|
1
|
Bellanti F, di Bello G, Tamborra R, Amatruda M, Lo Buglio A, Dobrakowski M, Kasperczyk A, Kasperczyk S, Serviddio G, Vendemiale G. Impact of senescence on the transdifferentiation process of human hepatic progenitor-like cells. World J Stem Cells 2021; 13:1595-1609. [PMID: 34786160 PMCID: PMC8567448 DOI: 10.4252/wjsc.v13.i10.1595] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/26/2021] [Revised: 06/14/2021] [Accepted: 08/23/2021] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Senescence is characterized by a decline in hepatocyte function, with impairment of metabolism and regenerative capacity. Several models that duplicate liver functions in vitro are essential tools for studying drug metabolism, liver diseases, and organ regeneration. The human HepaRG cell line represents an effective model for the study of liver metabolism and hepatic progenitors. However, the impact of senescence on HepaRG cells is not yet known.
AIM To characterize the effects of senescence on the transdifferentiation capacity and mitochondrial metabolism of human HepaRG cells.
METHODS We compared the transdifferentiation capacity of cells over 10 (passage 10 [P10]) vs P20. Aging was evaluated by senescence-associated (SA) beta-galactosidase activity and the comet assay. HepaRG transdifferentiation was analyzed by confocal microscopy and flow cytometry (expression of cluster of differentiation 49a [CD49a], CD49f, CD184, epithelial cell adhesion molecule [EpCAM], and cytokeratin 19 [CK19]), quantitative PCR analysis (expression of albumin, cytochrome P450 3A4 [CYP3A4], γ-glutamyl transpeptidase [γ-GT], and carcinoembryonic antigen [CEA]), and functional analyses (albumin secretion, CYP3A4, and γ-GT). Mitochondrial respiration and the ATP and nicotinamide adenine dinucleotide (NAD+)/NAD with hydrogen (NADH) content were also measured.
RESULTS SA β-galactosidase staining was higher in P20 than P10 HepaRG cells; in parallel, the comet assay showed consistent DNA damage in P20 HepaRG cells. With respect to P10, P20 HepaRG cells exhibited a reduction of CD49a, CD49f, CD184, EpCAM, and CK19 after the induction of transdifferentiation. Furthermore, lower gene expression of albumin, CYP3A4, and γ-GT, as well as reduced albumin secretion capacity, CYP3A4, and γ-GT activity were reported in transdifferentiated P20 compared to P10 cells. By contrast, the gene expression level of CEA was not reduced by transdifferentiation in P20 cells. Of note, both cellular and mitochondrial oxygen consumption was lower in P20 than in P10 transdifferentiated cells. Finally, both ATP and NAD+/NADH were depleted in P20 cells with respect to P10 cells.
CONCLUSION SA mitochondrial dysfunction may limit the transdifferentiation potential of HepaRG cells, with consequent impairment of metabolic and regenerative properties, which may alter applications in basic studies.
Collapse
Affiliation(s)
- Francesco Bellanti
- Department of Medical and Surgical Sciences, University of Foggia, Foggia 71122, Italy
| | - Giorgia di Bello
- Department of Medical and Surgical Sciences, University of Foggia, Foggia 71122, Italy
| | - Rosanna Tamborra
- Department of Medical and Surgical Sciences, University of Foggia, Foggia 71122, Italy
| | - Marco Amatruda
- Department of Medical and Surgical Sciences, University of Foggia, Foggia 71122, Italy
| | - Aurelio Lo Buglio
- Department of Medical and Surgical Sciences, University of Foggia, Foggia 71122, Italy
| | - Michał Dobrakowski
- Department of Biochemistry, Medical University of Silesia, Zabrze 41-808, Poland
| | | | - Sławomir Kasperczyk
- Department of Biochemistry, Medical University of Silesia, Zabrze 41-808, Poland
| | - Gaetano Serviddio
- Department of Medical and Surgical Sciences, University of Foggia, Foggia 71122, Italy
| | - Gianluigi Vendemiale
- Department of Medical and Surgical Sciences, University of Foggia, Foggia 71122, Italy
| |
Collapse
|
|
2
|
Stevens LJ, Donkers JM, Dubbeld J, Vaes WHJ, Knibbe CAJ, Alwayn IPJ, van de Steeg E. Towards human ex vivo organ perfusion models to elucidate drug pharmacokinetics in health and disease. Drug Metab Rev 2020; 52:438-454. [DOI: 10.1080/03602532.2020.1772280] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Affiliation(s)
- Lianne J. Stevens
- Department of Surgery, Division of Transplantation, Leiden University Medical Centre (LUMC), Leiden, The Netherlands
- The Netherlands Organisation for Applied Scientific Research (TNO), Zeist, The Netherlands
| | - Joanne M. Donkers
- The Netherlands Organisation for Applied Scientific Research (TNO), Zeist, The Netherlands
| | - Jeroen Dubbeld
- Department of Surgery, Division of Transplantation, Leiden University Medical Centre (LUMC), Leiden, The Netherlands
| | - Wouter H. J. Vaes
- The Netherlands Organisation for Applied Scientific Research (TNO), Zeist, The Netherlands
| | - Catherijne A. J. Knibbe
- Division of Systems Biomedicine and Pharmacology, Leiden Academic Centre for Drug Research (LACDR), Leiden, The Netherlands
| | - Ian P. J. Alwayn
- Department of Surgery, Division of Transplantation, Leiden University Medical Centre (LUMC), Leiden, The Netherlands
| | - Evita van de Steeg
- The Netherlands Organisation for Applied Scientific Research (TNO), Zeist, The Netherlands
| |
Collapse
|
|
3
|
Jang M, Kleber A, Ruckelshausen T, Betzholz R, Manz A. Differentiation of the human liver progenitor cell line (HepaRG) on a microfluidic-based biochip. J Tissue Eng Regen Med 2019; 13:482-494. [PMID: 30746894 DOI: 10.1002/term.2802] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2017] [Revised: 12/26/2018] [Accepted: 01/09/2019] [Indexed: 12/13/2022]
Abstract
HepaRG is a bipotent stem cell line that can be differentiated towards hepatocyte-like and biliary-like cells. The entire cultivation process requires 1 month and relies on the addition of 2% dimethyl sulfoxide (DMSO) to the culture. Our motivation in this research is to differentiate HepaRG cells (progenitor cells and undifferentiated cells) towards hepatocyte-like cells by minimizing the cultivation time and without using DMSO treatment by instead using a microfluidic device combined with the following strategies: (a) comparison of extracellular matrices (matrigel and collagen I), (b) types of flow (one or both sides), and (c) effects of DMSO. Our results demonstrate that matrigel promotes the differentiation of progenitor cells towards hepatocytes and biliary-like cells. Moreover, the frequent formation of HepaRG cell clusters was observed by a supply of both sides of flow, and the cell viability and liver specific functions were influenced by DMSO. Finally, differentiated HepaRG progenitor cells cultured in a microfluidic device for 14 days without DMSO treatment yielded 70% of hepatocyte-like cells with a highly polarized organization that reacted to stimulation with IL-6 to produce C-reactive protein (CRP). This culture model has high potential for investigating cell differentiation and liver pathophysiology research.
