Cytochrome P450 Assays

Enock Delaporte1, A. David Rodrigues1

1 Merck Research Labs, West Point, Pennsylvania
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 3.9
DOI:  10.1002/0471141755.ph0309s15
Online Posting Date:  February, 2002
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Abstract

Cytochrome P450s (CYPs) play a major role in drug detoxification, and inhibition of CYP‐mediated metabolism may lead to accumulation of toxic drug levels in the plasma. To prevent adverse drug‐drug interactions, new drug candidates are routinely tested for their ability to inhibit these enzymes. This unit describes a variety of protocols for evaluating new chemical entities as inhibitors of CYP activity. An example protocol illustrates a high‐throughput screening format using a fluorogenic probe, and a method for evaluating a test compound as a time‐dependent inhibitor of CYP is also described.

     
 
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Table of Contents

  • Basic Protocol 1: Evaluation of a Test Compound as an Inhibitor of CYP1A2 Activity Using Phenacetin
  • Basic Protocol 2: Evaluation of a Test Compound as an Inhibitor of CYP2A6 Activity Using Coumarin
  • Basic Protocol 3: Evaluation of a Test Compound as an Inhibitor of CYP2B6 and CYP2C19 Activities Using S‐Mephenytoin
  • Basic Protocol 4: Evaluation of a Test Compound as an Inhibitor of CYP2C8 Activity Using Paclitaxel
  • Basic Protocol 5: Evaluation of a Test Compound as an Inhibitor of CYP2C9 Activity Using Tolbutamide
  • Basic Protocol 6: Evaluation of a Test Compound as an Inhibitor of CYP2D6 Activity Using Dextromethorphan
  • Basic Protocol 7: Evaluation of a Test Compound as an Inhibitor of CYP2E1 Activity Using Chlorzoxazone
  • Basic Protocol 8: Evaluation of a Test Compound as an Inhibitor of CYP3A4 Activity Using Testosterone
  • Alternate Protocol 1: High‐Throughput Screening for CYP Inhibition Using 7‐Benzyloxyquinoline
  • Alternate Protocol 2: High‐Throughput Screening for CYP Inhibition Using Testosterone
  • Alternate Protocol 3: Evaluate a Test Compound as a Time‐Dependent Inhibitor of CYP Activity
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Evaluation of a Test Compound as an Inhibitor of CYP1A2 Activity Using Phenacetin

  Materials
  • Microsomes (IIAM, Human Biologics International, GENTEST, Xenotech; store at –70°C)
  • 1.0 M potassium phosphate buffer, pH 7.4 ( appendix 2A)
  • 1.6 mM phenacetin (see recipe)
  • Test compound
  • 10 mM NADPH or NADPH‐generating system containing:
  •  10,000 IU/ml glucose‐6‐phosphate dehydrogenase
  •  100 mM glucose‐6‐phosphate
  •  10 mM NADP+
  •  100 mM MgCl 2
  • 3 µg/ml temazepam in acetonitrile
  • HPLC‐grade acetonitrile containing 0.05% acetic acid
  • HPLC‐grade water containing 0.05% acetic acid
  • 0.5% (v/v) mM phosphoric acid in H 2O
  • HPLC‐grade methanol containing 0.5% (v/v) phosphoric acid
  • 12 × 75–mm borosilicate glass tubes
  • Shaking water bath
  • Tabletop centrifuge
  • HPLC equipped with autosampler and UV detector
  • SB‐C8 column (3.5 µm particle size, 4.6 × 50 mm, MAC‐MOD Analytical)
  • API 150 MCA Mass Spectrometer (Sciex)
  • Zorbax Phenyl column (3.5 µm particle size, 4.6 × 150 mm, Agilent Technologies)
  • Software for nonlinear regression analysis (e.g., SigmaPlot)

Basic Protocol 2: Evaluation of a Test Compound as an Inhibitor of CYP2A6 Activity Using Coumarin

