In Vitro Drug Metabolism Using Liver Microsomes

Jeanette R. Hill1

1 Ricerca Biosciences, Concord, Ohio
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 7.8
DOI:  10.1002/0471141755.ph0708s23
Online Posting Date:  February, 2004
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Abstract

Metabolic biotransformation of a drug can increase the rate of elimination from the body and have a significant effect on efficacy and safety. Drug candidates are screened early in the discovery process for metabolic stability using liver microsomes. Methods for microsome isolation and characterization and the determination of metabolic stability are presented.

Keywords: in vitro metabolism; metabolic stability; microsome

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Compound Metabolic Stability Using Liver Microsomes
  • Alternate Protocol 1: Higher‐Throughput Screening for Metabolic Stability
  • Support Protocol 1: Preparation of Microsomes from Rat Liver
  • Support Protocol 2: Determination of Cytochrome P450 (CYP) Content in 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 phosphate buffer, pH 7.4, pre‐warmed to 37°C
  • 10 mM NADPH freshly prepared in 0.1 M phosphate buffer, pH 7.4 or an NADPH‐regenerating system (see recipe)
  • Positive control (e.g., 0.5 mM testosterone, see recipe)
  • Human or animal microsomes (see protocol 3 or purchased commercially from BD GENTEST, In Vitro Technologies, Panvera)
  • Methanol, ice cold
  • 1.5‐ml microcentrifuge polypropylene tubes
  • 37°C shaking water bath (∼150 rpm)
  • Tabletop centrifuge
  • Additional reagents and equipment for analytical method such as LC/MS or HPLC

Alternate Protocol 1: Higher‐Throughput Screening for Metabolic Stability

  • 96‐well plates (polystyrene or polypropylene)
  • Multi‐channel pipet and reagent reservoirs
  • Centrifuge with a plate holder‐capable rotor
  • 96‐well autosampler for LC/MS or HPLC

Support Protocol 1: Preparation of Microsomes from Rat Liver

  Materials
  • Rats
  • Homogenizing buffer (see recipe), ice cold
  • Pyrophosphate buffer (see recipe), ice cold
  • Microsome buffer (see recipe), ice cold
  • 0.1 M phosphate buffer, pH 7.4
  • 10‐ml syringe and 22‐G needle
  • Ice bath
  • Surgical scissors
  • Hand‐held blender (e.g., Bio Homogenizer)
  • Teflon pestle (Potter type), motor driven
  • Centrifuge tubes, ice cold
  • Ultracentrifuge tubes
  • Ultracentrifuge, refrigerated
  • Additional reagents and equipment for determining protein concentration by standard methods ( appendix 3A)

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

  Materials
  • Microsomal preparation (see protocol 3)
  • 0.2 M phosphate buffer, pH 7.4
  • Crystalline dithionite (Na 2S 2O 4 or sodium hydrosulfite)
  • Carbon monoxide (CO) in a tank with a two‐stage valve
  • Spectrophotometric cuvettes with 1‐cm light path (quartz or plastic)
  • UV/VIS spectrophotometer, preferably a dual‐beam instrument
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Figures

  •   FigureFigure 7.8.1 Sample plate layout shown for 8 compounds; up to 32 compounds can be assayed per plate.
  •   FigureFigure 7.8.2 Methanol plate layout shown for 8 compounds.

Videos

Literature Cited

   Anonymous, 1997. Guidance for Industry. Drug Metabolism/Drug Interaction Studies in the Drug Development Process: Studies In Vitro. U.S. FDA, Center for Drug Evaluation and Research.
   Bogaards, J.J.P., Bertrand, M., Jackson, P., Oudshoorn, M.J., Weaver, R.J., van Bladeren, P.J., and Walther, B. 2000. Determining the best animal model for human cytochrome P450 activities: A comparison of mouse, rat, rabbit, dog, micropig, monkey and man. Xenobiotica 30:1131–1152.
   Chauret, N., Gauthier, A., Martin, J., and Nicoll‐Griffith, D.A. 1997. In vitro comparison of cytochrome P450‐mediated metabolic activities in human, dog, cat and horse. Drug Metab. Disp. 25:1130–1136.
   De Graaf, I.A.M., Van Meijeren, C.E., Pektas, F., and Koster, H.J. 2002. Comparison of in vitro preparations for semi‐quantitative prediction of in vivo drug metabolism. Drug Metab. Disp. 30:1129–1136.
   Gibson, G.G. and Skett, P. 1986. Introduction to Drug Metabolism, Chapman and Hall, London.
   Glue, P. and Clement, R.P. 1999. Cytochrome P450 enzymes and drug metabolism—Basic concepts and methods of assessment. Cell. Mol. Neurobiol. 19:309–323.
   Guengerich, F.P. 1989. Analysis and characterization of enzymes. In Principles and Methods of Toxicology (A.W. Hayes, ed.) pp. 777–814. Raven Press, New York.
   Iwatsubo, T., Hirota, N., Ooie, T., Suzuki, H., Shimada, N., Chiba, K., Ishizaki, T., Green, C.E., Tyson, C.A., and Sugiyama, Y. 1997. Prediction of in vivo drug metabolism in the human liver from in vitro metabolism data. Pharmacol. Ther. 73:147–171.
   Korfmacher, W.A., Palmer, C.A., Nardo, C., Dunn‐Meynell, K., Grotz, D., Cox, K., Lin, C‐C., Elicone, C., Liu, C., and Duchoslav, E. 1999. Development of an automated mass spectrometry system for the quantitative analysis of liver microsomal incubation samples: A tool for rapid screening of new compounds for metabolic stability. Rapid Comm. Mass Spec. 13:901–907.
   McGinnity, D.F., Parker, A.J., Soars, M., and Riley, R.J. 2000. Automated definintion of the enzymology of drug oxidation by the major human drug metabolizing cytochrome P450s. Drug Metab. Disp. 28:1327–1334.
   Omura, T. and Sato, R. 1964. The carbon monoxide‐binding pigment in liver microsomes. J. Biol. Chem. 239:2379–2385.
   Omura, T., Siekevitz, P., and Palade, G.E. 1967. Turnover of constituents of the endoplasmic reticulum membranes of rat hepatocytes. J. Biol. Chem. 242:2389–96.
   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.
   Woolf, T.F. (ed.) 1999. Handbook of Drug Metabolism. Marcel Dekker, New York.
   Wrighton, S.A., Ring, B.J., and VandenBranden, M. 1995. The use of in vitro metabolism techniques in the planning and interpretation of drug safety studies. Toxicol. Path. 23:199–208.
Internet Resources
  http://medicine.iupui.edu/flockhart/
  Provides drug‐drug interaction tables.
  http://www.imm.ki.se/CYPalleles/
  Provides nomenclature and alleles for CYPs.
  http://bioresearch.ac.uk/nb/e02ea9321b94a9deb45349f052d34457.html
  Overview and links to CYP resources.
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