Collapse
Affiliation(s)
- Mi Jang
- Department of system engineering, Saarland University, Saarbrücken, Germany.,Microfluidics group, KIST Europe, Saarbrücken, Germany.,Department of Neuroscience, Korea University College of Medicine, Seoul, Korea
| | - Astrid Kleber
- Rhineland Palantinate Centre of Excellence for climate Change Impacts, Trippstadt, Germany
| | - Thomas Ruckelshausen
- Dynamic Biomaterial group, INM - Leibniz-Institut für Neue Materialien GmbH, Saarbrücken, Germany.,Service and Support group, PicoQuant, Rudower Chaussee 29, Berlin, Germany
| | - Ralf Betzholz
- School of Physics, Huazhong University of Science and Technology, Wuhan, China
| | - Andreas Manz
- Department of system engineering, Saarland University, Saarbrücken, Germany.,Microfluidics group, KIST Europe, Saarbrücken, Germany
| |
Collapse
|
|
4
|
Li AP, Ho MCD, Amaral K, Loretz C. A Novel In Vitro Experimental System for the Evaluation of Drug Metabolism: Cofactor-Supplemented Permeabilized Cryopreserved Human Hepatocytes (MetMax Cryopreserved Human Hepatocytes). Drug Metab Dispos 2018; 46:1608-1616. [PMID: 29363498 DOI: 10.1124/dmd.117.079657] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2017] [Accepted: 01/22/2018] [Indexed: 12/14/2022] Open
Abstract
We report here a novel experimental system, cryopreserved MetMax human hepatocytes (MMHHs), for in vitro drug metabolism studies. MMHHs consist of cofactor-supplemented permeabilized cryopreserved human hepatocytes. The use procedures for MMHHs are significantly simplified from that for conventional cryopreserved human hepatocytes (CCHHs): 1) storage at -80°C instead of in liquid nitrogen and 2) usage directly after thawing without centrifugation and microscopic evaluation of cell density and viability and cell density adjustment. In this study, we compared MMHHs and CCHHs in CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4, CYP2J2, monoamine oxidase A, aldehyde oxidase, flavin-containing monooxygenase, UDP-glucuronyl transferase, SULT, N-acetyltransferase 1, and acetaminophen glutathione (GSH) conjugation activities based on liquid chromatography-tandem mass spectrometry quantification of substrate metabolism. MMHHs were prepared from CCHHs consisting of hepatocytes pooled from 10 individual donors. The drug metabolizing enzyme activities of both CCHHs and MMHHs were cell concentration and time dependent, with specific activities of MMHHs ranging from 27.2% (carboxylesterase 2) to 234.2% (acetaminophen GSH conjugation) of that for CCHHs. As observed in CCHHs, sequential oxidation and conjugation was observed in MMHHs for coumarin, 7-ethoxycoumarin, and acetaminophen. 7-Hydroxycoumarin conjugation results showed that metabolic pathways in MMHHs could be selected via the choice of cofactors, with glucuronidation but not sulfation observed in the presence of UDP-glucuronic acid and not 3-phosphoadenosine-5-phosphosulfate, and vice versa. Results with noncytotoxic and cytotoxic concentrations of acetaminophen showed that drug metabolism was compromised in CCHHs but not in MMHHs. Our results suggest that the MMHHs system represents a convenient and robust in vitro experimental system for the evaluation of drug metabolism.
Collapse
Affiliation(s)
- Albert P Li
- In Vitro ADMET Laboratories Inc., Columbia, Maryland (A.P.L., C.L.) and In Vitro ADMET Laboratories Inc., Malden, Massachusetts (M.-C.D.H., K.A.)
| | - Ming-Chih David Ho
- In Vitro ADMET Laboratories Inc., Columbia, Maryland (A.P.L., C.L.) and In Vitro ADMET Laboratories Inc., Malden, Massachusetts (M.-C.D.H., K.A.)
| | - Kirsten Amaral
- In Vitro ADMET Laboratories Inc., Columbia, Maryland (A.P.L., C.L.) and In Vitro ADMET Laboratories Inc., Malden, Massachusetts (M.-C.D.H., K.A.)
| | - Carol Loretz
- In Vitro ADMET Laboratories Inc., Columbia, Maryland (A.P.L., C.L.) and In Vitro ADMET Laboratories Inc., Malden, Massachusetts (M.-C.D.H., K.A.)
| |
Collapse
|
|
5
|
Forkuo AD, Ansah C, Pearson D, Gertsch W, Cirello A, Amaral A, Spear J, Wright CW, Rynn C. Identification of cryptolepine metabolites in rat and human hepatocytes and metabolism and pharmacokinetics of cryptolepine in Sprague Dawley rats. BMC Pharmacol Toxicol 2017; 18:84. [PMID: 29273084 PMCID: PMC5741962 DOI: 10.1186/s40360-017-0188-8] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2016] [Accepted: 12/05/2017] [Indexed: 01/04/2023] Open
Abstract
BACKGROUND This study aims at characterizing the in vitro metabolism of cryptolepine using human and rat hepatocytes, identifying metabolites in rat plasma and urine after a single cryptolepine dose, and evaluating the single-dose oral and intravenous pharmacokinetics of cryptolepine in male Sprague Dawley (SD) rats. METHODS The in vitro metabolic profiles of cryptolepine were determined by LC-MS/MS following incubation with rat and human hepatocytes. The in vivo metabolic profile of cryptolepine was determined in plasma and urine samples from Sprague Dawley rats following single-dose oral administration of cryptolepine. Pharmacokinetic parameters of cryptolepine were determined in plasma and urine from Sprague Dawley rats after single-dose intravenous and oral administration. RESULTS Nine metabolites were identified in human and rat hepatocytes, resulting from metabolic pathways involving oxidation (M2-M9) and glucuronidation (M1, M2, M4, M8, M9). All human metabolites were found in rat hepatocyte incubations except glucuronide M1. Several metabolites (M2, M6, M9) were also identified in the urine and plasma of rats following oral administration of cryptolepine. Unchanged cryptolepine detected in urine was negligible. The Pharmacokinetic profile of cryptolepine showed a very high plasma clearance and volume of distribution (Vss) resulting in a moderate average plasma half-life of 4.5 h. Oral absorption was fast and plasma exposure and oral bioavailability were low. CONCLUSIONS Cryptolepine metabolism is similar in rat and human in vitro with the exception of direct glucuronidation in human. Clearance in rat and human is likely to include a significant metabolic contribution, with proposed primary human metabolism pathways hydroxylation, dihydrodiol formation and glucuronidation. Cryptolepine showed extensive distribution with a moderate half-life.