  Materials
  • Microsomes (IIAM, Human Biologics International, GENTEST, Xenotech; store at −70°C)
  • 1.0 M potassium phosphate buffer, pH 7.4 ( appendix 2A)
  • 1 mM coumarin (see recipe)
  • Test compound
  • 10 mM NADPH or NADPH‐generating system containing:
  •  10,000 IU/ml glucose‐6‐phosphate dehydrogenase
  •  100 mM glucose‐6‐phosphate
  •  10 mM NADP+
  •  100 mM MgCl 2
  • 15% (w/v) trichloroacetic acid (TCA), ice‐cold
  • Chloroform
  • 10 mM sodium hydroxide/1 M sodium chloride
  • 12 × 75–mm borosilicate glass tubes
  • Shaking water bath
  • Tabletop centrifuge
  • Spectrofluorimeter
  • Software for nonlinear regression analysis (e.g., SigmaPlot)

Basic Protocol 3: Evaluation of a Test Compound as an Inhibitor of CYP2B6 and CYP2C19 Activities Using S‐Mephenytoin

  Materials
  • Microsomes (IIAM, Human Biologics International, GENTEST, Xenotech; store at –70°C)
  • 1.0 M potassium phosphate buffer, pH 7.4 ( appendix 2A)
  • 40 mM S‐mephenytoin (see recipe)
  • Test compound
  • 10 mM NADPH or NADPH‐generating system containing:
  •  10,000 IU/ml glucose‐6‐phosphate dehydrogenase
  •  100 mM glucose‐6‐phosphate
  •  10 mM NADP+
  •  100 mM MgCl 2
  • HPLC‐grade methanol
  • 100 µM tolbutamide in methanol
  • 10 mM potassium phosphate, pH 7.0 ( appendix 2A)
  • 12 × 75–mm borosilicate glass tubes
  • Shaking water bath
  • Tabletop centrifuge
  • HPLC equipped with autosampler and UV detector
  • Supelcosil LC‐18 reversed‐phase C 18 column (5 µm particle size, 4.6 × 150 mm), with LC‐18 guard column
  • Software for nonlinear regression analysis (e.g., SigmaPlot)

Basic Protocol 4: Evaluation of a Test Compound as an Inhibitor of CYP2C8 Activity Using Paclitaxel

  Materials
  • Microsomes (IIAM, Human Biologics International, GENTEST, Xenotech; store at –70°C)
  • 1.0 M potassium phosphate buffer, pH 7.4 ( appendix 2A)
  • 500 µM paclitaxel (see recipe)
  • Test compound
  • 10 mM NADPH or NADPH‐generating system containing:
  •  10,000 IU/ml glucose‐6‐phosphate dehydrogenase
  •  100 mM glucose‐6‐phosphate
  •  10 mM NADP+
  •  100 mM MgCl 2
  • Ethyl acetate
  • 35% acetonitrile in H 2O
  • Solution A (10% methanol)
  • Solution B (100% methanol)
  • 12 × 75–mm borosilicate glass tubes
  • Shaking water bath and 40°C water bath
  • Tabletop centrifuge
  • Nitrogen source
  • Curosil‐G column (Phenomenex), 6 µm particle size, 250 × 3.2 mm
  • Waters Bondapak guard column C 18/Corasil, 20 × 4 mm, 37‐ to 50‐µm
  • HPLC equipped with autosampler and UV detector
  • Nucleosil C 18 column (Supelco), 5 µm particle size, 4.6 × 250 mm
  • Waters 3.9 × 20 mm 60A 4‐µm Sentry guard column
  • Software for nonlinear regression analysis (e.g., SigmaPlot)

Basic Protocol 5: Evaluation of a Test Compound as an Inhibitor of CYP2C9 Activity Using Tolbutamide

