Use of Cocktail Probe Drugs for Indexing Cytochrome P450 Enzymes in Clinical Pharmacology Studies – Review of Case Studies

Author(s): Poonam Giri, Harilal Patel, Nuggehally R. Srinivas*

Journal Name: Drug Metabolism Letters

Volume 13 , Issue 1 , 2019

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Graphical Abstract:


Background: The cocktail approach of probing drug metabolizing enzymes, in particular cytochrome P450 (CYP) enzymes, is a cornerstone in clinical pharmacology studies. The first report of the famous “Pittsburg cocktail” has led the way for the availability of numerous cocktail substrate mixtures that provide options for indexing of CYP enzymes and/or evaluating the perpetrator capacity of the drug.

Objective: The key objectives were: 1) To collate, tabulate, and discuss the various cocktail substrates to determine specific CYP enzyme activity in clinical pharmacology studies with specific case studies; 2) To introspect on how the cocktail approach has withstood the test of time and evolved for enabling key decision(s); 3) To provide some futuristic views on the use of cocktail in drug discovery and development.

Method: The review was compiled after consultation with databases such as PubMed (NCBI database) and Google scholar to source various published literature on cocktail approaches in drug development.

Results: In the reviewed case studies, CYP indexing was achieved using a single time point (differing for specific CYP enzyme) plasma determination of the metabolite to parent ratio for all CYP enzymes with the exception of CYP3A4/5, where multiple time points were required for exposure measurement of midazolam and its metabolite. Likewise, a single void of urine, for a specific time duration, has been utilized for the recovery measurements of parent and metabolite for CYP indexing purposes.

Conclusion: The review provides a comprehensive list of various types of cocktail approaches and discusses some key considerations including the evolution of the cocktail approaches over time, perspectives and futuristic views for the use of probe drugs to aid the execution of clinical pharmacology studies and data interpretation.

Keywords: Cocktail probe drugs, cytochrome P450, clinical pharmacology, CYP enzymes, pittsburg cocktail, perpetrator capacity.