Collapse
Affiliation(s)
- Arnold Donkor Forkuo
- Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Science, College of Health Sciences Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
| | - Charles Ansah
- Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Science, College of Health Sciences Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
| | - David Pearson
- Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Postfach, CH-4002, Basel, Switzerland
| | - Werner Gertsch
- Analytical Sciences and Imaging, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Postfach, CH-4002, Basel, Switzerland
| | - Amanda Cirello
- Analytical Sciences and Imaging, Novartis Institutes for BioMedical Research, 250 Massachusetts Ave Cambridge, 02139, Cambridge, MA, USA
| | - Adam Amaral
- Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, 250 Massachusetts Ave Cambridge, 02139, Cambridge, MA, USA
| | - Jaimie Spear
- Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, 250 Massachusetts Ave Cambridge, 02139, Cambridge, MA, USA
| | - Colin W Wright
- School of Pharmacy, University of Bradford, West Yorkshire, BD7 1DP, Bradford, USA
| | - Caroline Rynn
- Metabolism and Pharmacokinetics, Novartis Institute for BioMedical Research, Novartis Pharma AG, Postfach, CH-4002, Basel, Switzerland
| |
Collapse
|
|
6
|
Ho MCD, Ring N, Amaral K, Doshi U, Li AP. Human Enterocytes as an In Vitro Model for the Evaluation of Intestinal Drug Metabolism: Characterization of Drug-Metabolizing Enzyme Activities of Cryopreserved Human Enterocytes from Twenty-Four Donors. Drug Metab Dispos 2017; 45:686-691. [PMID: 28396528 DOI: 10.1124/dmd.116.074377] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2016] [Accepted: 04/05/2017] [Indexed: 01/09/2023] Open
Abstract
We report in this work successful isolation and cryopreservation of enterocytes from human small intestine. The enterocytes were isolated by enzyme digestion of the intestinal lumen, followed by partial purification via differential centrifugation. The enterocytes were cryopreserved directly after isolation without culturing to maximize retention of in vivo drug-metabolizing enzyme activities. Post-thaw viability of the cryopreserved enterocytes was consistently over 80% based on trypan blue exclusion. Cryopreserved enterocytes pooled from eight donors (four male and four female) were evaluated for their metabolism of 14 pathway-selective substrates: CYP1A2 (phenacetin hydroxylation), CYP2A6 (coumarin 7-hydroxylation), CYP2B6 (bupropion hydroxylation), CYP2C8 (paclitaxel 6α-hydroxylation), CYP2C9 (diclofenac 4-hydroxylation), CYP2C19 (S-mephenytoin 4-hydroxylation), CYP2D6 (dextromethorphan hydroxylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4 (midazolam 1'-hydroxylation and testosterone 6β-hydroxylation), CYP2J2 (astemizole O-demethylation), UDP-glucuronosyltransferase (UGT; 7-hydroxycoumarin glucuronidation), sulfotransferase (SULT; 7-hydroxycoumarin sulfation), and carboxylesterase 2 (CES2; irinotecan hydrolysis) activities. Quantifiable activities were observed for CYP2C8, CYP2C9, CYP2C19, CYP2E1, CYP3A4, CYPJ2, CES2, UGT, and SULT, but not for CYP1A2, CYP2A6, CYP2B6, and CYP2D6. Enterocytes from all 24 donors were then individually evaluated for the quantifiable drug metabolism pathways. All demonstrated quantifiable activities with the expected individual variations. Our results suggest that cryopreserved human enterocytes represent a physiologically relevant and convenient in vitro experimental system for the evaluation of intestinal metabolism, akin to cryopreserved human hepatocytes for hepatic metabolism.
Collapse
Affiliation(s)
| | | | | | | | - Albert P Li
- In Vitro ADMET Laboratories, Columbia, Maryland
| |
Collapse
|
|
7
|
Ferreira A, Rodrigues M, Falcão A, Alves G. A Rapid and Sensitive HPLC–DAD Assay to Quantify Lamotrigine, Phenytoin and Its Main Metabolite in Samples of Cultured HepaRG Cells. J Chromatogr Sci 2016; 54:1352-8. [DOI: 10.1093/chromsci/bmw088] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2015] [Indexed: 12/29/2022]
|
|
8
|
Ferreira A, Rodrigues M, Falcão A, Alves G. HPLC–DAD Method for the Quantification of Carbamazepine, Oxcarbazepine and their Active Metabolites in HepaRG Cell Culture Samples. Chromatographia 2016. [DOI: 10.1007/s10337-016-3063-7] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
|
|
9
|
Gayathri L, Dhanasekaran D, Akbarsha MA. Scientific concepts and applications of integrated discrete multiple organ co-culture technology. J Pharmacol Pharmacother 2015; 6:63-70. [PMID: 25969651 PMCID: PMC4419250 DOI: 10.4103/0976-500x.155481] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2014] [Revised: 09/06/2014] [Accepted: 12/17/2014] [Indexed: 01/04/2023] Open
Abstract
Over several decades, animals have been used as models to investigate the human-specific drug toxicity, but the outcomes are not always reliably extrapolated to the humans in vivo. Appropriate in vitro human-based experimental system that includes in vivo parameters is required for the evaluation of multiple organ interaction, multiple organ/organ-specific toxicity, and metabolism of xenobiotic compounds to avoid the use of animals for toxicity testing. One such versatile in vitro technology in which human primary cells could be used is integrated discrete multiple organ co-culture (IdMOC). IdMOC system adopts wells-within-well concept that facilitates co-culture of cells from different organs in a discrete manner, separately in the respective media in the smaller inner wells which are then interconnected by an overlay of a universal medium in the large containing well. This novel in vitro approach mimics the in vivo situation to a great extent, and employs cells from multiple organs that are physically separated but interconnected by a medium that mimics the systemic circulation and provides for multiple organ interaction. Applications of IdMOC include assessment of multiple organ toxicity, drug distribution, organ-specific toxicity, screening of anticancer drugs, metabolic cytotoxicity, etc.
Collapse
Affiliation(s)
- Loganathan Gayathri
- Department of Microbiology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India ; Mahatma Gandhi Doerenkamp-Center, Bharathidasan University, Tiruchirappalli - 620024, Tamil Nadu, India
| | | | - Mohammad A Akbarsha
- Mahatma Gandhi Doerenkamp-Center, Bharathidasan University, Tiruchirappalli - 620024, Tamil Nadu, India
| |
Collapse
|
|
10
|
Li AP, Uzgare A, LaForge YS. Definition of metabolism-dependent xenobiotic toxicity with co-cultures of human hepatocytes and mouse 3T3 fibroblasts in the novel integrated discrete multiple organ co-culture (IdMOC) experimental system: results with model toxicants aflatoxin B1, cyclophosphamide and tamoxifen. Chem Biol Interact 2012; 199:1-8. [PMID: 22640811 DOI: 10.1016/j.cbi.2012.05.003] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2012] [Revised: 05/04/2012] [Accepted: 05/07/2012] [Indexed: 11/28/2022]
Abstract
The integrated discrete multiple organ co-culture system (IdMOC) allows the co-culturing of multiple cell types as physically separated cells interconnected by a common overlying medium. We report here the application of IdMOC with two cell types: the metabolically competent primary human hepatocytes, and a metabolically incompetent cell line, mouse 3T3 fibroblasts, in the definition of the role of hepatic metabolism on the cytotoxicity of three model toxicants: cyclophosphamide (CPA), aflatoxin B1 (AFB) and tamoxifen (TMX). The presence of hepatic metabolism in IdMOC with human hepatocytes was demonstrated by the metabolism of the P450 isoform 3A4 substrate, luciferin-IPA. The three model toxicants showed three distinct patterns of cytotoxic profile: TMX was cytotoxic to 3T3 cells in the absence of hepatocytes, with slightly lower cytotoxicity towards both 3T3 cells and hepatocytes in the IdMOC. AFB was selective toxic towards the human hepatocytes and relatively noncytotoxic towards 3T3 cells both in the presence and absence of the hepatocytes. CPA cytotoxicity to the 3T3 cells was found to be significantly enhanced by the presence of the hepatocytes, with the cytotoxicity dependent of the number of hepatocytes, and with the cytotoxicity attenuated by the presence of a non-specific P450 inhibitor, 1-aminobenzotriazole. We propose here the following classification of toxicants based on the role of hepatic metabolism as defined by the human hepatocyte-3T3 cell IdMOC assay: type I: direct-acting cytotoxicants represented by TMX as indicated by cytotoxicity in 3T3 cells in the absence of hepatocytes; type II: metabolism-dependent cytotoxicity represented by AFB1 with effects localized within the site of metabolic activation (i. e. hepatocytes); and type III: metabolism-dependent cytotoxicity with metabolites that can diffuse out of the hepatocytes to cause toxicity in cells distal from the site of metabolism, as exemplified by CPA.
