In Vitro Species Comparisons and Metabolite Identification

Paul Dow1

1 Covance Laboratories, Inc., Madison, Wisconsin
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 7.10
DOI:  10.1002/0471141755.ph0710s34
Online Posting Date:  October, 2006
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Abstract

The metabolism and elimination of a drug has a direct bearing on its potential to cause toxicity in humans as well as to influence its clinical activity. The confidence with which data from preclinical safety studies, particularly toxicology studies, can be extrapolated to humans depends upon knowing whether humans are exposed to the same chemical entities (i.e., a parent drug and its metabolites) as the laboratory animals used to study toxicity and pharmacological responses. To address this issue, the FDA Guidance for Industry (1997) recommends the use of in vitro interspecies metabolic comparisons at an early stage in the drug development process. Knowledge about the metabolism of a compound is also important from a drug discovery perspective. In vitro studies provide a convenient and cost‐effective way to obtain this information. This unit presents protocols for comparing metabolite profiles across several species, as well as methods for identifying metabolites.

Keywords: interspecies comparison; metabolite profiles; in vitro metabolism; metabolite identification; LC‐MS; NMR

     
 
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Table of Contents

  • Basic Protocol 1: In Vitro Species Comparison of Test Compound Metabolites Using Radiolabeled Material
  • Alternate Protocol 1: In Vitro Species Comparison of test compound Metabolite Profiles Using Nonradiolabeled Material
  • Basic Protocol 2: Metabolite Identification
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: In Vitro Species Comparison of Test Compound Metabolites Using Radiolabeled Material

  Materials
  • 1 mM test article, preferably radiolabeled to high specific activity
  • Positive control (e.g., [3H]‐testosterone or 7‐ethoxycoumarin), preferably radiolabeled to high specific activity (e.g., GE Healthcare)
  • 20 mg/ml microsomal protein from liver microsomal preparations from different species (BD‐Gentest, In Vitro Technologies, Xenotech; also see unit 7.8) or other preparations such as hepatocytes, liver slices, homogenates, or purified enzymes
  • Boiled microsome preparation (for negative control): boil for 5 min
  • 10 mM NADPH
  • 0.1 M sodium phosphate buffer, pH 7.4 ( appendix 2A)
  • Precipitant (e.g., ice cold acetonitrile, methanol, 4% perchloric acid)
  • Scintillation cocktail
  • Mobile phase A: water containing 0.01% formic acid
  • Mobile phase B: acetonitrile containing 0.01% formic acid
  • Incubation tubes (e.g., 1‐ml microcentrifuge tubes)
  • 37°C water bath
  • Refrigerated centrifuge
  • Plastic vials (e.g., 1‐ml microcentrifuge tubes)
  • Balance accurate to four or five decimal places
  • Sample concentration unit (e.g., N‐Evap, Turbovap)
  • Liquid scintillation vials
  • Gradient HPLC system equipped with:
    • Reversed‐phase column (e.g., Zorbax RX C 8, 5‐µm particle size, 150‐mm × 4.6‐mm‐i.d.)
    • Variable wavelength UV (210 nm) and radiochemical (e.g., Packard Radiomatic liquid cell) detectors

Alternate Protocol 1: In Vitro Species Comparison of test compound Metabolite Profiles Using Nonradiolabeled Material

  • Test article
  • Positive control (e.g., 7‐ethoxycoumarin)

Basic Protocol 2: Metabolite Identification

  • Radio‐flow detector using solid scintillant cell (e.g., Packard)
  • Additional reagents and equipment for LC‐MS and LC‐NMR
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Figures

Videos

Literature Cited

   Baillie, T.A., Cayen, M.N., Fouda, H., Gerson, R.J., Green, J.D., Grossman, S.J., Klunk, L.J., LeBlanc, B., Perkins, D.G., and Shipley, L.A. 2002. Drug metabolites in safety testing. Toxicol. Appl. Pharmacol. 182:188‐196.
   Parkinson, A. 2001. Biotransformation of Xenobiotics. In Casarett & Doull's Toxicology, 6th ed. (C.D. Klaassen, ed.) pp. 133‐224. The McGraw‐Hill Companies, New York.
   Salmon, A.G., 1989. In vitro studies on metabolism, In Xenobiotic Metabolism and Disposition: The Design of Studies on Novel Compounds (H.P.A. Illing, ed.) pp. 171‐191. CRC Press, Boca Raton, Fla.
   U.S. Food and Drug Administration 1997. Guidance for industry: Drug metabolism/drug interaction studies in the drug development process: Studies in vitro. U.S. Food and Drug Administration, Rockville, Md.
Internet Resources
   http://www.fda.gov/cder/guidance/clin3.pdf
  Guidance for industry: Drug metabolism/drug interaction studies in the drug development process: Studies in vitro.
   http://www.nhlbi.nih.gov/funding/policies/repos‐gl.htm
  Guidelines for the Human Tissue Repository.
   http://www.law.upenn.edu/bll/ulc/fnact99/uaga87.pdf
  Uniform Anatomical Gift Act (1987).
  http://www.hhs.gov/ohrp/irb/irb_guidebook.htm
  Office for Human Research Protections (OHRP) IRB guidebook.
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