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Simultaneous Determination of Multiple CYP Inhibition Constants using a Cocktail-Probe Approach

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Cytochrome P450 Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 987))

Abstract

To identify cytochrome P450 (CYP) drug–drug interaction (DDI) potential of a new chemical entity, the use of a specific clinically relevant probe substrate in the presence of a test compound is common place. In early discovery of new chemical entities, a balance of rigor, the ability to predict clinical DDI, and throughput is desired in an in vitro assay. This chapter describes a high-throughput CYP-mediated DDI assay method that balances these characteristics. The method utilizes a cassette approach using a cocktail of five selective probe substrates for the major clinically relevant CYPs involved in drug interactions. CYP1A2, 2C9, 2C19, 2D6, and 3A activities are assessed with liquid chromatography/tandem mass spectrometry (LC-MS/MS) quantification of metabolite formation. The method also outlines specific inhibitors to evaluate dynamic range and as a positive control. The benefits and needs for caution of this method are noted and discussed.

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References

  1. Smith CC, Bennett PM, Pearce HM, Harrison PI, Reynolds DJ, Aronson JK, Grahame-Smith DG (1996) Adverse drug reactions in a hospital general medical unit meriting notification to the Committee on Safety of Medicines. Br J Clin Pharmacol 42:423–9

    Article  PubMed  CAS  Google Scholar 

  2. Issa AM, Phillips KA, Van Bebber S, Nidmarthy HG, Lasser KE, Haas JS, Alldredge BK, Wachter RM, Bates DW (2007) Drug withdrawals in the United States: a systematic review of the evidence and analysis of trends. Curr Drug Saf 2:177–185

    Article  PubMed  CAS  Google Scholar 

  3. Bjornsson TD, Callaghan JT, Einolf HJ, Fischer V, Gan L, Grimm S, Kao J, King SP, Miwa G, Ni L, Kumar G, McLeod J, Obach RS, Roberts S, Roe A, Shah A, Snikeris F, Sullivan JT, Tweedie D, Vega JM, Walsh J, Wrighton SA (2003) The conduct of in vitro and in vivo drug-drug interaction studies: a Pharmaceutical Research and Manufacturers of America (PhRMA) perspective. Drug Metabol Dispos 31:815–32

    Article  CAS  Google Scholar 

  4. Huang S, Stifano T (2006) C.f.D.E.a.R. (CDER), C.f.B.E.a.R. (CBER) (ed) Draft guidance for industry: drug interaction studies—study design, data analysis, and implications for dosing and labeling. U.S. Department of Health and Human Services Food and Drug Administration.

    Google Scholar 

  5. Tucker G, Houston B, Huang S (2001) Optimizing drug development: strategies to assess drug metabolism/transporter interaction potential—toward a consensus. Pharmaceut Res V18:1071–1080

    Article  Google Scholar 

  6. Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerich FP (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270:414–23

    PubMed  CAS  Google Scholar 

  7. Yuan R, Madani S, Wei XX, Reynolds K, Huang SM (2002) Evaluation of cytochrome P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions. Drug Metab Dispos 30:1311–9

    Article  PubMed  CAS  Google Scholar 

  8. Williams JA, Hyland R, Jones BC, Smith DA, Hurst S, Goosen TC, Peterkin V, Koup JR, Ball SE (2004) Drug-drug interactions for UDP-glucuronosyltransferase substrates: a pharmacokinetic explanation for typically observed low exposure (AUCi/AUC) ratios. Drug Metab Dispos 32:1201–8

    Article  PubMed  CAS  Google Scholar 

  9. Bu HZ, Knuth K, Magis L, Teitelbaum P (2000) High-throughput cytochrome P450 inhibition screening via cassette probe-dosing strategy. IV. Validation of a direct injection on-line guard cartridge extraction/tandem mass spectrometry method for simultaneous CYP3A4, 2D6 and 2E1 inhibition assessment. Rapid Commun Mass Spectrom 14:1943–8

    Article  PubMed  CAS  Google Scholar 

  10. Bu HZ, Knuth K, Magis L, Teitelbaum P (2001) High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy: III. Validation of a direct injection/on-line guard cartridge extraction-tandem mass spectrometry method for CYP2C19 inhibition evaluation. J Pharm Biomed Anal 25:437–42

    Article  PubMed  CAS  Google Scholar 

  11. Bu HZ, Knuth K, Magis L, Teitelbaum P (2001) High-throughput cytochrome P450 (CYP) inhibition screening via a cassette probe-dosing strategy. V. Validation of a direct injection/on-line guard cartridge extraction–tandem mass spectrometry method for CYP1A2 inhibition assessment. Eur J Pharm Sci 12:447–52

    Article  PubMed  CAS  Google Scholar 

  12. Bu HZ, Magis L, Knuth K, Teitelbaum P (2000) High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. I. Development of direct injection/on-line guard cartridge extraction/tandem mass spectrometry for the simultaneous detection of CYP probe substrates and their metabolites. Rapid Commun Mass Spectrom 14:1619–24