Streetman, D.S.; Bertino, J.S.; Nafziger, A.N. Phenotyping of drug-metabolizing enzymes in adults: A review of in-vivo cytochrome P450 phenotyping probes. Pharmacogenetics, 2000, 10, 187-216.
Frye, R.F. Probing the world of cytochrome p450 enzymes. Mol. Interv., 2004, 4, 157-162.
Breimer, D.D.; Schellens, J.H.A. ‘cocktail’ strategy to assess in vivo oxidative drug metabolism in humans. Trends Pharmacol. Sci., 1990, 11(6), 223-225.
Frye, R.F.; Matzke, G.R.; Adedoyin, A.; Porter, J.A.; Branch, R.A. Validation of the five-drug “Pittsburgh cocktail” approach for assessment of selective regulation of drug metabolizing enzymes. Clin. Pharmacol. Ther., 1997, 62(4), 365-376.
Tucker, G.T.; Houston, J.B.; Huang, S.M. Optimising drug development: Strategies to assess drug metabolism/transporter interaction - toward a consensus. Clin. Pharmacol. Ther., 2001, 70(2), 103-114.
Zanger, U.M.; Schwab, M. Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol. Ther., 2013, 138(1), 103-141.
Preissner, S.C.; Hoffmann, M.F.; Preissner, R.; Dunkel, M.; Gewiess, A.; Preissner, S. Polymorphic cytochrome P450 enzymes (CYPs) and their role in personalized therapy. PLoS One, 2013, 8(12), e82562.
Xie, F.; Ding, X.; Zhang, Q.Y. An update on the role of intestinal cytochrome P450 enzymes in drug disposition. Acta Pharm. Sin. B, 2016, 6(5), 374-383.
Adedoyin, A.; Frye, R.F.; Mauro, K.; Branch, R.A. Chloroquine modulation of specific metabolizing enzymes activities: investigation with selective five drug cocktail. Br. J. Clin. Pharmacol., 1998, 46(3), 215-219.
Zgheib, N.K.; Frye, R.F.; Tracy, T.S.; Romkes, M.; Branch, R.A. Validation of incorporating flurbiprofen into the Pittsburgh cocktail. Clin. Pharmacol. Ther., 2006, 80(3), 0257-0263.
Brynne, N.; Böttiger, Y.; Hallén, B.; Bertilsson, L. Tolterodine does not affect the human in vivo metabolism of the probe drugs caffeine, debrisoquine and omeprazole. Br. J. Clin. Pharmacol., 1999, 47(2), 145-150.
Damkier, P.; Hansen, L.L.; Brosen, K. Effect of diclofenac, disulfiram, itraconazole, grapefruit juice and erythromycin on the pharmacokinetics of quinidine. Br. J. Clin. Pharmacol., 1999, 48(6), 829-838.
Damkier, P.; Brosen, K. Quinidine as a probe for CYP3A4/5 activity: intrasubject variability and lack of correlation with probe-based assays for CYP1A2, CYP2C9, CYP2C19, and CYP2D6. Clin. Pharmacol. Ther., 2000, 68(2), 199-209.
Scott, R.J.; Palmer, J.; Lewis, I.A.; Pleasance, S. Determination of a ‘GW cocktail’ of cytochrome P450 probe substrates and their metabolites in plasma and urine using automated solid phase extraction and fast gradient liquid chromatography tandem mass spectrometry. Rapid Commun. Mass Spectrom., 1999, 13(23), 2305-2319.
Streetman, D.S.; Bleakley, J.F.; Kim, J.S.; Nafziger, A.N.; Leeder, J.S.; Gaedigk, A.; Gotschall, R.; Kearns, G.L.; Bertino, J.S., Jr Combined phenotypic assessment of CYP1A2, CYP2C19, CYP2D6, CYP3A, N-acetyltransferase-2, and xanthine oxidase with the “Cooperstown cocktail”. Clin. Pharmacol. Ther., 2000, 68(4), 375-383.
Wang, Z.; Gorski, J.C.; Hamman, M.A.; Huang, S.M.; Lesko, L.J.; Hall, S.D. The effects of St John’s wort (Hypericum perforatum) on human cytochrome P450 activity. Clin. Pharmacol. Ther., 2001, 70(4), 317-326.
Palmer, J.L.; Scott, R.J.; Gibson, A.; Dickins, M.; Pleasance, S. An interaction between the cytochrome P450 probe substrates chlorzoxazone (CYP2E1) and midazolam (CYP3A). Br. J. Clin. Pharmacol., 2001, 52(5), 555-561.
