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1
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Three-dimensional (3D) liver cell models - a tool for bridging the gap between animal studies and clinical trials when screening liver accumulation and toxicity of nanobiomaterials. Drug Deliv Transl Res 2022; 12:2048-2074. [PMID: 35507131 PMCID: PMC9066991 DOI: 10.1007/s13346-022-01147-0] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/03/2022] [Indexed: 12/13/2022]
Abstract
Despite the exciting properties and wide-reaching applications of nanobiomaterials (NBMs) in human health and medicine, their translation from bench to bedside is slow, with a predominant issue being liver accumulation and toxicity following systemic administration. In vitro 2D cell-based assays and in vivo testing are the most popular and widely used methods for assessing liver toxicity at pre-clinical stages; however, these fall short in predicting toxicity for NBMs. Focusing on in vitro and in vivo assessment, the accurate prediction of human-specific hepatotoxicity is still a significant challenge to researchers. This review describes the relationship between NBMs and the liver, and the methods for assessing toxicity, focusing on the limitations they bring in the assessment of NBM hepatotoxicity as one of the reasons defining the poor translation for NBMs. We will then present some of the most recent advances towards the development of more biologically relevant in vitro liver methods based on tissue-mimetic 3D cell models and how these could facilitate the translation of NBMs going forward. Finally, we also discuss the low public acceptance and limited uptake of tissue-mimetic 3D models in pre-clinical assessment, despite the demonstrated technical and ethical advantages associated with them.
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2
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Fu D. Collagen Sandwich Culture of Primary Hepatocytes for Image-Based Investigations. Methods Mol Biol 2022; 2544:159-169. [PMID: 36125717 DOI: 10.1007/978-1-0716-2557-6_11] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/15/2023]
Abstract
Collagen sandwich culture of hepatocytes retains many in vivo-like properties and is used for many investigations in liver cell biology, and hepatic pharmacology and toxicology. This chapter describes the method of establishing collagen sandwich culture of hepatocytes in a glass bottom dish for image-based studies.
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Affiliation(s)
- Dong Fu
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
- In vivo Pharmacology and Toxicology, Frontera Therapeutics, Bedford, MA, USA.
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3
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Hadjittofi C, Feretis M, Martin J, Harper S, Huguet E. Liver regeneration biology: Implications for liver tumour therapies. World J Clin Oncol 2021; 12:1101-1156. [PMID: 35070734 PMCID: PMC8716989 DOI: 10.5306/wjco.v12.i12.1101] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/27/2021] [Revised: 06/22/2021] [Accepted: 11/28/2021] [Indexed: 02/06/2023] Open
Abstract
The liver has remarkable regenerative potential, with the capacity to regenerate after 75% hepatectomy in humans and up to 90% hepatectomy in some rodent models, enabling it to meet the challenge of diverse injury types, including physical trauma, infection, inflammatory processes, direct toxicity, and immunological insults. Current understanding of liver regeneration is based largely on animal research, historically in large animals, and more recently in rodents and zebrafish, which provide powerful genetic manipulation experimental tools. Whilst immensely valuable, these models have limitations in extrapolation to the human situation. In vitro models have evolved from 2-dimensional culture to complex 3 dimensional organoids, but also have shortcomings in replicating the complex hepatic micro-anatomical and physiological milieu. The process of liver regeneration is only partially understood and characterized by layers of complexity. Liver regeneration is triggered and controlled by a multitude of mitogens acting in autocrine, paracrine, and endocrine ways, with much redundancy and cross-talk between biochemical pathways. The regenerative response is variable, involving both hypertrophy and true proliferative hyperplasia, which is itself variable, including both cellular phenotypic fidelity and cellular trans-differentiation, according to the type of injury. Complex interactions occur between parenchymal and non-parenchymal cells, and regeneration is affected by the status of the liver parenchyma, with differences between healthy and diseased liver. Finally, the process of termination of liver regeneration is even less well understood than its triggers. The complexity of liver regeneration biology combined with limited understanding has restricted specific clinical interventions to enhance liver regeneration. Moreover, manipulating the fundamental biochemical pathways involved would require cautious assessment, for fear of unintended consequences. Nevertheless, current knowledge provides guiding principles for strategies to optimise liver regeneration potential.
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Affiliation(s)
- Christopher Hadjittofi
- University Department of Surgery, Addenbrookes Hospital, NIHR Comprehensive Biomedical Research and Academic Health Sciences Center, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, United Kingdom
| | - Michael Feretis
- University Department of Surgery, Addenbrookes Hospital, NIHR Comprehensive Biomedical Research and Academic Health Sciences Center, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, United Kingdom
| | - Jack Martin
- University Department of Surgery, Addenbrookes Hospital, NIHR Comprehensive Biomedical Research and Academic Health Sciences Center, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, United Kingdom
| | - Simon Harper
- University Department of Surgery, Addenbrookes Hospital, NIHR Comprehensive Biomedical Research and Academic Health Sciences Center, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, United Kingdom
| | - Emmanuel Huguet
- University Department of Surgery, Addenbrookes Hospital, NIHR Comprehensive Biomedical Research and Academic Health Sciences Center, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, United Kingdom
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4
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Jazaeri F, Sheibani M, Nezamoleslami S, Moezi L, Dehpour AR. Current Models for Predicting Drug-induced Cholestasis: The Role of Hepatobiliary Transport System. IRANIAN JOURNAL OF PHARMACEUTICAL RESEARCH : IJPR 2021; 20:1-21. [PMID: 34567142 PMCID: PMC8457732 DOI: 10.22037/ijpr.2020.113362.14254] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
Drug-induced cholestasis is the main type of liver disorder accompanied by high morbidity and mortality. Evidence for the role of hepatobiliary pumps in the cholestasis patho-mechanism is constantly increasing. Recognition of the interactions of chemical agents with these transporters at the initial phases of drug discovery can help develop new drug candidates with low cholestasis potential. This review delivers an outline of the role of these transport proteins in bile creation. It addresses the pathophysiological mechanism for drug-induced cholestasis. In-vitro models, including cell-based and membrane-based approaches and In-vivo models such as genetic knockout animals, are considered. The benefits and restrictions of each model are discussed in this review. Current understandings into the cellular and molecular process that control the activity of hepatobiliary pumps have directed to a better understanding of the pathophysiology of drug-induced cholestasis. A combination of in-vitro monitoring for transport interaction, in-silico predicting systems, and consideration of and metabolic and physicochemical properties must cause more effective monitoring of possible liver problems.
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Affiliation(s)
- Farahnaz Jazaeri
- Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran.,Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.,F. J. and M. Sh. contributed equally to this work
| | - Mohammad Sheibani
- Department of Pharmacology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.,F. J. and M. Sh. contributed equally to this work
| | - Sadaf Nezamoleslami
- Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran.,Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Leila Moezi
- Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Ahmad-Reza Dehpour
- Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran.,Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
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5
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Garzel B, Zhang L, Huang SM, Wang H. A Change in Bile Flow: Looking Beyond Transporter Inhibition in the Development of Drug-induced Cholestasis. Curr Drug Metab 2020; 20:621-632. [PMID: 31288715 DOI: 10.2174/1389200220666190709170256] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2019] [Revised: 05/22/2019] [Accepted: 06/12/2019] [Indexed: 12/17/2022]
Abstract
BACKGROUND Drug-induced Liver Injury (DILI) has received increasing attention over the past decades, as it represents the leading cause of drug failure and attrition. One of the most prevalent and severe forms of DILI involves the toxic accumulation of bile acids in the liver, known as Drug-induced Cholestasis (DIC). Traditionally, DIC is studied by exploring the inhibition of hepatic transporters such as Bile Salt Export Pump (BSEP) and multidrug resistance-associated proteins, predominantly through vesicular transport assays. Although this approach has identified numerous drugs that alter bile flow, many DIC drugs do not demonstrate prototypical transporter inhibition, but rather are associated with alternative mechanisms. METHODS We undertook a focused literature search on DIC and biliary transporters and analyzed peer-reviewed publications over the past two decades or so. RESULTS We have summarized the current perception regarding DIC, biliary transporters, and transcriptional regulation of bile acid homeostasis. A growing body of literature aimed to identify alternative mechanisms in the development of DIC has been evaluated. This review also highlights current in vitro approaches used for prediction of DIC. CONCLUSION Efforts have continued to focus on BSEP, as it is the primary route for hepatic biliary clearance. In addition to inhibition, drug-induced BSEP repression or the combination of these two has emerged as important alternative mechanisms leading to DIC. Furthermore, there has been an evolution in the approaches to studying DIC including 3D cell cultures and computational modeling.
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Affiliation(s)
- Brandy Garzel
- Office of Clinical Pharmacology, Office of Translational Sciences, Center for Drug Evaluation and Research, Food and Drug Administration (FDA), Silver Spring, MD 20993, United States.,Becton Dickinson, 54 Loveton Circle, Sparks, MD 21152, United States
| | - Lei Zhang
- Office of Clinical Pharmacology, Office of Translational Sciences, Center for Drug Evaluation and Research, Food and Drug Administration (FDA), Silver Spring, MD 20993, United States.,Office of Research and Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, FDA, Silver Spring, MD 20993, United States
| | - Shiew-Mei Huang
- Office of Clinical Pharmacology, Office of Translational Sciences, Center for Drug Evaluation and Research, Food and Drug Administration (FDA), Silver Spring, MD 20993, United States
| | - Hongbing Wang
- Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, United States
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6
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Oorts M, Keemink J, Deferm N, Adriaensen R, Richert L, Augustijns P, Annaert P. Extra collagen overlay prolongs the differentiated phenotype in sandwich-cultured rat hepatocytes. J Pharmacol Toxicol Methods 2018; 90:31-38. [DOI: 10.1016/j.vascn.2017.10.007] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2017] [Revised: 09/16/2017] [Accepted: 10/20/2017] [Indexed: 02/03/2023]
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7
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Keemink J, Deferm N, De Bruyn T, Augustijns P, Bouillon T, Annaert P. Effect of Cryopreservation on Enzyme and Transporter Activities in Suspended and Sandwich Cultured Rat Hepatocytes. AAPS JOURNAL 2018; 20:33. [PMID: 29468289 DOI: 10.1208/s12248-018-0188-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/01/2017] [Accepted: 01/03/2018] [Indexed: 12/11/2022]
Abstract
Freshly-isolated rat hepatocytes are commonly used as tools for hepatic drug disposition. From an ethical point of view, it is important to maximize the use of isolated hepatocytes by cryopreservation. The present study compared overall hepatocyte functionality as well as activity of the organic anion transporting polypeptide (Oatp), multidrug resistance-associated protein 2 (Mrp2), and UDP-glucuronosyltransferase 1 (Ugt1), in in vitro models established with cryopreserved and freshly-isolated hepatocytes. A similar culture time-dependent decline in cellular functionality, as assessed by urea production, was observed in sandwich-cultured hepatocytes (SCH) obtained from freshly-isolated and cryopreserved cells. Concentration-dependent uptake kinetics of the Oatp substrate sodium fluorescein in suspended hepatocytes (SH) or SCH were not significantly affected by cryopreservation. Mrp2-mediated biliary excretion of 5 (and 6)-carboxy-2',7'-dichlorofluorescein by SCH was assessed with semi-quantitative fluorescence imaging: biliary excretion index values increased between day 3 and day 4, but did not differ significantly between cryopreserved and freshly-isolated hepatocytes. Finally, telmisartan disposition was evaluated in SCH to simultaneously explore Oatp, Ugt1, and Mrp2 activity. In order to distinguish between the susceptibilities of the individual disposition pathways to cryopreservation, a mechanistic cellular disposition model was developed. Basolateral and canalicular efflux as well as glucuronidation of telmisartan were affected by cryopreservation. In contrast, the disposition parameters of telmisartan-glucuronide were not impacted by cryopreservation. Overall, the relative contribution of the rate-determining processes (uptake, metabolism, efflux) remained unaltered between cryopreserved and freshly-isolated hepatocytes, indicating that cryopreserved hepatocytes are a suitable alternative for freshly-isolated hepatocytes when studying these cellular disposition pathways.
