Jump to ContentJump to Main Navigation
Show Summary Details
More options …

Drug Metabolism and Personalized Therapy

Official journal of the European Society of Pharmacogenomics and Personalised Therapy

Editor-in-Chief: Llerena, Adrián

Editorial Board: Benjeddou, Mongi / Chen, Bing / Dahl, Marja-Liisa / Devinsky, Ferdinand / Hirata, Rosario / Hubacek, Jaroslav A. / Ingelman-Sundberg, Magnus / Maitland-van der Zee, Anke-Hilse / Manolopoulos, Vangelis G. / Marc, Janja / Melichar, Bohuslav / Meyer, Urs A. / Nair, Sujit / Nofziger, Charity / Peiro, Ana / Sadee, Wolfgang / Salazar, Luis A. / Simmaco, Maurizio / Turpeinen, Miia / Schaik, Ron / Shin, Jae-Gook / Visvikis-Siest, Sophie / Zanger, Ulrich M.

CiteScore 2017: 1.46

SCImago Journal Rank (SJR) 2017: 0.531
Source Normalized Impact per Paper (SNIP) 2017: 0.645

See all formats and pricing
More options …
Volume 31, Issue 4


Improvement of the chemical inhibition phenotyping assay by cross-reactivity correction

Nicholas M. Njuguna
  • Corresponding author
  • The National Quality Control Laboratory, P. O. Box 29726 – 00202, KNH, Nairobi, Kenya, Phone: +254 722 904 908, Fax: +254 20 2718073
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Ken-ichi Umehara
  • Department of Drug Metabolism and Pharmacokinetics, Integrated Drug Disposition Section, Novartis Institutes for BioMedical Research, Basel, Switzerland
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Felix Huth
  • Department of Drug Metabolism and Pharmacokinetics, Integrated Drug Disposition Section, Novartis Institutes for BioMedical Research, Basel, Switzerland
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Hilmar Schiller
  • Department of Drug Metabolism and Pharmacokinetics, Integrated Drug Disposition Section, Novartis Institutes for BioMedical Research, Basel, Switzerland
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Kelly Chibale / Gian Camenisch
  • Department of Drug Metabolism and Pharmacokinetics, Integrated Drug Disposition Section, Novartis Institutes for BioMedical Research, Basel, Switzerland
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2016-10-08 | DOI: https://doi.org/10.1515/dmpt-2016-0028



The fraction of an absorbed drug metabolized by the different hepatic cytochrome P450 (CYP) enzymes, relative to total hepatic CYP metabolism (fmCYP), can be estimated by measuring the inhibitory effects of presumably selective CYP inhibitors on the intrinsic metabolic clearance of a drug using human liver microsomes. However, the chemical inhibition data are often affected by cross-reactivities of the chemical inhibitors used in this assay.


To overcome this drawback, the cross-reactivities exhibited by six chemical inhibitors (furafylline, montelukast, sulfaphenazole, ticlopidine, quinidine and ketoconazole) were quantified using specific CYP enzyme marker reactions. The determined cross-reactivities were used to correct the in vitro fmCYPs of nine marketed drugs. The corrected values were compared with reference data obtained by physiologically based pharmacokinetics simulation using the software SimCYP.


Uncorrected in vitro fmCYPs of the nine drugs showed poor linear correlation with their reference data (R2=0.443). Correction by factoring in inhibitor cross-reactivities significantly improved the correlation (R2=0.736).


Correcting in vitro chemical inhibition results for cross-reactivities appear to offer a straightforward and easily adoptable approach to provide improved fmCYP data for a drug.

Keywords: chemical inhibition; cross-reactivity; CYP; enzyme phenotyping; fmCYP; physiologically based pharmacokinetics (PBPK) model


  • 1.

    Bjornsson TD, Callaghan JT, Einolf HJ, Fischer V, Gan L, Grimm S, et al. The conduct of in vitro and in vivo drug-drug interaction studies: a Pharmaceutical Research and Manufacturers of America (PhRMA) perspective. Drug Metab Dispos 2003;31:815–32.Google Scholar

  • 2.

