Cytochrome P450 Inhibition

It is important to examine if an NME is an inhibitor of cytochrome P450s not involved in the metabolism of the drug. For this type of study, the effect of NME on the metabolism of probe substrate for each of the individual cytochrome P450 (see Table 1) is evaluated, usually in human liver microsomes, although individual recombinant human cytochrome P450 enzymes have also been used. The incubation conditions should be such that initial rates could be measured. To determine the Ki value for any specific cytochrome P450, at least four to five probe substrate concentrations and two to three NME concentrations should be used in the assays. Substrate concentrations should cover a wide range (preferably 10-20-fold) with the number of concentrations evenly distributed below and above the Km value. The importance of proper selection of both substrate and inhibitor concentrations in these studies is well illustrated in the paper by Madan et al. [22]. The rates of metabolite formation of probe substrate are determined in the presence and absence of the NME inhibitor and the data are displayed in graphical representation to determine Ki and the type of inhibition [22]. Substrate-dependent inhibition has been reported earlier for CYP3A [49, 51]. Two or more substrates may be needed when evaluating inhibitors of CYP3A using in vitro methods [21, 47, 49]. Because of significant solvent effects (particularly when concentration >1%) reported for various CYP enzyme studies, low solvent concentrations should be used in these in vitro studies [47].

In addition to reversible inhibition, time-dependent inhibition of cytochrome P450 activity by a drug candidate may also be examined to determine if the NME is a mechanism-based inhibitor. For this type of study, an NME, at various concentrations (covering a 10-20-fold range), is preincubated with human liver microsomes with and without NADPH for various lengths of time (e.g., 0, 10, 20, 30, 45, and 60min) to allow the generation of reactive metabolites that inhibit cytochrome P450 activity irreversibly or quasi-irreversibly [22]. At various incubation time points, an aliquot of the samples is removed and diluted several folds with fresh assay buffer. The activity of the remaining cytochrome P450 is determined by the reaction rates of a probe substrate, and the data are displayed in graphical representation to determine the Ki and Kinact values [22, 31].

If an NME and clinically co-administered drugs are metabolized by the same cytochrome P450 isoform, inhibition of this cytochrome P450 can lead to the accumulation of either of the drugs and thereby cause potential serious drug-drug interactions. This potential can be evaluated using an in vitro system of human liver microsomes in the presence of both the drugs. The importance in the proper use of concentrations of either of the drugs is as described in the preceding section. The Ki value for either of the drugs can be determined and the potential of drug-drug interaction of co administered drugs can be evaluated.

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