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Title: Two compartment model of diazepam biotransformation in an organotypical culture of primary human hepatocytes

Abstract

Drug biotransformation is one of the most important parameters of preclinical screening tests for the registration of new drug candidates. Conventional existing tests rely on nonhuman models which deliver an incomplete metabolic profile of drugs due to the lack of proper CYP450 expression as seen in human liver in vivo. In order to overcome this limitation, we used an organotypical model of human primary hepatocytes for the biotransformation of the drug diazepam with special reference to metabolites in both the cell matrix phase and supernatant and its interaction of three inducers (phenobarbital, dexamethasone, aroclor 1254) in different time responses (1, 2, 4, 8, 24 h). Phenobarbital showed the strongest inducing effect in generating desmethyldiazepam and induced up to a 150 fold increase in oxazepam-content which correlates with the increased availability of the precursor metabolites (temazepam and desmethyldiazepam). Aroclor 1254 and dexamethasone had the strongest inducing effect on temazepam and the second strongest on oxazepam. The strong and overlapping inductive role of phenobarbital strengthens the participation of CYP2B6 and CYP3A in diazepam N-demethylation and CYP3A in temazepam formation. Aroclor 1254 preferentially generated temazepam due to the interaction with CYP3A and potentially CYP2C19. In parallel we represented these data in the formmore » of a mathematical model with two compartments explaining the dynamics of diazepam metabolism with the effect of these other inducers in human primary hepatocytes. The model consists of ten differential equations, with one for each concentration c{sub i,j} (i = diazepam, temazepam, desmethyldiazepam, oxazepam, other metabolites) and one for each compartment (j = cell matrix phase, supernatant), respectively. The parameters p{sub k} (k = 1, 2, 3, 4, 13) are rate constants describing the biotransformation of diazepam and its metabolites and the other parameters (k = 5, 6, 7, 8, 9, 10, 11, 12, 14, 15) explain the concentration changes in the two compartments.« less

Authors:
 [1];  [2]; ;  [1];  [3];  [4]
  1. Center for Biotechnology and Biomedicine, Cell Techniques and Applied Stem Cell Biology, Universitaet Leipzig, Deutscher Platz 5, D-04103 Leipzig (Germany)
  2. (Germany)
  3. Molecular and Applied Microbiology, Beutenbergstrasse 11a, 07745 Jena Leipniz Institute for Natural Product Research and INFECTION BIOLOGY Hans-Knoll-Institute, Gena (Germany)
  4. Center for Biotechnology and Biomedicine, Cell Techniques and Applied Stem Cell Biology, Universitaet Leipzig, Deutscher Platz 5, D-04103 Leipzig (Germany), E-mail: augustinus.bader@bbz.uni-leipzig.de
Publication Date:
OSTI Identifier:
21182704
Resource Type:
Journal Article
Resource Relation:
Journal Name: Toxicology and Applied Pharmacology; Journal Volume: 234; Journal Issue: 2; Other Information: DOI: 10.1016/j.taap.2008.09.029; PII: S0041-008X(08)00413-4; Copyright (c) 2008 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; DEXAMETHASONE; DIFFERENTIAL EQUATIONS; HUMAN POPULATIONS; IN VIVO; LIVER; LIVER CELLS; METABOLISM; METABOLITES; PHENOBARBITAL; REACTION KINETICS