  Materials
  • Microsomes (IIAM, Human Biologics International, GENTEST, Xenotech; store at –70°C)
  • 1.0 M Tris⋅Cl, pH 7.4 ( appendix 2A)
  • 1 M KCl
  • Test compound
  • 10 mM sodium tolbutamide in 100 mM NaOH (see recipe)
  • 10 mM NADPH or NADPH‐generating system containing:
  •  10,000 IU/ml glucose‐6‐phosphate dehydrogenase
  •  100 mM glucose‐6‐phosphate
  •  10 mM NADP+
  •  100 mM MgCl 2
  • 43% (v/v) H 3PO 4
  • 100 µg/ml chlorpropamide (internal standard)
  • Methylene chloride
  • 40% (v/v) acetonitrile:60% 0.05 (v/v) H 3PO 4
  • 12 × 75–mm borosilicate glass tubes
  • Shaking water bath and 40°C bath
  • Tabletop centrifuge
  • Nitrogen source
  • Zorbax ODS reversed‐phase C 18 column (Agilent Technologies), 5 µm particle size, 4.6 × 250 mm, preceded by a C 18guard column
  • HPLC equipped with autosampler and UV detector
  • Software for nonlinear regression analysis (e.g., SigmaPlot)

Basic Protocol 6: Evaluation of a Test Compound as an Inhibitor of CYP2D6 Activity Using Dextromethorphan

  Materials
  • Microsomes (IIAM, Human Biologics International, GENTEST, Xenotech; store at –70°C)
  • 1.0 M potassium phosphate, pH 7.4 ( appendix 2A)
  • 10 mM dextromethorphan (see recipe)
  • Test compound
  • 10 mM NADPH or NADPH‐generating system containing:
  •  10,000 IU/ml glucose‐6‐phosphate dehydrogenase
  •  100 mM glucose‐6‐phosphate
  •  10 mM NADP+
  •  100 mM MgCl 2
  • Dichloromethane
  • 20 µM benz[a]anthracene 5,6‐dihydrodiol (internal standard; see recipe)
  • HPLC‐grade acetonitrile containing 0.05% (v/v) acetic acid
  • HPLC‐grade water containing 0.05% (v/v) acetic acid
  • 12 × 75–mm borosilicate glass tubes
  • Shaking water bath and 40°C bath
  • Tabletop centrifuge
  • Nitrogen source
  • Zorbax SB‐C18 column, 5 µm particle, 4.6 × 150 mm (Agilent Technologies)
  • HPLC equipped with autosampler and fluorescence detector
  • Software for nonlinear regression analysis (e.g., Sigma Plot)

Basic Protocol 7: Evaluation of a Test Compound as an Inhibitor of CYP2E1 Activity Using Chlorzoxazone

  Materials
  • Microsomes (IIAM, Human Biologics International, GENTEST, Xenotech; store at –70°C)
  • 1.0 M potassium phosphate, pH 7.4 ( appendix 2A)
  • 25 mM chlorzoxazone in 60 mM KOH (freshly prepared, see recipe)
  • Test compound
  • 10 mM NADPH or NADPH‐generating system containing:
  •  10,000 IU/ml glucose‐6‐phosphate dehydrogenase
  •  100 mM glucose‐6‐phosphate
  •  10 mM NADP+
  •  100 mM MgCl 2
  • 500 µM zoxazolamine in 30% perchloric acid (see recipe)
  • Solution A: 90:10 mixture of 20 mM sodium perchlorate (pH 2.5) and acetonitrile
  • Solution B: 75:25 mixture of 20 mM sodium perchlorate (pH 2.5) and acetonitrile
  • 12 × 75–mm borosilicate glass tubes
  • Shaking water bath
  • Tabletop centrifuge
  • Zorbax ODS reversed‐phase C 18 column (Agilent Technologies), 5 µm particle, 4.6 × 250 mm, preceded by a C 18 guard column
  • HPLC equipped with autosampler and UV detector
  • Software for linear regression (e.g., Sigma Plot)

