In Vitro Drug Metabolism Using Liver Microsomes

Kathleen M. Knights1, David M. Stresser2, John O. Miners3, Charles L. Crespi4

1 Department of Clinical Pharmacology, School of Medicine, Flinders University, Adelaide, South Australia, 2 Corning Gentest Contract Research, Corning Incorporated Life Sciences, Woburn, Massachusetts, 3 Department of Clinical Pharmacology and Flinders Centre for Innovation in Cancer, School of Medicine, Flinders University, Adelaide, South Australia, 4 Corning Incorporated, Tewksbury, Massachusetts
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 7.8
DOI:  10.1002/cpph.9
Online Posting Date:  September, 2016
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Abstract

Knowledge of the metabolic stability of newly discovered drug candidates eliminated by metabolism is essential for predicting the pharmacokinetic (PK) parameters that underpin dosing and dosage frequency. Further, characterization of the enzyme(s) responsible for metabolism (reaction phenotyping) allows prediction, at least at the qualitative level, of factors (including metabolic drug‐drug interactions) likely to alter the clearance of both new chemical entities (NCEs) and established drugs. Microsomes are typically used as the enzyme source for the measurement of metabolic stability and for reaction phenotyping because they express the major drug‐metabolizing enzymes cytochrome P450 (CYP) and UDP‐glucuronosyltransferase (UGT), along with others that contribute to drug metabolism. Described in this unit are methods for microsome isolation, as well as for the determination of metabolic stability and metabolite formation (including kinetics). © 2016 by John Wiley & Sons, Inc.

Keywords: in vitro drug metabolism; liver microsomes; metabolic stability; microsome isolation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Compound Metabolic Stability Using Liver Microsomes
  • Alternate Protocol 1: Higher‐Throughput Screening for Metabolic Stability Using Liver Microsomes
  • Alternate Protocol 2: Higher‐Throughput Screening for Metabolic Stability Using Liver Microsomes Supplemented with Nadph and UDP‐GlcUA
  • Alternate Protocol 3: Measurment of Metabolite Formation in Incubations Using Liver Microsomes
  • Support Protocol 1: Preparation of Microsomes from Human Liver
  • Support Protocol 2: Determination of Cytochrome P450 (P450) Content of Microsomes
  • Support Protocol 3: Determination of the Amount of Microsomal Protein Per Gram of Liver (MPPGL)
  • Support Protocol 4: Measurement of the Kinetics of Drug Glucuronidation by Human Liver Microsomes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Compound Metabolic Stability Using Liver Microsomes

  Materials
  • Test compound(s)
  • 0.1 M potassium phosphate buffer, pH 7.4 ( appendix 2A), pre‐warmed to 37°C
  • 10 mM NADPH freshly prepared in 0.1 M potassium phosphate buffer, pH 7.4, or NADPH‐generating system (see recipe).
  • Positive control (e.g., 10 mM DL‐propranolol, see recipe)
  • Human or animal microsomes [see protocol 5 or purchase commercially from Corning Life Sciences (http://www.corning.com), Bioreclamation/IVT (http://www.bioreclamationivt.com), or XenoTech (http://www.xenotech.com)]
  • Acetonitrile
  • 1.5‐ml microcentrifuge polypropylene tubes
  • 37°C water bath
  • Tabletop centrifuge
  • Additional reagents and equipment for analytical method, e.g., LC/MS/MS (Korfmacher, )

Alternate Protocol 1: Higher‐Throughput Screening for Metabolic Stability Using Liver Microsomes

  Additional Materials (also see protocol 1Basic Protocol)
  • 96‐well plates (polypropylene; Axygen Scientific, cat. no. P‐DW‐20‐C)
  • Multichannel pipettor (Rainin Instrument, LLC)
  • Reagent reservoirs for multichannel pipetting (e.g., 50‐ml polystyrene; Corning Costar, cat. no. 4870)
  • Centrifuge with a plate holder–capable rotor
  • 96‐well autosampler for LC/MS/MS

Alternate Protocol 2: Higher‐Throughput Screening for Metabolic Stability Using Liver Microsomes Supplemented with Nadph and UDP‐GlcUA

  Additional Materials (also see Basic Protocol 1)
  • 500 mM magnesium chloride
  • 25 μg/ml alamethicin (see recipe)
  • UDP‐GlcUA (see recipe)
  • 96‐well plates (polypropylene; Axygen Scientific, cat. no. P‐DW‐20‐C)
  • Multichannel pipettor (Rainin Instrument LLC)
  • Reagent reservoirs (e.g., 50 ml polystyrene, Corning Costar catalog no. 4870)
  • Centrifuge with a plate holder–capable rotor
  • 96‐well auto sampler for LC/MS/MS

