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Metabolic capability of upcyte human hepatocytes in long-term sandwich culture and utility for clearance prediction
Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S22eS26
(UGT). In order to predict their impact on drug disposition, data on their absolute intestinal and hepatic abundance as well as metabolic capacity are required. In contrast to the liver, few respective protein data have been published for the intestine. Hence, it was the aim of the current study to determine the absolute amount of clinically relevant metabolizing enzymes and their functional activity along the human intestine. Methods: 8 jejunal, 8 ileum and 8 colon samples were obtained from patients undergoing intestinal surgery. After tissue disruption and homogenization, the microsomal fraction was isolated and subjected to a tryptic digest. A validated LC-MS/MS method has been applied for the simultaneous and absolute quantification of nine CYP (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) and four UGT enzymes (UGT1A1, UGT1A3, UGT2B7 and UGT2B15) which are considered to be of clinical relevance in human drug metabolism. Protein quantification has been conducted by targeted proteomics using enzyme specific tryptic peptides and stable isotope-labelled peptides as internal standard. In addition, the respective gene expression was determined using quantitative real-time RT-PCR. In parallel, the isolated microsomal fraction has been incubated with diclofenac (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6) and midazolam (CYP3A4) as surrogate drugs for assessing the metabolic capacity. Their respective metabolites 4-OH-diclofenac, 5-OH-omeprazole, dextrorphan and 1-OHmidazolam have been quantified using a validated LC-MS/MS method. Results: Generally, four CYP (CYP2C9, CYP2C19, CYP2D6 and CYP3A4) as well as three UGT enzymes (UGT1A1, UGT1A3 and UGT2B7) of the monitored 13 proteins could be found along the human intestine. Almost all of these enzymes showed the highest abundance in the jejunum, lesser amounts in the ileum and the lowest extent in the colon, which was in accordance with the gene expression data. At the functional level, metabolism of the probe drugs took place in correspondence to the protein found in each intestinal section. Conclusion: Our study reports a comprehensive overview about the absolute protein abundance of clinical relevant enzymes along the human intestine in combination with their respective metabolism of probe drugs. The enzyme distribution was markedly different between the investigated intestinal sections which may affect the intestinal drug absorption. A4 METABOLIC CAPABILITY OF UPCYTE HUMAN HEPATOCYTES IN LONGTERM SANDWICH CULTURE AND UTILITY FOR CLEARANCE PREDICTION Michelle Schaefer 1, Akiko Matsui 2, Gaku Morinaga 2, Asami anzle 1, Daniel Bischoff 1, Roderich Saito 2, Shinobu Suzuki 2, Gerhard Sch€ D. Süssmuth 3. 1 Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany; 2 Nippon Boehringer Ingelheim, Kobe, Japan; 3 €t Berlin, Berlin, Germany Technische Universita Cultured primary human hepatocytes are a well-established in vitro model for studying hepatic drug metabolism during drug discovery and development. The purpose of this study was to evaluate recently introduced upcyte human hepatocytes (UHH) as an alternative hepatic in vitro system. UHH, derived from primary human hepatocytes (PHH) by lentiviral transduction of proliferation stimulating genes, provide a virtually unlimited source for non-transformed primary hepatocyte cultures. Differentiated UHH express adult hepatic markers and are responsive to prototypical inducers of cytochrome P450 enzymes1,2. We assessed the metabolic capability of UHH cultured in sandwich configuration for up to 21 days, focussing on functional in situ enzyme activity of selected phase I and phase II enzymes using specific probe substrates, accompanied by relative mRNA expression analysis. Absolute cytochrome P450 protein expression was determined by LC-MS/MS quantification of specific tryptic fragments released by trypsinization from microsomal preparations. Furthermore, depletion of parent compound was measured for a set of slowly and intermediate cleared reference drugs, including alprazolam, diazepam, oxazepam, tolbutamide, warfarin, risperidone, lidocaine and midazolam. In vitro intrinsic clearance (CLint) values were scaled to in vivo hepatic clearance (CLH). Data from the above experiments was compared to those from sandwich cultures of common cryopreserved PHH. As a result, UHH exhibited overall comparable enzymatic activities for major