Collapse
Affiliation(s)
- Albert P Li
- In Vitro ADMET Laboratories LLC, 9221 Rumsey Rd, Suite 8, Columbia, MD 21045, USA.
| | | | | |
Collapse
|
|
11
|
Sharma R, Strelevitz TJ, Gao H, Clark AJ, Schildknegt K, Obach RS, Ripp SL, Spracklin DK, Tremaine LM, Vaz ADN. Deuterium isotope effects on drug pharmacokinetics. I. System-dependent effects of specific deuteration with aldehyde oxidase cleared drugs. Drug Metab Dispos 2012; 40:625-34. [PMID: 22190693 DOI: 10.1124/dmd.111.042770] [Citation(s) in RCA: 79] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/13/2025] Open
Abstract
The pharmacokinetic properties of drugs may be altered by kinetic deuterium isotope effects. With specifically deuterated model substrates and drugs metabolized by aldehyde oxidase, we demonstrate how knowledge of the enzyme's reaction mechanism, species differences in the role played by other enzymes in a drug's metabolic clearance, and differences in systemic clearance mechanisms are critically important for the pharmacokinetic application of deuterium isotope effects. Ex vivo methods to project the in vivo outcome using deuterated carbazeran and zoniporide with hepatic systems demonstrate the importance of establishing the extent to which other metabolic enzymes contribute to the metabolic clearance mechanism. Differences in pharmacokinetic outcomes in guinea pig and rat, with the same metabolic clearance mechanism, show how species differences in the systemic clearance mechanism can affect the in vivo outcome. Overall, to gain from the application of deuteration as a strategy to alter drug pharmacokinetics, these studies demonstrate the importance of understanding the systemic clearance mechanism and knowing the identity of the metabolic enzymes involved, the extent to which they contribute to metabolic clearance, and the extent to which metabolism contributes to the systemic clearance.
Collapse
Affiliation(s)
- Raman Sharma
- Department of Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and Development, Groton, Connecticut 06340, USA
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
12
|
Fagerholm U. Prediction of human pharmacokinetics—evaluation of methods for prediction of hepatic metabolic clearance. J Pharm Pharmacol 2010; 59:803-28. [PMID: 17637173 DOI: 10.1211/jpp.59.6.0007] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Abstract
Methods for prediction of hepatic clearance (CLH) in man have been evaluated. A physiologically-based in-vitro to in-vivo (PB-IVIV) method with human unbound fraction in blood (fu,bl) and hepatocyte intrinsic clearance (CLint)-data has a good rationale and appears to give the best predictions (maximum ∼2-fold errors; < 25% errors for half of CL-predictions; appropriate ranking). Inclusion of an empirical scaling factor is, however, needed, and reasons include the use of cryopreserved hepatocytes with low activity, and inappropriate CLint- and fu,bl-estimation methods. Thus, an improvement of this methodology is possible and required. Neglect of fu,bl or incorporation of incubation binding does not seem appropriate. When microsome CLint-data are used with this approach, the CLH is underpredicted by 5- to 9-fold on average, and a 106-fold underprediction (attrition potential) has been observed. The poor performance could probably be related to permeation, binding and low metabolic activity. Inclusion of scaling factors and neglect of fu,bl for basic and neutral compounds improve microsome predictions. The performance is, however, still not satisfactory. Allometry incorrectly assumes that the determinants for CLH relate to body weight and overpredicts human liver blood flow rate. Consequently, allometric methods have poor predictability. Simple allometry has an average overprediction potential, > 2-fold errors for ∼1/3 of predictions, and 140-fold underprediction to 5800-fold overprediction (potential safety risk) range. In-silico methodologies are available, but these need further development. Acceptable prediction errors for compounds with low and high CLH should be ∼50 and ∼10%, respectively. In conclusion, it is recommended that PB-IVIV with human hepatocyte CLint and fu,bl is applied and improved, limits for acceptable errors are decreased, and that animal CLH-studies and allometry are avoided.
Collapse
Affiliation(s)
- Urban Fagerholm
- Clinical Pharmacology, AstraZeneca R&D Södertälje, S-151 85 Södertälje, Sweden.
| |
Collapse
|
|
13
|
Miranda JP, Leite SB, Muller-Vieira U, Rodrigues A, Carrondo MJT, Alves PM. Towards an extended functional hepatocyte in vitro culture. Tissue Eng Part C Methods 2009; 15:157-67. [PMID: 19072051 DOI: 10.1089/ten.tec.2008.0352] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Primary cultures of human hepatocytes are a reference cellular model, because they maintain key features of liver cells in vivo, such as expression of drug-metabolizing enzymes, response to enzyme inducers, and generation of hepatic metabolites. However, there is a restricted availability of primary hepatocytes, and they show phenotypic instability in culture. Thus, different alternatives have been developed to overcome the culture limitations and to mimic in vivo tissue material. Herein, culture conditions, such as medium composition, impeller type, and cell inoculum concentration, were optimized in stirred culture vessels and applied to a three-dimensional (3D) bioreactor system. Cultures of rat hepatocytes as 3D structures on bioreactor, better resembling in vivo cellular organization, were compared to traditional monolayer cultures. Liver-specific functions, such as albumin and urea secretion, phase I and phase II enzyme activities, and the capacity to metabolize diphenhydramine and troglitazone, were measured over time. Hepatocyte functions were preserved for longer time in the 3D bioreactor than in the monolayer system. Moreover, rat hepatocytes grown in 3D system maintained the ability to metabolize such compounds, as well as in vivo. Our results indicate that hepatocytes cultured as 3D structures are a qualified model system to study hepatocyte drug metabolism over a long period of time. Moreover, these cultures can be used as feeding systems to obtain cells for other tests in a short time.