Alternate Protocol 3: Measurment of Metabolite Formation in Incubations Using Liver Microsomes

  Materials
  • Test compound(s)
  • 0.1 M potassium phosphate buffer, pH 7.4 ( appendix 2A), pre‐warmed to 37°C
  • 10 mM NADPH freshly prepared in 0.1 M potassium phosphate buffer, pH 7.4, or an NADPH‐generating system (see recipe)
  • Positive control (e.g., 20.8 mM testosterone and 3 mM 6β‐hydroxytestosterone, see reciperecipes)
  • Human or animal microsomes [see protocol 5 or purchase commercially from Corning Life Sciences (http://www.corning.com), Bioreclamation/IVT (http://www.bioreclamationivt.com), or XenoTech (http://www.xenotech.com)]
  • Acetonitrile
  • 1.5‐ml microcentrifuge polypropylene tubes
  • 37°C water bath
  • Tabletop centrifuge
  • Additional reagents and equipment for analytical method such as LC/MS (Korfmacher, )

Support Protocol 1: Preparation of Microsomes from Human Liver

  Materials
  • Human liver tissue (1 gm of tissue per 5 ml of microsome preparation buffer)
  • Microsome preparation buffer (see recipe)
  • Microsome storage buffer (see recipe)
  • Open‐top polycarbonate centrifuge tubes (50 ml)
  • Surgical scissors
  • Tissue homogenizer (e.g., Ultra Turrax T25, http://www.ika.com)
  • Potter‐Elvehjem tissue grinder (30 ml) with Teflon pestle (motor driven)
  • Refrigerated centrifuge
  • Syringe (50 ml) with blunt‐end aspiration needle
  • Ultracentrifuge tubes (∼25 ml; e.g., Beckman‐Coulter)
  • Ultracentrifuge, refrigerated to 4°C before use
  • Ultracentrifuge rotors, maintained at 4°C for at least 16 hr before use.
  • Plastic round‐ended probe
  • Potter‐Elvehjem tissue grinder (10 ml) with Teflon pestle (hand held)
  • Glass storage vials (suitable for −70°C)
  • Additional reagents and equipment for determining protein concentration ( appendix 3A; Olson, )

Support Protocol 2: Determination of Cytochrome P450 (P450) Content of Microsomes

  Materials
  • Crystalline sodium dithionite (Na 2S 2O 4)
  • Carbon monoxide (CO; e.g., BOC, Praxair) in a tank with a two‐stage regulator; attach flexible tubing with the appropriate end outlet (e.g., blunt end needle, fine Pasteur pipet) to the outlet of the regulator.
  • Microsome storage buffer (see recipe)
  • Semi‐micro quartz spectrophotometric cuvettes (1.4 ml) with 10‐mm light path
  • UV/vis scanning spectrophotometer, preferably a dual‐beam instrument (e.g., Agilent, Eppendorf, JASCO)
  • Additional reagents and equipment for preparation of human liver microsomes (HLM; see protocol 5)

Support Protocol 3: Determination of the Amount of Microsomal Protein Per Gram of Liver (MPPGL)

  Materials
  • Human liver microsomes (HLM; protocol 5)
  • 0.1 M potassium phosphate buffer, pH 7.4 ( appendix 2A),
  • Alamethicin (from Trichoderma viride; A.G. Scientific; http://www.agscientific.com)
  • 40 mM magnesium chloride (MgCl 2)
  • 3′‐azido‐3′ deoxythymidine (AZT; Sigma‐Aldrich)
  • 50 mM UDP‐GlcUA (trisodium salt; Sigma‐Aldrich)
  • 11.6 M perchloric acid
  • AZT‐β‐D‐glucuronide (Sigma‐Aldrich) for standards
  • Tabletop centrifuge
  • Additional reagents and equipment for the analytical method (typically LC/MS or HPLC; Uchaipichat et al., ; Rowland et al., )
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Figures

Videos

Literature Cited

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Internet Resources
  http://medicine.iupui.edu/flockhart/
  Provides drug‐drug interaction tables.
  http://www.cypalleles.ki.se/
  Provides nomenclature and alleles for CYPs.
  http://www.flinders.edu.au/medicine/sites/clinical‐pharmacology/udp‐glucuronosyltransferases.cfm
  Provides information on UGT sequences.
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