Basic Protocol 8: Evaluation of a Test Compound as an Inhibitor of CYP3A4 Activity Using Testosterone

  Materials
  • Microsomes (IIAM, Human Biologics International, GENTEST, Xenotech; store at –70°C)
  • 1.0 M potassium phosphate, pH 7.4 ( appendix 2A)
  • 7.5 mM testosterone (see recipe)
  • Test compound
  • 10 mM NADPH or NADPH‐generating system containing:
  •  10,000 IU/ml glucose‐6‐phosphate dehydrogenase
  •  100 mM glucose‐6‐phosphate
  •  10 mM NADP+
  •  100 mM MgCl 2
  • Dichloromethane
  • 1 mM corticosterone (see recipe)
  • Methanol
  • 6β‐hydroxytestosterone (Steraloids, Sigma‐Aldrich)
  • 7.5% tetrahydrofuran in HPLC‐grade methanol
  • 7.5% tetrahydrofuran in HPLC‐grade water
  • 12 × 75–mm borosilicate glass tubes
  • 37°C shaking water bath and 40°C bath
  • Tabletop centrifuge
  • Nitrogen source
  • Zorbax ODS reversed‐phase C 18 column (Agilent Technologies), 5 µm particle, 4.6 × 250 mm
  • HPLC equipped with autosampler and UV detector
  • Software for linear regression analysis (e.g., SigmaPlot)

Alternate Protocol 1: High‐Throughput Screening for CYP Inhibition Using 7‐Benzyloxyquinoline

  Materials
  • 0.5 M potassium phosphate, pH 7.4 ( appendix 2A)
  • 20 × cofactor solution (see recipe)
  • Glucose‐6‐phosphate dehydrogenase (2000 IU/2.9 ml, Sigma‐Aldrich)
  • Acetonitrile
  • 20 mM 7‐benzyloxyquinoline in acetonitrile (see recipe)
  • Test compounds
  • 20 mg/ml microsomes (IIAM, Human Biologics International, GENTEST, Xenotech; store at –70°C)
  • STOP solution (see recipe)
  • 1 mM 7‐hydroxyquinoline (see recipe)
  • 96‐well plate (Costar # 3585)
  • Automated liquid handling station (Tecan Genesis RSP 150/8) with either a heating block at 37°C or microtiter plate incubator (The Jitterbug, Boekel Scientific)
  • 0.5‐ to10‐µl multichannel pipettor (e.g., Eppendorf)
  • 30‐ to 300‐µl multichannel pipettor (e.g., Eppendorf)
  • Reservoir (Matrix Technologies)
  • Fluorescence plate reader (SpectraMax Gemini, Molecular Devices)
  • Software for linear regression analysis (e.g., SigmaPlot)

Alternate Protocol 2: High‐Throughput Screening for CYP Inhibition Using Testosterone

  Materials
  • 0.5 M potassium phosphate, pH 7.4 ( appendix 2A)
  • 20× cofactor solution (see recipe)
  • 2000 IU/2.9 ml glucose‐6‐phosphate dehydrogenase
  • Acetonitrile
  • 15 mM testosterone (see recipe)
  • 0.267 mM 6β‐hydroxytestosterone (see recipe)
  • 5 mM test compound prepared in 50% acetonitrile
  • 20 mg/ml microsomes (IIAM, Human Biologics International, GENTEST, Xenotech; store at –70°C)
  • 600 ng/ml cortisone in acetonitrile containing 0.1% TFA
  • Disposable cluster tube system (incubation plates, 1.2 ml; Fisher)
  • 0.5‐ to10‐µl multichannel pipettor (e.g., Eppendorf)
  • 30‐ to 300‐µl multichannel pipettor (e.g., Eppendorf)
  • Automated liquid handling station (Tecan Genesis RSP 150/8)
  • 37°C water bath
  • Benchtop centrifuge (Eppendorf 5810R)
  • Tecan orbital shaker 40‐250
  • Bibby Sterlin 96‐well V‐bottom plate (Dynalab)
  • Packard plate sealer Micromate 496
  • Packard TopSeal‐S, microplate heat sealing film
  • HPLC system consisting of:
  •  BDS Hypersil C 8, 50 × 2 mm, 5 µm particles (Keystone Scientific)
  •  Perkin Elmer Series 200 Micro HPLC pump
  •  Leap HTS PAL autosampler equipped with a 20‐µl loop
  • Sciex API2000 mass spectrometer with heated nebulizer interface (APCI)
  • Software for linear regression analysis (e.g., Sigma Plot)
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Figures