Collapse
Affiliation(s)
- Joana P Miranda
- Animal Cell Technology Laboratory, IBET/ITQB-UNL, Apartado 12, Oeiras, Portugal
| | | | | | | | | | | |
Collapse
|
|
14
|
Blanchard N, Alexandre E, Abadie C, Lavé T, Heyd B, Mantion G, Jaeck D, Richert L, Coassolo P. Comparison of clearance predictions using primary cultures and suspensions of human hepatocytes. Xenobiotica 2008; 35:1-15. [PMID: 15788364 DOI: 10.1080/00498250400021820] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
Various incubation conditions of human hepatocytes were compared for their accuracy in predicting the in vivo hepatic clearance (CL(H)) of model compounds. The test compounds were the highly cleared, low protein bound naloxone (in vivo CL(H) = 25 ml min(-1) kg(-1); free fraction = 0.6), the medium clearance, highly protein bound midazolam (CL(H) = 12 ml min(-1) kg(-1); free fraction = 0.04) and the low clearance, highly protein bound bosentan (CL(H) = 3.9 ml min(-1) kg(-1); free fraction = 0.02). Each compound was tested in three 'hepatocyte systems', using resections from three donors, in the presence and absence of human serum. Those hepatocyte systems were: conventional primary cultures, freshly isolated suspensions and cryopreserved suspended hepatocytes. Except for a twofold overestimated CL(H) for bosentan from conventional primary cultures, and despite variable cryopreservation recoveries, similar predictions of CL(H) were recorded with all hepatocyte systems. Moreover, the CL(H) values obtained with cryopreserved suspended hepatocytes were similar to those obtained with freshly isolated suspensions. For midazolam and bosentan, the predicted in vivo CL(H) was markedly higher in the presence of serum, whereas serum had little influence on the scaled-up CL(H) of naloxone. In vivo, CL(H) was properly approached for naloxone and bosentan (particularly from experiments in the presence of serum), but it was strongly underestimated for midazolam (particularly in the absence of serum). Additional compounds need to be investigated to confirm the above findings as well as to assess why the clearances of some highly protein-bound compounds are still considerably underestimated.
Collapse
Affiliation(s)
- N Blanchard
- F. Hoffmann-La Roche AG, Pharmaceuticals Division, CH-4070 Basel, Switzerland
| | | | | | | | | | | | | | | | | |
Collapse
|
|
15
|
Abstract
Metabolism of melatonin (MEL) in mouse was evaluated through a metabolomic analysis of urine samples from control and MEL-treated mice. Besides identifying seven known MEL metabolites (6-hydroxymelatonin glucuronide, 6-hydroxymelatonin sulfate, N-acetylserotonin glucuronide, N-acetylserotonin sulfate, 6-hydroxymelatonin, 2-oxomelatonin, 3-hydroxymelatonin), principal components analysis of urinary metabolomes also uncovered seven new MEL metabolites, including MEL glucuronide, cyclic MEL, cyclic N-acetylserotonin glucuronide, cyclic 6-hydroxymelatonin; 5-hydroxyindole-3-acetaldehyde, di-hydroxymelatonin and its glucuronide conjugate. However, N(1)-acetyl-N(2)-formyl-5-methoxy-kynuramine and N(1)-acetyl-5-methoxy-kynuramine, known as MEL antioxidant products, were not detected in mouse urine. Metabolite profiling of MEL further indicated that 6-hydroxymelatonin glucuronide was the most abundant MEL metabolite in mouse urine, which comprised 75, 65, and 88% of the total MEL metabolites in CBA, C57/BL6, and 129Sv mice, respectively. Chemical identity of 6-hydroxymelatonin glucuronide was confirmed by deconjugation reactions using beta-glucuronidase and sulfatase. Compared with wild-type and CYP1A2-humanized mice, Cyp1a2-null mice yielded much less 6-hydroxymelatonin glucuronide (approximately 10%) but more N-acetylserotonin glucuronide (approximately 195%) and MEL glucuronide (approximately 220%) in urine. In summary, MEL metabolism in mouse was recharacterized by using a metabolomic approach, and the MEL metabolic map was extended to include seven known and seven novel pathways. This study also confirmed that 6-hydroxymelatonin glucuronide was the major MEL metabolite in the mouse, and suggested that there was no interspecies difference between humans and mice with regard to CYP1A2-mediated metabolism of MEL, but a significant difference in phase II conjugation, yielding 6-hydroxymelatonin glucuronide in the mouse and 6-hydroxymelatonin sulfate in humans.
Collapse
Affiliation(s)
- Xiaochao Ma
- Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA
| | | | | | | | | |
Collapse
|
|
16
|
Schmitmeier S, Langsch A, Jasmund I, Bader A. Development and characterization of a small-scale bioreactor based on a bioartificial hepatic culture model for predictive pharmacological in vitro screenings. Biotechnol Bioeng 2007; 95:1198-206. [PMID: 16807928 DOI: 10.1002/bit.21089] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
A vast majority of pharmacons are beset by possible interactions and side effects which have usually been tested in laboratory animals. However, better methods are needed to reduce the number of animal experiments and interspecies differences with respect to drug metabolism, as well as to provide a faster and more cost-effective way of analysis. These facts have led to the development of in vitro models based on isolated primary hepatocytes to better assess drug metabolism, interactions, and toxicity. A new small-scale bioreactor with the hepatic sandwich model and a gas-permeable membrane at the bottom allowing a definable oxygen exchange, has been constructed and compared with the conventional well plates. Compared to hepatocytes cultured in conventional systems, the cells exhibited a stronger liver-specific capacity and remained in a differentiated state in the small-scale bioreactor over a cultivation period of 17 days. This in vitro model could serve as a tool to predict the liver response to newly developed drugs.
Collapse
Affiliation(s)
- Stephanie Schmitmeier
- Biotechnological-Biomedical Center, Cell Techniques and Applied Stem Cell Biology, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
| | | | | | | |
Collapse
|
|
17
|
Cardoso HS, Bicalho B, Genari P, Santagada V, Caliendo G, Perissutti E, Donato JL, De Nucci G. In vitro mutagenicity of anti-inflammatory parsalmide analogues PA7, PA10, and PA31 triggered by biotransformation into hydroxy derivatives. Eur J Med Chem 2006; 41:408-16. [PMID: 16417948 DOI: 10.1016/j.ejmech.2005.10.019] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2005] [Revised: 10/21/2005] [Accepted: 10/26/2005] [Indexed: 11/21/2022]
Abstract
In this study, the mutagenicity of the anti-inflammatory parsalmide [5-amino-N-butyl-2-(2-propynyloxy)-benzamide] analogues PA7 [5-amino-N-butyl-2-cyclohexyloxy-benzamide], PA10 [5-amino-N-butyl-2-phenoxy-benzamide] and PA31 [5-amino-N-butyl-2-(p-tolyloxy)-benzamide] was determined by an Ames Salmonella assay. The experiments were performed by preincubating the compounds in the absence and presence of a post-mitochondrial fraction (S9) of rat liver homogenate from phenobarbital/beta-naphtoflavone treated rats. No mutagenic effect was observed after direct testing (no S9 added) in Salmonella typhymurium strains TA98, TA100, TA102, TA1535 and TA1537. However, in the presence of S9, the test substances triggered mutagenic responses in strains TA100 and TA98. PA31 presented the strongest mutagenic potential. The reversion rates in the presence of PA31 were about 2-19 fold higher than spontaneous mutation rates. In the presence of PA7, the reversion increased 2-14-fold over spontaneous rates. While PA10 showed a relatively mild mutagenic potential, as the number of revertants did not exceed 2.5 times the number of spontaneous mutations. Mass spectrometric analysis of the in vitro biotransformation showed that S9 converted (%), regioselectively, PA7 (19%), PA10 (7%) and PA31 (12%) into hydroxy-derivatives.