Sharma, A.; Pilote, S.; Bélanger, P.M.; Arsenault, M.; Hamelin, B.A. A convenient five-drug cocktail for the assessment of major drug metabolizing enzymes: a pilot study. Br. J. Clin. Pharmacol., 2004, 58(3), 288-297.
Zhu, B.; Ou-Yang, D.S.; Chen, X.P.; Huang, S.L.; Tan, Z.R.; He, N.; Zhou, H.H. Assessment of cytochrome P450 activity by a five-drug cocktail approach. Clin. Pharmacol. Ther., 2001, 70(5), 455-461.
Christensen, M.; Andersson, K.; Dalén, P. The Karolinska cocktail for phenotyping of five human cytochrome P450 enzymes. Clin. Pharmacol. Ther., 2003, 73, 517-528.
Chainuvati, S.; Nafziger, A.N.; Leeder, J.S.; Gaedigk, A.; Kearns, G.L.; Sellers, E.; Zhang, Y.; Kashuba, A.D.; Rowland, E.; Bertino, J.S., Jr Combined phenotypic assessment of cytochrome p450 1A2, 2C9, 2C19, 2D6, and 3A, N-acetyltransferase-2, and xanthine oxidase activities with the “Cooperstown 5+1 cocktail”. Clin. Pharmacol. Ther., 2003, 74(5), 437-447.
Jerdi, M.C.; Daali, Y.; Oestreicher, M.K.; Cherkaoui, S.; Dayer, P. A simplified analytical method for a phenotyping cocktail of major CYP450 biotransformation routes. J. Pharm. Biomed. Anal., 2004, 35(5), 1203-1212.
Blakey, G.E.; Lockton, J.A.; Perrett, J.; Norwood, P.; Russell, M.; Aherne, Z.; Plume, J. Pharmacokinetic and pharmacodynamic assessment of a five-probe metabolic cocktail for CYPs 1A2, 3A4, 2C9, 2D6 and 2E1. Br. J. Clin. Pharmacol., 2004, 57(2), 162-169.
Yin, O.Q.; Lam, S.S.; Lo, C.M.; Chow, M.S. Rapid determination of five probe drugs and their metabolites in human plasma and urine by liquid chromatography/tandem mass spectrometry: application to cytochrome P450 phenotyping studies. Rapid Commun. Mass Spectrom., 2004, 18(23), 2921-2933.
Tomalik-Scharte, D.; Jetter, A. Kinzig-Schippers., M.; Skott, A.; Sörgel, F.; Klaassen, T.; Kasel, D.; Harlfinger, S.; Doroshyenko, O.; Frank, D.; Kirchheiner, J.; Bräter, M.; Richter, K.; Gramatté, T.; Fuhr, U. Effect of propiverine on cytochrome P450 enzymes: a cocktail interaction study in healthy volunteers. Drug Metab. Dispos., 2005, 33(12), 1859-1866.
Krösser, S.; Neugebauer, R.; Dolgos, H.; Fluck, M.; Rost, K.L.; Kovar, A. Investigation of sarizotan’s impact on the pharmacokinetics of probe drugs for major cytochrome P450 isoenzymes: a combined cocktail trial. Eur. J. Clin. Pharmacol., 2006, 62(4), 277-284.
Ryu, J.Y.; Song, I.S.; Sunwoo, Y.E.; Shon, J.H.; Liu, K.H.; Cha, I.J.; Shin, J.G. Development of the “Inje cocktail” for high-throughput evaluation of five human cytochrome P450 isoforms in vivo. Clin. Pharmacol. Ther., 2007, 82(5), 531-540.
Klaassen, T.; Jetter, A.; Tomalik-Scharte, D.; Kasel, D.; Kirchheiner, J.; Jaehde, U.; Fuhr, U. Assessment of urinary mephenytoin metrics to phenotype for CYP2C19 and CYP2B6 activity. Eur. J. Clin. Pharmacol., 2008, 64(4), 387-398.
Petsalo, A.; Turpeinen, M.; Pelkonen, O.; Tolonen, A. Analysis of nine drugs and their cytochrome P450-specific probe metabolites from urine by liquid chromatography-tandem mass spectrometry utilizing sub 2 microm particle size column. J. Chromatogr. A, 2008, 1215(1-2), 107-115.
Ghassabian, S.; Chetty, M.; Tattam, B.N.; Chem, M.C.; Glen, J.; Rahme, J.; Stankovic, Z.; Ramzan, I.; Murray, M.; McLachlan, A.J. A high-throughput assay using liquid chromatography-tandem mass spectrometry for simultaneous in vivo phenotyping of 5 major cytochrome p450 enzymes in patients. Ther. Drug Monit., 2009, 31(2), 239-246.
