P450 enzyme inhibition assay

Several higher throughput in vitro assays may be used to assess various DMPK properties of NCEs. One common parameter is that HPLC/MS/MS is the method of choice for the analytical step.11 17 26 These higher throughput assays include the Caco-2 assay, p450 enzyme inhibition assay, and in vitro stability assay. Each assay has different requirements and solutions and they will be described individually. 

Higher throughput screening with human cytochrome P450 to study P450-medi-ated metabolism is now available [38-40]. Similarly, rapid microhtre plate assays to conduct for example, P450 enzyme inhibition studies have been developed, using individually expressed CYP enzymes (Supersomes) [41]. 

C. Nomeir, A. A. Validation of higher-throughput high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry assays to conduct cytochrome P450s CYP2D6 and CYP3A4 enzyme inhibition studies in human liver microsomes. Rapid Commun Mass Spectrom 2000, 14, 207-214. 

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. 

Palamanda, J.R. Eavreau, L. Lin, C.C. Nomeir, A.A. Validation of a rapid microtiter plate assay to conduct cytochrome P450 2D6 enzyme inhibition studies. Drug Discov. Today 1998, 5, 466-470. 

Rainville, P.D. et al., Sub one minute inhibition assays for the major cytochrome P450 enzymes utilizing ultra-performance liquid chromatography/tandem mass spectrometry, Rapid Commun. Mass Spectrom., 22(9), 1345, 2008. 

Yao, M. et al., Development and full validation of six inhibition assays for five major cytochrome P450 enzymes in human liver microsomes using an automated 96-well microplate incubation format and LC-MS/MS analysis, J. Pharm. Biomed. Anal., 44, 211, 2007. 

Figure 7.31), is a relatively selective and very potent inhibitor of human placental aro-matase. Its (-I-) enantiomer has a lower IC5Q value than the (—) enantiomer when assayed against human placental aromatase and exhibits no appreciable inhibition of other steroidogenic enzymes or liver microsomal P450 enzymes at concentrations up to 1,000-times the aromatase... 

Prior to P450 reaction phenotyping or inhibition experiments, it is important to determine enzyme kinetic parameters such as Km and Umax for the formation of selected metabolites that are subjected to quantitative analysis by LC-MS. For example, -warfarin is catalyzed by CYP2C9 to a specific metabolite, 7-hydroxy-5 -warfarin (Fig. 15.13). Thus, a CYP2C9 inhibition assay is developed based on the reaction. In the assay, Y-warfarin is incubated with HLM in the presence of a test compound, followed by quantification of 7-hydroxy-5 -warfarin by LC—MS (Zhang et ah, 2001). To set up this assay in our lab, enzyme kinetics for the formation of 7-hydroxy-iS-warfarin in HLM was determined. In this experiment, warfarin was incubated at concentrations from 0 to 250 >M with HLM at optimized conditions. Rates of 7-hydroxy-S -warfarin formation at various substrate concentrations were determined as shown in Figure 15.13a, from which Km and Umax values were calculated. The warfarin assay represented an analytical challenge since the turnover of warfarin in the HLM system was extremely low. To be able to quantitatively determine low concentrations of 7-hydroxy-5 -warfarin in the incubations, a very sensitive LC—MS method that used MRM with a 4000 QTRAP has been developed (Fig. 15.13a). 

Estabrook, demonstrated the involvement of cytochrome P450 enzymes in steroid biochemistry when they showed that carbon monoxide inhibited the 21-hydroxylase activity in adrenal microsomes [3], Radiolabeled steroids became commercially available in the 1960s, which fa-cihtated experiments to confirm precursor-product relationships. Despite these initial advances, further progress was slow, due to low abundance of enzymes, the need to obtain animal adrenals as the source, the tedious nature of the assays, species-specific variations in the pathways, and the inability to purify the enzymes, which limited the interpretation of messy experiments. 

Biological/Medical Applications As a substrate for measuring cytochrome P 450 enzymes activity cytochrome P450 inhibition assays drug design - drug metabolism " treating viral or parasite infections " ... 

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