Collapse
Affiliation(s)
- H S Cardoso
- Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas, SP, Brazil
| | | | | | | | | | | | | | | |
Collapse
|
|
18
|
Ponsoda X, Donato MT, Perez-Cataldo G, Gómez-Lechón MJ, Castell JV. Drug metabolism by cultured human hepatocytes: how far are we from the in vivo reality? Altern Lab Anim 2005; 32:101-10. [PMID: 15601238 DOI: 10.1177/026119290403200207] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
The investigation of metabolism is an important milestone in the course of drug development. Drug metabolism is a determinant of drug pharmacokinetics variability in human beings. Fundamental to this are phenotypic differences, as well as genotypic differences, in the expression of the enzymes involved in drug metabolism. Genotypic variability is easy to identify by means of polymerase chain reaction-based or DNA chip-based methods, whereas phenotypic variability requires direct measurement of enzyme activities in liver, or, indirectly, measurement of the rate of metabolism of a given compound in vivo. There is a great deal of phenotypic variability in human beings, only a minor part being attributable to gene polymorphisms. Thus, enzyme activity measurements in a series of human livers, as well as in vivo studies with human volunteers, show that phenotypic variability is, by far, much greater than genotypic variability. In vitro models are currently used to investigate the hepatic metabolism of new compounds. Cultured human hepatocytes are considered to be the closest model to the human liver. However, the fact that hepatocytes are placed in a microenvironment that differs from that of the cells in the liver raises the question of to what extent drug metabolism variability observed in vitro actually reflects that in the liver in vivo. This issue has been examined by investigating the metabolism of the model compound, aceclofenac (an approved analgesic/anti-inflammatory drug), both in vitro and in vivo. Hepatocytes isolated from programmed liver biopsies were incubated with aceclofenac, and the metabolites formed were investigated by HPLC. The patients were given the drug during the course of clinical recovery, and the metabolites, largely present in urine, were analysed. In vitro and in vivo data from the same individual were compared. There was a good correlation between the in vitro and in vivo relative abundance of oxidised metabolites (4'-OH-aceclofenac + 4'-OH-diclofenac; Spearman's rho = 0.855), and the hydrolysis of aceclofenac (diclofenac + 4'-OH-aceclofenac + 4'-OH-diclofenac; rho = 0.691), while the conjugation of the drug in vitro was somewhat lower than in vivo. Globally, the metabolism of aceclofenac in vitro correlated with the amount of metabolites excreted in urine after 16 hours (rho = 0.95). Overall, although differing among assays, the in vitro/in vivo metabolism data for each patient were surprisingly similar. Thus, the variability observed in vitro appears to reflect genuine phenotypic variability among the donors.
Collapse
Affiliation(s)
- Xavier Ponsoda
- Unit of Experimental Hepatology, Research Centre, University Hospital La Fe, Avda. Campanar 21, 46009 Valencia, Spain
| | | | | | | | | |
Collapse
|
|
19
|
Gao J, Ann Garulacan L, Storm SM, Hefta SA, Opiteck GJ, Lin JH, Moulin F, Dambach DM. Identification of in vitro protein biomarkers of idiosyncratic liver toxicity. Toxicol In Vitro 2004; 18:533-41. [PMID: 15130611 DOI: 10.1016/j.tiv.2004.01.012] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2003] [Accepted: 01/28/2004] [Indexed: 10/26/2022]
Abstract
Drug-induced idiosyncratic hepatotoxicity continues to be an important safety issue for the pharmaceutical industry. This toxicity is due, in part, to the limited predictive nature of current pre-clinical study systems. A hypothesis was formed that treatment of existing in vitro hepatocyte cultures with drugs clinically linked to idiosyncratic hepatotoxicity would result in the release of extracellular protein biomarkers indicative of liver toxicity. To test this hypothesis, a combination of proteomic and immunological techniques were used to first identify, and subsequently verify, components of the protein-laden conditioned culture media from immortalized human hepatocytes which overexpressed cytochrome p450 3A4. These cells were treated separately with seven individual compounds made up of a combination of thiazolidinedione and l-tyrosine PPARgamma agonists and HIV protease inhibitors, plus a vehicle control (dimethyl sulfoxide). For each drug class, clinically determined hepatotoxic and non-hepatotoxic compounds were compared. Two proteins, BMS-PTX-265 and BMS-PTX-837, were reproducibly and significantly increased in the conditioned media from cells treated with each of the toxic compounds as compared to media from cells treated with the non-toxic compounds (and vehicle). This result supported the hypothesis, and so a series of successive assays (western blots and enzyme linked immunosorbent assays) were used to measure the response of these two proteins as a function of an expanded set of 20 compounds. For all 20 drugs, elevations of BMS-PTX-265 correlated exactly with the known safety profile; whereas changes in BMS-PTX-837 correctly predicted the safety profile in 19 of 20 drugs (one false negative). In summary, the data supports both the pre-clinical in vitro method as a means to identify new biomarkers of liver toxicity, as well as the validity of the biomarkers themselves.
Collapse
Affiliation(s)
- Ji Gao
- Clinical Discovery, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, Princeton, NJ, USA
| | | | | | | | | | | | | | | |
Collapse
|
|
20
|
Deglmann CJ, Ebner T, Ludwig E, Happich S, Schildberg FW, Koebe HG. Protein binding capacity in vitro changes metabolism of substrates and influences the predictability of metabolic pathways in vivo. Toxicol In Vitro 2004; 18:835-40. [PMID: 15465650 DOI: 10.1016/j.tiv.2004.04.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2003] [Accepted: 04/02/2004] [Indexed: 10/26/2022]
Abstract
UNLABELLED Large numbers of lipophilic molecules are attached to fractions of serum protein, e.g. albumin, in vivo. Cell culture medium of most in vitro hepatocyte models for the prediction of metabolism does not contain albumin. Consequently, in vitro availability and metabolism of substrates could differ significantly from the in vivo situation. The influence of albumin on the in vitro metabolism was tested on a new lipophilic compound. METHODS Primary human and rat hepatocytes were cultured in a collagen sandwich configuration and incubated with (14)C-labeled compound X127 that is known to exhibit a high propensity to bind to plastic surfaces. Groups contained either 1% (w/v) BSA or none. Substrates as well as metabolism products were determined with radio-HPLC and radioactivity levels in the medium were recorded. RESULTS Quantitative differences were seen in the distribution of the compound in BSA and non BSA containing groups, thus indicating a substantial binding of the compound to polystyrol surfaces of cell culture dishes. Metabolic radio-HPLC profiles showed different patterns after 24 h of incubation between the two species as well as between the BSA- and non-BSA groups within the species. CONCLUSIONS With addition of albumin the adherance of lipophilic substrates and metabolites to cell culture dish surfaces can be neutralized and in vitro systems can more closely mimic the in vivo situation.
