Description of a 96-well plate assay to measure cytochrome P4503A inhibition in human liver microsomes using a selective fluorescent probe

The standard method to evaluate CYP3A inhibition is to study the conversion of the specific CYP3A probe testosterone to its 6 beta-hydroxy metabolite in human liver microsomes, in the absence and presence of potential inhibit ors. Quantification of the 6 beta-hydroxy metabolite is achieved by HPLC re sulting in a tedious and time-consuming assay. In order to increase the P45 0 inhibition throughput, efforts were made to find a CYP3A probe that would produce a fluorescent metabolite. This paper reports the discovery of DFB as a potential CYP3A fluorescent probe. DFB was significantly metabolized i n human microsomes (similar to 1-2 nmol/(min mg protein)) to give the fluor escent compound DFH. The involvement of: CYP3A in the metabolism of DFB was determined using multiple approaches. First, incubations conducted with mi crosomes made from cell lines expressing single CYPs (Gentest Supersomes) i ndicated that CYP3A played a major role in the metabolism of DFB. Secondly, immunoinhibition studies conducted with CYP3A antibody resulted in >95% in hibition of DFB metabolism in HLM. Thirdly, inhibition studies with specifi c CYP1A1, 1A2, 2C8/9, 2C19, 2D6, and 2E1 chemical inhibitors did not suppre ss DFB activity in HLM. However, ketoconazole, miconazole, nicardipine, and nifedipine, all known CYP3A inhibitors, completely abolished the formation of DFH in HLM. The potency of several inhibitors determined using DFB and testosterone as CYP3A probes was consistent (R = 0.98). Finally, a good agr eement was obtained for the formation of DFH and production of 6 beta-hydro xytestosterone when DFB and testosterone were incubated separately with var ious human liver microsome preparations (R = 0.94, N = 11). In order to use DFH as a fluorescent CYP3A marker in a 96-well plate format, it was import ant to remove the excess of NADPH at the end of the incubation because the fluorescence of NADPH interferes with DFH detection. This was achieved by a dding oxidized glutathione and glutathione reductase to convert NADPH to NA DP(+) which is not fluorescent. The liquid-handling steps were fully automa ted in a 96-well plate format and a template was designed to generate IC50 curves and to address potential fluorescent interferences from the test com pounds. The assay was found to be reproducible (intraday variability <10% a nd interday variability indicated less than a 2-fold variation in the IC50 values) and is now routinely used in our laboratory to evaluate CYP3A inhib ition of NCEs. (C) 1999 Academic Press.