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Affiliation(s)
- Janneke Keemink
- Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, Gasthuisberg O&N2 Herestraat 49-box 921, 3000, Leuven, Belgium.,Drug Delivery, Department of Pharmacy, Uppsala University, Uppsala, Sweden
| | - Neel Deferm
- Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, Gasthuisberg O&N2 Herestraat 49-box 921, 3000, Leuven, Belgium
| | - Tom De Bruyn
- Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, Gasthuisberg O&N2 Herestraat 49-box 921, 3000, Leuven, Belgium.,Genentech, Inc, South San Francisco, California, USA
| | - Patrick Augustijns
- Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, Gasthuisberg O&N2 Herestraat 49-box 921, 3000, Leuven, Belgium
| | - Thomas Bouillon
- Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, Gasthuisberg O&N2 Herestraat 49-box 921, 3000, Leuven, Belgium
| | - Pieter Annaert
- Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, Gasthuisberg O&N2 Herestraat 49-box 921, 3000, Leuven, Belgium.
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8
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Yamasaki Y, Kobayashi K, Inaba A, Uehara D, Tojima H, Kakizaki S, Chiba K. Indirect activation of pregnane X receptor in the induction of hepatic CYP3A11 by high-dose rifampicin in mice. Xenobiotica 2017; 48:1098-1105. [PMID: 29095659 DOI: 10.1080/00498254.2017.1400128] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
Rifampicin (RIF), a typical ligand of human pregnane X receptor (PXR), powerfully induces the expression of cytochrome P450 3A4 (CYP3A4) in humans. Although it is thought that RIF is not a ligand of rodent PXR, treatment with high-dose RIF (e.g. more than 20 mg/kg) increases the expression of CYP3A in the mouse liver. In this study, we investigated whether the induction of CYP3A by high-dose RIF in the mouse liver is mediated via indirect activation of mouse PXR (mPXR). The results showed that high-dose RIF increased the expression of CYP3A11 and other PXR-target genes in the liver of wild-type mice but not PXR-knockout mice. However, the results of reporter gene and ligand-dependent assembly assays showed that RIF does not activate mPXR in a ligand-dependent manner. In addition, high-dose RIF stimulated nuclear accumulation of mPXR in the mouse liver, and geldanamycin and okadaic acid attenuated the induction of Cyp3a11 and other PXR-target genes in primary hepatocytes, suggesting that high-dose RIF triggers nuclear translocation of mPXR. In conclusion, the present study suggests that high-dose RIF stimulates nuclear translocation of mPXR in the liver of mice by indirect activation, resulting in the transactivation of Cyp3a11 and other PXR-target genes.
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Affiliation(s)
- Yuki Yamasaki
- a Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University , Chiba , Japan and
| | - Kaoru Kobayashi
- a Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University , Chiba , Japan and
| | - Asumi Inaba
- a Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University , Chiba , Japan and
| | - Daisuke Uehara
- b Division of Gastroenterology and Hepatology, Department of Internal Medicine, Graduate School of Medicine, Gunma University , Maebashi , Japan
| | - Hiroki Tojima
- b Division of Gastroenterology and Hepatology, Department of Internal Medicine, Graduate School of Medicine, Gunma University , Maebashi , Japan
| | - Satoru Kakizaki
- b Division of Gastroenterology and Hepatology, Department of Internal Medicine, Graduate School of Medicine, Gunma University , Maebashi , Japan
| | - Kan Chiba
- a Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University , Chiba , Japan and
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9
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Kaschek D, Sharanek A, Guillouzo A, Timmer J, Weaver RJ. A Dynamic Mathematical Model of Bile Acid Clearance in HepaRG Cells. Toxicol Sci 2017; 161:48-57. [DOI: 10.1093/toxsci/kfx199] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
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10
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Dai Y, Ma BL, Zheng M, Shi R, Li YY, Wang TM, Ma YM. Identification of drug transporters involved in the uptake and efflux of rhein in hepatocytes. RSC Adv 2017. [DOI: 10.1039/c6ra28205a] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Rhein is an herbal medicine with various bioactivities and is derived from an anthraquinone compound. In this study, we aimed to identify drug transporters involved in the uptake and efflux of rhein in hepatocytes.
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Affiliation(s)
- Yan Dai
- Department of Pharmacology
- School of Pharmacy
- Shanghai University of Traditional Chinese Medicine
- Shanghai
- China
| | - Bing-Liang Ma
- Department of Pharmacology
- School of Pharmacy
- Shanghai University of Traditional Chinese Medicine
- Shanghai
- China
| | - Min Zheng
- Department of Pharmacology
- School of Pharmacy
- Shanghai University of Traditional Chinese Medicine
- Shanghai
- China
| | - Rong Shi
- Department of Pharmacology
- School of Pharmacy
- Shanghai University of Traditional Chinese Medicine
- Shanghai
- China
| | - Yuan-Yuan Li
- Department of Pharmacology
- School of Pharmacy
- Shanghai University of Traditional Chinese Medicine
- Shanghai
- China
| | - Tian-Ming Wang
- Department of Pharmacology
- School of Pharmacy
- Shanghai University of Traditional Chinese Medicine
- Shanghai
- China
| | - Yue-Ming Ma
- Department of Pharmacology
- School of Pharmacy
- Shanghai University of Traditional Chinese Medicine
- Shanghai
- China
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11
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Sandwich-Cultured Hepatocytes as a Tool to Study Drug Disposition and Drug-Induced Liver Injury. J Pharm Sci 2016; 105:443-459. [PMID: 26869411 DOI: 10.1016/j.xphs.2015.11.008] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2015] [Revised: 11/06/2015] [Accepted: 11/09/2015] [Indexed: 12/21/2022]
Abstract
Sandwich-cultured hepatocytes (SCH) are metabolically competent and have proper localization of basolateral and canalicular transporters with functional bile networks. Therefore, this cellular model is a unique tool that can be used to estimate biliary excretion of compounds. SCH have been used widely to assess hepatobiliary disposition of endogenous and exogenous compounds and metabolites. Mechanistic modeling based on SCH data enables estimation of metabolic and transporter-mediated clearances, which can be used to construct physiologically based pharmacokinetic models for prediction of drug disposition and drug-drug interactions in humans. In addition to pharmacokinetic studies, SCH also have been used to study cytotoxicity and perturbation of biological processes by drugs and hepatically generated metabolites. Human SCH can provide mechanistic insights underlying clinical drug-induced liver injury (DILI). In addition, data generated in SCH can be integrated into systems pharmacology models to predict potential DILI in humans. In this review, applications of SCH in studying hepatobiliary drug disposition and bile acid-mediated DILI are discussed. An example is presented to show how data generated in the SCH model were used to establish a quantitative relationship between intracellular bile acids and cytotoxicity, and how this information was incorporated into a systems pharmacology model for DILI prediction.
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12
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Yoshikado T, Yoshida K, Kotani N, Nakada T, Asaumi R, Toshimoto K, Maeda K, Kusuhara H, Sugiyama Y. Quantitative Analyses of Hepatic OATP-Mediated Interactions Between Statins and Inhibitors Using PBPK Modeling With a Parameter Optimization Method. Clin Pharmacol Ther 2016; 100:513-523. [PMID: 27170342 DOI: 10.1002/cpt.391] [Citation(s) in RCA: 79] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2016] [Revised: 04/20/2016] [Accepted: 05/03/2016] [Indexed: 01/01/2023]
Abstract
This study aimed to construct a widely applicable method for quantitative analyses of drug-drug interactions (DDIs) caused by the inhibition of hepatic organic anion transporting polypeptides (OATPs) using physiologically based pharmacokinetic (PBPK) modeling. Models were constructed for pitavastatin, fluvastatin, and pravastatin as substrates and cyclosporin A (CsA) and rifampicin (RIF) as inhibitors, where enterohepatic circulations (EHC) of statins were incorporated. By fitting to clinical data, parameters that described absorption, hepatic elimination, and EHC processes were optimized, and the extent of these DDIs was explained satisfactorily. Similar in vivo inhibition constant (Ki ) values of each inhibitor against OATPs were obtained, regardless of the substrates. Estimated Ki values of CsA were comparable to reported in vitro values with the preincubation of CsA, while those of RIF were smaller than reported in vitro values (coincubation). In conclusion, this study proposes a method to optimize in vivo PBPK parameters in hepatic uptake transporter-mediated DDIs.
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Affiliation(s)
- T Yoshikado
- Sugiyama Laboratory, RIKEN Innovation Center, RIKEN, Yokohama, Kanagawa, Japan
| | - K Yoshida
- Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan
| | - N Kotani
- Clinical Pharmacology Strategy Group, Translational Clinical Research Science & Strategy Dept., Chugai Pharmaceutical Co., Tokyo, Japan
| | - T Nakada
- DMPK Research Laboratories Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma, Chiba, Japan
| | - R Asaumi
- Pharmacokinetic Research Laboratories, Ono Pharmaceutical Co., Ibaraki, Japan
| | - K Toshimoto
- Sugiyama Laboratory, RIKEN Innovation Center, RIKEN, Yokohama, Kanagawa, Japan
| | - K Maeda
- Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan
| | - H Kusuhara
- Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan
| | - Y Sugiyama
- Sugiyama Laboratory, RIKEN Innovation Center, RIKEN, Yokohama, Kanagawa, Japan.
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13
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Ramboer E, Rogiers V, Vanhaecke T, Vinken M. Effects of Trichostatin A on drug uptake transporters in primary rat hepatocyte cultures. EXCLI JOURNAL 2015; 14:567-76. [PMID: 26648816 PMCID: PMC4669911 DOI: 10.17179/excli2015-220] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/11/2015] [Accepted: 03/30/2015] [Indexed: 11/10/2022]
Abstract
The present study was set up to investigate the effects of Trichostatin A (TSA), a prototypical epigenetic modifier, on the expression and activity of hepatic drug uptake transporters in primary cultured rat hepatocytes. To this end, the expression of the sinusoidal transporters sodium-dependent taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptide 4 (Oatp4) was monitored by real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblotting. The activity of the uptake transporters was analyzed using radiolabeled substrates and chemical inhibitors. Downregulation of the expression and activity of Oatp4 and Ntcp was observed as a function of the cultivation time and could not be counteracted by TSA. In conclusion, the epigenetic modifier TSA does not seem to exert a positive effect on the expression and activity of the investigated uptake transporters in primary rat hepatocyte cultures.