Collapse
Affiliation(s)
- C J Deglmann
- Department of Surgery, Klinikum Grosshadern, LMU-Munich, Marchioninistr, 15, 81377 Munich, Germany.
| | | | | | | | | | | |
Collapse
|
|
21
|
Blanchard N, Richert L, Notter B, Delobel F, David P, Coassolo P, Lavé T. Impact of serum on clearance predictions obtained from suspensions and primary cultures of rat hepatocytes. Eur J Pharm Sci 2004; 23:189-99. [PMID: 15451007 DOI: 10.1016/j.ejps.2004.07.007] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2004] [Revised: 06/28/2004] [Accepted: 07/08/2004] [Indexed: 11/23/2022]
Abstract
The objective of the present study was to compare two configurations of the hepatocyte model namely suspensions (SH) and conventional primary cultures (CPC) for their ability to predict the hepatic clearance in vivo in the rat and, to investigate the impact of serum on the prediction accuracy. The metabolic competences of several cytochrome P450 isoenzymes were investigated both in CPC and SH in the presence or absence of serum. Under the same conditions, the in vitro intrinsic clearance of six test compounds metabolised by a variety of phase I and phase II enzymes (antipyrine, RO-X, mibefradil, midazolam, naloxone and oxazepam) were derived from Vmax/Km scaled up to the corresponding in vivo hepatic metabolic clearance. CYP activities were shown to be stable in both CPC and SH for up to 6 h of incubation, except for the CYP 3A1 activity that decreased in CPC even in the presence of serum. Moreover, the clearances predicted from SH in the presence of serum were closer to the in vivo values than those obtained from CPC. SH represent a convenient model to assess the hepatic metabolism of xenobiotics, the presence of serum in the incubation medium significantly improved in several instances the quality of the predictions.
Collapse
Affiliation(s)
- Nadège Blanchard
- Pharma Research Basel (70/131), F. Hoffmann-LaRoche Ltd., Pharmaceuticals Division, Grenzacherstrasse No. 124, CH 4070 Basel, Switzerland
| | | | | | | | | | | | | |
Collapse
|
|
22
|
Ponsoda X, Pareja E, Gómez-Lechón MJ, Fabra R, Carrasco E, Trullenque R, Castell JV. Drug biotransformation by human hepatocytes. In vitro/in vivo metabolism by cells from the same donor. J Hepatol 2001; 34:19-25. [PMID: 11211902 DOI: 10.1016/s0168-8278(00)00085-4] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
BACKGROUND/AIMS Cultured human hepatocytes are considered a close model to human liver. However, the fact that hepatocytes are placed in a microenvironment that differs from that of the cell in the liver raises the question: to what extent does drug metabolism in vitro reflect that of the liver in vivo? This issue was examined by investigating the in vitro and in vivo metabolism of aceclofenac, an analgesic/anti-inflammatory drug. METHODS Hepatocytes isolated from programmed liver biopsies were incubated with aceclofenac, and the metabolites formed were investigated by HPLC. During the course of clinical recovery, patients were given the drug, and the metabolites, largely present in the urine, were analyzed. In vitro and in vivo data of the same individual were compared. RESULTS The relative abundance of oxidized metabolites in vitro (i.e. 4'OH-aceclofenac + 4'OH-diclofenac vs. total hydroxylated metabolites; Spearman's p = 0.855), as well the hydrolysis of aceclofenac (4'OH-diclofenac vs. 4'OH-aceclofenac + 4'OH-diclofenac; p = 0.691) correlated well with in vivo data. The conjugation of the drug in vitro (24.6 +/- 7.6%) was lower than that in vivo (44.9 +/- 5.3%). The rate of 4'OH-aceclofenac formation in vitro correlated with the amount of metabolites excreted in urine after 16 h (p = 0.95). CONCLUSIONS The in vitro/in vivo metabolism of the drug was surprisingly similar in each patient. The variability observed in vitro reflected an existing phenotypic variability among donors.
Collapse
Affiliation(s)
- X Ponsoda
- Unidad de Hepatología Experimental, Centro de Investigación, Hospital Universitario la Fe, Valencia, Spain
| | | | | | | | | | | | | |
Collapse
|
|
23
|
Chapman D, Michener S, Powis G. Adenine and pyridine nucleotide concentrations and relationships to 2,6-dinitrotoluene metabolism in cultured rat liver slices. Toxicol In Vitro 1994; 8:343-9. [DOI: 10.1016/0887-2333(94)90155-4] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/1993] [Revised: 10/12/1993] [Indexed: 11/26/2022]
|
|
24
|
Ramsby ML, Makowski GS, Khairallah EA. Differential detergent fractionation of isolated hepatocytes: biochemical, immunochemical and two-dimensional gel electrophoresis characterization of cytoskeletal and noncytoskeletal compartments. Electrophoresis 1994; 15:265-77. [PMID: 8026443 DOI: 10.1002/elps.1150150146] [Citation(s) in RCA: 100] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Two-dimensional (2-D) gel electrophoresis is often used in toxicologic and metabolic studies to assess treatment- or stage-specific changes in protein synthesis, degradation or posttranslational modification. When combined with cell fractionation studies the detectability of low abundance proteins is enhanced, and changes in subcellular distribution of proteins can also be monitored. Detergent fractionation is a simpler alternative to differential pelleting, which partitions cellular constituents into functionally distinct populations while preserving cytoskeletal integrity. We defined and characterized a differential detergent fractionation (DDF) protocol to enable protein dynamics in cytoskeletal and noncytoskeletal compartments of isolated hepatocytes to be monitored simultaneously. Rat hepatocytes were maintained in suspension culture and fractionated by sequential extraction with detergent-containing buffers (digitonin/EDTA, Triton/EDTA, Tween/deoxycholate). DDF reproducibly yielded four electrophoretically distinct fractions enriched in cytosolic, membrane-organelle, nuclear membrane and cytoskeletal-matrix markers, respectively. Immunoblotting with over 20 different antibodies corroborated the selectivity of fractionation and was used to characterize the distribution profiles of cytoskeletal (actin, tubulins, cytokeratins, vinculin, myosin, desmoplakins, fodrin, nuclear lamins) and noncytoskeletal proteins (heat-shock 70 proteins, glutathione-S-transferase, calpains, carbamoyl phosphate synthetase, etc.), as well as to identify spots in 2-D gels. Detergent buffers were compatible with equilibrium or nonequilibrium 2-D gel electrophoretic analysis. Extensive 2-D maps of acidic and basic proteins in each fraction were generated along with a tabular listing of M(r) and pI. Thus, DDF reproducibly partitions hepatocytic proteins into functionally distinct cytoskeletal and noncytoskeletal compartments that are readily analyzed by 2-D gel electrophoresis. DDF is simple, applicable to use with other cell types or culture systems and is especially useful when biomaterial is limited (i.e., clinical studies).
Collapse
Affiliation(s)
- M L Ramsby
- Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269
| | | | | |
Collapse
|
|
25
|
Sandker GW, Vos RM, Delbressine LP, Slooff MJ, Meijer DK, Groothuis GM. Metabolism of three pharmacologically active drugs in isolated human and rat hepatocytes: analysis of interspecies variability and comparison with metabolism in vivo. Xenobiotica 1994; 24:143-55. [PMID: 8017089 DOI: 10.3109/00498259409043228] [Citation(s) in RCA: 52] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
1. The metabolism of the three drugs (Org GB 94, Org 3770 and Org OD 14) was studied in isolated human and rat hepatocytes. The metabolic profiles in rat and human hepatocytes were compared with the available in vivo data in both species. 2. All three drugs were metabolized extensively under the conditions used, both in human and rat hepatocytes, showing both extensive phase I and II metabolism. 3. During 3-h incubation with rat hepatocytes the three compounds were metabolized completely, whereas incubation with human hepatocytes only resulted in partial metabolism, amounting for 58% (Org GB 94), 36% (Org 3770) and 94% (Org OD 14) of the dose. In addition, rat hepatocytes excreted relatively more of the formed metabolites than human hepatocytes. 4. For both species, the metabolites formed in the isolated cells were quite similar to those found in vivo. With respect to Org GB 94 and Org 3770, metabolites were detected in man in vivo and in isolated human hepatocytes that were not found in any of the animal species studied previously. 5. The reflection of interspecies differences in isolated hepatocytes, with respect to both metabolite profiles and human-specific metabolites, renders isolated human hepatocytes a very valuable tool during preclinical drug development.