Turpault, S.; Brian, W.; Van Horn, R.; Santoni, A.; Poitiers, F.; Donazzolo, Y.; Boulenc, X. Pharmacokinetic assessment of a five-probe cocktail for CYPs 1A2, 2C9, 2C19, 2D6 and 3A. Br. J. Clin. Pharmacol., 2009, 68(6), 928-935.
Wohlfarth, A.; Naue, J.; Lutz-Bonengel, S.; Dresen, S.; Auwärter, V. Cocktail approach for in vivo phenotyping of 5 major CYP450 isoenzymes: development of an effective sampling, extraction, and analytical procedure and pilot study with comparative genotyping. J. Clin. Pharmacol., 2012, 52(8), 1200-1214.
Zadoyan, G.; Rokitta, D.; Klement, S.; Dienel, A.; Hoerr, R.; Gramatté, T.; Fuhr, U. Effect of Ginkgo biloba special extract EGb 761® on human cytochrome P450 activity: a cocktail interaction study in healthy volunteers. Eur. J. Clin. Pharmacol., 2012, 68(5), 553-560.
Doroshyenko, O.; Rokitta, D.; Zadoyan, G.; Klement, S.; Schläfke, S.; Dienel, A.; Gramatté, T.; Lück, H.; Fuhr, U. Drug cocktail interaction study on the effect of the orally administered lavender oil preparation silexan on cytochrome P450 enzymes in healthy volunteers. Drug Metab. Dispos., 2013, 41(5), 987-993.
de Andrés, F.; Sosa-Macías, M.; Lazalde-Ramos, B.P.; Naranjo, M.E.; Tarazona-Santos, E.; Llerena, A. CEIBA.FP Consortium. Evaluation of drug-metabolizing enzyme hydroxylation phenotypes in Hispanic populations: the CEIBA cocktail. Drug Metabol. Drug Interact., 2013, 28(3), 135-146.
Snyder, B.D.; Rowland, A.; Polasek, T.M.; Miners, J.O.; Doogue, M.P. Evaluation of felodipine as a potential perpetrator of pharmacokinetic drug-drug interactions. Eur. J. Clin. Pharmacol., 2014, 70(9), 1115-1122.
Tanaka, S.; Uchida, S.; Inui, N.; Takeuchi, K.; Watanabe, H.; Namiki, N. Simultaneous LC-MS/MS analysis of the plasma concentrations of a cocktail of 5 cytochrome P450 substrate drugs and their metabolites. Biol. Pharm. Bull., 2014, 37(1), 18-25.
Bosilkovska, M.; Clément, M.; Dayer, P.; Desmeules, J.; Daali, Y. Incorporation of flurbiprofen in a 4-drug cytochrome p450 phenotyping cocktail. Basic Clin. Pharmacol. Toxicol., 2014, 115(5), 465-466.
Bosilkovska, M.; Samer, C.F.; Déglon, J.; Rebsamen, M.; Staub, C.; Dayer, P.; Walder, B.; Desmeules, J.A.; Daali, Y. Geneva cocktail for cytochrome p450 and P-glycoprotein activity assessment using dried blood spots. Clin. Pharmacol. Ther., 2014, 96(3), 349-359.
Bosilkovska, M.; Samer, C.; Déglon, J.; Thomas, A.; Walder, B.; Desmeules, J.; Daali, Y. Evaluation of mutual drug-drug interaction within Geneva cocktail for cytochrome P450 phenotyping using innovative dried blood sampling method. Basic Clin. Pharmacol. Toxicol., 2016, 119(3), 284-290.
Donzelli, M.; Derungs, A.; Serratore, M.G.; Noppen, C.; Nezic, L.; Krähenbühl, S.; Haschke, M. The basel cocktail for simultaneous phenotyping of human cytochrome P450 isoforms in plasma, saliva and dried blood spots. Clin. Pharmacokinet., 2014, 53(3), 271-282.
Derungs, A.; Donzelli, M.; Berger, B.; Noppen, C.; Krähenbühl, S.; Haschke, M. Effects of cytochrome P450 inhibition and induction on the phenotyping metrics of the basel cocktail: A randomized crossover study. Clin. Pharmacokinet., 2016, 55(1), 79-91.
Lenuzza, N.; Duval, X.; Nicolas, G.; Thévenot, E.; Job, S.; Videau, O.; Narjoz, C.; Loriot, M.A.; Beaune, P.; Becquemont, L.; Mentre, F.; Funck-Brentano, C.; Alavoine, L.; Arnaud, P.; Delaforge, M.; Bénech, H. Safety and pharmacokinetics of the CIME combination of drugs and their metabolites after a single oral dosing in healthy volunteers. Eur. J. Drug Metab. Pharmacokinet., 2016, 41(2), 125-138.
