Measurement of In Vitro Cytochrome P450 2B6 Activity

Robert L. Walsky1, R. Scott Obach1

1 Pfizer Global Research and Development, Groton, Connecticut
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.27
DOI:  10.1002/0471140856.tx0427s39
Online Posting Date:  February, 2009
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Cytochrome P450 2B6 (CYP2B6) is responsible for the metabolism of a number of therapeutically relevant drugs and environmental chemicals. While not as frequently involved in xenobiotic metabolism as other more commonly studied P450 enzymes such as CYP3A, CYP2D6, or CYP2C9, its importance is becoming increasingly realized. Bupropion hydroxylase activity is an excellent indicator of in vitro CYP2B6 activity, and the metabolite formed is easily measured by HPLC/MS/MS. This assay allows the use of very low concentrations of human liver microsomes or recombinant enzyme, and it represents a selective, sensitive approach to assessing in vitro CYP2B6 activity, with minimal sample preparation. Curr. Protoc. Toxicol. 39:4.27.1‐4.27.11. © 2009 by John Wiley & Sons, Inc.

Keywords: CYP2B6; HPLC/MS/MS; P450

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Table of Contents

  • Basic Protocol 1: In Vitro Bupropion Hydroxylation by Cytochrome P450 2B6
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: In Vitro Bupropion Hydroxylation by Cytochrome P450 2B6

  • Inhibitors: e.g., felodipine (example in protocol), raloxifene, sertraline (Sigma)
  • 50:50 and 95:5 acetonitrile/H 2O: prepare in bulk and store up to 6 months at room temperature
  • 100 µM hydroxybupropion standard curve stock solution (see recipe)
  • 100 mM potassium phosphate buffer, pH 7.40 ±0.02 (see recipe)
  • 165 mM magnesium chloride stock (see recipe)
  • 20 mg/ml pooled human liver microsomes (BD Gentest) or 9 mg/ml recombinantly expressed human CYP2B6 (rhCYP2B6) + reductase (Supersomes, BD Gentest)
  • 80 mM bupropion HCl stock solution: prepared by dissolving 10 mg bupropion HCl (BD Gentest) in 0.449 ml 5:95 acetonitrile/water; store up to 3 months at −10°C
  • 13 mM NADPH stock solution (see recipe)
  • Acidified internal standard solution (see recipe), for incubation termination
  • Internal standard: 100 µM [D 6]hydroxybupropion internal stock solution (see recipe)
  • Mobile phase A: 5 mM aqueous ammonium formate, 0.05% (v/v) formic acid
  • Mobile phase B: 95:5 acetonitrile/methanol, 0.05% (v/v) formic acid
  • 15‐ and 50‐ml polypropylene screw‐cap centrifuge tubes
  • 96‐well polypropylene microtiter plates
  • Multichannel micropipettors
  • Parafilm
  • 96‐well polypropylene PCR plates (e.g., Axygen)
  • 37°C heating block
  • Polypropylene film (e.g., Whatman)
  • 96‐well 0.45‐µm MultiScreen‐HA filter plates (Millipore)
  • HPLC/MS/MS system (e.g., Micromass Ultima, Waters)
  • Quantitation software (e.g., Micromass QuanLynx, Waters)
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  •   FigureFigure 4.27.1 Structures of bupropion, hydroxybupropion (resulting from CYP2B6 activity), and [D6]hydroxybupropion (internal standard).
  •   FigureFigure 4.27.2 Elution profiles of hydroxybupropion at its LLOQ and [D6]hydroxybupropion (internal standard).
  •   FigureFigure 4.27.3 Inhibition of rhCYP2B6 bupropion hydroxylase activity by felodipine, as seen by a reduction in activity with increasing felodipine concentration relative to the solvent control. The horizontal dashed line represents 50% of control activity and corresponds to the calculated IC50 concentration.


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Key References
   Walsky and Obach, 2004. See above.
  Describes in detail the analytical and biological methodology for inhibition studies.
   Faucette et al., 2000. See above.
  Describes the specificity of bupropion hydroxylation as a marker of in vitro CYP2B6 activity.
   Hesse et al., 2000. See above.
Internet Resources
  FDA, 2006. Draft Guidance for Industry: Drug Interaction Studies – Study Design, Data Analysis, and Implications for Dosing and Labeling.
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