Citation Formats

Acikgoez, Ali, Department of Surgery, Universitaet Leipzig, Liebig Str. 20, D-04103 Leipzig, Karim, Najibulla, Giri, Shibashish, Schmidt-Heck, Wolfgang, and Bader, Augustinus. Two compartment model of diazepam biotransformation in an organotypical culture of primary human hepatocytes. United States: N. p., 2009. Web. doi:10.1016/j.taap.2008.09.029.
Acikgoez, Ali, Department of Surgery, Universitaet Leipzig, Liebig Str. 20, D-04103 Leipzig, Karim, Najibulla, Giri, Shibashish, Schmidt-Heck, Wolfgang, & Bader, Augustinus. Two compartment model of diazepam biotransformation in an organotypical culture of primary human hepatocytes. United States. doi:10.1016/j.taap.2008.09.029.
Acikgoez, Ali, Department of Surgery, Universitaet Leipzig, Liebig Str. 20, D-04103 Leipzig, Karim, Najibulla, Giri, Shibashish, Schmidt-Heck, Wolfgang, and Bader, Augustinus. 2009. "Two compartment model of diazepam biotransformation in an organotypical culture of primary human hepatocytes". United States. doi:10.1016/j.taap.2008.09.029.
@article{osti_21182704,
title = {Two compartment model of diazepam biotransformation in an organotypical culture of primary human hepatocytes},
author = {Acikgoez, Ali and Department of Surgery, Universitaet Leipzig, Liebig Str. 20, D-04103 Leipzig and Karim, Najibulla and Giri, Shibashish and Schmidt-Heck, Wolfgang and Bader, Augustinus},
abstractNote = {Drug biotransformation is one of the most important parameters of preclinical screening tests for the registration of new drug candidates. Conventional existing tests rely on nonhuman models which deliver an incomplete metabolic profile of drugs due to the lack of proper CYP450 expression as seen in human liver in vivo. In order to overcome this limitation, we used an organotypical model of human primary hepatocytes for the biotransformation of the drug diazepam with special reference to metabolites in both the cell matrix phase and supernatant and its interaction of three inducers (phenobarbital, dexamethasone, aroclor 1254) in different time responses (1, 2, 4, 8, 24 h). Phenobarbital showed the strongest inducing effect in generating desmethyldiazepam and induced up to a 150 fold increase in oxazepam-content which correlates with the increased availability of the precursor metabolites (temazepam and desmethyldiazepam). Aroclor 1254 and dexamethasone had the strongest inducing effect on temazepam and the second strongest on oxazepam. The strong and overlapping inductive role of phenobarbital strengthens the participation of CYP2B6 and CYP3A in diazepam N-demethylation and CYP3A in temazepam formation. Aroclor 1254 preferentially generated temazepam due to the interaction with CYP3A and potentially CYP2C19. In parallel we represented these data in the form of a mathematical model with two compartments explaining the dynamics of diazepam metabolism with the effect of these other inducers in human primary hepatocytes. The model consists of ten differential equations, with one for each concentration c{sub i,j} (i = diazepam, temazepam, desmethyldiazepam, oxazepam, other metabolites) and one for each compartment (j = cell matrix phase, supernatant), respectively. The parameters p{sub k} (k = 1, 2, 3, 4, 13) are rate constants describing the biotransformation of diazepam and its metabolites and the other parameters (k = 5, 6, 7, 8, 9, 10, 11, 12, 14, 15) explain the concentration changes in the two compartments.},
doi = {10.1016/j.taap.2008.09.029},
journal = {Toxicology and Applied Pharmacology},
number = 2,
volume = 234,
place = {United States},
year = 2009,
month = 1
}
  • Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine modelmore » along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes.« less
  • An alkaline unwinding assay was used to quantitate the induction of DNA strand breaks (DNA SB) in the livers of rats and mice treated in vivo, in rodent hepatocytes in primary culture, and in CCRF-CEM cells, a human lymphoblastic leukemia cell line, following treatment with tri-(TCA), di-(CA), and mono-(MCA) chloroacetic acid and their corresponding aldehydes, tri-(chloralhydrate, CH), di(DCAA) and mono-(CAA) chloroacetaldehyde. None of the chloracetic acids induced DNA SB in the livers of rats at 4 hr following a single administration of 1-10 mmole/kg. TCA (10 mmole/kg) and DCA (5 and 10 mmole/kg) did produce a small amount of strandmore » breakage in mice (7% at 