Albert P. Li

ID 19303 Poster Board 374 Evaluation of metabolic fates of drug candidates is a major activity in drug development, with results critical to the assessment of efficacy and safety. Due to the routine application of high throughput assays for hepatic clearance using in vitro systems such as human liver microsomes and human hepatocytes in the early phases of drug discovery and development, pharmaceutical companies have accumulated a collection of slowly metabolized compounds (SMC) as potential drug candidates. The metabolic stability of the SMC poses a challenge for further development as their metabolic fates cannot be readily defined using conventional approaches. To overcome this challenge, we have developed a plated hepatocyte relay assay (PHRA) with which SMC can be evaluated for intrinsic hepatic clearance as well as metabolite formation. PHRA is similar to the suspension hepatocyte relay assay developed by Di et al (2012) except that plated hepatocytes are employed in lieu of suspension hepatocytes, thereby extending each relay from 3 h to 24 h, and the elimination of the laborious centrifugation step required for medium transfer. The principle of the assay is to subject the SMC to metabolism by hepatocytes plated in a 96-well plate (4 hours after plating to allow cell attachment) on the day of evaluation, with medium transferred to freshly plated hepatocytes in subsequent days. Examples of the accumulative incubation durations in PHRA after each relay are as follows: 24 h (initial incubation), 48 h (1^st relay), and 72 h (2^nd relay), 96 h (3^rd relay), and 120 h (4^th relay). A validation study with 15 compounds with known human in vivo hepatic clearance which included the ultra low clearance compounds (CL_non-renal < 1 mL/min/kg) meloxicam, tolbutamide, warfarin, disopyramide, and tenoxicam; and the low clearance compounds (CL_non-renal 1-5.1 mL/min/kg) glimepiride, clozapine, riluzole, ibuprofen, prednisolone, voriconazole, quinidine, prednisone, dexamethasone and risperidone. Linear time-dependent disappearance of the parent compounds was observed for all compounds up to the longest incubation duration of 120 hrs. We further evaluated the potential applications of PHRA towards the derivation of enzyme kinetic parameters and metabolite formation of SMC using the model ultra low clearance compound, warfarin. Warfarin was evaluated at concentrations of 1, 2, 5, 10, 20 and 50 uM for incubation durations of 24, 48 and 72 hrs in the relay assay. Linear time-dependent warfarin clearance was observed at all concentrations, with concentration-dependent metabolic clearance consistent with Michalis Menten enzyme kinetics, allowing the derivation of apparent Km and Vmax values. Furthermore, time-dependent formation of warfarin metabolites, 7-hydroxy and 7-glucuronide warfarin, were observed. Our results suggest the PHRA can be readily applied towards to evaluation of intrinsic hepatic clearance, enzyme kinetics, as well as metabolite production of SMC.

We report here a novel in vitro experimental system, the metabolism-dependent cytotoxicity assay (MDCA), for the definition of the roles of hepatic drug metabolism in toxicity. MDCA employs permeabilized cofactor-supplemented cryopreserved human hepatocytes (MetMax{trade mark, serif} human hepatocytes, MMHH), as an exogenous metabolic activating system, and HEK-293 cells, a cell line devoid of drug metabolizing enzyme activity, as target cells for the quantification of drug toxicity. The assay was performed in the presence and absence of cofactors for key drug metabolism pathways known to play key roles in drug toxicity: NADPH/NAD+ for phase 1 oxidation, UDPGA for UGT mediated glucuronidation, PAPS for SULT mediated sulfation, and GSH for GST mediated GSH conjugation. Six drugs with clinically significant hepatoxicity, resulting in liver failure or a need for liver transplantation: acetaminophen, amiodarone, cyclophosphamide, ketoconazole, nefazodone and troglitazone were evaluated. All six drugs exhibited cytotoxicity enhancement by NADPH, suggesting metabolic activation via phase 1 oxidation. Attenuation of cytotoxicity by UDPGA was observed for acetaminophen, ketoconazole and troglitazone, by PAPS for acetaminophen, ketoconazole and troglitazone, and by GSH for all six drugs. Our results suggest that MDCA can be applied towards the elucidation of metabolic activation and detoxification pathways, providing information that can be applied in drug development to guide structure optimization to reduce toxicity and to aid the assessment of metabolism-based risk factors for drug toxicity. GSH detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites, a key property of drugs with idiosyncratic hepatotoxicity. Significance Statement Application of the metabolism-dependent cytotoxicity assay (MDCA) for the elucidation of the roles of metabolic activation and detoxification pathways in drug toxicity may provide information to guide structure optimization in drug development to reduce hepatotoxic potential, and to aid the assessment of metabolism-based risk factors. GSH detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites may be applied towards the evaluation of idiosyncratic hepatotoxicity.