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Affiliation(s)
- Eva Ramboer
- In Vitro Toxicology and Dermato-cosmetology research group, Center for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel
| | - Vera Rogiers
- In Vitro Toxicology and Dermato-cosmetology research group, Center for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel
| | - Tamara Vanhaecke
- In Vitro Toxicology and Dermato-cosmetology research group, Center for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel
| | - Mathieu Vinken
- In Vitro Toxicology and Dermato-cosmetology research group, Center for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel
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14
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Yang K, Pfeifer ND, Köck K, Brouwer KLR. Species differences in hepatobiliary disposition of taurocholic acid in human and rat sandwich-cultured hepatocytes: implications for drug-induced liver injury. J Pharmacol Exp Ther 2015; 353:415-23. [PMID: 25711339 PMCID: PMC4407722 DOI: 10.1124/jpet.114.221564] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2014] [Accepted: 02/20/2015] [Indexed: 12/28/2022] Open
Abstract
The bile salt export pump (BSEP) plays an important role in bile acid excretion. Impaired BSEP function may result in liver injury. Bile acids also undergo basolateral efflux, but the relative contributions of biliary (CLBile) versus basolateral efflux (CLBL) clearance to hepatocellular bile acid excretion have not been determined. In the present study, taurocholic acid (TCA; a model bile acid) disposition was characterized in human and rat sandwich-cultured hepatocytes (SCH) combined with pharmacokinetic modeling. In human SCH, biliary excretion of TCA predominated (CLBile = 0.14 ± 0.04 ml/min per g liver; CLBL = 0.042 ± 0.019 ml/min per g liver), whereas CLBile and CLBL contributed approximately equally to TCA hepatocellular excretion in rat SCH (CLBile = 0.34 ± 0.07 ml/min per g liver; CLBL = 0.26 ± 0.07 ml/min per g liver). Troglitazone decreased TCA uptake, CLBile, and CLBL; membrane vesicle assays revealed for the first time that the major metabolite, troglitazone sulfate, was a noncompetitive inhibitor of multidrug resistance-associated protein 4, a basolateral bile acid efflux transporter. Simulations revealed that decreased CLBile led to a greater increase in hepatic TCA exposure in human than in rat SCH. A decrease in both excretory pathways (CLBile and CLBL) exponentially increased hepatic TCA in both species, suggesting that 1) drugs that inhibit both pathways may have a greater risk for hepatotoxicity, and 2) impaired function of an alternate excretory pathway may predispose patients to hepatotoxicity when drugs that inhibit one pathway are administered. Simulations confirmed the protective role of uptake inhibition, suggesting that a drug's inhibitory effects on bile acid uptake also should be considered when evaluating hepatotoxic potential. Overall, the current study precisely characterized basolateral efflux of TCA, revealed species differences in hepatocellular TCA efflux pathways, and provided insights about altered hepatic bile acid exposure when multiple transport pathways are impaired.
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Affiliation(s)
- Kyunghee Yang
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
| | - Nathan D Pfeifer
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
| | - Kathleen Köck
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
| | - Kim L R Brouwer
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
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Yang K, Pfeifer ND, Köck K, Brouwer KLR. Species differences in hepatobiliary disposition of taurocholic acid in human and rat sandwich-cultured hepatocytes: implications for drug-induced liver injury. J Pharmacol Exp Ther 2015. [PMID: 25711339 DOI: 10.1124/jpet.] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The bile salt export pump (BSEP) plays an important role in bile acid excretion. Impaired BSEP function may result in liver injury. Bile acids also undergo basolateral efflux, but the relative contributions of biliary (CLBile) versus basolateral efflux (CLBL) clearance to hepatocellular bile acid excretion have not been determined. In the present study, taurocholic acid (TCA; a model bile acid) disposition was characterized in human and rat sandwich-cultured hepatocytes (SCH) combined with pharmacokinetic modeling. In human SCH, biliary excretion of TCA predominated (CLBile = 0.14 ± 0.04 ml/min per g liver; CLBL = 0.042 ± 0.019 ml/min per g liver), whereas CLBile and CLBL contributed approximately equally to TCA hepatocellular excretion in rat SCH (CLBile = 0.34 ± 0.07 ml/min per g liver; CLBL = 0.26 ± 0.07 ml/min per g liver). Troglitazone decreased TCA uptake, CLBile, and CLBL; membrane vesicle assays revealed for the first time that the major metabolite, troglitazone sulfate, was a noncompetitive inhibitor of multidrug resistance-associated protein 4, a basolateral bile acid efflux transporter. Simulations revealed that decreased CLBile led to a greater increase in hepatic TCA exposure in human than in rat SCH. A decrease in both excretory pathways (CLBile and CLBL) exponentially increased hepatic TCA in both species, suggesting that 1) drugs that inhibit both pathways may have a greater risk for hepatotoxicity, and 2) impaired function of an alternate excretory pathway may predispose patients to hepatotoxicity when drugs that inhibit one pathway are administered. Simulations confirmed the protective role of uptake inhibition, suggesting that a drug's inhibitory effects on bile acid uptake also should be considered when evaluating hepatotoxic potential. Overall, the current study precisely characterized basolateral efflux of TCA, revealed species differences in hepatocellular TCA efflux pathways, and provided insights about altered hepatic bile acid exposure when multiple transport pathways are impaired.
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Affiliation(s)
- Kyunghee Yang
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
| | - Nathan D Pfeifer
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
| | - Kathleen Köck
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
| | - Kim L R Brouwer
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
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Murray JW, Han D, Wolkoff AW. Hepatocytes maintain greater fluorescent bile acid accumulation and greater sensitivity to drug-induced cell death in three-dimensional matrix culture. Physiol Rep 2014; 2:2/12/e12198. [PMID: 25524275 PMCID: PMC4332201 DOI: 10.14814/phy2.12198] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/05/2022] Open
Abstract
Primary hepatocytes undergo phenotypic dedifferentiation upon isolation from liver that typically includes down regulation of uptake transporters and up regulation of efflux transporters. Culturing cells between layers of collagen in a three‐dimensional (3D) “sandwich” is reported to restore hepatic phenotype. This report examines how 3D culturing affects accumulation of fluorophores, the cytotoxic response to bile acids and drugs, and whether cell to cell differences in fluorescent anion accumulation correlate with differences in cytotoxicity. Hepatocytes were found to accumulate fluorescent bile acid (FBA) at significantly higher levels than the related fluorophores, carboxyfluorescein diacetate, (4.4‐fold), carboxyfluorescein succinimidyl ester (4.8‐fold), and fluorescein (30‐fold). In 2D culture, FBA accumulation decreased to background levels by 32 h, Hoechst nuclear accumulation strongly decreased, and nuclear diameter increased, indicative of an efflux phenotype. In 3D culture, FBA accumulation was maintained through 168 h but at 1/3 the original intensity. Cell to cell differences in accumulated FBA did not correlate with levels of liver zonal markers L‐FBAP (zone 1) or glutamine synthetase (zone 3). Cytotoxic response to hydrophobic bile acids, acetaminophen, and phalloidin was maintained in 3D culture, and cells with higher FBA accumulation showed 12–18% higher toxicity than the total population toward hydrophobic bile acids (P < 0.05). Long‐term imaging showed oscillations in the accumulation of FBA over periods of hours. Overall, the studies suggest that high accumulation of FBA can indicate the sensitivity of cultured hepatocytes to hydrophobic bile acids and other toxins. These studies use automated image analysis and fluorescent dye accumulation to demonstrate that 3D culturing enhances organic anion accumulation and cytotoxic response in long‐term hepatocyte cultures. The level of anion accumulation was found to vary through days in culture and also between single cells, and higher fluorescent bile acid accumulation correlated with higher toxic response to hydrophobic bile acids.
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Affiliation(s)
- John W Murray
- Department of Anatomy and Structural Biology, Division of Gastroenterology and Liver Diseases, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York
| | - Dennis Han
- Department of Anatomy and Structural Biology, Division of Gastroenterology and Liver Diseases, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York
| | - Allan W Wolkoff
- Department of Anatomy and Structural Biology, Division of Gastroenterology and Liver Diseases, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York
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Hosey CM, Broccatelli F, Benet LZ. Predicting when biliary excretion of parent drug is a major route of elimination in humans. AAPS JOURNAL 2014; 16:1085-96. [PMID: 25004821 DOI: 10.1208/s12248-014-9636-1] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/29/2014] [Accepted: 05/27/2014] [Indexed: 01/29/2023]
Abstract
Biliary excretion is an important route of elimination for many drugs, yet measuring the extent of biliary elimination is difficult, invasive, and variable. Biliary elimination has been quantified for few drugs with a limited number of subjects, who are often diseased patients. An accurate prediction of which drugs or new molecular entities are significantly eliminated in the bile may predict potential drug-drug interactions, pharmacokinetics, and toxicities. The Biopharmaceutics Drug Disposition Classification System (BDDCS) characterizes significant routes of drug elimination, identifies potential transporter effects, and is useful in understanding drug-drug interactions. Class 1 and 2 drugs are primarily eliminated in humans via metabolism and will not exhibit significant biliary excretion of parent compound. In contrast, class 3 and 4 drugs are primarily excreted unchanged in the urine or bile. Here, we characterize the significant elimination route of 105 orally administered class 3 and 4 drugs. We introduce and validate a novel model, predicting significant biliary elimination using a simple classification scheme. The model is accurate for 83% of 30 drugs collected after model development. The model corroborates the observation that biliarily eliminated drugs have high molecular weights, while demonstrating the necessity of considering route of administration and extent of metabolism when predicting biliary excretion. Interestingly, a predictor of potential metabolism significantly improves predictions of major elimination routes of poorly metabolized drugs. This model successfully predicts the major elimination route for poorly permeable/poorly metabolized drugs and may be applied prior to human dosing.