    Article  PubMed  CAS  Google Scholar 

  13. Bu HZ, Magis L, Knuth K, Teitelbaum P (2001) High-throughput cytochrome P450 (CYP) inhibition screening via a cassette probe-dosing strategy. VI. Simultaneous evaluation of inhibition potential of drugs on human hepatic isozymes CYP2A6, 3A4, 2C9, 2D6 and 2E1. Rapid Commun Mass Spectrom 15:741–8

    Article  PubMed  CAS  Google Scholar 

  14. Bu HZ, Magis L, Knuth K, Teitelbaum P (2001) High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. II. Validation of a direct injection/on-line guard cartridge extraction-tandem mass spectrometry method for CYP2D6 inhibition assessment. J Chromatogr B Biomed Sci Appl 753:321–6

    Article  PubMed  CAS  Google Scholar 

  15. Gao F, Johnson DL, Ekins S, Janiszewski J, Kelly KG, Meyer RD, West M (2002) Optimizing higher throughput methods to assess drug-drug interactions for CYP1A2, CYP2C9, CYP2C19, CYP2D6, rCYP2D6, and CYP3A4 in vitro using a single point IC(50). J Biomol Screen 7:373–82

    PubMed  CAS  Google Scholar 

  16. Obach RS, Walsky RL, Venkatakrishnan K, Houston JB, Tremaine LM (2005) In vitro cytochrome P450 inhibition data and the prediction of drug-drug interactions: qualitative relationships, quantitative predictions, and the rank-order approach. Clin Pharmacol Ther 78:582–92

    Article  PubMed  CAS  Google Scholar 

  17. Smith D, Sadagopan N, Zientek M, Reddy A, Cohen L (2007) Analytical approaches to determine cytochrome P450 inhibitory potential of new chemical entities in drug discovery. J Chromatogr B Analyt Technol Biomed Life Sci 850:455–63

    Article  PubMed  CAS  Google Scholar 

  18. Walsky RL, Obach RS (2004) Validated assays for human cytochrome P450 activities. Drug Metab Dispos 32:647–60

    Article  PubMed  CAS  Google Scholar 

  19. Youdim KA, Lyons R, Payne L, Jones BC, Saunders K (2008) An automated, high-throughput, 384 well Cytochrome P450 cocktail IC50 assay using a rapid resolution LC-MS/MS end-point. J Pharm Biomed Anal 48:92–9

    Article  PubMed  CAS  Google Scholar 

  20. Zientek M, Miller H, Smith D, Dunklee M, Heinle L, Thurston A, Lee C, Hyland R, Fahmi O, Burdette D (2008) Development of an in vitro drug-drug interaction assay to simultaneously monitor five cytochrome P450 isoforms and performance assessment using drug library compounds. J Pharmacol Toxicol Methods 58:206–214

    Article  PubMed  CAS  Google Scholar 

  21. Cawley GF, Batie CJ, Backes WL (1995) Substrate-dependent competition of different P450 isozymes for limiting NADPH-cytochrome P450 reductase. Biochemistry 34:1244–7

    Article  PubMed  CAS  Google Scholar 

  22. West SB, Lu AY (1972) Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds and endogenous substrates. V. Competition between cytochromes P-450 and P-448 for reductase in 3,4-benzpyrene hydroxylation. Arch Biochem Biophys 153:298–303

    Article  PubMed  CAS  Google Scholar 

  23. Weaver R, Graham KS, Beattie IG, Riley RJ (2003) Cytochrome P450 inhibition using recombinant proteins and mass spectrometry/multiple reaction monitoring technology in a cassette incubation. Drug Metab Dispos 31:955–66

    Article  PubMed  CAS  Google Scholar 

  24. Hurley JH, Dean AM, Koshland DE Jr, Stroud RM (1991) Catalytic mechanism of NADP(+)-dependent isocitrate dehydrogenase: implications from the structures of magnesium-isocitrate and NADP+ complexes. Biochemistry 30:8671–8

    Article  PubMed  CAS  Google Scholar 

  25. Busby WF Jr, Ackermann JM, Crespi CL (1999) Effect of methanol, ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450. Drug Metab Dispos 27:246–9

    PubMed  CAS  Google Scholar 

  26. Easterbrook J, Lu C, Sakai Y, Li AP (2001) Effects of organic solvents on the activities of cytochrome P450 isoforms, UDP-dependent glucuronyl transferase, and phenol sulfotransferase in human hepatocytes. Drug Metab Dispos 29:141–4

    PubMed  CAS  Google Scholar 

  27. Chauret N, Gauthier A, Nicoll-Griffith DA (1998) Effect of common organic solvents on in vitro cytochrome P450-mediated metabolic activities in human liver microsomes. Drug Metab Dispos 26:1–4

