PT - JOURNAL ARTICLE AU - Veera Raghava Choudary Palacharla AU - Prathyusha Chunduru AU - Devender Reddy Ajjala AU - Gopinadh Bhyrapuneni AU - Ramakrishna Nirogi AU - Albert P Li TI - Development and Validation of a Higher Throughput Cytochrome P450 Inhibition Assay with the Novel Cofactor-Supplemented Permeabilized Cryopreserved Human Hepatocytes (MetMax Human Hepatocytes) AID - 10.1124/dmd.119.088237 DP - 2019 Jan 01 TA - Drug Metabolism and Disposition PG - dmd.119.088237 4099 - http://dmd.aspetjournals.org/content/early/2019/08/02/dmd.119.088237.short 4100 - http://dmd.aspetjournals.org/content/early/2019/08/02/dmd.119.088237.full AB - We report here the application of a novel hepatocyte system, the cofactor-supplemented permeabilized cryopreserved human hepatocytes (MetMax human hepatocytes (MMHHs)) in a higher throughput 384-well plate assay for the evaluation of P450 inhibition. The assay was developed to develop physiologically relevant P450 inhibition information, taking advantage of the complete organelle composition and their associated drug metabolizing enzymes of the MMHH, but with the ease of use of human liver microsomes including storage at -80 deg C instead of liquid nitrogen, and thaw and use without centrifugation and microscopic evaluation as required for intact hepatocytes. Nine key cytochrome P450 (CYP) isoforms for drug metabolism: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, were evaluated using multiple isoform-selective inhibitors. Results with MMHH were found to be comparable to that obtained with intact cryopreserved human hepatocytes (CHHs). Isoform-selective drug metabolizing enzyme pathways evaluated were phenacetin O-deethylation (CYP1A2), coumarin hydroxylation (CYP2A6), bupropion hydroxylation (CYP2B6), amodiaquine N-deethylation (CYP2C8), diclofenac hydroxylation (CYP2C9), mephenytoin hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone hydroxylation (CYP2E1), midazolam and testosterone hydroxylation (CYP3A4). The Km values obtained with MMHHs were comparable with those reported in the literature for CHHs. Using substrate concentrations at or near Km values, the IC50 values for the standard inhibitors against the P450 activities were found to be comparable between MMHHs and CHHs, with 73% and 84% of values falling within two-fold and three-fold, respectively, from the line of unity. The results indicate that MMHHs can be an efficient experimental system for the evaluation of P450 inhibition in hepatocytes.SIGNIFICANCE STATEMENT We report here the development of a higher throughput hepatocyte-based P450 inhibition assay using a novel human hepatocyte system, the cofactor-supplemented permeabilized human hepatocytes (MetMax Human Hepatocytes; MMHH) (Li et al., 2018). MMHHs have the desirable properties of both intact hepatocytes and HLMs - the completeness of the DME’s in hepatocytes and the robustness and ease of handling of HLMs including storage at -80 deg. C freezer instead of liquid nitrogen, and use directly after thawing without a need for centrifugation and microscopic examination as required for CHHs. Our results show that MMHH can be used in a 384-well plate format for the evaluation of P450 inhibition, yielding results similar to that obtained with intact human hepatocytes. The MMHH 384-well plate P450 inhibition assay may represent an effective approach for the early screening of DDI potential of NCEs.