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Affiliation(s)
- Chelsea M Hosey
- Department of Bioengineering and Therapeutic Sciences, University of California, 533 Parnassus Ave., Room U-68, San Francisco, California, 94143-0912, USA
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Lundquist P, Lööf J, Fagerholm U, Sjögren I, Johansson J, Briem S, Hoogstraate J, Afzelius L, Andersson TB. Prediction of in vivo rat biliary drug clearance from an in vitro hepatocyte efflux model. Drug Metab Dispos 2014; 42:459-68. [PMID: 24396143 DOI: 10.1124/dmd.113.054155] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/13/2025] Open
Abstract
Well-established techniques are available to predict in vivo hepatic uptake and metabolism from in vitro data, but predictive models for biliary clearance remain elusive. Several studies have verified the expression and activity of ATP-binding cassette (ABC) efflux transporters central to biliary clearance in freshly isolated rat hepatocytes, raising the possibility of predicting biliary clearance from in vitro efflux measurements. In the present study, short-term plated rat hepatocytes were evaluated as a model to predict biliary clearance from in vitro efflux measurements before major changes in transporter expression known to take place in long-term hepatocyte cultures. The short-term cultures were carefully characterized for their uptake and metabolic properties using a set of model compounds. In vitro efflux was studied using digoxin, fexofenadine, napsagatran, and rosuvastatin, representing compounds with over 100-fold differences in efflux rates in vitro and 60-fold difference in measured in vivo biliary clearance. The predicted biliary clearances from short-term plated rat hepatocytes were within 2-fold of measured in vivo values. As in vitro efflux includes both basolateral and canalicular effluxes, pronounced basolateral efflux may introduce errors in predictions for some compounds. In addition, in vitro rat hepatocyte uptake rates corrected for simultaneous efflux predicted rat in vivo hepatic clearance of the biliary cleared compounds with less than 2-fold error. Short-term plated hepatocytes could thus be used to quantify hepatocyte uptake, metabolism, and efflux of compounds and considerably improve the prediction of hepatic clearance, especially for compounds with a large biliary clearance component.
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Affiliation(s)
- Patrik Lundquist
- CNS and Pain Innovative Medicines DMPK, AstraZeneca R&D, Södertälje (P.L., J.L., U.F., I.S., J.J., S.B., J.H., L.A.); Cardiovascular and Metabolic Diseases Innovative Medicines DMPK, AstraZeneca R&D, Mölndal (P.L., T.B.A.); Department of Pharmacy, Uppsala University, Uppsala (P.L.); and Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm (T.B.A.), Sweden
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Chatterjee S, Bijsmans IT, van Mil SW, Augustijns P, Annaert P. Toxicity and intracellular accumulation of bile acids in sandwich-cultured rat hepatocytes: Role of glycine conjugates. Toxicol In Vitro 2014; 28:218-30. [DOI: 10.1016/j.tiv.2013.10.020] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2013] [Revised: 10/24/2013] [Accepted: 10/30/2013] [Indexed: 02/06/2023]
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20
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Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, Bode JG, Bolleyn J, Borner C, Böttger J, Braeuning A, Budinsky RA, Burkhardt B, Cameron NR, Camussi G, Cho CS, Choi YJ, Craig Rowlands J, Dahmen U, Damm G, Dirsch O, Donato MT, Dong J, Dooley S, Drasdo D, Eakins R, Ferreira KS, Fonsato V, Fraczek J, Gebhardt R, Gibson A, Glanemann M, Goldring CEP, Gómez-Lechón MJ, Groothuis GMM, Gustavsson L, Guyot C, Hallifax D, Hammad S, Hayward A, Häussinger D, Hellerbrand C, Hewitt P, Hoehme S, Holzhütter HG, Houston JB, Hrach J, Ito K, Jaeschke H, Keitel V, Kelm JM, Kevin Park B, Kordes C, Kullak-Ublick GA, LeCluyse EL, Lu P, Luebke-Wheeler J, Lutz A, Maltman DJ, Matz-Soja M, McMullen P, Merfort I, Messner S, Meyer C, Mwinyi J, Naisbitt DJ, Nussler AK, Olinga P, Pampaloni F, Pi J, Pluta L, Przyborski SA, Ramachandran A, Rogiers V, Rowe C, Schelcher C, Schmich K, Schwarz M, Singh B, Stelzer EHK, Stieger B, Stöber R, Sugiyama Y, Tetta C, Thasler WE, Vanhaecke T, Vinken M, Weiss TS, Widera A, Woods CG, Xu JJ, Yarborough KM, Hengstler JG. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol 2013; 87:1315-530. [PMID: 23974980 PMCID: PMC3753504 DOI: 10.1007/s00204-013-1078-5] [Citation(s) in RCA: 965] [Impact Index Per Article: 80.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2013] [Accepted: 05/06/2013] [Indexed: 12/15/2022]
Abstract
This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.
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Affiliation(s)
- Patricio Godoy
- Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139 Dortmund, Germany
| | | | - Ute Albrecht
- Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany
| | - Melvin E. Andersen
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Nariman Ansari
- Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
| | - Sudin Bhattacharya
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Johannes Georg Bode
- Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany
| | - Jennifer Bolleyn
- Department of Toxicology, Centre for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Christoph Borner
- Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany
| | - Jan Böttger
- Institute of Biochemistry, Faculty of Medicine, University of Leipzig, 04103 Leipzig, Germany
| | - Albert Braeuning
- Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Wilhelmstr. 56, 72074 Tübingen, Germany
| | - Robert A. Budinsky
- Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, MI USA
| | - Britta Burkhardt
- BG Trauma Center, Siegfried Weller Institut, Eberhard Karls University Tübingen, 72076 Tübingen, Germany
| | - Neil R. Cameron
- Department of Chemistry, Durham University, Durham, DH1 3LE UK
| | - Giovanni Camussi
- Department of Medical Sciences, University of Torino, 10126 Turin, Italy
| | - Chong-Su Cho
- Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921 Korea
| | - Yun-Jaie Choi
- Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921 Korea
| | - J. Craig Rowlands
- Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, MI USA
| | - Uta Dahmen
- Experimental Transplantation Surgery, Department of General Visceral, and Vascular Surgery, Friedrich-Schiller-University Jena, 07745 Jena, Germany
| | - Georg Damm
- Department of General-, Visceral- and Transplantation Surgery, Charité University Medicine Berlin, 13353 Berlin, Germany
| | - Olaf Dirsch
- Institute of Pathology, Friedrich-Schiller-University Jena, 07745 Jena, Germany
| | - María Teresa Donato
- Unidad de Hepatología Experimental, IIS Hospital La Fe Avda Campanar 21, 46009 Valencia, Spain
- CIBERehd, Fondo de Investigaciones Sanitarias, Barcelona, Spain
- Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Valencia, Spain
| | - Jian Dong
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Steven Dooley
- Department of Medicine II, Section Molecular Hepatology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
| | - Dirk Drasdo
- Interdisciplinary Center for Bioinformatics (IZBI), University of Leipzig, 04107 Leipzig, Germany
- INRIA (French National Institute for Research in Computer Science and Control), Domaine de Voluceau-Rocquencourt, B.P. 105, 78153 Le Chesnay Cedex, France
- UPMC University of Paris 06, CNRS UMR 7598, Laboratoire Jacques-Louis Lions, 4, pl. Jussieu, 75252 Paris cedex 05, France
| | - Rowena Eakins
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - Karine Sá Ferreira
- Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany
- GRK 1104 From Cells to Organs, Molecular Mechanisms of Organogenesis, Faculty of Biology, University of Freiburg, Freiburg, Germany
| | - Valentina Fonsato
- Department of Medical Sciences, University of Torino, 10126 Turin, Italy
| | - Joanna Fraczek
- Department of Toxicology, Centre for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Rolf Gebhardt
- Institute of Biochemistry, Faculty of Medicine, University of Leipzig, 04103 Leipzig, Germany
| | - Andrew Gibson
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - Matthias Glanemann
- Department of General-, Visceral- and Transplantation Surgery, Charité University Medicine Berlin, 13353 Berlin, Germany
| | - Chris E. P. Goldring
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - María José Gómez-Lechón
- Unidad de Hepatología Experimental, IIS Hospital La Fe Avda Campanar 21, 46009 Valencia, Spain
- CIBERehd, Fondo de Investigaciones Sanitarias, Barcelona, Spain
| | - Geny M. M. Groothuis
- Department of Pharmacy, Pharmacokinetics Toxicology and Targeting, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands
| | - Lena Gustavsson
- Department of Laboratory Medicine (Malmö), Center for Molecular Pathology, Lund University, Jan Waldenströms gata 59, 205 02 Malmö, Sweden
| | - Christelle Guyot
- Department of Clinical Pharmacology and Toxicology, University Hospital, 8091 Zurich, Switzerland
| | - David Hallifax
- Centre for Applied Pharmacokinetic Research (CAPKR), School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT UK
| | - Seddik Hammad
- Department of Forensic Medicine and Veterinary Toxicology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt
| | - Adam Hayward
- Biological and Biomedical Sciences, Durham University, Durham, DH13LE UK
| | - Dieter Häussinger
- Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany
| | - Claus Hellerbrand
- Department of Medicine I, University Hospital Regensburg, 93053 Regensburg, Germany
| | | | - Stefan Hoehme
- Interdisciplinary Center for Bioinformatics (IZBI), University of Leipzig, 04107 Leipzig, Germany
| | - Hermann-Georg Holzhütter
- Institut für Biochemie Abteilung Mathematische Systembiochemie, Universitätsmedizin Berlin (Charité), Charitéplatz 1, 10117 Berlin, Germany
| | - J. Brian Houston
- Centre for Applied Pharmacokinetic Research (CAPKR), School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT UK
| | | | - Kiyomi Ito
- Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo, 202-8585 Japan
| | - Hartmut Jaeschke
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 USA
| | - Verena Keitel
- Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany
| | | | - B. Kevin Park
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - Claus Kordes
- Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany
| | - Gerd A. Kullak-Ublick
- Department of Clinical Pharmacology and Toxicology, University Hospital, 8091 Zurich, Switzerland
| | - Edward L. LeCluyse
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Peng Lu
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | | | - Anna Lutz
- Department of Pharmaceutical Biology and Biotechnology, University of Freiburg, Freiburg, Germany
| | - Daniel J. Maltman
- Reinnervate Limited, NETPark Incubator, Thomas Wright Way, Sedgefield, TS21 3FD UK
| | - Madlen Matz-Soja
- Institute of Biochemistry, Faculty of Medicine, University of Leipzig, 04103 Leipzig, Germany
| | - Patrick McMullen
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Irmgard Merfort
- Department of Pharmaceutical Biology and Biotechnology, University of Freiburg, Freiburg, Germany
| | | | - Christoph Meyer
- Department of Medicine II, Section Molecular Hepatology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
| | - Jessica Mwinyi
- Department of Clinical Pharmacology and Toxicology, University Hospital, 8091 Zurich, Switzerland
| | - Dean J. Naisbitt
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - Andreas K. Nussler
- BG Trauma Center, Siegfried Weller Institut, Eberhard Karls University Tübingen, 72076 Tübingen, Germany
| | - Peter Olinga
- Division of Pharmaceutical Technology and Biopharmacy, Department of Pharmacy, University of Groningen, 9713 AV Groningen, The Netherlands
| | - Francesco Pampaloni
- Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
| | - Jingbo Pi
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Linda Pluta
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Stefan A. Przyborski
- Reinnervate Limited, NETPark Incubator, Thomas Wright Way, Sedgefield, TS21 3FD UK
- Biological and Biomedical Sciences, Durham University, Durham, DH13LE UK
| | - Anup Ramachandran
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 USA
| | - Vera Rogiers
- Department of Toxicology, Centre for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Cliff Rowe
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - Celine Schelcher
- Department of Surgery, Liver Regeneration, Core Facility, Human in Vitro Models of the Liver, Ludwig Maximilians University of Munich, Munich, Germany
| | - Kathrin Schmich
- Department of Pharmaceutical Biology and Biotechnology, University of Freiburg, Freiburg, Germany
| | - Michael Schwarz
- Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Wilhelmstr. 56, 72074 Tübingen, Germany
| | - Bijay Singh
- Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921 Korea
| | - Ernst H. K. Stelzer
- Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
| | - Bruno Stieger
- Department of Clinical Pharmacology and Toxicology, University Hospital, 8091 Zurich, Switzerland
| | - Regina Stöber
- Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139 Dortmund, Germany
| | - Yuichi Sugiyama
- Sugiyama Laboratory, RIKEN Innovation Center, RIKEN, Yokohama Biopharmaceutical R&D Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045 Japan
| | - Ciro Tetta
- Fresenius Medical Care, Bad Homburg, Germany
| | - Wolfgang E. Thasler
- Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich, Germany
| | - Tamara Vanhaecke
- Department of Toxicology, Centre for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Mathieu Vinken
- Department of Toxicology, Centre for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Thomas S. Weiss
- Department of Pediatrics and Juvenile Medicine, University of Regensburg Hospital, Regensburg, Germany
| | - Agata Widera
- Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139 Dortmund, Germany
| | - Courtney G. Woods
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | | | | | - Jan G. Hengstler
- Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139 Dortmund, Germany
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De Bruyn T, Chatterjee S, Fattah S, Keemink J, Nicolaï J, Augustijns P, Annaert P. Sandwich-cultured hepatocytes: utility for in vitro exploration of hepatobiliary drug disposition and drug-induced hepatotoxicity. Expert Opin Drug Metab Toxicol 2013; 9:589-616. [PMID: 23452081 DOI: 10.1517/17425255.2013.773973] [Citation(s) in RCA: 98] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
INTRODUCTION The sandwich-cultured hepatocyte (SCH) model has become an invaluable in vitro tool for studying hepatic drug transport, metabolism, biliary excretion and toxicity. The relevant expression of many hepatocyte-specific functions together with the in vivo-like morphology favor SCHs over other preclinical models for evaluating hepatobiliary drug disposition and drug-induced hepatotoxicity. AREAS COVERED In this review, the authors highlight recommended procedures required for reproducibly culturing hepatocytes in sandwich configuration. It also provides an overview of the SCH model characteristics as a function of culture time. Lastly, the article presents a summary of the most prominent applications of the SCH model, including hepatic drug clearance prediction, drug-drug interaction potential and drug-induced hepatotoxicity. EXPERT OPINION When human (cryopreserved) hepatocytes are used to establish sandwich cultures, the model appears particularly valuable to quantitatively investigate clinically relevant mechanisms related to in vivo hepatobiliary drug disposition and hepatotoxicity. Nonetheless, the SCH model would largely benefit from better insight into the fundamental cell signaling mechanisms that are critical for long-term in vitro maintenance of the hepatocytic phenotype. Studies systematically exploring improved cell culture conditions (e.g., co-cultures or extracellular matrix modifications), as well as in vitro work identifying key transcription factors involved in hepatocyte differentiation are currently emerging.