Hepatic gene expression as a function of culture duration was evaluated in prolonged cultured human hepatocytes. Human hepatocytes from 7 donors were maintained as near-confluent collagen-matrigel sandwich cultures, with messenger RNA expression for genes responsible for key hepatic functions quantified by real time polymerase chain reaction at culture durations of 0 (day of plating), 2, 7, 9, 16, 23, 26, 29, 36 and 43 days. Key hepatocyte genes were evaluated including the differentiation markers albumin (ALB), transferrin (TR) and transthyretin (TTR); the hepatocyte-specific asialoglycoprotein receptor (ASGR1); cytochrome P450 isoforms CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A7; uptake transporter isoforms SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, SLCO2B1; efflux transporter isoforms ABCB1, ABCB11, ABCC2, ABCC3, ABCC4, ABCG2; as well as the nonspecific housekeeping gene hypoxanthine ribosyl transferase (HPRT1). The well-established dedifferentiation phenomenon was observed on day 2, with substantial (>80%) decreases in gene expression in day 2 cultures observed for all genes evaluated except HPRT1 and efflux transporters ABCB1, ABCC2, ABCC3 ( 400% increase in expression), and ABCG2 (no decrease in expression). All genes with a >80% decrease in expression were found to have increased levels of expression on day 7, with peak expression observed on either day 7 or day 9, followed by a gradual decrease in expression up to the longest duration evaluated of 43 days. Our results provide evidence that cultured human hepatocytes undergo redifferentiation upon prolonged culturing. Significance StatementWe report that while human hepatocytes underwent dedifferentiation upon 2 days of culture, prolonged culturing resulted in redifferentiation based on gene expression of differentiation markers, uptake and efflux transporters, and P450 isoforms. The observed redifferentiation suggests that prolonged (>7 days) culturing of human hepatocyte cultures may represent an experimental approach to overcome the initial dedifferentiation process, resulting in “stabilized” hepatocytes that can be applied towards the evaluation of drug properties requiring an extended period of treatment and evaluation.

Commercial formulations of 29 commonly used herbal supplements (HS) and grapefruit juice were evaluated for drug interaction potential via quantification of their CYP3A inhibitory potential in two in vitro experimental models of human small intestine, cryopreserved human intestinal mucosa (CHIM) and cryopreserved human enterocytes (CHE). Two CYP3A substrates were used - in the studies with CHIM, CYP3A activity was quantified via LC/MS-MS quantification of midazolam 19-hydroxylation while in CHE, luciferin IPA metabolism to luciferin was quantified by luminescence. Upon treatment of CHIM with the estimated lumen concentration of the HS upon each oral administration (manufacturers9 recommended dosage dissolved in 200 mL of culture medium), >80% CYP3A inhibition was observed for green tea extract, St. John9s wort, valerian root, horehound and grapefruit juice. Less than 50% inhibition was observed for fenugreek, aloe vera, guarana, soy isoflavone, maca, echinacea, spirulina, evening primrose, milk thistle, cranberry, red yeast rice, rhodiola, ginkgo biloba, turmeric, curcumin, white kidney bean,, garlic, cinnamon, saw palmetto berries, panax ginseng, black elderberry, wheat grass juice, flaxseed oil, black cohosh, and ginger root. The results were confirmed in a a dose-response study with HS obtained from three suppliers for the four inhibitory HS (green tea extract, horehound, St. John9s wort, valerian root), and three representative non-inhibitory HS (black cohosh, black elderberry, echinacea). Similar results were obtained with the inhibitory HS in CHE. The results illustrate that CHIM and CHE represent physiologically relevant in vitro experimental models for the evaluation of drug interaction potential of herbal supplements. Significance Statement In vitro evaluation of 29 popular herbal supplements in cryopreserved human intestinal mucosa identified green tea extract, horehound, St. John’s wort, valerian root to have CYP3A inhibitory potential similar to that for grapefruit juice, suggesting their potential to have clinically-significant pharmacokinetic interaction with orally administered drugs that are CYP3A substrates. The results suggest that CHIM can be used for in vitro evaluation of drug interactions involving enteric drug metabolism.