Collapse
Affiliation(s)
- G W Sandker
- Department of Pharmacology and Therapeutics, University Centre for Pharmacy, Groningen, The Netherlands
| | | | | | | | | | | |
Collapse
|
|
26
|
Azer SA, Stacey NH. Differential effects of cyclosporin A on the transport of bile acids by human hepatocytes. Biochem Pharmacol 1993; 46:813-9. [PMID: 8373435 DOI: 10.1016/0006-2952(93)90489-j] [Citation(s) in RCA: 30] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Cyclosporin A (CsA) treatment has been reported to cause rises in serum bile acids both in humans and rats. It has also been shown to suppress bile flow in situ in rats and inhibit the transport of bile salts by rat hepatocytes. The purpose of this study was to examine the influence of CsA on uptake of radiolabelled cholate (CA), glycocholate (GC) and taurocholate (TC) by isolated human hepatocytes. CsA did not significantly change Vmax for CA uptake [0.23 +/- 0.01 vs 0.25 +/- 0.02 nmol/mg protein/min for control and CsA (10 microM), respectively], but significantly increased Km (37 +/- 2 vs 86 +/- 8 microM). Similarly, Vmax for TC uptake was not affected (0.51 +/- 0.02 vs 0.67 +/- 0.05 nmol/mg protein/min) while Km was significantly increased [46 +/- 3 vs 109 +/- 11 microM for control and CsA (10 microM), respectively]. On the other hand, neither Vmax nor Km for GC uptake was affected by CsA. The data indicate a competitive pattern of inhibition induced by CsA on CA and TC uptake. Furthermore, CsA was found to cause a dose-related inhibition of accumulation of both cholate and taurocholate, but not GC accumulation. None of the concentrations of CsA showed a significant effect on the integrity of the human hepatocytes as assessed by ALT (alanine aminotransferase), AST (aspartate aminotransferase) and LDH (lactate dehydrogenase) release. The findings, in human hepatocytes, are generally consistent with the observations reported from rodent studies. They strongly support the contention that serum bile acid increases in CsA-treated patients are due to interference with the hepatocellular transport and accumulation of particular bile acids.
Collapse
Affiliation(s)
- S A Azer
- National Institute of Occupational Health and Safety, University of Sydney, Australia
| | | |
Collapse
|
|
27
|
Dauphin JF, Graviere C, Bouzard D, Rohou S, Chesne C, Guillouzo A. Comparative metabolism of tosufloxacin and BMY 43748 in hepatocytes from rat, dog, monkey and man. Toxicol In Vitro 1993; 7:499-503. [DOI: 10.1016/0887-2333(93)90054-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
|
|
28
|
Lavrijsen K, van Houdt J, van Dyck D, Hendrickx J, Bockx M, Hurkmans R, Meuldermans W, Le Jeune L, Lauwers W, Heykants J. Comparative metabolism of flunarizine in rats, dogs and man: an in vitro study with subcellular liver fractions and isolated hepatocytes. Xenobiotica 1992; 22:815-36. [PMID: 1455902 DOI: 10.3109/00498259209053143] [Citation(s) in RCA: 31] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
1. The biotransformation of 3H-flunarizine ((E)-1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propenyl)piperazine dihydrochloride, FLUN) was studied in subcellular liver fractions (microsomes and 12,000 g fraction) and in suspensions or primary cell cultures of isolated hepatocytes of rats, dogs and man. The major in vitro metabolites were characterized by h.p.l.c. co-chromatography and/or by mass spectrometric analysis. 2. The kinetics of FLUN metabolism was studied in microsomes of dog and man. The metabolism followed linear Michaelis-Menten kinetics over the concentration range 0.1-20 microM FLUN. 3. A striking sex difference was observed for the in vitro metabolism of FLUN in rat. In male rats, oxidative N-dealkylation at one of the piperazine nitrogens, resulting in bis(4-fluorophenyl) methanol, was a major metabolic pathway, whereas aromatic hydroxylation at the phenyl of the cinnamyl moiety, resulting in hydroxy-FLUN, was a major metabolic pathway in female rats. In incubates with hepatocytes, these two metabolites were converted to the corresponding glucuronides. 4. In human subcellular fractions, aromatic hydroxylation to hydroxy-FLUN was the major metabolic pathway. In primary cell cultures of human hepatocytes, oxidative N-dealkylation at the 1- and 4-piperazine nitrogen and glucuronidation of bis(4-fluorophenyl)methanol were observed. The in vitro metabolism of FLUN in humans, resembled more than in female rats and in dogs than that in male rats. 5. The present in vitro results are compared with data of previous in vivo studies in rats and dogs. The use of subcellular fractions and/or isolated hepatocytes for the study of species differences in the biotransformation of xenobiotics is discussed.
Collapse
Affiliation(s)
- K Lavrijsen
- Department of Drug Metabolism and Pharmacokinetics, Janssen Research Foundation, Beerse, Belgium
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
29
|
Berry MN, Halls HJ, Grivell MB. Techniques for pharmacological and toxicological studies with isolated hepatocyte suspensions. Life Sci 1992; 51:1-16. [PMID: 1614269 DOI: 10.1016/0024-3205(92)90212-8] [Citation(s) in RCA: 66] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Since its introduction in 1969, the high-yield preparation of isolated hepatocytes has become a frequently used tool for the study of hepatic uptake, excretion, metabolism and toxicity of drugs and other xenobiotics. Basic preparative methods are now firmly established involving perfusion of the liver with a balanced-saline solution containing collagenase. Satisfactory procedures are available for determining cell yields, for expressing cellular activities and for establishing optimal incubation conditions. Gross cellular damage can be detected by means of trypan blue or by measuring enzyme leakage, and damaged cells can be removed from the preparation. Specialized techniques are available for preparing hepatocyte couplets and suspensions enriched with periportal or perivenous hepatocytes. The isolated hepatocyte preparation is particularly convenient for the study of the kinetics of hepatic drug uptake and excretion because the cells can be rapidly separated from the incubation medium. Isolated liver cells have also proved valuable for investigating drug metabolism since they show many of the features of the intact liver. However, they also show important differences such as losses of membrane specialization, some degree of cell polarity and the capacity to form bile. The many consequences of the hepatic toxicity of xenobiotics including lipid peroxidation, free radical formation, glutathione depletion, and covalent binding to macromolecules are also readily studied with the isolated liver cell preparation. A particular advantage is the ease with which morphological changes as a result of drug exposure can be observed in isolated hepatocytes. However, it must be remembered that the isolation procedure inevitably introduces changes that may make the cells more susceptible than the normal liver to damage by xenobiotic agents. Despite its limitations, the isolated hepatocyte preparation is now firmly established in the armamentarium of the investigator examining the interaction of the liver with xenobiotics.
Collapse
Affiliation(s)
- M N Berry
- Department of Medical Biochemistry, School of Medicine, Flinders University of South Australia, Adelaide
| | | | | |
Collapse
|