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Affiliation(s)
- Tom De Bruyn
- Drug Delivery and Disposition, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, O&N2, Herestraat 49-bus-921, 3000 Leuven, Belgium
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22
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Sinclair J, Henderson C, Tettey J, Grant M. The influence of the choice of digestion enzyme used to prepare rat hepatocytes on xenobiotic uptake and efflux. Toxicol In Vitro 2013; 27:451-7. [DOI: 10.1016/j.tiv.2012.07.012] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2011] [Revised: 07/23/2012] [Accepted: 07/31/2012] [Indexed: 10/28/2022]
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Ramboer E, Vanhaecke T, Rogiers V, Vinken M. Primary hepatocyte cultures as prominent in vitro tools to study hepatic drug transporters. Drug Metab Rev 2013; 45:196-217. [PMID: 23368091 DOI: 10.3109/03602532.2012.756010] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Before any drug can be placed on the market, drug efficacy and safety must be ensured through rigorous testing. Animal models are used for this purpose, though currently increasing attention goes to the use of alternative in vitro systems. In particular, liver-based testing platforms that allow the prediction of pharmacokinetic (PK) and pharmacotoxicological properties during the early phase of drug development are of interest. They also enable the screening of potential effects on hepatic drug transporters. The latter are known to affect drug metabolism and disposition, thereby possibly underlying drug-drug interactions, which, in turn, may result in liver toxicity. Clearly, stable in vivo-like functional expression of drug transporters in hepatic in vitro settings is a prerequisite to be applicable in routine PK and pharmacotoxicological testing. In the first part of the article, an updated overview of hepatic drug transporters is provided, followed by a state-of-the-art review of drug-transporter production and activity in primary hepatocyte cultures (PHCs), being the gold-standard in vitro system. Specific focus is hereby put on strategies to maintain long-term functional expression, in casu of drug transporters, in these systems. In the second part, the use of PHCs to assess hepatobiliary transport and transporter-mediated interactions is outlined.
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Affiliation(s)
- Eva Ramboer
- Department of Toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Brussels, Belgium.
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24
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Griffin LM, Watkins PB, Perry CH, St Claire RL, Brouwer KLR. Combination lopinavir and ritonavir alter exogenous and endogenous bile acid disposition in sandwich-cultured rat hepatocytes. Drug Metab Dispos 2013; 41:188-96. [PMID: 23091188 PMCID: PMC3533430 DOI: 10.1124/dmd.112.047225] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2012] [Accepted: 10/22/2012] [Indexed: 02/06/2023] Open
Abstract
Inhibition of the bile salt export pump (BSEP) can cause intracellular accumulation of bile acids and is a risk factor for drug-induced liver injury in humans. Antiretroviral protease inhibitors lopinavir (LPV) and ritonavir (RTV) are reported BSEP inhibitors. However, the consequences of LPV and RTV, alone and combined (LPV/r), on hepatocyte viability, bile acid transport, and endogenous bile acid disposition in rat hepatocytes have not been examined. The effect of LPV, RTV, and LPV/r on cellular viability and the disposition of [(3)H]taurocholic acid (TCA) and [(14)C]chenodeoxycholic acid (CDCA) was determined in sandwich-cultured rat hepatocytes (SCRH) and suspended rat hepatocytes. Lactate dehydrogenase and ATP assays revealed a concentration-dependent effect of LPV and RTV on cellular viability. LPV (5 µM), alone and combined with 5 µM RTV, significantly decreased [(3)H]TCA accumulation in cells + bile of SCRHs compared with control. LPV/r significantly increased [(3)H]TCA cellular accumulation (7.7 ± 0.1 pmol/mg of protein) compared with vehicle and 5 µM LPV alone (5.1 ± 0.7 and 5.0 ± 0.5 pmol/mg of protein). The [(3)H]TCA biliary clearance was reduced significantly by LPV and RTV and further reduced by LPV/r. LPV and RTV did not affect the initial uptake rates of [(3)H]TCA or [(14)C]CDCA in suspended rat hepatocytes. LPV (50 µM), RTV (5 µM), and LPV/r (5 and 50 µM/5 µM) significantly decreased the accumulation of total measured endogenous bile acids (TCA, glycocholic acid, taurochenodeoxycholic acid, glycochenodeoxycholic acid, and α/β-tauromuricholic acid) in SCRH. Quantification of endogenous bile acids in SCRH may reveal important adaptive responses associated with exposure to known BSEP inhibitors.
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Affiliation(s)
- LaToya M Griffin
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
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Yin J, Meng Q. Use of primary rat hepatocytes in the gel entrapment culture to predictin vivobiliary excretion. Xenobiotica 2011; 42:417-28. [DOI: 10.3109/00498254.2011.633716] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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Jemnitz K, Veres Z, Szabo M, Baranyai Z, Jakab F, Vereczkey L. Differential inhibitory effect of cyclosporin A and bosentan on taurocholate uptake in human and rat hepatocytes as a function of culturing time. Toxicol In Vitro 2011; 26:174-81. [PMID: 22119333 DOI: 10.1016/j.tiv.2011.11.005] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2011] [Revised: 11/02/2011] [Accepted: 11/09/2011] [Indexed: 01/11/2023]
Abstract
Bile salt transport across hepatocytes requires a coordinate action of transporters, which is thought to be a target for drug-induced cholestasis. Hepatocytes provide the most competent in vitro model to predict transporter-related toxic drug effects. The aim of this study was to show a correlation between inhibitory potential of drugs and the change of rate, as well as of the active to passive ratio of taurocholate uptake in these cells. In rat hepatocytes, along with a significant decrease of uptake (86.4% by 72h), and the shift of saturable/unsaturable transport (from 92/8 to 55/45 in a 24-72h time interval), the efficacy of taurocholate uptake inhibition was highly reduced (IC(50) cyclosporin A 3.9 to >100μM, and bosentan 9.1-49.8μM at 1 and 72h, respectively). In contrast, 5-day-old human hepatocytes preserved 70% of their taurocholate uptake capacity with a 2-fold higher active than passive transport, which resulted in a more efficient inhibition by drugs (IC(50) cyclosporin A, 2.4 to ∼10μM and bosentan 28.9-45.5μM at 1h and 5days, respectively). Our results support that reliable drug interaction studies might be performed in 5-day-old human hepatocyte cultures, while experiments using rat hepatocytes at more than 24h after seeding will highly underestimate the probability of drug interaction.
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Affiliation(s)
- Katalin Jemnitz
- Institute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences, 1025 Budapest, Pusztaszeri ut 59-67, Hungary.
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Time-course activities of Oct1, Mrp3, and cytochrome P450s in cultures of cryopreserved rat hepatocytes. Eur J Pharm Sci 2011; 44:427-36. [DOI: 10.1016/j.ejps.2011.09.001] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2011] [Revised: 08/13/2011] [Accepted: 09/05/2011] [Indexed: 11/23/2022]
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Kotani N, Maeda K, Watanabe T, Hiramatsu M, Gong LK, Bi YA, Takezawa T, Kusuhara H, Sugiyama Y. Culture period-dependent changes in the uptake of transporter substrates in sandwich-cultured rat and human hepatocytes. Drug Metab Dispos 2011; 39:1503-10. [PMID: 21673128 DOI: 10.1124/dmd.111.038968] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/13/2025] Open
Abstract
Sandwich-cultured hepatocytes (SCH) are a useful tool for evaluating hepatobiliary drug transport in vitro. Some studies have investigated the in vitro-in vivo correlations of the biliary clearance of drugs using SCH. In most cases, the biliary clearance observed in vivo correlated well with the predicted clearance, but the predicted absolute values were underestimated when based on in vitro experiments with SCH. We hypothesized that the down-regulated function of uptake transporters is one of the causes of this underestimation. Therefore, the uptake of taurocholate, digoxin, pravastatin, and rosuvastatin was investigated in sandwich-cultured rat hepatocytes (SCRH) cultured for 5, 24, 48, and 96 h, and the predicted hepatic clearance from in vitro uptake clearance (CL(H, vitro)) was calculated with a dispersion model. In SCRH cultured for 96 h, the saturable uptake of taurocholate, digoxin, pravastatin, and rosuvastatin decreased to 7.5, 3.3, 64, and 23%, respectively, of their uptake in hepatocytes cultured for 5 h, and a better prediction of in vivo hepatic clearance (CL(H, vivo)) was achieved when based on CL(H, vitro) of 5-h-cultured hepatocytes. These results suggest that the uptake activity is considerably reduced in cell culture, even in a sandwich-culture format. In a similar study, we also examined taurocholate and rosuvastatin in sandwich-cultured human hepatocytes (SCHH). Unlike in SCRH, the saturable uptake of these compounds did not differ markedly in SCHH cultured for 5 or 96 h. Thus, the uptake activity in SCHH was maintained relatively well compared with that in SCRH.