Kim, D.S.; Kim, Y.; Jeon, J.Y.; Kim, M.G. Effect of Red Ginseng on cytochrome P450 and P-glycoprotein activities in healthy volunteers. J. Ginseng Res., 2016, 40(4), 375-381.
Williams, D.; Tao, X.; Zhu, L.; Stonier, M.; Lutz, J.D.; Masson, E.; Zhang, S.; Ganguly, B.; Tzogas, Z.; Lubin, S.; Murthy, B. Use of a cocktail probe to assess potential drug interactions with cytochrome P450 after administration of belatacept, a costimulatory immunomodulator. Br. J. Clin. Pharmacol., 2017, 83(2), 370-380.
Armani, S.; Ting, L.; Sauter, N.; Darstein, C.; Tripathi, A.P.; Wang, L.; Zhu, B.; Gu, H.; Chun, D.Y.; Einolf, H.J.; Kulkarni, S. Drug interaction potential of osilodrostat (LCI699) based on its effect on the pharmacokinetics of probe drugs of cytochrome P450 enzymes in healthy adults. Clin. Drug Investig., 2017, 37(5), 465-472.
Garimella, T.; Tao, X.; Sims, K.; Chang, Y.T.; Rana, J.; Myers, E.; Wind-Rotolo, M.; Bhatnagar, R.; Eley, T.; LaCreta, F.; AbuTarif, M. Effects of a fixed-dose co-formulation of daclatasvir, asunaprevir, and beclabuvir on the pharmacokinetics of a cocktail of cytochrome p450 and drug transporter substrates in healthy subjects. Drugs R D., 2018, 18(1), 55-65.
Lancaster, D.L.; Adio, R.A.; Tai, K.K.; Simooya, O.O.; Broadhead, G.D.; Tucker, G.T.; Lennard, M.S. Inhibition of metoprolol metabolism by chloroquine and other antimalarial drugs. J. Pharm. Pharmacol., 1990, 42(4), 267-271.
Gill, H.J.; Tingle, M.D.; Park, B.K. N-hydroxylation of dapsone by multiple enzymes of cytochrome P450: implications for inhibition of haemotoxicity. Br. J. Clin. Pharmacol., 1995, 40(6), 531-538.
Jetter, A.; Kinzig-Schippers, M.; Skott, A.; Tomalik-Scharte, D.; Kirchheiner, J.; Walchner-Bonjean, M.; Hering, U.; Jakob, V.; Rodamer, M.; Jabrane, W.; Kasel, D.; Brockmöller, J.; Fuhr, U.; Sörgel, F. Cytochrome P450 2C9 phenotyping using low-dose tolbutamide. Eur. J. Clin. Pharmacol., 2004, 60(3), 165-171.
Bruce, M.A.; Hall, S.D.; Haehner-Daniels, B.D.; Gorski, J.C. In vivo effect of clarithromycin on multiple cytochrome P450s. Drug Metab. Dispos., 2001, 29(7), 1023-1028.
Choi, J.H.; Lee, M.G.; Cho, J.Y.; Lee, J.E.; Kim, K.H.; Park, K. Influence of OATP1B1 genotype on the pharmacokinetics of rosuvastatin in Koreans. Clin. Pharmacol. Ther., 2008, 83(2), 251-257.
Prueksaritanont, T.; Chu, X.; Evers, R.; Klopfer, S.O.; Caro, L.; Kothare, P.A.; Dempsey, C.; Rasmussen, S.; Houle, R.; Chan, G.; Cai, X.; Valesky, R.; Fraser, I.P.; Stoch, S.A. Pitavastatin is a more sensitive and selective organic anion-transporting polypeptide 1B clinical probe than rosuvastatin. Br. J. Clin. Pharmacol., 2014, 78(3), 587-598.
Schellens, J.H.; Ghabrial, H.; van der Wart, H.H.; Bakker, E.N.; Wilkinson, G.R.; Breimer, D.D. Differential effects of quinidine on the disposition of nifedipine, sparteinee, and mephenytoin in humans. Clin. Pharmacol. Ther.,, 1991, 50(5-part 1), 520-528.
Henschel, L.; Hoffmann, A. Assessment of biotransformation capacity following oral administration of various model substances as cocktail. Z. Gastroenterol., 1991, 29(12), 645-649.
Setiabudy, R.; Kusaka, M.; Chiba, K.; Darmansjah, I.; Ishizaki, T. Dapsone N-acetylation, metoprolol alpha-hydroxylation, and S-mephenytoin 4-hydroxylation polymorphisms in an Indonesian population: a cocktail and extended phenotyping assessment trial. Clin. Pharmacol. Ther., 1994, 56(2), 142-153.
