangold BLK, Wheeler C, Beilan HS, and Ortiz de Montellano PR (1983) The cytochrome P-450 active site. Regiospecificity of prosthetic heme alkylation by olefins and acetylenes. J Biol. Chem. 258:4202–4207.Google Scholar
  29. Kunze KL and Trager WF (1993) Isoform-selective mechanism-based inhibition of human cytochrome P450 1A2 by furafylline. Chem Res Toxicol 6:649–656.CrossRefPubMedGoogle Scholar
  30. Lamberg TS, Kivisto KT and Neuvonen PJ (1998) Effects of verapamil and diltiazem on the pharmacokinetics and pharmacodynamics of buspirone. Clin Pharmacol Ther 63:640–645.CrossRefPubMedGoogle Scholar
  31. Lightning LK, Jones JP, Friedberg T, Pritchard MP, Shou M, Rushmore TH and Trager WF (2000) Mechanism-based inactivation of cytochrome P450 3A4 by L-754,394. Biochemistry 39:4276–4287.CrossRefPubMedGoogle Scholar
  32. Lin HL, Kent UM and Hollenberg PF (2002) Mechanism-based inactivation of cytochrome P450 3A4 by 17 alpha-ethynylestradiol: evidence for heme destruction and covalent binding to protein. J Pharmacol Exp Ther 301:160–167.CrossRefPubMedGoogle Scholar
  33. Lopez-Garcia MP, Dansette PM and Mansuy D (1994) Thiophene derivatives as new mechanism-based inhibitors of cytochromes P-450: inactivation of yeast-expressed human liver cytochrome P-450 2C9 by tienilic acid. Biochemistry 33:166–175.CrossRefPubMedGoogle Scholar
  34. Lu P, Schrag ML, Slaughter DE, Raab CE, Shou M and Rodrigues AD (2003) Mechanism-based inhibition of human liver microsomal cytochrome P450 1A2 by zileuton, a 5-lipoxygenase inhibitor. Drug Metab Dispos 31:1352–1360.CrossRefPubMedGoogle Scholar
  35. Margolis JM and Obach RS (2003) Impact of nonspecific binding to microsomes and phospholipid on the inhibition of cytochrome P4502D6: implications for relating in vitro inhibition data to in vivo drug interactions. Drug Metab Dispos 31:606–611.CrossRefPubMedGoogle Scholar
  36. Maurer T and Fung HL (2000) Comparison of methods for analyzing kinetic data from mechanism-based enzyme inactivation: application to nitric oxide synthase. AAPS Pharm Sci 2:E8.CrossRefGoogle Scholar
  37. Mayhew BS, Jones DR and Hall SD (2000) An in vitro model for predicting in vivo inhibition of cytochrome P450 3A4 by metabolic intermediate complex formation. Drug Metab Dispos 28:1031–1037.PubMedGoogle Scholar
  38. McConn DJ, Lin YS, Allen K, Kunze KL and Thummel KE (2004) Differences in the inhibition of cytochromes P450 3A4 and 3A5 by metabolite-inhibitor complex-forming drugs. Drug Metab Dispos 32:1083–1091.PubMedGoogle Scholar
  39. Obach RS, Walsky RL and Venkatakrishnan K (2007) Mechanism-based inactivation of human cytochrome p450 enzymes and the prediction of drug–drug interactions. Drug Metab Dispos 35:246–255.CrossRefPubMedGoogle Scholar
  40. Obach RS, Walsky RL, Venkatakrishnan K, Gaman EA, Houston JB and Tremaine LM (2006) The utility of in vitro cytochrome P450 inhibition data in the prediction of drug–drug interactions. J Pharmacol Exp Ther 316:336–348.CrossRefPubMedGoogle Scholar
  41. O‘Donnell JP, Dalvie DK, Kalgutkar AS and Obach RS (2003) Mechanism-based inactivation of human recombinant P450 2C9 by the nonsteroidal anti-inflammatory drug suprofen. Drug Metab Dispos 31:1369–1377.CrossRefPubMedGoogle Scholar
  42. O‘Reilly RA (1982) Ticrynafen-racemic warfarin interaction: hepatotoxic or stereoselective?. Clin Pharmacol Ther 32:356–361.CrossRefPubMedGoogle Scholar
  43. Pearson JT, Wahlstrom JL, Dickmann LJ, Kumar S, Halpert JR, Wienkers LC, Foti RS and Rock DA (2007) Differential time-dependent inactivation of P450 3A4 and P450 3A5 by raloxifene: a key role for C239 in quenching reactive intermediates. Chem Res Toxicol 20:1778–1786.CrossRefPubMedGoogle Scholar
  44. Polasek TM, Elliot DJ, Somogyi AA, Gillam EMJ, Lewis BC, and Miners JO (2006) An evaluation of potential mechanism-based inactivation of human drug metabolizing cytochromes P450 by monoamine oxidase inhibitors, including isoniazid. Br. J. Clin. Pharmacol. 61:570–584.Google Scholar
  45. Polasek TM and Miners JO (2007) In vitro approaches to investigate mechanism-based inactivation of CYP enzymes. Expert Opin Drug Metab Toxicol 3:321–329.CrossRefPubMedGoogle Scholar
  46. Racha JK, Rettie AE and Kunze KL (1998) Mechanism-based inactivation of human cytochrome P450 1A2 by furafylline: detection of a 1:1 adduct to protein and evidence for the formation of a novel imidazomethide intermediate. Biochemistry 37:7407–7419.CrossRefPubMedGoogle Scholar