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Affiliation(s)
- Naoki Kotani
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan
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Murray JW, Thosani AJ, Wang P, Wolkoff AW. Heterogeneous accumulation of fluorescent bile acids in primary rat hepatocytes does not correlate with their homogenous expression of ntcp. Am J Physiol Gastrointest Liver Physiol 2011; 301:G60-8. [PMID: 21474652 PMCID: PMC3129936 DOI: 10.1152/ajpgi.00035.2011] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Sodium taurocholate-cotransporting polypeptide (ntcp) is considered to be a major determinant of bile acid uptake into hepatocytes. However, the regulation of ntcp and the degree that it participates in the accumulation of specific substrates are not well understood. We utilized fluorescent bile acid derivatives and direct quantitation of fluorescent microscopy images to examine the regulation of ntcp and its role in the cell-to-cell variability of fluorescent bile acid accumulation. Primary-cultured rat hepatocytes rapidly accumulated the fluorescent bile acids, chenodeoxycholylglycylamidofluorescein (CDCGamF), 7-β- nitrobenzoxadiazole 3-α hydroxy 5-β cholan-24-oic acid (NBD-CA), and cholyl-glycylamido-fluorescein (CGamF). However, in stably transfected HeLa cells, ntcp preferred CDCGamF, whereas the organic anion transporter, organic anion transporting polypeptide 1 (oatp1a1), preferred NBD-CA, and neither ntcp nor oatp1a1 showed strong accumulation of CGamF by these methods. Ntcp-mediated transport of CDCGamF was inhibited by taurocholate, cyclosporin, actin depolymerization, and an inhibitor of atypical PKC-ζ. The latter two agents altered the cellular distribution of ntcp as visualized in ntcp-green fluorescent protein-transfected cells. Although fluorescent bile acid accumulation was reproducible by the imaging assays, individual cells showed variable accumulation that was not attributable to changes in membrane permeability or cell viability. In HeLa cells, this was accounted for by variable levels of ntcp, whereas, in hepatocytes, ntcp expression was uniform, and low accumulation was seen in a large portion of cells despite the presence of ntcp. These studies indicate that single-cell imaging can provide insight into previously unrecognized details of anion transport in the complex environment of polarized hepatocytes.
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Affiliation(s)
- John W. Murray
- 1Department of Anatomy and Structural Biology, ,2Marion Bessin Liver Research Center and Division of Gastroenterology and Liver Diseases, Albert Einstein College of Medicine, Bronx, New York
| | - Amar J. Thosani
- 2Marion Bessin Liver Research Center and Division of Gastroenterology and Liver Diseases, Albert Einstein College of Medicine, Bronx, New York
| | - Pijun Wang
- 1Department of Anatomy and Structural Biology, ,2Marion Bessin Liver Research Center and Division of Gastroenterology and Liver Diseases, Albert Einstein College of Medicine, Bronx, New York
| | - Allan W. Wolkoff
- 1Department of Anatomy and Structural Biology, ,2Marion Bessin Liver Research Center and Division of Gastroenterology and Liver Diseases, Albert Einstein College of Medicine, Bronx, New York
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Determination of OATP-, NTCP- and OCT-mediated substrate uptake activities in individual and pooled batches of cryopreserved human hepatocytes. Eur J Pharm Sci 2011; 43:297-307. [DOI: 10.1016/j.ejps.2011.05.002] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2010] [Revised: 04/01/2011] [Accepted: 05/07/2011] [Indexed: 01/11/2023]
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31
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Rapid and enhanced repolarization in sandwich-cultured hepatocytes on an oxygen-permeable membrane. Biochem Eng J 2010. [DOI: 10.1016/j.bej.2010.08.018] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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Morgan RE, Trauner M, van Staden CJ, Lee PH, Ramachandran B, Eschenberg M, Afshari CA, Qualls CW, Lightfoot-Dunn R, Hamadeh HK. Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development. Toxicol Sci 2010; 118:485-500. [PMID: 20829430 DOI: 10.1093/toxsci/kfq269] [Citation(s) in RCA: 243] [Impact Index Per Article: 16.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The bile salt export pump (BSEP) is an efflux transporter, driving the elimination of endobiotic and xenobiotic substrates from hepatocytes into the bile. More specifically, it is responsible for the elimination of monovalent, conjugated bile salts, with little or no assistance from other apical transporters. Disruption of BSEP activity through genetic disorders is known to manifest in clinical liver injury such as progressive familial intrahepatic cholestasis type 2. Drug-induced disruption of BSEP is hypothesized to play a role in the development of liver injury for several marketed or withdrawn therapeutics. Unfortunately, preclinical animal models have been poor predictors of the liver injury associated with BSEP interference observed for humans, possibly because of interspecies differences in bile acid composition, differences in hepatobiliary transporter modulation or constitutive expression, as well as other mechanisms. Thus, a BSEP-mediated liver liability may go undetected until the later stages of drug development, such as during clinical trials or even postlicensing. In the absence of a relevant preclinical test system for BSEP-mediated liver injury, the toxicological relevance of available in vitro models to human health rely on the use of benchmark compounds with known clinical outcomes, such as marketed or withdrawn drugs. In this study, membrane vesicles harvested from BSEP-transfected insect cells were used to assess the activity of more than 200 benchmark compounds to thoroughly investigate the relationship between interference with BSEP function and liver injury. The data suggest a relatively strong association between the pharmacological interference with BSEP function and human hepatotoxicity. Although the most accurate translation of risk would incorporate pharmacological potency, pharmacokinetics, clearance mechanisms, tissue distribution, physicochemical properties, indication, and other drug attributes, the additional understanding of a compound's potency for BSEP interference should help to limit or avoid BSEP-related liver liabilities in humans that are not often detected by standard preclinical animal models.
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Affiliation(s)
- Ryan E Morgan
- Department of Comparative Biology and Safety Sciences Amgen Inc., Thousand Oaks, California 91320, USA
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33
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Li N, Singh P, Mandrell KM, Lai Y. Improved extrapolation of hepatobiliary clearance from in vitro sandwich cultured rat hepatocytes through absolute quantification of hepatobiliary transporters. Mol Pharm 2010; 7:630-41. [PMID: 20438085 DOI: 10.1021/mp9001574] [Citation(s) in RCA: 66] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Previously we have reported that hepatobiliary transporter expressions in sandwich cultured hepatocytes (SCH) are altered 2- to 5-fold. This change could limit the model's predictive power for in vivo biliary clearance. The present study was designed to better establish in vitro to in vivo correlation (IVIVC) of biliary clearance. Eleven compounds representing the substrates of Mrp2/Abcc2, Bcrp/Abcg2 and Bsep/Abcb11 were tested in the sandwich cultured rat hepatocyte (SCRH) model. Simultaneously, the absolute difference of hepatobiliary transporters between rat livers and SCRH at day 5 post culture was determined by LC-MS/MS. This difference was integrated into the well-stirred hepatic prediction model. A correction factor named "g_factor" was mathematically defined to reflect the difference in hepatobiliary transporter expressions between the SCRH model and in vivo models, as well as the contribution of multiple transporters. When the g_factor correction was applied, the in vivo biliary clearance prediction was significantly improved. In addition, for those compounds which are poorly permeable and/or undergo transporter-dependent active uptake, the known intracellular concentrations of substrates were used to estimate intrinsic bile clearance. This led to further improvement in the prediction of in vivo bile secretion. While the rate-limiting processes of uptake transporters in the SCRH model remain to be further determined, we showed that integration of the absolute difference of hepatobiliary transporter proteins and transport contributions could improve the predictability of SCRH model. This integration is fundamental for increased confidence in the IVIVC of human biliary clearance.
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Affiliation(s)
- Na Li
- Department of Pharmacokinetics, Dynamics, and Drug Metabolism, Pfizer Global Research & Development, Pfizer Inc, Groton, Connecticut 06340, USA
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34
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Ye ZW, Camus S, Augustijns P, Annaert P. Interaction of eight HIV protease inhibitors with the canalicular efflux transporter ABCC2 (MRP2) in sandwich-cultured rat and human hepatocytes. Biopharm Drug Dispos 2010; 31:178-88. [PMID: 20238377 DOI: 10.1002/bdd.701] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Hepatotoxicity has been reported as a side-effect in some patients on HIV protease inhibitors (PI). Since transporter interaction has been implicated as a mechanism underlying drug-mediated hepatotoxicity and drug-drug interactions, the interaction of PI with the hepatic canalicular efflux transporter ABCC2 (MRP2; multidrug resistance associated protein-2) was studied. Interaction with ABCC2/Abcc2 was evaluated in human and rat sandwich-cultured hepatocytes using 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) as substrate. In rat hepatocytes, interaction with estradiol-17-beta-D-glucuronide (E17G) efflux was also studied. In human hepatocytes, saquinavir, ritonavir and atazanavir were the most efficient inhibitors of ABCC2-mediated biliary excretion of CDF, whereas in rat hepatocytes indinavir, lopinavir and nelfinavir were the most efficient. No species-similarity was found for ABCC2/Abcc2 inhibition. In rat hepatocytes, the effects on Abcc2 were substrate-dependent as inhibition of biliary excretion of E17G was most pronounced for saquinavir (completely blocked), amprenavir (82% inhibition) and indinavir (68% inhibition). In conclusion, several HIV PI showed substantial ABCC2 inhibition, which, combined with the effects of PI on other hepatobiliary disposition mechanisms, will determine the clinical relevance of these in vitro interaction data.
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Affiliation(s)
- Zhi-Wei Ye
- Laboratory for Pharmacotechnology and Biopharmacy, Department of Pharmaceutical Sciences, Katholieke Universiteit Leuven, O&N2, Herestraat 49-bus-921, 3000 Leuven, Belgium
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Swift B, Pfeifer ND, Brouwer KLR. Sandwich-cultured hepatocytes: an in vitro model to evaluate hepatobiliary transporter-based drug interactions and hepatotoxicity. Drug Metab Rev 2010; 42:446-71. [PMID: 20109035 PMCID: PMC3097390 DOI: 10.3109/03602530903491881] [Citation(s) in RCA: 290] [Impact Index Per Article: 19.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Sandwich-cultured hepatocytes (SCH) are a powerful in vitro tool that can be utilized to study hepatobiliary drug transport, species differences in drug transport, transport protein regulation, drug-drug interactions, and hepatotoxicity. This review provides an up-to-date summary of the SCH model, including a brief history of, and introduction to, the use of SCH, as well as methodology to evaluate hepatobiliary drug disposition. A summary of the literature that has utilized this model to examine the interplay between drug-metabolizing enzymes and transport proteins, drug-drug interactions at the transport level, and hepatotoxicity as a result of altered hepatic transport also is provided.
