Cytochrome P450 Inhibition Assays Using Traditional and Fluorescent Substrates

Elsa Paradise1, Prasoon Chaturvedi1, Elena Ter‐Ovanesyan1

1 Surface Logix, Inc., Brighton, Massachusetts
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 7.11
DOI:  10.1002/0471141755.ph0711s39
Online Posting Date:  December, 2007
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Abstract

A key liability in transitioning a new chemical entity (NCE) to a development candidate is NCE‐related inhibition (or induction) of cytochrome P450 enzymes, a superfamily of heme‐containing oxygenases that are the major route of first‐pass metabolism for the majority of marketed drugs. The drawback of a drug/NCE that modulates CYP450 enzyme activity occurs when the compound is co‐administered with another drug that relies on the same P450 enzyme for its metabolism. This could result in overdose of the second drug in the case of inhibition, or more rapid metabolism of one or both drugs accompanied by loss of efficacy in the case of enzyme induction. Screening for the inhibition of CYP450 enzymes is now routine in the early stages of evaluating NCEs. This unit describes two inhibition assays using traditional and fluorescent substrates. Curr. Protoc. Pharmacol. 39:7.11.1‐7.11.12. © 2007 by John Wiley & Sons, Inc.

Keywords: cytochrome P450 (CYP450); substrate; inhibitor; inhibition constant (IC50); HPLC; LC‐MS/MS

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: CYP450 Inhibition Assays Using Fluorescent Substrates
  • Basic Protocol 2: CYP450 Inhibition Assays Using Traditional Substrates
  • Support Protocol 1: Analytical Methods for CYP450 Inhibition Assays Using Traditional Substrates
  • Support Protocol 2: Computation of IC50 Values for Fluorescent and Traditional CYP50 Inhibition Assays
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: CYP450 Inhibition Assays Using Fluorescent Substrates

  Materials
  • Fluorescence dilution buffer (FDB; see recipe)
  • Substrates and inhibitors (see Table 7.11.1)
    Table 7.1.1   MaterialsSubstrates and Inhibitors for Fluorescent Assays

    Reagent a Purpose Source
    25 mM 7‐benzyloxy‐4‐(trifluoromethyl)‐coumarin (BFC) Substrate for 3A4 BD Bioscience
    25 mM 7‐benzyloxyquinoline (BQ) Substrate for 3A4 BD Bioscience
    1 mM dibenzylfluorescein (DBF) Substrate for 3A4 BD Bioscience
    25 mM 7‐methoxy‐(trifluoromethyl)‐coumarin (MFC) Substrate for 2C9 and 2E1 BD Bioscience
    20 mM 3‐cyano‐7‐ethoxycoumarin (CEC) Substrate for 2C19 and 1A2 BD Bioscience
    1 mM 3‐[2‐(N,N‐diethyl‐N‐methylammonium)ethyl]‐7‐methoxy‐4‐methylcoumarin (AMMC) Substrate for 2D6 BD Bioscience
    10 mM ketoconazole Inhibitor for 3A4 BD Bioscience
    10 mM sulfaphenazole Inhibitor for 2C9 Sigma‐Aldrich
    20 mM tranylcypromine Inhibitor for 2C19 Sigma‐Aldrich
    20 mM furafylline Inhibitor for 1A2 Sigma‐Aldrich
    10 mM quinidine Inhibitor for 2D6 BD Bioscience
    20 mM diethyldithiocarbamate Inhibitor for 2E1 Sigma‐Aldrich

     aThese reagents may be stored up to 1 year at −20°C.
  • 100% dimethyl sulfoxide (DMSO)
  • Recombinant human CYP450 3A4, 2C9, 2C19, 1A2, 2D6, and 2E1 enzymes (e.g., Supersomes from BD Bioscience; Supersomes typically have a CYP450 content of 1000 pmol/ml; prepare 50‐µl aliquots of CYP450 enzymes and store up to 1 year at −80°C)
  • 0.5 M potassium phosphate (KH 2PO 4), pH 7.4 ( appendix 2A)
  • Acetonitrile
  • 15‐ and 50‐ml conical tubes
  • 96‐well V‐bottom polypropylene microplate
  • Black 96‐well flat‐bottom microplate
  • Plate seals
  • 37°C plate incubator
  • 8‐channel multi‐pipettors
  • Fluorescence plate reader

Basic Protocol 2: CYP450 Inhibition Assays Using Traditional Substrates

  Materials
  • Traditional dilution buffer (TDB; see recipe)
  • Substrates and inhibitors (see Table 7.11.5)
    Table 7.1.5   MaterialsSubstrates and Inhibitors for Traditional CYP450 Inhibition Assays

    Reagent g Purpose Source
    20 mM testosterone Substrate for 3A4 Sigma
    20 mM 6‐β‐hydroxytestosterone Product of 3A4/testosterone reaction Sigma
    20 mM diclofenac Substrate for 2C9 Sigma
    20 mM 4‐hydroxydiclofenac Product of 2C9/diclofenac reaction Sigma
    20 mM mephenytoin Substrate for 2C19 BD Bioscience
    20 mM 4‐hydroxymephenytoin Product of 2C19/mephenytoin reaction BD Bioscience
    20 mM phenacetin Substrate for 1A2 Sigma‐Aldrich
    20 mM acetaminophen Product of 1A2/phenacetin reaction Sigma‐Aldrich
    20 mM bufuralol Substrate for 2D6 Sigma‐Aldrich
    20 mM 1‐hydroxybufuralol Product of 2D6/bufuralol reaction Sigma‐Aldrich
    20 mM chlorzoxazone Substrate for 2E1 Sigma‐Aldrich
    20 mM 6‐hydroxychlorzoxazone Product of 2E1/chlorzoxazone reaction Sigma‐Aldrich
    10 mM ketoconazole Inhibitor for 3A4 BD Bioscience
    10 mM sulfaphenazole Inhibitor for 2C9 Sigma‐Aldrich
    20 mM ticlopidine Inhibitor for 2C19 Sigma‐Aldrich
    20 mM furafylline Inhibitor for 1A2 Sigma‐Aldrich
    10 mM quinidine Inhibitor for 2D6 BD Bioscience
    20 mM diethyldithiocarbamate Inhibitor for 2E1 Sigma‐Aldrich