    PubMed  CAS  Google Scholar 

  28. Leemann T, Transon C, Dayer P (1993) Cytochrome P450TB (CYP2C): a major monooxygenase catalyzing diclofenac 4′-hydroxylation in human liver. Life Sci 52:29–34

    Article  PubMed  CAS  Google Scholar 

  29. Wrighton SA, Stevens JC, Becker GW, VandenBranden M (1993) Isolation and characterization of human liver cytochrome P450 2C19: correlation between 2C19 and S-mephenytoin 4′-hydroxylation. Arch Biochem Biophys 306:240–5

    Article  PubMed  CAS  Google Scholar 

  30. Goldstein JA, Faletto MB, Romkes-Sparks M, Sullivan T, Kitareewan S, Raucy JL, Lasker JM, Ghanayem BI (1994) Evidence that CYP2C19 is the MAJOR (S)-mephenytoin 4′-hydroxylase in humans. Biochemistry 33:1743–1752

    Article  PubMed  CAS  Google Scholar 

  31. Rodrigues AD (1996) Measurement of human liver microsomal cytochrome P450 2D6 activity using (O-methyl-14C)dextromethorphan as substrate. Methods Enzymol 272:186–95

    Article  PubMed  CAS  Google Scholar 

  32. Kronbach T, Mathys D, Umeno M, Gonzalez FJ, Meyer UA (1989) Oxidation of midazolam and triazolam by human liver cytochrome P450IIIA4. Mol Pharmacol 36:89–96

    PubMed  CAS  Google Scholar 

  33. Dierks EA, Stams KR, Lim HK, Cornelius G, Zhang H, Ball SE (2001) A method for the simultaneous evaluation of the activities of seven major human drug-metabolizing cytochrome P450s using an in vitro cocktail of probe substrates and fast gradient liquid chromatography tandem mass spectrometry. Drug Metab Dispos 29:23–9

    PubMed  CAS  Google Scholar 

  34. Obach RS, Reed-Hagen AE (2002) Measurement of Michaelis constants for cytochrome P450-mediated biotransformation reactions using a substrate depletion approach. Drug Metab Dispos 30:831–7

    Article  PubMed  CAS  Google Scholar 

  35. Becquemont L, Le Bot MA, Riche C, Funck-Brentano C, Jaillon P, Beaune P (1998) Use of heterologously expressed human cytochrome P450 1A2 to predict tacrine-fluvoxamine drug interaction in man. Pharmacogenetics 8:101–8

    Article  PubMed  CAS  Google Scholar 

  36. Cheng Y, Prusoff WH (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22:3099–108

    Article  PubMed  CAS  Google Scholar 

  37. Walsky RL, Obach RS, Gaman EA, Gleeson JP, Proctor WR (2005) Selective inhibition of human cytochrome P4502C8 by montelukast. Drug Metab Dispos 33:413–8

    Article  PubMed  CAS  Google Scholar 

  38. Margolis JM, 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 31:606–11

    Article  PubMed  CAS  Google Scholar 

  39. Jenkins KM, Angeles R, Quintos MT, Xu R, Kassel DB, Rourick RA (2004) Automated high throughput ADME assays for metabolic stability and cytochrome P450 inhibition profiling of combinatorial libraries. J Pharm Biomed Anal 34:989–1004

    Article  PubMed  CAS  Google Scholar 

  40. Hewavitharana AK (2011) Matrix matching in liquid chromatography-mass spectrometry with stable isotope labelled internal standards—is it necessary? J Chromatogr A 1218:359–61

    Article  PubMed  CAS  Google Scholar 

  41. Youdim KA, Saunders KC (2010) A review of LC-MS techniques and high-throughput approaches used to investigate drug metabolism by cytochrome P450s. J Chromatogr B Analyt Technol Biomed Life Sci 878:1326–36

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Pfizer Inc., La Jolla, CA, USA

    Michael Zientek

  2. F. Hoffmann-La Roche AG, Basel, Switzerland

    Kuresh Youdim

Authors
  1. Michael Zientek
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  2. Kuresh Youdim
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Editor information

Editors and Affiliations

  1. Sch. of Biological & Chemical Sciences, Queen Mary, University of London, Mile End Road, London, E1 4NS, United Kingdom

    Ian R. Phillips

  2. , Department of Structural and Molecular B, University College London, Gower Street, London, WC1E 6BT, United Kingdom

    Elizabeth A. Shephard

  3. University of California, 16th St 600, San Francisco, 94158-2517, California, USA

    Paul R. Ortiz de Montellano

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Zientek, M., Youdim, K. (2013). Simultaneous Determination of Multiple CYP Inhibition Constants using a Cocktail-Probe Approach. In: Phillips, I., Shephard, E., Ortiz de Montellano, P. (eds) Cytochrome P450 Protocols. Methods in Molecular Biology, vol 987. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-321-3_2

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  • DOI: https://doi.org/10.1007/978-1-62703-321-3_2

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-320-6

  • Online ISBN: 978-1-62703-321-3

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