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Affiliation(s)
- Brandon Swift
- University of North Carolina at Chapel Hill, 27599-7569, USA
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Swift B, Yue W, Brouwer KLR. Evaluation of (99m)technetium-mebrofenin and (99m)technetium-sestamibi as specific probes for hepatic transport protein function in rat and human hepatocytes. Pharm Res 2010; 27:1987-98. [PMID: 20652625 DOI: 10.1007/s11095-010-0203-x] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2010] [Accepted: 06/21/2010] [Indexed: 01/09/2023]
Abstract
PURPOSE This study characterized 99mTc-Mebrofenin (MEB) and 99mTc-Sestamibi (MIBI) hepatic transport and preferential efflux routes (canalicular vs. basolateral) in rat and human sandwich-cultured hepatocytes (SCH). METHODS 99mTc-MEB and 99mTc-MIBI disposition was determined in suspended hepatocytes and in SCH in the presence and absence of inhibitors and genetic knockdown of breast cancer resistance protein (Bcrp). RESULTS The general organic anion transporting polypeptide (Oatp/OATP) inhibitor rifamycin SV reduced initial 99mTc-MEB uptake in rat and human suspended hepatocytes. Initial 99mTc-MIBI uptake in suspended rat hepatocytes was not Na+-dependent or influenced by inhibitors. Multidrug resistance-associated protein (Mrp2/MRP2) inhibitors decreased 99mTc-MEB canalicular efflux in rat and human SCH. 99mTc-MEB efflux in human SCH was predominantly canalicular (45.8 +/- 8.6%) and approximately 3-fold greater than in rat SCH. 99mTc-MIBI canalicular efflux was similar in human and rat SCH; basolateral efflux was 37% greater in human than rat SCH. 99mTc-MIBI cellular accumulation, biliary excretion index and in vitro biliary clearance in rat SCH were unaffected by Bcrp knockdown. CONCLUSION 99mTc-MEB hepatic uptake is predominantly Oatp-mediated with biliary excretion by Mrp2. 99mTc-MIBI appears to passively diffuse into hepatocytes; biliary excretion is mediated by P-gp. The SCH model is useful to investigate factors that may alter the route and/or extent of hepatic basolateral and canalicular efflux of substrates.
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Affiliation(s)
- Brandon Swift
- Division of Pharmacotherapy and Experimental Therapeutics UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7569, USA
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37
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Ye ZW, Van Pelt J, Camus S, Snoeys J, Augustijns P, Annaert P. Species-Specific Interaction of HIV Protease Inhibitors With Accumulation of Cholyl-Glycylamido-Fluorescein (CGamF) in Sandwich-Cultured Hepatocytes. J Pharm Sci 2010; 99:2886-98. [DOI: 10.1002/jps.22018] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
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Swift B, Brouwer KL. Influence of seeding density and extracellular matrix on bile Acid transport and mrp4 expression in sandwich-cultured mouse hepatocytes. Mol Pharm 2010; 7:491-500. [PMID: 19968322 PMCID: PMC3235796 DOI: 10.1021/mp900227a] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
This study was undertaken to examine the influence of seeding density, extracellular matrix and days in culture on bile acid transport proteins and hepatobiliary disposition of the model bile acid taurocholate. Mouse hepatocytes were cultured in a sandwich configuration on six-well Biocoat plates with an overlay of Matrigel (BC/MG) or gelled-collagen (BC/GC) for 3 or 4 days at seeding densities of 1.0, 1.25, or 1.5 x 10(6) cells/well. The lower seeding densities of 1.0 and 1.25 x 10(6) cells/well resulted in good hepatocyte morphology and bile canalicular network formation, as visualized by 5-(and 6)-carboxy-2',7'-dichlorofluorescein accumulation. In general, taurocholate cellular accumulation tended to increase as a function of seeding density in BC/GC; cellular accumulation was significantly increased in hepatocytes cultured in BC/MG compared to BC/GC at the same seeding density on both days 3 and 4 of culture. In general, in vitro intrinsic biliary clearance of taurocholate was increased at higher seeding densities. Levels of bile acid transport proteins on days 3 and 4 were not markedly influenced by seeding density or extracellular matrix except for multidrug resistance protein 4 (Mrp4), which was inversely related to seeding density. Mrp4 levels decreased approximately 2- to 3-fold between seeding densities of 1.0 x 10(6) and 1.25 x 10(6) cells/well regardless of extracellular matrix; an additional approximately 3- to 5-fold decrease in Mrp4 protein was noted in BC/GC between seeding densities of 1.25 x 10(6) and 1.5 x 10(6) cells/well. Results suggest that seeding density, extracellular matrix and days in culture profoundly influence Mrp4 expression in sandwich-cultured mouse hepatocytes. Primary mouse hepatocytes seeded in a BC/MG configuration at densities of 1.25 x 10(6) cells/well and 1.0 x 10(6), and cultured for 3 days, yielded optimal transport based on the probes studied. This work demonstrates the applicability of the sandwich-cultured model to mouse hepatocytes.
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Affiliation(s)
- Brandon Swift
- Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599-7569
| | - Kim L.R. Brouwer
- Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599-7569
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Lee JK, Marion TL, Abe K, Lim C, Pollock GM, Brouwer KLR. Hepatobiliary disposition of troglitazone and metabolites in rat and human sandwich-cultured hepatocytes: use of Monte Carlo simulations to assess the impact of changes in biliary excretion on troglitazone sulfate accumulation. J Pharmacol Exp Ther 2010; 332:26-34. [PMID: 19801447 PMCID: PMC2802476 DOI: 10.1124/jpet.109.156653] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2009] [Accepted: 10/01/2009] [Indexed: 01/23/2023] Open
Abstract
This study examined the hepatobiliary disposition of troglitazone (TGZ) and metabolites [TGZ sulfate (TS), TGZ glucuronide (TG), and TGZ quinone (TQ)] over time in rat and human sandwich-cultured hepatocytes (SCH). Cells were incubated with TGZ; samples were analyzed for TGZ and metabolites by liquid chromatography-tandem mass spectrometry. SCH mimicked the disposition of TGZ/metabolites in vivo in rats and humans; TGZ was metabolized primarily to TS and to a lesser extent to TG and TQ. In human SCH, the biliary excretion index (BEI) was negligible for TGZ and TQ, approximately 16% for TS, and approximately 43% for TG over the incubation period; in rat SCH, the BEI for TS and TG was approximately 13 and approximately 41%, respectively. Hepatocyte accumulation of TS was extensive, with intracellular concentrations ranging from 132 to 222 microM in rat SCH; intracellular TGZ concentrations ranged from 7.22 to 47.7 microM. In human SCH, intracellular TS and TGZ concentrations ranged from 136 to 160 microM and from 49.4 to 84.7 microM, respectively. Pharmacokinetic modeling and Monte Carlo simulations were used to evaluate the impact of modulating the biliary excretion rate constant (K(bile)) for TS on TS accumulation in hepatocytes and medium. Simulations demonstrated that intracellular concentrations of TS may increase up to 3.1- and 5.7-fold when biliary excretion of TS was decreased 2- and 10-fold, respectively. It is important to note that altered hepatobiliary transport and the extent of hepatocyte exposure may not always be evident based on medium concentrations (analogous to systemic exposure in vivo). Pharmacokinetic modeling/simulation with data from SCH is a useful approach to examine the impact of altered hepatobiliary transport on hepatocyte accumulation of drug/metabolites.
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Affiliation(s)
- Jin Kyung Lee
- Division of Pharmacotherapy and Experimental Therapeutics, The University of North Carolina at Chapel Hill Eshelman School of Pharmacy, North Carolina 27599-7360, USA
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Abstract
Increasingly, research suggests that for certain systems, animal models are insufficient for human toxicology testing. The development of robust, in vitro models of human toxicity is required to decrease our dependence on potentially misleading in vivo animal studies. A critical development in human toxicology testing is the use of human primary hepatocytes to model processes that occur in the intact liver. However, in order to serve as an appropriate model, primary hepatocytes must be maintained in such a way that they persist in their differentiated state. While many hepatocyte culture methods exist, the two-dimensional collagen "sandwich" system combined with a serum-free medium, supplemented with physiological glucocorticoid concentrations, appears to robustly maintain hepatocyte character. Studies in rat and human hepatocytes have shown that when cultured under these conditions, hepatocytes maintain many markers of differentiation including morphology, expression of plasma proteins, hepatic nuclear factors, phase I and II metabolic enzymes. Functionally, these culture conditions also preserve hepatic stress response pathways, such as the SAPK and MAPK pathways, as well as prototypical xenobiotic induction responses. This chapter will briefly review culture methodologies but will primarily focus on hallmark hepatocyte structural, expression and functional markers that characterize the differentiation status of the hepatocyte.
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Affiliation(s)
- Katy M Olsavsky Goyak
- Center for Molecular Toxicology & Carcinogenesis and Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA, USA
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Use of cassette dosing in sandwich-cultured rat and human hepatocytes to identify drugs that inhibit bile acid transport. Toxicol In Vitro 2009; 24:297-309. [PMID: 19706322 DOI: 10.1016/j.tiv.2009.08.009] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2009] [Revised: 08/18/2009] [Accepted: 08/19/2009] [Indexed: 01/13/2023]
Abstract
Hepatocellular accumulation of bile acids due to inhibition of the canalicular bile salt export pump (BSEP/ABCB11) is one proposed mechanism of drug-induced liver injury (DILI). Some hepatotoxic compounds also are potent inhibitors of bile acid uptake by Na(+)-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1). This study used a cassette dosing approach in rat and human sandwich-cultured hepatocytes (SCH) to determine whether known or suspected hepatotoxic drugs inhibit bile acid transport individually or in combination. [(3)H]-Taurocholate served as the NTCP/BSEP probe substrate. Individually, cyclosporin A and rifampin decreased taurocholate in vitro biliary clearance (Cl(biliary)) and biliary excretion index (BEI) by more than 20% in rat SCH, suggesting that these drugs primarily inhibited canalicular efflux. In contrast, ampicillin, carbenicillin, cloxacillin, nafcillin, oxacillin, carbamazepine, pioglitazone, and troglitazone decreased the in vitro Cl(biliary) by more than 20% with no notable change in BEI, suggesting that these drugs primarily inhibited taurocholate uptake. Cassette dosing (n=2-4 compounds per cassette) in rat SCH yielded similar findings, and results in human SCH were consistent with rat SCH. In summary, cassette dosing in SCH is a useful in vitro approach to identify compounds that inhibit the hepatic uptake and/or excretion of bile acids, which may cause DILI.
