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CYTOCHROME P450 INHIBITION USING RECOMBINANT PROTEINS AND MASS SPECTROMETRY/MULTIPLE REACTION MONITORING TECHNOLOGY IN A CASSETTE INCUBATION

Physical & Metabolic Science, AstraZeneca R&D Charnwood, Loughborough, United Kingdom

Detailed cytochrome P450 (P450) inhibition profiles are now required for the registration of novel molecular entities. This method uses combined substrates (phenacetin, diclofenac, S-mephenytoin, bufuralol, and midazolam) with combined recombinant P450 enzymes (CYP1A2, 2C9, 2C19, 2D6, and 3A4) in an attempt to limit interactions with other more minor P450s and associated reductases. Kinetic analysis of single substrate with single P450 (sP450) yielded apparent K_m values of 25, 2, 20, 9, and 3 µM, for CYP1A2, 2C9, 2C19, 2D6, and 3A4, respectively. Combined substrates with combined P450s (cP450) yielded apparent K_m values of 65, 4, 19, 7, and 2 µM. Selectivity of the substrates for each P450 isoform was checked. Phenacetin proved to be the least selective substrate. However, the ratio of the various P450s was modified in the final assay such that metabolism of phenacetin by other enzymes was 20% of the metabolism by CYP1A2. IC₅₀ determinations with -naphthoflavone (0.04 µM), sulfaphenazole (0.26 µM), tranylcypromine (9 µM), quinidine (0.02 µM), and ketoconazole (0.01 µM) were similar for sP450 and cP450 enzymes. The assay was further evaluated with 11 literature compounds and 52 in-house new chemical entities, and the data compared with radiometric/fluorescent values. The overall protein level of the assay was reduced from the original starting point, as this led to some artificially high IC₅₀ measurements when compared with existing lower protein assays (radiometric/fluorometric). This method offers high throughput P450 inhibition profiling with potential advantages over current radiometric or fluorometric methods.

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