  47. Richter T, Murdter TE, Heinkele G, Pleiss J, Tatzel S, Schwab M, Eichelbaum M and Zanger UM (2004) Potent mechanism-based inhibition of human CYP2B6 by clopidogrel and ticlopidine. J Pharmacol Exp Ther 308:189–197.CrossRefPubMedGoogle Scholar
  48. Riley RJ, Grime K and Weaver R (2007) Time-dependent CYP inhibition. Expert Opin Drug Metab Toxicol 3:51–66.CrossRefPubMedGoogle Scholar
  49. Rostami-Hodjegan A and Tucker GT (2007) Simulation and prediction of in vivo drug metabolism in human populations from in vitro data. Nat Rev Drug Discov 6:140–148.CrossRefPubMedGoogle Scholar
  50. Sahali-Sahly Y, Balani SK, Lin JH and Baillie TA (1996) In vitro studies on the metabolic activation of the furanopyridine L-754,394, a highly potent and selective mechanism-based inhibitor of cytochrome P450 3A4. Chem Res Toxicol 9:1007–1012.CrossRefPubMedGoogle Scholar
  51. Schenkman JB and Jansson I (1998) Spectral analyses of cytochromes P450. Meth. Molec. Biol. 107:25–33.Google Scholar
  52. Sinal C and Bend JR (1996) Kinetics and selectivity of mechanism-based inhibition of guinea pig hepatic and pulmonary cytochrome P450 by N-benzyl-1-aminobenzotriazole and N-.alpha.-methylbenzyl-1-aminobenzotriazole. Drug Metab. Dispos. 24:996–1001.Google Scholar
  53. Shou M, Hayashi M, Pan Y, Xu Y, Morrissey K, Xu L and Skiles GL (2008) Modeling, prediction, and in vitro in vivo correlation of CYP3A4 induction. Drug Metab Dispos 36:2355–2370.CrossRefPubMedGoogle Scholar
  54. Turpeinen M, Tolonen A, Uusitalo J, Jalonen J, Pelkonen O and Laine K (2005) Effect of clopidogrel and ticlopidine on cytochrome P450 2B6 activity as measured by bupropion hydroxylation. Clin Pharmacol Ther 77:553–559.CrossRefPubMedGoogle Scholar
  55. Van LM, Heydari A, Yang J, Hargreaves J, Rowland-Yeo K, Lennard MS, Tucker GT and Rostami-Hodjegan A (2006) The impact of experimental design on assessing mechanism-based inactivation of CYP2D6 by MDMA (Ecstasy). J Psychopharmacol 20:834–841.Google Scholar
  56. Venkatakrishnan K and Obach RS (2005) In vitro-in vivo extrapolation of CYP2D6 inactivation by paroxetine: prediction of nonstationary pharmacokinetics and drug interaction magnitude. Drug Metab Dispos 33:845–852.CrossRefPubMedGoogle Scholar
  57. Venkatakrishnan K and Obach RS (2007) Drug–drug interactions via mechanism-based cytochrome P450 inactivation: points to consider for risk assessment from in vitro data and clinical pharmacologic evaluation. Curr Drug Metab 8:449–462.CrossRefPubMedGoogle Scholar
  58. Venkatakrishnan K, Obach RS and Rostami-Hodjegan A (2007) Mechanism-based inactivation of human cytochrome P450 enzymes: strategies for diagnosis and drug–drug interaction risk assessment. Xenobiotica 37:1225–1256.CrossRefPubMedGoogle Scholar
  59. Walsky RL and Boldt SE (2008) In vitro cytochrome P450 inhibition and induction. Curr Drug Metab 9:928–939.CrossRefPubMedGoogle Scholar
  60. Walsky RL and Obach RS (2007) A comparison of 2-phenyl-2-(1-piperidinyl)propane (ppp), 1,1',1-phosphinothioylidynetrisaziridine (thioTEPA), clopidogrel, and ticlopidine as selective inactivators of human cytochrome P450 2B6. Drug Metab Dispos 35:2053–2059.CrossRefPubMedGoogle Scholar
  61. Wang YH, Jones DR and Hall SD (2004) Prediction of cytochrome P450 3A inhibition by verapamil enantiomers and their metabolites. Drug Metab Dispos 32:259–266.CrossRefPubMedGoogle Scholar
  62. Williams JA, Hurst SI, Bauman J, Jones BC, Hyland R, Gibbs JP, Obach RS and Ball SE (2003) Reaction phenotyping in drug discovery: moving forward with confidence?. Curr Drug Metab 4:527–534.CrossRefPubMedGoogle Scholar
  63. Yang J, Jamei M, Yeo KR, Tucker GT and Rostami-Hodjegan A (2007) Theoretical assessment of a new experimental protocol for determining kinetic values describing mechanism (time)-based enzyme inhibition. Eur J Pharm Sci 31:232–241.CrossRefPubMedGoogle Scholar
  64. Zhou S, Yung CS, Cher GB, Chan E, Duan W, Huang M and McLeod HL (2005) Mechanism-based inhibition of cytochrome P450 3A4 by therapeutic drugs. Clin Pharmacokinet 44:279–304.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • R. Scott Obach
    • 1
  • Odette A. Fahmi
    • 1
  • Robert L. Walsky
    • 1
  1. 1.Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer Inc.Global Research & DevelopmentGrotonUSA

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