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Li N, Bi YA, Duignan DB, Lai Y. Quantitative Expression Profile of Hepatobiliary Transporters in Sandwich Cultured Rat and Human Hepatocytes. Mol Pharm 2009; 6:1180-9. [DOI: 10.1021/mp900044x] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Affiliation(s)
- Na Li
- Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research & Development, St. Louis Laboratories, and Groton Laboratories, Pfizer Inc, St. Louis, Missouri
| | - Yi-An Bi
- Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research & Development, St. Louis Laboratories, and Groton Laboratories, Pfizer Inc, St. Louis, Missouri
| | - David B. Duignan
- Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research & Development, St. Louis Laboratories, and Groton Laboratories, Pfizer Inc, St. Louis, Missouri
| | - Yurong Lai
- Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research & Development, St. Louis Laboratories, and Groton Laboratories, Pfizer Inc, St. Louis, Missouri
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Wolf KK, Brouwer KR, Pollack GM, Brouwer KLR. Effect of albumin on the biliary clearance of compounds in sandwich-cultured rat hepatocytes. Drug Metab Dispos 2008; 36:2086-92. [PMID: 18653747 PMCID: PMC2574866 DOI: 10.1124/dmd.108.020842] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022] Open
Abstract
The purpose of the present study was to evaluate the effects of bovine serum albumin (BSA) and essentially fatty acid-free BSA (BSA-FAF) on the biliary clearance of compounds in sandwich-cultured rat hepatocytes. Unbound fraction, biliary excretion index (BEI), and unbound intrinsic biliary clearance (intrinsic Clbiliary') were determined for digoxin, pravastatin, and taurocholate in the absence or presence of BSA or BSA-FAF. BSA had little effect on the BEI or intrinsic Clbiliary' of these compounds. Surprisingly, BSA-FAF decreased both BEI and intrinsic Clbiliary' for digoxin and pravastatin, which represent low and moderately bound compounds, respectively. The BEI and intrinsic Clbiliary' of taurocholate, a highly bound compound, were not altered significantly by BSA-FAF. Neither BSA nor BSA-FAF had a discernable effect on the bile canalicular networks based on carboxydichlorofluorescein retention. Neither the addition of physiological concentrations of calcium nor the addition of fatty acids to BSA-FAF was able to restore the BEI or intrinsic Clbiliary' of the model compounds to similar values in the absence or presence of BSA. Careful consideration is warranted when selecting the type of BSA for addition to in vitro systems such as sandwich-cultured rat hepatocytes.
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Affiliation(s)
- Kristina K Wolf
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, CB #7360, Kerr Hall Room 3205, Chapel Hill, NC 27599, USA
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Zhang S, Xia L, Kang CH, Xiao G, Ong SM, Toh YC, Leo HL, van Noort D, Kan SH, Tang HH, Yu H. Microfabricated silicon nitride membranes for hepatocyte sandwich culture. Biomaterials 2008; 29:3993-4002. [DOI: 10.1016/j.biomaterials.2008.06.024] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2008] [Accepted: 06/21/2008] [Indexed: 11/30/2022]
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Ye ZW, Augustijns P, Annaert P. Cellular accumulation of cholyl-glycylamido-fluorescein in sandwich-cultured rat hepatocytes: kinetic characterization, transport mechanisms, and effect of human immunodeficiency virus protease inhibitors. Drug Metab Dispos 2008; 36:1315-21. [PMID: 18420783 DOI: 10.1124/dmd.107.019398] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/13/2025] Open
Abstract
The present study was aimed at characterizing the in vitro cellular uptake mechanism and kinetics of the bile salt analog cholylglycylamido-fluorescein (CGamF) in sandwich-cultured rat hepatocytes (SCRHs). Concentration-dependent inhibition of active CGamF accumulation by seven human immunodeficiency virus (HIV) protease inhibitors (PIs) was also determined and compared with inhibition data obtained with taurocholate (TC) as a substrate. A K(m) value of 9.3 +/- 2.6 microM was obtained for saturable CGamF accumulation in SCRHs. The organic anion-transporting polypeptide (Oatp) inhibitor rifampicin (100 microM) inhibited CGamF (1 microM) accumulation in SCRHs by 72%; sodium depletion did not further reduce CGamF accumulation. In contrast, TC accumulation was reduced by only 25% in the presence of rifampicin, whereas additional sodium depletion resulted in a complete loss of TC accumulation. These data imply that Oatp(s) and sodium taurocholate-cotransporting polypeptide preferentially mediate hepatic uptake of CGamF and TC, respectively. Coincubation of CGamF with HIV PIs (amprenavir, atazanavir, darunavir, indinavir, nelfinavir, ritonavir, saquinavir) revealed that five of them had a concentration-dependent inhibitory effect on CGamF accumulation in SCRHs, with IC(50) values between 0.25 +/- 0.07 and 43 +/- 12 microM. The rank order for inhibition of CGamF accumulation in SCRHs was: ritonavir >> saquinavir > atazanavir > darunavir > amprenavir. Indinavir (up to 100 microM) did not alter CGamF accumulation, whereas nelfinavir solubility was limited to 10 microM. Taken together, these findings illustrate the utility of CGamF as a suitable probe (complementary to TC) for rapid in vitro determination of interaction potential with sodium-independent uptake mechanisms (likely Oatps) in rat liver.
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Affiliation(s)
- Zhi-Wei Ye
- Laboratory for Pharmacotechnology and Biopharmacy, Department of Pharmaceutical Sciences, Katholieke Universiteit Leuven, O&N2, Herestraat 49-bus 921, 3000 Leuven, Belgium
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Oshima H, Kon J, Ooe H, Hirata K, Mitaka T. Functional expression of organic anion transporters in hepatic organoids reconstructed by rat small hepatocytes. J Cell Biochem 2008; 104:68-81. [DOI: 10.1002/jcb.21601] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
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Lee JK, Leslie EM, Zamek-Gliszczynski MJ, Brouwer KLR. Modulation of trabectedin (ET-743) hepatobiliary disposition by multidrug resistance-associated proteins (Mrps) may prevent hepatotoxicity. Toxicol Appl Pharmacol 2007; 228:17-23. [PMID: 18191164 DOI: 10.1016/j.taap.2007.11.020] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2007] [Revised: 11/20/2007] [Accepted: 11/24/2007] [Indexed: 01/02/2023]
Abstract
Trabectedin is a promising anticancer agent, but dose-limiting hepatotoxicity was observed during phase I/II clinical trials. Dexamethasone (DEX) has been shown to significantly reduce trabectedin-mediated hepatotoxicity. The current study was designed to assess the capability of sandwich-cultured primary rat hepatocytes (SCRH) to predict the hepato-protective effect of DEX against trabectedin-mediated cytotoxicity. The role of multidrug resistance-associated protein 2 (Mrp2; Abcc2) in trabectedin hepatic disposition also was examined. In SCRH from wild-type Wistar rats, cytotoxicity was observed after 24-h continuous exposure to trabectedin. SCRH pretreated with additional DEX (1 microM) exhibited a 2- to 3-fold decrease in toxicity at 100 nM and 1000 nM trabectedin. Unexpectedly, toxicity in SCRH from Mrp2-deficient (TR(-)) compared to wild-type Wistar rats was markedly reduced. Depletion of glutathione from SCRH using buthionine sulfoximine (BSO) mitigated trabectedin toxicity associated with 100 nM and 1000 nM trabectedin. Western blot analysis demonstrated increased levels of CYP3A1/2 and Mrp2 in SCRH pretreated with DEX; interestingly, Mrp4 expression was increased in SCRH after BSO exposure. Trabectedin biliary recovery in isolated perfused livers from TR(-) rats was decreased by approximately 75% compared to wild-type livers. In conclusion, SCRH represent a useful in vitro model to predict the hepatotoxicity of trabectedin observed in vivo. The protection by DEX against trabectedin-mediated cytotoxicity may be attributed, in part, to enhanced Mrp2 biliary excretion and increased metabolism by CYP3A1/2. Decreased trabectedin toxicity in SCRH from TR(-) rats, and in SCRH pretreated with BSO, may be due to increased basolateral excretion of trabectedin by Mrp3 and/or Mrp4.
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Affiliation(s)
- Jin Kyung Lee
- School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360, USA
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Marion TL, Leslie EM, Brouwer KLR. Use of Sandwich-Cultured Hepatocytes To Evaluate Impaired Bile Acid Transport as a Mechanism of Drug-Induced Hepatotoxicity. Mol Pharm 2007; 4:911-8. [DOI: 10.1021/mp0700357] [Citation(s) in RCA: 76] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Affiliation(s)
- Tracy L. Marion
- Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7, and School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
| | - Elaine M. Leslie
- Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7, and School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
| | - Kim L. R. Brouwer
- Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7, and School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
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Bi YA, Kazolias D, Duignan DB. Use of cryopreserved human hepatocytes in sandwich culture to measure hepatobiliary transport. Drug Metab Dispos 2006; 34:1658-65. [PMID: 16782767 DOI: 10.1124/dmd.105.009118] [Citation(s) in RCA: 158] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Fresh hepatocytes cultured in a sandwich configuration allow for the development of intact bile canaliculi and the ability to measure hepatic uptake and biliary clearance. A disadvantage of this model is its dependence upon hepatocytes from fresh tissue. Therefore, the ability to use cryopreserved human hepatocytes in this model would be a great advantage. Multiple variables were tested, and the recommended conditions for culturing cryopreserved human hepatocytes in a sandwich configuration in 24-well plates are as follows: BioCoat plates, a cell density of 0.35 x 10(6) cells/well in 500 microl, an overlay of Matrigel and InVitroGRO media. These conditions resulted in good hepatocyte morphology and the formation of distinct bile canaliculi. The function of multiple uptake and efflux transporters was tested in multiple lots of cryopreserved and fresh human hepatocytes. For taurocholate [Na+ taurocholate cotransporting polypeptide/organic anion transporting polypeptide (OATP) uptake/bile salt export pump efflux], the average apparent uptake, apparent intrinsic biliary clearance, and biliary excretion index among five cryopreserved hepatocyte lots was high, ranging from 11 to 17 pmol/min/mg protein, 5.8 to 10 microl/min/mg protein, and 41 to 63%, respectively. The corresponding values for digoxin (OATP-8 uptake/multidrug resistance protein 1 efflux) were 0.69 to 1.5 pmol/min/mg protein, 0.60 to 1.5 microl/min/mg protein, and 37 to 63%. Both substrates exhibited similar results when fresh human hepatocytes were used. In addition, substrates of breast cancer resistance protein and multidrug resistance-associated protein 2 were also tested in this model, and all cryopreserved lots showed functional transport of these substrates. The use of cryopreserved human hepatocytes in 24-well sandwich culture to form intact bile canaliculi and to exhibit functional uptake and efflux transport has been successfully demonstrated.
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Affiliation(s)
- Yi-an Bi
- ADME Technology Group, Department of Pharmacokinetics, Dynamics & Metabolism, Groton/New London Laboratories, Pfizer, Inc., Groton, CT 06340, USA
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