A Novel In Vitro Experimental System for the Evaluation of Drug Metabolism: Cofactor-Supplemented Permeabilized Cryopreserved Human Hepatocytes (MetMax Cryopreserved Human Hepatocytes)

Li, Albert P.; Ho, Ming-Chih David; Amaral, Kirsten; Loretz, Carol

doi:10.1124/dmd.117.079657

Cited by 13 publications

(14 citation statements)

References 34 publications

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“…Thawing Procedures for MMHHs and CHHs. The MMHH vial was thawed and used directly without the need for centrifugation or cell viability determination (Li et al, 2018). In brief, MMHH vials were removed from the freezer (280°C), thawed, and the contents were transferred to cofactor N10 vials (IVAL) with gentle mixing.…”

Section: Methodsmentioning

confidence: 99%

“…Here, we report the development of a higher-throughput hepatocytebased P450 inhibition assay using a novel human hepatocyte system, the cofactor-supplemented permeabilized human hepatocytes [MetMax human hepatocytes (MMHHs)] (Li et al, 2018). MMHHs have the desirable properties of both intact hepatocytes and HLMs-the completeness of the drug metabolizing enzymess in hepatocytes and the robustness and ease of handling of HLMs, including storage in a 280°C freezer instead of liquid nitrogen, and use directly after thawing without a need for centrifugation and microscopic examination as required for CHHs.…”

Section: Introductionmentioning

confidence: 99%

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Development and Validation of a Higher-Throughput Cytochrome P450 Inhibition Assay with the Novel Cofactor-Supplemented Permeabilized Cryopreserved Human Hepatocytes (MetMax Human Hepatocytes)

et al. 2019

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Here, we report 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 cytochrome P450 (P450) inhibition. The assay was created 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 280°C instead of in liquid nitrogen, and thaw and use without centrifugation and microscopic evaluation as required for intact hepatocytes. Nine key P450 isoforms for drug metabolism (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) were evaluated using multiple isoformselective inhibitors. Results with MMHH were found to be comparable to those obtained with intact cryopreserved human hepatocytes (CHHs). Isoform-selective drug-metabolizing enzyme pathways evaluated were phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), bupropion hydroxylation (CYP2B6), amodiaquine N-deethylation (CYP2C8), diclofenac 4-hydroxylation (CYP2C9), s-mephenytoin 49-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and midazolam 19-hydroxylation and testosterone 6b-hydroxylation (CYP3A4). The K m values obtained with MMHHs were comparable with those reported in the literature for CHHs. Using substrate concentrations at or near K m values, the IC 50 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 2-fold and 3-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 MetMax human hepatocytes (MMHHs) are cofactor-supplemented cryopreserved human hepatocytes with the complete drug-metabolizing enzyme pathways of the conventional hepatocytes but with the convenience of human liver microsomes, including storage at 280°C instead of in liquid nitrogen, and direct thaw and use without a need for centrifugation and microscopic examination. Here, we report the application of MMHH in a high-throughput assay in a 384-well plate format for the evaluation of cytochrome P450 (P450) inhibition. Our results show that data obtained with MMHH are similar to those with conventional hepatocytes, suggesting that the MMHH 384-well P450 inhibition assay can be used routinely for the evaluation of drug-drug interaction potential of new chemical entities in drug development.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Development and Validation of a Higher-Throughput Cytochrome P450 Inhibition Assay with the Novel Cofactor-Supplemented Permeabilized Cryopreserved Human Hepatocytes (MetMax Human Hepatocytes)

et al. 2019

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“…As a differentiation compared to intestinal mucosa, permeabilized enterocytes offer an increased cellular permeability and biochemical assay environment (i.e. no need for cell counting and culturing) (Li et al, 2018a;Li et al, 2018c), possibly enabling drug metabolism studies with poorly permeable and cytotoxic compounds (e.g. cancer chemotherapeutics).…”

Section: Downloaded Frommentioning

confidence: 99%

Evaluation of In Vitro Models for Assessment of Human Intestinal Metabolism in Drug Discovery

