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CYP450 Metabolism-Dependent Inhibition in Human Liver Microsomes

Inhibition of cytochrome P450 enzymes is a well-recognized cause of drug-drug interactions and occurs by two general mechanisms: direct inhibition and metabolism-dependent inhibition (MDI).1  In typical drug discovery and development, and in line with FDA and EMEA requirements, MDI is evaluated in vitro in human liver microsomes by determining if the inhibitory potency of a test compound increases following an incubation period. 
MDI requires biotransformation of an inhibitor in the presence of cofactor (NADPH) and results in an increased inhibitory potency. The IC50 is determined following pre-incubation with test compound in the presence and absence of NADPH.  A positive MDI result is inferred from a decrease in IC50 following pre-incubation with NADPH.
   Assay description

   CYP450 isoform
      CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4

   Compound concentration
      0-100µM (IC50)

   Compound requirements
      1-2 mg of dry matter

   Incubation details
      isoform specific substrate (Table 1)
      isoform specific time of incubation

   Assay controls
      isoform specific controls (Table 1)

   Detection method
      LC-MS/MS with internal standard

    1 Parkinson et al 2011, Drug Metab Dispos 39, 1370

Figure 1. IC50 fold shift for midazolam 4-hydroxylation by:
(a) troleandomycin (metabolism-dependent inhibitor) and
(b) ketoconazole (reversible inhibitor)


Assay controls

  Table 1. CYP450 isoform specific substrates and controls

Assay details adjustable to client’s and/or project specific requests

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