Gass, R.J.; Gal, J.; Fogle, P.W.; Detmar-Hanna, D.; Gerber, J.G. Neither dapsone hydroxylation nor cortisol 6beta-hydroxylation detects the inhibition of CYP3A4/5 by HIV-1 protease inhibitors. Eur. J. Clin. Pharmacol., 1998, 54(9-10), 741-747.
Pauli-Magnus, C.; Rekersbrink, S.; Klotz, U.; Fromm, M.F. Interaction of omeprazole, lansoprazole and pantoprazole with P-glycoprotein. Naunyn Schmiedebergs Arch. Pharmacol., 2001, 364(6), 551-557.
Zhang, Y.; Blouin, R.A.; McNamara, P.J.; Steinmetz, J.; Wedlund, P.J. Limitation to the use of the urinary S-/R-mephenytoin ratio in pharmacogenetic studies. Br. J. Clin. Pharmacol., 1991, 31(3), 350-352.
Srinivas, N.R. Unsuspected and paradoxical potential for drug interaction by rifampin: things to ponder with antiretroviral therapy. J. Infect. Dis., 2009, 199(5), 766-767.
Srinivas, N.R. Pharmacokinetic interaction of rifampicin with oral versus intravenous anticancer drugs: Challenges, dilemmas and paradoxical effects due to multiple mechanisms. Drugs R D., 2016, 16(2), 141-148.
Inui, N.; Akamatsu, T.; Uchida, S.; Tanaka, S.; Namiki, N.; Karayama, M.; Chida, K.; Watanabe, H. Chronological effects of rifampicin discontinuation on cytochrome P450 activity in healthy Japanese volunteers, using the cocktail method. Clin. Pharmacol. Ther., 2013, 94(6), 702-708.
Link, B.; Haschke, M.; Grignaschi, N.; Bodmer, M.; Aschmann, Y.Z.; Wenk, M.; Krähenbühl, S. Pharmacokinetics of intravenous and oral midazolam in plasma and saliva in humans: usefulness of saliva as matrix for CYP3A phenotyping. Br. J. Clin. Pharmacol., 2008, 66(4), 473-484.
Dash, R.P.; Rais, R.; Srinivas, N.R. Key pharmacokinetic essentials of fixed-dosed combination products: Case studies and perspectives. Clin. Pharmacokinet., 2018, 57(4), 419-426.
Dahlinger, D.; Duechting, S.; Nuecken, D.; Sydow, K.; Fuhr, U.; Frechen, S. Development and validation of an in vitro, seven-in-one human cytochrome P450 assay for evaluation of both direct and time-dependent inhibition. J. Pharmacol. Toxicol. Methods, 2016, 77, 66-75.
Derks, M.; Fowler, S.; Kuhlmann, O. In vitro and in vivo assessment of the effect of dalcetrapib on a panel of CYP substrates. Curr. Med. Res. Opin., 2009, 25(4), 891-902.
Dahlinger, D.; Duechting, S.; Nuecken, D.; Sydow, K.; Fuhr, U.; Frechen, S. Development and validation of an in vitro, seven-in-one human cytochrome P450 assay for evaluation of both direct and time-dependent inhibition. J. Pharmacol. Toxicol. Methods, 2016, 77, 66-75.
Kumar, S.; Samuel, K.; Subramanian, R.; Braun, M.P.; Stearns, R.A.; Chiu, S.H.; Evans, D.C.; Baillie, T.A. Extrapolation of diclofenac clearance from in vitro microsomal metabolism data: role of acyl glucuronidation and sequential oxidative metabolism of the acyl glucuronide. J. Pharmacol. Exp. Ther., 2002, 303(3), 969-978.
Lee, C.R.; Pieper, J.A.; Frye, R.F.; Hinderliter, A.L.; Blaisdell, J.A.; Goldstein, J.A. Tolbutamide, flurbiprofen, and losartan as probes of CYP2C9 activity in humans. J. Clin. Pharmacol., 2003, 43(1), 84-91.
Magalhães, P.; De Andrés, F.; Falcao, A. LLerena, A.; Alves, G. Can the CEIBA cocktail designed for human cytochrome p450 enzymes be used in the rat for drug interaction studies? J. Pharm. Pharm. Sci., 2016, 19(4), 520-529.

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Year: 2019
Published on: 29 April, 2019
Page: [3 - 18]
Pages: 16
DOI: 10.2174/1872312812666181119154734

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