Videos

Literature Cited

Literature Cited
   Arlotto, M.P. and Parkinson, A. 1989. Identification of cytochrome P450a (P450IIA1) as the principal testosterone 7α‐hydroxylase in rat liver microsomes and its regulation by thyroid hormones. Arch. Biochem. Biophys. 270:458‐471.
   Arlotto, M.P., Sonderfan, A.J., Klaassen, C.D., and Parkinson, A. 1987. Studies on the pregnenolone‐16I‐carbonitrile‐inducible form of rat liver microsomal cytochrome P‐450 and UDP‐glucuronosyl transferase. Biochem. Pharmacol. 36:3859‐3866.
   Ayrton, J., Plumb, R., Leavens, W.J., Mallett, D., Dickins, M., and Dear, G.J. 1998. Application of a generic fast gradient liquid chromatography tandem mass spectometry method for the analysis of cytochrome P450 probe substrates. . Rapid Commun. Mass. Spec. 12:217‐224.
   Bloomer, J.C., Clarke, S.E., and Chenery, R.J. 1995. Determination of P4501A2 activity in human liver microsomes using [3‐14C‐methyl]caffeine. Xenobiotica 25:917‐927.
   Busby, W.F. Jr., Ackermann, J.A., and Crespi, C.L. 1999. Effect of methanol, ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of CDNA‐expressed human cytochromes P‐450. Drug Metab. Dispos. 27:246‐249.
   Chauret, N., Gauthier, A., and Nicoll‐Griffith, D.A. 1998. Effect of common solvents on in vitro cytochrome P450‐mediated metabolic activities in human liver microsomes. Drug Metab. Dispos. 26:1‐4.
   Draper, A.J., Madan, A., Smith, K., and Parkinson, A. 1998. Development of a non‐high pressure liquid chromatography assay to determine testosterone hydroxylase (CYP3A) activity in human liver microsomes. Drug Metab. Dispos. 26:299‐304.
   Draper, A.J., Madan, A., Latham, J., and Parkinson, A. 1998. Development of a non‐high pressure liquid chromatography assay to determine [14C]chlorzoxazone 6‐hydroxylase (CYP2E1) activity in human liver microsomes. Drug Metab. Dispos. 26:305‐312.
   Fuhr, U., Wolff, T., Harder, S., Schymanski, P., and Staib, A.H. 1990. Quinolone inhibition of cytochrome P‐450‐dependent caffeine metabolism in human liver microsomes. Drug Metab. Dispos. 16:1005‐1010.
   Guengerich, F.P. 1994. Analysis and characterization of enzymes. In Principles and Methods of Toxicology (W.A. Hayes, ed.), pp. 1259‐1313. Raven Press, New York.
   Ioannides, C. (ed.) 1996. Cytochrome P450 Metabolic and Toxicological Aspects. CRC Press, Boca Raton, Fla.
   Korzekwa, K.R., Krishnamachary, N., Shou, M., Ogai, A., Parise, R.A., Rettie, A.E., Gonzalez, F.J., and Tracy, T.S. 1998. Evaluation of atypical cytochrome P450 kinetics with two‐substrate models: Evidence that multiple substrates can simultaneously bind to cytochrome P450 active sites. Biochemistry 37:4137‐4147.
   Lin, J.H. and Rodrigues, A.D. 2000. In vitro models for early studies of drug metabolism. In Pharmacokinetics Optimization in Drug Research: Biological, Physicochemical and Computational Strategies (B. Testa, H. van de Waterbeemd, G. Folkers, and R. Guy, Eds.) pp. 217‐243. John Wiley & Sons, New York.