S23
cytochrome P450s as seen for cryopreserved cultured primary human hepatocytes, and activity data correlated well with absolute protein expression. Sandwich cultured UHH showed well maintained activity levels over the investigated culture time, thus allowing for prolonged depletion experiments of up to 120 hours with low metabolic turnover reference compounds. Clearance prediction using UHH was found superior for the subset of low CL drugs, whereas sandwich cultured cryopreserved PHH returned better prediction results for intermediate CL drugs. In conclusion, UHH showed highly reproducible functional activity levels and predicted hepatic CL values, thereby suggesting that UHH are a promising alternative model for studying hepatic in vitro drug metabolism during discovery research. 1. Burkard, A. et al. (2012) Generation of proliferating human hepatocytes using Upcyte® technology: characterization and applications in induction and cytotoxicity assays. Xenobiotica 42: 939-956. 2. Levy, G. et al. (2015) Long-term culture and expansion of primary human hepatocytes. Nat.Biotechnol. 33: 1264-1271. A5 COMPARISON OF THE INHIBITORY EFFECTS OF SELECTIVE ESTROGEN RECEPTOR MODULATORS, TYROSINE KINASE INHIBITORS, AND STEROIDAL DRUGS ON HUMAN ALDEHYDE OXIDASE ACTIVITY Shiyan Chen, Aik Jiang Lau. National University of Singapore, Singapore, Singapore Aldehyde oxidase (AO) is a member of molybdo-flavoenzymes involved in phase I drug metabolism. It metabolizes not only aldehydes, but also azaheterocycles and oxo-heterocycles present in many clinically-used drugs and drug candidates. In recent years, AO is gaining importance because it plays a role in the toxicity of drug candidates. To date, the potent inhibitors of human AO include raloxifene, which is a second generation selective estrogen receptor modulator (SERM), and several steroid compounds, such as estradiol and estrone. In addition, a tyrosine kinase inhibitor, GDC-0834, was shown to be both a substrate and potent inhibitor of AO recently. However, it is not known whether other members in these three classes of drugs would also influence the AO activity. In the present study, we compared the inhibition of AO activity by new members of SERMs (acolbifene, bazedoxifene, lasofoxifene), and a selected group of tyrosine kinase/kinase inhibitors (crizotinib, gefitinib, palbociclib, sunitinib, tofacitinib) and steroidal drugs (cyproterone acetate, danazol, exemestane, spironolactone). AO activity was assessed by carbazeran 4-oxidation, which was quantified using ultra-high performance liquid chromatography-tandem mass spectrometry. The enzyme assay was performed under linear conditions with respect to the amount of cytosolic protein and incubation time. Carbazeran 4-oxidation by pooled mixed-gender human liver cytosol (150 donors) yielded an apparent Km of 3.96 ± 0.55 mM, Vmax of 1450 ± 257 pmol/min/mg protein, Vmax/apparent Km of 336.16 ml/min/mg protein, and the kinetics followed Michaelis-Menten model. The rank-order for the inhibition of human liver cytosol-catalyzed carbazeran 4oxidation by 25 mM of the drugs was bazedoxifene > lasofoxifene > gefitinib > crizotinib > sunitinib > spironolactone > exemestane > danazol > cyproterone acetate > palbociclib > tofacitinib > acolbifene. In contrast to bazedoxifene and lasofoxifene, acolbifene (a fourth generation SERM that is in clinical development) inhibited AO activity minimally by 11.7 ± 10.7%. At a lesser concentration of 1 mM, lasofoxifene, bazedoxifene, gefitinib, crizotinib, and sunitinib decreased AO activity by 83.6 ± 2.7%, 83.3 ± 4.4%, 32.3 ± 6.4%, 17 ± 10.1%, and 12.9 ± 6.6%, respectively. Comparatively, 1 mM raloxifene, 25 mM hydralazine, and 25 mM estrone, which are positive controls, decreased the activity by 96.5 ± 1.5%, 71.9 ± 4.6%, and 93.2 ± 0.5%, respectively. Among the drugs investigated, the most potent human AO inhibitors were bazedoxifene and lasofoxifene. The minimum concentration for the inhibition by both drugs was 0.03 mM. Dose-response analysis indicated that the IC50 was 0.19 ± 0.04 mM for bazedoxifene and 0.30 ± 0.04 mM for lasofoxifene. In conclusion, bazedoxifene, lasofoxifene, gefitinib, crizotinib, sunitinib, spironolactone, exemestane, danazol, cyproterone acetate, palbociclib, tofacitinib, and acolbifene differentially decreased AO activity. Our findings suggest potential effects of these therapeutic drugs on AO-mediated metabolism of other drugs and endogenous chemicals.
