Assessment of the Time‐Dependent Inhibition (TDI) Potential of Test Compounds with Human Liver Microsomes by IC50 Shift Method Using a Nondilution Approach

Lian de Ron1, Ganesh Rajaraman1

1 NoAb BioDiscoveries, Mississauga, Ontario, Canada
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 7.14
DOI:  10.1002/0471141755.ph0714s58
Online Posting Date:  September, 2012
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Abstract

Time‐dependent inhibition (TDI) of hepatic cytochrome P450 (CYP) enzymes is increasingly implicated in the majority of clinically relevant drug‐drug interactions (DDIs). A time‐dependent inhibitor or its reactive metabolite irreversibly inactivates CYP enzymes, thereby inhibiting the metabolism of other drugs. As the majority of marketed drugs are metabolized by CYP enzymes, their inhibition has important clinical consequences, such as in decreasing the metabolic clearance of a co‐administered drug (victim drug). This could be life threatening, as such an effect narrows the therapeutic index for drugs such as warfarin and other potentially toxic agents. Therefore, it is essential to examine new chemical entities for their potential to cause TDI to minimize adverse drug reactions during human studies and use. This unit presents an in vitro procedure utilizing a nondilution method in human liver microsomes for determining the TDI potential of test compounds. Curr. Protoc. Pharmacol. 58:7.14.1‐7.14.13. © 2012 by John Wiley & Sons, Inc.

Keywords: time‐dependent inhibition; mechanism‐based inhibition; human liver microsomes; cytochrome P450

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Solutions 1, 2, 2A, 5, and 5A (see recipe for incubation solutions), freshly prepared
  • Positive control (PC) inhibitor stock solution for the CYP isoform to be tested (see Table 7.14.3)
  • NCE stock solution (see recipe)
  • Solutions 3, 4, 6, and 7 (see recipe for assay‐specific solutions), freshly prepared
  • Organic solvent (see Table 7.14.3)
  • Multichannel pipet (Eppendorf Research Plus, 12‐channel)
  • Dilution plates: 96‐well 500‐µl polypropylene plates (VWR)
  • Assay plates: 96‐well 2‐ml polypropylene plates (VWR)
  • Orbital shaker/incubator (Lab‐Line Enviro Shaker)
  • 96‐well mat, square, 100% silicone with PTFE coating (Canadian Life Science)
  • Microplate shaker (EAS 2/4, SLT Lab Instruments)
  • Parafilm
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Figures

Videos

Literature Cited

Literature Cited
   European Medicines Agency (draft 2010). Guideline on the Investigation of Drug Interactions. http://www.ema.euorpa.eu.
   FDA Guidance for the Industry (draft 2006). Drug interaction study study design, data analysis, and implications for dosing and labeling draft guidance. U.S. Department of Health and Human Services, Food and Drug Administration. http://www.fda.cder/guidance/index.htm.
   Fowler, H. and Zhang, S. 2008. In vitro evaluation of reversible and irreversible cytochrome P450 inhibition: Current status on methodologies and their utility for predicting drug–drug interactions. AAPS J. 10:410‐424.
   Grimm, S.W., Einolf, H.J., Hall, S.D., He, K., Lim, H‐K., Ling, K‐H., Lu, C., Nomeir, A.A, Seibert, E., Skordos, K.W., Tonn, G.R., Van Horn, R., Wang, R.W., Wong, Y.N., Yang, T.J., and Obach, R.S. 2009. The conduct of in vitro Studies to address time‐dependent inhibition of drug‐metabolizing enzymes: A perspective of the Pharmaceutical Research and Manufacturers of America. Drug Metab. Dispos. 37:1355‐1370.
   Omura, T. and Sato, R. 1964. The carbon monoxide‐binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239:2370‐2378.
   Parkinson, A., Kazmi, F., Buckley, D.B., Yerino, P., Paris, B.L., Holsapple, J., Toren, P., Otradovec, S. M., and Ogilvie, B.W. 2011. An evaluation of the dilution method for identifying metabolism‐dependent inhibitors (MBIs) of Cytochrome P450 (CYP) enzymes. Drug Metab. Dispos. 39:1370‐1387.
   Shou, M., Mei, Q., Ettore, M.W. Jr., Dai, R., Baillie, T.A., and Rushmore, T.H. 1999. Sigmoidal kinetic model for two co‐operative substrate‐binding sites in a cytochrome P450 3A4 active site: An example of the metabolism of diazepam and its derivatives. Biochem. J. 340:845‐863.
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