CYP, inhibition studies

The additional recommendations contained in the draft guidance and on the FDA s website as they relate specifically to the design of in vitro CYP inhibition studies can be summarized as follows ... 

The system chosen to conduct CYP inhibition studies should be well characterized. This procedure requires initial time-course experiments and determination of linearity of metabolite formation with the chosen incubation time and enzyme concentration. After these experiments, the kinetic parameters (i.e., Km and Vmax) for each substrate used with six or more concentrations spanning from 1/3 to 3 A), and inhibition potencies (i.e., IC50 or K,) of typical inhibitors should be determined. This characterization does not need to be repeated for each batch or lot of test system. 

Additional Industry Perspectives on the Conduct of In Vitro CYP Inhibition Studies... 

All three assumptions can be violated in the case of CYP enzymes, depending on the design of the in vitro CYP inhibition study. The first assumption can be potentially violated if the drug being tested is a time-dependent inhibitor (e.g., one with a slow on rate see below). The potency of some inhibitors (e.g., the CYP3A inhibitors ketoconazole and clotrimazole) is such that the free concentration of the inhibitor tends to approach the concentration of the enzyme (40), a violation of the second assumption. In the case of such tight-binding inhibition, an apparent A) value (A i a ) )) can be estimated, as follows ... 

For most cases encountered in drug development, it is not necessary to definitively prove all of the above criteria. Ideally, the experimental design of a definitive in vitro CYP inhibition study should allow the following questions to be answered in a single initial experiment ... 

Figure 5 Flowchart for initial and possible follow-up CYP inhibition studies. The first box represents the IC50 experiment with and without a 30-minute preincubation with NADPH (the highest concentration of test article is also preincubated for 30 minutes without NADPH). Remaining boxes depict possible outcomes and follow-up experiments. Abbreviations CYP, cytochrome P450 IC50, concentration of inhibition causing 50% inhibition.

Table 6 Typical In Vitro Incubation Conditions for CYP Inhibition Studies in Human Liver Microsomes and Selected Analytical and Kinetic Parameters... 

Positive control inhibitors for each of the major CYP enzymes should also be included to further demonstrate that the test system is performing as expected. The direct-acting inhibitors used in our laboratory are summarized in Table 7, along with the IC50 values determined during assay validation and a comparison with literature values. It is worth noting that the positive control inhibitors used for CYP inhibition studies need not necessarily be CYP-selective inhibitors, in contrast to those used for reaction phenotyping, which should be CYP-selective inhibitors. 

In addition to being prone to homotropic activation, CYP3A4 is also prone to heterotropic activation. The CYP1A2 inhibitor, a-naphthoflavone is an activator of certain CYP3A4-dependent reactions [a factor that complicates the use of this flavonoid in CYP inhibition studies (discussed later)]. CYP3A4-catalyzed... 

II.K.2 CYP Inhibition Studies Using Recombinant P450 Isoenzymes. 552... 

II.K.3 CYP Inhibition Studies Using Human Liver Microsomes 554... 

II.K.4 CYP Inhibition Studies Using Isolated/Cultured Hepatocytes or Liver Slices 557... 

Table 1 summarizes typical assay conditions for CYP inhibition studies of the most relevant P450 enzymes using recombinant P450 isoenzymes (Super-somes) which is applicable to 96 and 384 well format. Assay conditions for additional P450 isoenzymes can be found under www.gentest.com. 

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