(UGT). In order to predict their impact on drug disposition, data on their absolute intestinal and hepatic abundance as well as metabolic capacity are required. In contrast to the liver, few respective protein data have been published for the intestine. Hence, it was the aim of the current study to determine the absolute amount of clinically relevant metabolizing enzymes and their functional activity along the human intestine. Methods: 8 jejunal, 8 ileum and 8 colon samples were obtained from patients undergoing intestinal surgery. After tissue disruption and homogenization, the microsomal fraction was isolated and subjected to a tryptic digest. A validated LC-MS/MS method has been applied for the simultaneous and absolute quantification of nine CYP (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) and four UGT enzymes (UGT1A1, UGT1A3, UGT2B7 and UGT2B15) which are considered to be of clinical relevance in human drug metabolism. Protein quantification has been conducted by targeted proteomics using enzyme specific tryptic peptides and stable isotope-labelled peptides as internal standard. In addition, the respective gene expression was determined using quantitative real-time RT-PCR. In parallel, the isolated microsomal fraction has been incubated with diclofenac (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6) and midazolam (CYP3A4) as surrogate drugs for assessing the metabolic capacity. Their respective metabolites 4-OH-diclofenac, 5-OH-omeprazole, dextrorphan and 1-OHmidazolam have been quantified using a validated LC-MS/MS method. Results: Generally, four CYP (CYP2C9, CYP2C19, CYP2D6 and CYP3A4) as well as three UGT enzymes (UGT1A1, UGT1A3 and UGT2B7) of the monitored 13 proteins could be found along the human intestine. Almost all of these enzymes showed the highest abundance in the jejunum, lesser amounts in the ileum and the lowest extent in the colon, which was in accordance with the gene expression data. At the functional level, metabolism of the probe drugs took place in correspondence to the protein found in each intestinal section. Conclusion: Our study reports a comprehensive overview about the absolute protein abundance of clinical relevant enzymes along the human intestine in combination with their respective metabolism of probe drugs. The enzyme distribution was markedly different between the investigated intestinal sections which may affect the intestinal drug absorption. A4 METABOLIC CAPABILITY OF UPCYTE HUMAN HEPATOCYTES IN LONGTERM SANDWICH CULTURE AND UTILITY FOR CLEARANCE PREDICTION Michelle Schaefer 1, Akiko Matsui 2, Gaku Morinaga 2, Asami anzle 1, Daniel Bischoff 1, Roderich Saito 2, Shinobu Suzuki 2, Gerhard Sch€ D. Süssmuth 3. 1 Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany; 2 Nippon Boehringer Ingelheim, Kobe, Japan; 3 €t Berlin, Berlin, Germany Technische Universita Cultured primary human hepatocytes are a well-established in vitro model for studying hepatic drug metabolism during drug discovery and development. The purpose of this study was to evaluate recently introduced upcyte human hepatocytes (UHH) as an alternative hepatic in vitro system. UHH, derived from primary human hepatocytes (PHH) by lentiviral transduction of proliferation stimulating genes, provide a virtually unlimited source for non-transformed primary hepatocyte cultures. Differentiated UHH express adult hepatic markers and are responsive to prototypical inducers of cytochrome P450 enzymes1,2. We assessed the metabolic capability of UHH cultured in sandwich configuration for up to 21 days, focussing on functional in situ enzyme activity of selected phase I and phase II enzymes using specific probe substrates, accompanied by relative mRNA expression analysis. Absolute cytochrome P450 protein expression was determined by LC-MS/MS quantification of specific tryptic fragments released by trypsinization from microsomal preparations. Furthermore, depletion of parent compound was measured for a set of slowly and intermediate cleared reference drugs, including alprazolam, diazepam, oxazepam, tolbutamide, warfarin, risperidone, lidocaine and midazolam. In vitro intrinsic clearance (CLint) values were scaled to in vivo hepatic clearance (CLH). Data from the above experiments was compared to those from sandwich cultures of common cryopreserved PHH. As a result, UHH exhibited overall comparable enzymatic activities for major
S23