     gThese reagents may be stored up to 1 year at –20°C.
  • 100% dimethyl sulfoxide (DMSO)
  • Recombinant human CYP450 3A4, 2C9, 2C19, 1A2, 2D6, and 2E1 enzymes (e.g., Supersomes from BD Bioscience; Supersomes typically have a CYP450 content of 1000 pmol/ml; prepare 50‐µl aliquots and store up to 1 year at −80°C)
  • 1 M magnesium chloride (MgCl 2), store in aliquots up to 2 years at −20°C
  • Nicotinamide adenine dinucleotide phosphate (NADPH), reduced form
  • Acetonitrile
  • Acetic acid
  • 0.5 M potassium phosphate (KH 2PO 4), pH 7.4 (for 3A4, 2C19, 1A2, 2D6, and 2E1); see appendix 2A for recipe
  • 1 M Tris·Cl, pH 7.4 (for 2C9), at room temperature; see appendix 2A for recipe
  • 96‐well V‐bottom polypropylene microplate
  • 1.5‐ml polypropylene flip‐cap tubes
  • Tube rack
  • 37°C water bath
  • Microcentrifuge
  • HPLC or LC‐MS/MS system

Support Protocol 1: Analytical Methods for CYP450 Inhibition Assays Using Traditional Substrates

  Materials
  • GraphPad Prism software
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Figures

Videos

Literature Cited

   Amato, G.M., Longo, V., Mazzaccaro, A., and Gervasi, P. 1998. Chlorzoxazone 6‐hydroxylase and p‐nitrophenol hydroxylases: The most suitable activities for assaying cytochrome P450 2E1. Drug Metab. Dispos. 26:483‐489.
   Bjornsson, T.D., Callaghan, J.T., Einolf, H.J., Fischer, V., Gan, L., Grimm, S., Kao, J., King, S.P., Miwa, G., Ni, L., Kumar, G., McLeod, J., Obach, R.S., Roberts, S., Roe, A., Shah, A., Snikeris, F., Sullivan J., Tweedie, D., Vega, J.M., Walsh, J., and Wrighton, S.A. 2003. The conduct of in vitro and in vivo drug‐drug interaction studies: A Pharmaceutical Research and Manufacturers of America (PhRMA) perspective. Drug Metab. Dispos. 31:815‐832.
   Cohen, L.H., Remley, M.J., Raunig, D., and Vaz, A.D.N. 2003. In vitro drug interactions of cytochrome P450: An evaluation of fluorogenic to conventional substrates. Drug Metab. Dispos. 31:1005‐1015.
   Cornish‐Bowden, A. 1979. Fundamentals of Enzyme Kinetics. Butterworth Inc., London.
   Hosea, N., Miller, G., and Guengerich, P. 2000. Elucidation of distinct ligand binding sites for cytochrome P450 3A4. Biochemistry 39:5929‐5939.
   Omiecinski, C.J., Remmel, R.P., and Hosagrahara, V.P. 1999. Concise review of the cytochrome P450s and their roles in toxicology. Toxicol. Sci. 48:151‐156.
   Stresser, D.M., Blanchard, A.P., Turner, S.D., Erve, J.C., Dandeneau, A.A., Miller, V.P., and Crespi, C.L. 2000. Substrate‐dependent modulation of Cyp3A4 catalytic activity: Analysis of 27 test compounds with four fluorometric substrates. Drug Metab. Dispos. 28:1440‐1448.
   Tucker, G.T., Houston, J.B., and Huang, S.M. 2001. Optimising drug development: Strategies to assess drug metabolism/transporter interaction potential‐towards a consensus. Eur. J. Pharm. Sci. 13:417‐428.
   Weaver, R., Graham, K., Beattie, I., and Riley, R. 2003. Cytochrome P450 inhibition using recombinant proteins and mass spectroscopy/multiple reaction monitoring technology in a cassette incubation. Drug Metab. Dispos. 31:955‐966.
Key References
   Department of Health and Human Services. US Food and Drug Administration. Guidance for Industry: Drug Metabolism/Drug Interactions Studies in the Drug Development Process: Studies In Vitro. Center for Drug Evaluation and Research. Center for Biologics Evaluation and Research. Guidance for Industry. April 1997.
  The FDA guidances for industry are valuable resources because they provide suggestions on current in vivo and in vitro approaches to studying drug metabolism and drug interactions; although the study designs listed are not requirements, they provide insight into planning experiments by naming specifics (substrates, inhibitors) that are FDA preferred.
   Department of Health and Human Services. US Food and Drug Administration. Guidance for Industry: Drug Interaction Studies‐Study Design, Data Analysis and Implications for Dosing and Labeling. Center for Drug Evaluation and Research. Center for Biologics Evaluation and Research. Guidance for Industry. September 2006.
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