4hr) but not at 1 hr. N-nitrosodiethylamine (DENA), an established alkylating agent and a rodent hepatocarcinogen, produced DNA SB in the livers of both species. TCA, DCA, and MCA also failed to induce DNA strand breaks in splenocytes and epithelial cells derived from the stomach and duodenum of mice treated in vivo. None of the three chloroacetaldehydes induced DNA SB in either mouse or rat liver. These studies provide further evidence that the chloroacetic acids lack genotoxic activity not only in rodent liver, a tissue in that they induce tumors, but in a variety of other rodent tissues and cultured cell types. Two of the chloroacetaldehydes, DCAA and CAA, are direct acting DNA damaging agents in CCRF-CEM cells, but not in liver or splenocytes in vivo or in cultured hepatocytes. CH showed no activity in any system investigated. 58 refs., 6 figs., 2 tabs.« less
  • The pioneering studies of Berry and Friend on isolation of hepatocytes and that of Bissel et al on culturing hepatocytes in nonproliferating monolayers demonstrated the feasibility of using isolated parenchymal cells to study functional aspects of the liver. The obvious advantage of such a system is that one can study metabolic events in a defined, easily manipulated population of cells for periods of hours or even days. This review focuses on nonproliferating hepatic parenchymal cells in monolayer culture as a model for investigating lipoprotein synthesis and degradation. 60 references, 1 table.
  • Rat liver parenchymal cells were cultured with 1-48 ..mu..M Zn for 18 h. Lactate dehydrogenase activity of the medium showed that the zinc concentrations used did not cause cell leakage. Incubation with zinc caused increases in cellular metallothionein from 0.1 mM at 1 ..mu..M Zn to 1.0 mM at 48 ..mu..M. Free radical and peroxidation production over a subsequent 2h period induced in the hepatocytes by 3-methylindole (8mM,3MI), t-butylhydroperoxide (2mM, TBHP) or a mixture of ferric -NTA and ascorbic acid (each at 10 ..mu..M, FE + AA) were measured. Peroxidation was assessed by malondialdehyde (MDA) content and free radicals bymore » electron paramagnetic resonance (EPR) techniques. Supplemental zinc suppressed the production of both MDA and free radicals (as assessed by the amplitude of the EPR spectra) in the cells induced by either 3MI, TBHP or FE + AA. By virtue of its high sulfhydryl content, metallothionein may play a role in free radical scavenging. However, as the zinc concentration in the media was increased so the activity of the microsomal enzyme NADPH cytochrome c reductase was significantly decreased. Thus, it could be that the change in this enzyme activity reflects in part, the mechanism by which zinc is exerting its influence in suppression of free radical production.« less
  • Human {sup 125}I-plasminogen bound readily to rat hepatocytes in primary culture at 4 {degree}C and at 37{degree}C. Binding was inhibited by lysine and reversed by lysine, epsilon-aminocaproic acid, or nonradiolabeled plasminogen. The Kd for binding of {sup 125}I-plasminogen to hepatocytes was 0.59 +/- 0.16 mumol/L, as determined from the saturation isotherm by nonlinear regression (r2 = 0.99) and the Scatchard transformation by linear regression (r2 = 0.93). The number of sites per cell was 14.1 +/- 1.1 x 10(6). Fibrinogen synthesis and secretion by hepatocytes was insufficient to account for the major fraction of plasminogen binding, as determined by enzyme-linkedmore » immunosorbent assay (ELISA). Polyacrylamide gel electrophoresis and trichloroacetic acid precipitation studies demonstrated that plasminogen is neither activated nor degraded when bound to hepatocytes at 37{degree}C. Thin slices of whole rat liver (500 microns), isolated and prepared totally at 4{degree}C, bound {sup 125}I-plasminogen. Binding was inhibited by lysine. {sup 125}I-albumin binding to liver slices was minimal and not inhibited by lysine. Activation of plasminogen by tissue plasminogen activator (t-PA) was enhanced by hepatocytes in primary culture. When lysine was included in the media, the enhanced rate of activation was no longer observed. After activation with t-PA, much of the plasmin remained associated with hepatocyte surfaces and was partially protected from inhibition by alpha 2-antiplasmin. These studies suggest that hepatocyte plasminogen binding sites may provide important surface anticoagulant activity.« less