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Cytochrome P450 Inhibition Using Recombinant Enzymes

Background:

Co-administration of drugs can result in drug-drug interaction, as drugs compete for the same enzymes.  Inhibition of CYP450 enzymes is a principal mechanism of metabolism-based drug-drug interactions and a common cause of adverse drug events. Therefore, one of the crucial properties assessed in early drug discovery is the potential of the test compound to inhibit a specific CYP450 isoform. This data is useful for further investigations of clinical drug-drug interactions (DDI).

CYP450 inhibition can be evaluated using recombinant enzymes, a high-throughput in vitro  screening approach that includes the use of:
i)  modified microsomes that contain only one specific CYP450 isoform , and
ii) specific substrates whose metabolism is monitored by fluorescence.

   Assay description

   CYP450 isoforms
      CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4

   Compound concentration
      10µM (single point) or 0-100µM (IC50)

    Compound requirements
      50µl of 10mM stock solution or           
      1-2 mg of dry matter

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

   Assay controls
      known isoform specific positive control
      (see Table 1 and Figure 1)

   Detection method
      Fluorescence

Table 1. CYP450 isoform specific substrates and positive controls

Figure 1.
Comparison of IC50 values for CYP450 isoform specific positive control with literature values1
1 Crespi et al. 1997, Anal Biochem 248, 188
Assay details adjustable to client’s and/or project specific requests
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