cytochrome P450s as seen for cryopreserved cultured primary human hepatocytes, and activity data correlated well with absolute protein expression. Sandwich cultured UHH showed well maintained activity levels over the investigated culture time, thus allowing for prolonged depletion experiments of up to 120 hours with low metabolic turnover reference compounds. Clearance prediction using UHH was found superior for the subset of low CL drugs, whereas sandwich cultured cryopreserved PHH returned better prediction results for intermediate CL drugs. In conclusion, UHH showed highly reproducible functional activity levels and predicted hepatic CL values, thereby suggesting that UHH are a promising alternative model for studying hepatic in vitro drug metabolism during discovery research. 1. Burkard, A. et al. (2012) Generation of proliferating human hepatocytes using Upcyte® technology: characterization and applications in induction and cytotoxicity assays. Xenobiotica 42: 939-956. 2. Levy, G. et al. (2015) Long-term culture and expansion of primary human hepatocytes. Nat.Biotechnol. 33: 1264-1271. A5 COMPARISON OF THE INHIBITORY EFFECTS OF SELECTIVE ESTROGEN RECEPTOR MODULATORS, TYROSINE KINASE INHIBITORS, AND STEROIDAL DRUGS ON HUMAN ALDEHYDE OXIDASE ACTIVITY Shiyan Chen, Aik Jiang Lau. National University of Singapore, Singapore, Singapore Aldehyde oxidase (AO) is a member of molybdo-flavoenzymes involved in phase I drug metabolism. It metabolizes not only aldehydes, but also azaheterocycles and oxo-heterocycles present in many clinically-used drugs and drug candidates. In recent years, AO is gaining importance because it plays a role in the toxicity of drug candidates. To date, the potent inhibitors of human AO include raloxifene, which is a second generation selective estrogen receptor modulator (SERM), and several steroid compounds, such as estradiol and estrone. In addition, a tyrosine kinase inhibitor, GDC-0834, was shown to be both a substrate and potent inhibitor of AO recently. However, it is not known whether other members in these three classes of drugs would also influence the AO activity. In the present study, we compared the inhibition of AO activity by new members of SERMs (acolbifene, bazedoxifene, lasofoxifene), and a selected group of tyrosine kinase/kinase inhibitors (crizotinib, gefitinib, palbociclib, sunitinib, tofacitinib) and steroidal drugs (cyproterone acetate, danazol, exemestane, spironolactone). AO activity was assessed by carbazeran 4-oxidation, which was quantified using ultra-high performance liquid chromatography-tandem mass spectrometry. The enzyme assay was performed under linear conditions with respect to the amount of cytosolic protein and incubation time. Carbazeran 4-oxidation by pooled mixed-gender human liver cytosol (150 donors) yielded an apparent Km of 3.96 ± 0.55 mM, Vmax of 1450 ± 257 pmol/min/mg protein, Vmax/apparent Km of 336.16 ml/min/mg protein, and the kinetics followed Michaelis-Menten model. The rank-order for the inhibition of human liver cytosol-catalyzed carbazeran 4oxidation by 25 mM of the drugs was bazedoxifene > lasofoxifene > gefitinib > crizotinib > sunitinib > spironolactone > exemestane > danazol > cyproterone acetate > palbociclib > tofacitinib > acolbifene. In contrast to bazedoxifene and lasofoxifene, acolbifene (a fourth generation SERM that is in clinical development) inhibited AO activity minimally by 11.7 ± 10.7%. At a lesser concentration of 1 mM, lasofoxifene, bazedoxifene, gefitinib, crizotinib, and sunitinib decreased AO activity by 83.6 ± 2.7%, 83.3 ± 4.4%, 32.3 ± 6.4%, 17 ± 10.1%, and 12.9 ± 6.6%, respectively. Comparatively, 1 mM raloxifene, 25 mM hydralazine, and 25 mM estrone, which are positive controls, decreased the activity by 96.5 ± 1.5%, 71.9 ± 4.6%, and 93.2 ± 0.5%, respectively. Among the drugs investigated, the most potent human AO inhibitors were bazedoxifene and lasofoxifene. The minimum concentration for the inhibition by both drugs was 0.03 mM. Dose-response analysis indicated that the IC50 was 0.19 ± 0.04 mM for bazedoxifene and 0.30 ± 0.04 mM for lasofoxifene. In conclusion, bazedoxifene, lasofoxifene, gefitinib, crizotinib, sunitinib, spironolactone, exemestane, danazol, cyproterone acetate, palbociclib, tofacitinib, and acolbifene differentially decreased AO activity. Our findings suggest potential effects of these therapeutic drugs on AO-mediated metabolism of other drugs and endogenous chemicals.