   Parkinson, A. 1996. An overview of current cytochrome P450 technology for assessing the safety and efficacy of new materials. Toxicol. Pathol. 24:45‐57.
   Pearce, R., Greenway, D., and Parkinson, A. 1992. Species differences and interindividual variation in liver microsomal cytochrome P450 2A enzymes: Effects on coumarin, dicumarol and testosterone oxidation. Arch. Biochem. Biophys. 298:211‐225.
   Pearce, R.E., McIntyre, C.J., Madan, A., Sanzgiri, U., Draper, A.J., Bullock, P.L., Cook, D.C., Burton, L.A., Latham, J., Nevins, C., and Parkinson, A. 1996. Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity. Arch. Biochem. Biophys. 331:145‐169.
   Rodrigues, A.D., Kukulka, M.J., Surber, B.W., Thomas, S.B., Uchic, J.T., Rotert, G.A., Michel, G., Thome‐Kromer, B., and Machinist, J.M. 1994. Measurement of liver microsome cytochrome P450 (CYP2D6) activity using [O‐methyl 14C]dextromethorphan. Anal. Biochem. 219:309‐320.
   Rodrigues, A.D., Surber, B.W., Yao, Y., Wong, S.L., and Roberts, E.M. 1997. [O‐ethyl14C]phenacetin O‐deethylase activity in human liver microsomes. Drug Metab. Dispos. 25:1097‐1100.
   Segel, I.H. 1975. Enzyme kinetics: Behavior and analysis of rapid equilibrium and steady‐state enzyme systems. John Wiley & Sons, New York.
   Tang, C., Shou, M., and Rodrigues, A.D. 2000. Substrate‐dependent effect of acetonitrile on human liver microsomal cytochrome P40 2C9 (CYP2C9) activity. Drug Metab. Dispos. 28:567‐572.
   Ueng, Y‐F., Kuwabara, K., Chun, Y‐J., and Guengerich, F.P. 1997. Cooperativity in oxidations catalyzed by cytochrome P450 3A4. Biochemistry 36:370‐381.
   van der Weide, J. and Steijns, L.S. 1999. Cytochrome P450 enzyme polymorphisms and impact on clinical pharmacology. Ann. Clin. Biochem. 36:722‐729.
   Venkatakrishnan, K., Von Moltke, L.L., Obach, R.S., and Greenblatt, D.J., 2000. Microsomal binding of amitriptyline: Effect on estimation of enzyme kinetic parameters in vitro. J. Pharmacol. Exp. Ther. 293:343‐350.
   Walle, T. 1996. Assays of CYP2C8‐ and CYP3A4‐mediated metabolism of taxol in vivo and in vitro. Methods Enzymol. 272:145‐151.
   Zhang, X‐Y. and Thomas, P.E. 1996. Erythromycin as a specific substrate for cytochrome P4503A isozymes and identification of a high affinity erythromycin N‐demethylase in adult female rats. Drug Metab. Dispos. 24:23‐27.
Key References
  Ioannides, C. (ed.) 1996. See above
  A good review of cytochrome P450s.
   Johnson, E.F. and Waterman, M.R. (eds). 1996. Methods in Enzymology. Vol. 272 Cytochrome P450 Part B. Academic Press, New York.
  Detailed experimental procedures for investigatings 9450s.
   Wolff, T.F. (ed) 1999. Handbook of Drug Metabolism. Marcel Dekker, Inc., New York.
  A good drug metabolism reference.
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