    Williams JA, Hurst SI, Bauman J, Jones BC, Hyland R, Gibbs JP, et al. Reaction phenotyping in drug discovery: moving forward with confidence? Curr Drug Metab 2003;4:527–34.Google Scholar

  • 3.

    Harper TW, Brassil PJ. Reaction phenotyping: current industry efforts to identify enzymes responsible for metabolizing drug candidates. AAPS J 2008;10:200–7.CrossrefGoogle Scholar

  • 4.

    Food and Drug Administration. Guidance for industry: drug interaction studies study design, data analysis, implications for dosing, and labeling recommendations. Rockville, Maryland, 2012.Google Scholar

  • 5.

    Huang S-M, Strong JM, Zhang L, Reynolds KS, Nallani S, Temple R, et al. New era in drug interaction evaluation: US Food and Drug Administration update on CYP enzymes, transporters, and the guidance process. J Clin Pharmacol 2008;48:662–70.Google Scholar

  • 6.

    Khojasteh SC, Prabhu S, Kenny JR, Halladay JS, Lu AY. Chemical inhibitors of cytochrome P450 isoforms in human liver microsomes: a re-evaluation of P450 isoform selectivity. Eur J Drug Metab Pharmacokinet 2011;36:1–16.Google Scholar

  • 7.

    Obach RS. The utility of in vitro cytochrome P450 inhibition data in the prediction of drug-drug interactions. J Pharmacol Exp Ther 2005;316:336–48.Google Scholar

  • 8.

    Zhang H, Davis CD, Sinz MW, Rodrigues AD. Cytochrome P450 reaction-phenotyping: an industrial perspective. Expert Opin Drug Metab Toxicol 2007;3:667–87.Google Scholar

  • 9.

    Shou M, Lu AY. Antibodies as a probe in cytochrome P450 research. Drug Metab Dispos 2009;37:925–31.Google Scholar

  • 10.

    Grillo MP, Hensley TN, Lam TT, Li Y, Loewen GJ, Nassar AF, et al. In vitro approaches for studying the inhibition of drug-metabolizing enzymes and identifying the drug-metabolizing enzymes responsible for the metabolism of drugs (reaction phenotyping) with emphasis on cytochrome P450. In: Nassar AF, ed. Biotransformation and metabolite elucidation of xenobiotics: characterization and identification. Hoboken, New Jersey: John Wiley & Sons, Inc., 2011.Google Scholar

  • 11.

    Lu C, Miwa GT, Prakash SR, Gan LS, Balani SK. A novel model for the prediction of drug-drug interactions in humans based on in vitro cytochrome P450 phenotypic data. Drug Metab Dispos 2007;35:79–85.Google Scholar

  • 12.

    Peng Y, Wu H, Zhang X, Zhang F, Qi H, Zhong Y, et al. A comprehensive assay for nine major cytochrome P450 enzymes activities with 16 probe reactions on human liver microsomes by a single LC/MS/MS run to support reliable in vitro inhibitory drug–drug interaction evaluation. Xenobiotica 2015;8254:1–17.Google Scholar

  • 13.

    Eiermann B, Engel G, Johansson I, Zanger UM, Bertilsson L. The involvement of CYP1A2 and CYP3A4 in the metabolism of clozapine. Br J Clin Pharmacol 2003;44:439–46.Google Scholar

  • 14.

    Ball SE, Ahern D, Scatina J, Kao J. Venlafaxine: in vitro inhibition of CYP2D6 dependent imipramine and desipramine metabolism; comparative studies with selected SSRIs, and effects on human hepatic CYP3A4, CYP2C9 and CYP1A2. Br J Clin Pharmacol 1997;43:619–26.Google Scholar

  • 15.

    Von Moltke LL, Greenblatt DJ, Duan SX, Daily JP, Harmatz JS, Shader RI. Inhibition of desipramine hydroxylation (cytochrome P450-2D6) in vitro by quinidine and by viral protease inhibitors: relation to drug interactions in vivo. J Pharm Sci 1998;87:1184–9.Google Scholar

  • 16.

    Stresser DM, Broudy MI, Ho T, Cargill CE, Blanchard AP, Sharma R, et al. Highly selective inhibition of human CYP3A in vitro by azamulin and evidence that inhibition is irreversible. Drug Metab Dispos 2004;32:105–12.Google Scholar

  • 17.