et al. 2020

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Although intestinal metabolism plays an important role in drug disposition, early predictions of human outcomes are challenging, in-part due to limitations of available in vitro models. To address this, we have evaluated three in vitro models of human intestine (microsomes, permeabilized enterocytes, and cryopreserved intestinal mucosal epithelium) as tools to assess intestinal metabolism and estimate the fraction escaping gut metabolism (f g) in drug discovery. The models were tested with a chemically diverse set of 32 compounds, including substrates for oxidoreductive, hydrolytic, and conjugative enzymes. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) was used to quantify substrate disappearance (CL int) and qualify metabolite formation (QuanQual). Fraction unbound in the incubation (f u,inc) was determined by rapid-equilibrium dialysis (RED). Measured in vitro results [intrinsic clearance (CL int) and f u,inc ] were supplemented with literature data [passive Caco-2 A→B permeability, enterocyte blood flow (Q ent), and intestinal surface area (A)] and combined using a midazolam-calibrated Q gut model to predict human f g values. All three models showed reliable cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) activities, but enterocytes and mucosa may offer advantages for low clearance compounds and alternative pathways (e.g. sulfation, hydrolases, and flavin-containing monooxigenases). Early predictions of human f g values were acceptable for the high f g compounds (arbitrarily f g >0.7). However, predictions of low and moderate f g values (arbitrarily f g <0.7) remain challenging, indicating that further evaluation is needed (e.g. saturation effects and impact of transporters), but not immediate compound avoidance. Results suggest that tested models offer an additional value in drug discovery, especially for drug design and chemotype evaluation.

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“…Permeabilized cofactor-supplemented (MetMax) cryopreserved human enterocytes. MetMax cryopreserved human enterocytes (Li et al, 2018b) were prepared using a proprietary technology previously developed for MetMax cryopreserved human hepatocytes (Li et al, 2018c). MetMax cryopreserved human enterocytes are prepared via the permeabilization of the plasma membrane of intact cryopreserved human enterocytes.…”

Section: Primary Human Enteric Modelsmentioning

confidence: 99%

In Vitro Human Cell–Based Experimental Models for the Evaluation of Enteric Metabolism and Drug Interaction Potential of Drugs and Natural Products

2020

Drug Metab Dispos

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Elements of key enteric drug metabolism and disposition pathways are reviewed to aid the assessment of the applicability of current cell-based enteric experimental systems for the evaluation of enteric metabolism and drug interaction potential. Enteric nuclear receptors include vitamin D receptor, constitutive androstane receptor, pregnane X receptor, farnesoid X receptor, liver X receptor, aryl hydrocarbon receptor, and peroxisome proliferator-activated receptor. Enteric drug metabolizing enzyme pathways include both cytochrome P450 (P450) and non-P450 drug metabolizing enzymes based on gene expression, proteomics, and activity. Both uptake and efflux transporters are present in the small intestine, with P-glycoprotein found to be responsible for most drug-drug and food-drug interactions. The cell-based in vitro enteric systems reviewed are 1) immortalized cell line model: the human colon adenocarcinoma (Caco-2) cells; 2) human stem cell-derived enterocyte models: stem cell enteric systems, either from intestinal crypt cells or induced pluripotent stem cells; and 3) primary cell models: human intestinal slices, cryopreserved human enterocytes, permeabilized cofactor-supplemented (MetMax) cryopreserved human enterocytes, and cryopreserved human intestinal mucosa. The major deficiency with both immortalized cell lines and stem cell-derived enterocytes is that drug metabolizing enzyme activities, although they are detectable, are substantially lower than those for the intestinal mucosa in vivo. Human intestine slices, cryopreserved human enterocytes, MetMax cryopreserved human enterocytes, and cryopreserved human intestinal mucosa retain robust enteric drug metabolizing enzyme activity and represent appropriate models for the evaluation of metabolism and metabolism-dependent drug interaction potential of orally administered xenobiotics including drugs, botanical products, and dietary supplements. SIGNIFICANCE STATEMENT Enteric drug metabolism plays an important role in the bioavailability and metabolic fate of orally administered drugs as well as in enteric drugdrug and food-drug interactions. The current status of key enteric drug metabolism and disposition pathways and in vitro human cell-based enteric experimental systems for the evaluation of the metabolism and drug interaction potential of orally administered substances is reviewed.

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A Novel In Vitro Experimental System for the Evaluation of Drug Metabolism: Cofactor-Supplemented Permeabilized Cryopreserved Human Hepatocytes (MetMax Cryopreserved Human Hepatocytes)

Cited by 13 publications

References 34 publications

Development and Validation of a Higher-Throughput Cytochrome P450 Inhibition Assay with the Novel Cofactor-Supplemented Permeabilized Cryopreserved Human Hepatocytes (MetMax Human Hepatocytes)

Development and Validation of a Higher-Throughput Cytochrome P450 Inhibition Assay with the Novel Cofactor-Supplemented Permeabilized Cryopreserved Human Hepatocytes (MetMax Human Hepatocytes)

Evaluation of In Vitro Models for Assessment of Human Intestinal Metabolism in Drug Discovery

In Vitro Human Cell–Based Experimental Models for the Evaluation of Enteric Metabolism and Drug Interaction Potential of Drugs and Natural Products

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