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 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{trade mark, serif}) cryopreserved human enterocytes, and cryopreserved human intestinal mucosa. The major deficiency with both immortalized cell lines and stem cell-derived enterocytes is that the activities of drug metabolizing enzymes, while detectable, are substantially lower than that for the intestinal mucosa in vivo. Human intestine slices, cryopreserved human enterocytes, MetMax{trade mark, serif} cryopreserved human enterocytes, and cryopreserved human intestinal mucosa retain robust enteric drug metabolizing enzyme activities and represent the most appropriate models for the evaluation of metabolism and drug interaction potential of orally administered xenobiotics including drugs, botanical products, and dietary supplements. Significance Statement The current in vitro human cell-based enteric experimental systems are reviewed for applications toward the evaluation of the metabolism and drug interaction potential of orally administered substances. Due to the robust drug metabolizing enzyme activities, human intestinal slices, cryopreserved human enterocytes and cryopreserved human intestinal mucosa are concluded to represent appropriate experimental models for this application.

We have previously reported successful isolation and cryopreservation of human intestinal mucosa (CHIM) with retention of viability and drug metabolizing enzyme activities. Here we report the results of the quantification of drug metabolizing enzyme activities in CHIM from different regions of the small intestines from 14 individual donors. CHIM were isolated from the duodenum, jejunum, and ileum of 10 individuals, and from 10 consecutive 12‐inch segments starting from the pyloric sphincter of human small intestines from four additional individuals. P450 and non‐P450 drug metabolizing enzyme activities (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A, UGT, SULT, FMO, MAO, AO, NAT1, and NAT2) were quantified via incubation with pathway‐selective substrates. Quantifiable activities were observed for all pathways except for CYP2A6. Comparison of the duodenum, jejunum, and ileum in 10 donors shows jejunum had higher activities for CYP2C9, CYP3A, UGT, SULT, MAO, and NAT1. Further definition of regional variations with CHIM from ten 12‐inch segments of the proximal small intestine shows that the segments immediately after the first 12‐inch segment (duodenum) had the highest activity for most of the drug metabolizing enzymes but with substantial differences among the four donors. Our overall results demonstrate that there are substantial individual differences in drug metabolizing enzymes and that jejunum, especially the regions immediately after the duodenum, had the highest drug metabolizing enzyme activities.

In vitro metabolic stability of nine fragrance chemicals: p‐tolyl acetate, cashmeran, ethylene brassylate, celestolide, galaxolide, traseolide, ambretone, tonalide and pentadecanolide, was evaluated in trout and human hepatocytes. The compounds were incubated with trout hepatocytes at 12°C and human hepatocytes at 37°C. Quantification of compound disappearance with time was performed using gas chromatography/mass spectrometry. in vivo hepatic intrinsic clearance values were calculated from the in vitro data. Significant metabolism was observed with trout hepatocytes for five of the nine fragrance chemicals, while all nine were metabolized significantly with human hepatocytes. Previously published models were used to examine expected bioaccumulation and persistence in whole organisms. Calculated half‐lives due to metabolism of the nine chemicals are significantly shorter for humans than trout: <1 hour and <1 day, respectively. For all chemicals with demonstrated hepatic metabolism, the models indicate a lack of accumulation. For those where metabolism was demonstrated in trout, calculated bioconcentration factors would not be classified as bioaccumulative under prevailing regulatory systems.

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 -80°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 isoform-selective 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 4'-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and midazolam 1'-hydroxylation and testosterone 6β-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 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 -80°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.