    Niwa T, Shiraga T, Ishii I, Kagayama A, Takagi A. Contribution of human hepatic cytochrome P450 isoforms to the metabolism of psychotropic drugs. Biol Pharm Bull 2005;28:1711–16.Google Scholar

  • 18.

    VandenBrink BM, Foti RS, Rock DA, Wienkers LC, Wahlstrom JL. Evaluation of CYP2C8 inhibition in vitro: utility of montelukast as a selective CYP2C8 probe substrate. Drug Metab Dispos 2011;39:1546–54.Google Scholar

  • 19.

    Kajosaari LI, Laitila J, Neuvonen PJ, Backman JT. Metabolism of repaglinide by CYP2C8 and CYP3A4 in vitro: effect of fibrates and rifampicin. Basic Clin Pharmacol Toxicol 2005;97:249–56.Google Scholar

  • 20.

    Baldwin SJ, Clarke SE, Chenery RJ. Characterization of the cytochrome P450 enzymes involved in the in vitro metabolism of rosiglitazone. Br J Clin Pharmacol 1999;48:424–32.Google Scholar

  • 21.

    Postlind H, Danielson Å, Lindgren A, Andersson SH. Tolterodine, a new muscarinic receptor antagonist, is metabolized by cytochromes P450 2D6 and 3A in human liver microsomes. Drug Metab Dispos 1998;26:289–93.Google Scholar

  • 22.

    Von Moltke LL, Greenblatt DJ, Granda BW, Su XD, Grassi JM, Venkatakrishnan K, et al. Zolpidem metabolism in vitro: responsible cytochromes, chemical inhibitors, and in vivo correlations. Br J Clin Pharmacol 1999;48:89–97.Google Scholar

  • 23.

    Stresser DM, Blanchard AP, Turner SD, Erve JC, Dandeneau AA, Miller VP, et al. Substrate-dependent modulation of CYP3A4 catalytic activity: analysis of 27 test compounds with four fluorometric substrates. Drug Metab Dispos 2000;28:1440–8.Google Scholar

  • 24.

    Kenworthy KE, Bloomer JC, Clarke SE, Houston JB. CYP3A4 drug interactions: correlation of 10 in vitro probe substrates. Br J Clin Pharmacol 1999;48:716–27.Google Scholar

  • 25.

    Rowland Yeo K, Rostami-Hodjegan A, Tucker GT. Abundance of cytochromes P450 in human liver: a meta-analysis. Br J Clin Pharmacol 2004;57:687–8.Google Scholar

  • 26.

    He M, Korzekwa KR, Jones JP, Rettie AE, Trager WF. Structural forms of phenprocoumon and warfarin that are metabolized at the active site of CYP2C9. Arch Biochem Biophys 1999;372:16–28.Google Scholar

  • 27.

    Walsky RL, Obach RS, Gaman EA, Gleeson J-PR, Proctor WR. Selective inhibition of human cytochrome P4502C8 by montelukast. Drug Metab Dispos 2005;33:413–18.Google Scholar

  • 28.

    Otton SV, Inaba T, Kalow W. Competitive inhibition of sparteine oxidation in human liver by β-adrenoceptor antagonists and other cardiovascular drugs. Life Sci 1984;34:73–80.Google Scholar

  • 29.

    Yuan R, Madani S, Wei X-X, Reynolds K, Huang S-M. Evaluation of cytochrome P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions. Drug Metab Dispos 2002;30:1311–19.Google Scholar

About the article

Received: 2016-08-29

Accepted: 2016-09-13

Published Online: 2016-10-08

Published in Print: 2016-12-01

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding: Declaration or None declared.

Employment or leadership: Declaration or None declared.

Honorarium: Declaration or None declared.

Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

Citation Information: Drug Metabolism and Personalized Therapy, Volume 31, Issue 4, Pages 221–228, ISSN (Online) 2363-8915, ISSN (Print) 2363-8907, DOI: https://doi.org/10.1515/dmpt-2016-0028.

Export Citation

©2016 Walter de Gruyter GmbH, Berlin/Boston.Get Permission

Comments (0)

Please log in or register to comment.
Log in