KaLy Cell

Plobsheim, France

KaLy Cell

Plobsheim, France
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Pekthong D.,KaLy Cell | Pekthong D.,Naresuan University | Desbans C.,KaLy Cell | Martin H.,University of Franche Comte | And 2 more authors.
Drug Metabolism and Disposition | Year: 2012

Benzyloxyresorufin-O-dealkylation (BROD) is usually used as a marker of cytochrome P450 (P450) 2B1 in rat. However, some reports show that CYP1A2 is also highly implicated. The purpose of the present study was to establish bupropion (BUP) hydroxylation, but not BROD, as a selective in vitro marker of CYP2B1 catalytic activity. IC 50 for BROD and BUP hydroxylation were equivalent (40.8 ± 4.6 and 41.8 ± 3.4 μM, respectively) when using liver microsomes from β-naphthoflavone-pretreated rats in the presence of metyrapone (CYP2B1 inhibitor). When using the same microsomes in the presence of CYP1A1/2-selective inhibitor α-naphthoflavone, we found an IC 50 of 2.5 × 10 -3 ± 0.8 × 10 -3 μM for BROD and >100 μM for BUP hydroxylation. These results suggest that CYP2B1 is similarly involved in both activities, whereas CYP1A2 is involved in BROD activity but not in BUP hydroxylation. BUP hydroxylation was assessed in microsomes from baculovirus-infected insect cells coexpressing NADPH-P450 oxidoreductase, and 14 rat P450s and kinetic parameters (K m and V max) were determined. BUP hydroxylation was predominantly catalyzed by CYP2B1 (75% of total hydroxybupropion formation), low activity was detected with CYP2E1 and CYP2C11 (10.9 and 8.7% of total hydroxybupropion, respectively), and activity was almost undetectable with the other P450 isoforms at saturating substrate concentrations (2500 μM), thereby validating the use of BUP as a diagnostic in vitro marker of CYP2B1 catalytic activity in rat. Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics.


Oorts M.,Catholic University of Leuven | Richert L.,KaLy Cell | Richert L.,University of Franche Comte | Annaert P.,Catholic University of Leuven
Journal of Pharmacological and Toxicological Methods | Year: 2015

Introduction: In vitro identification of compounds that cause cholestasis in vivo still remains a problem in pharmaceutical R&D. Currently existing in vitro systems show poor predictivity towards the clinical situation. Recently, our research group developed a model, based on sandwich-cultured (rat) hepatocytes (SC(R)H), to detect compounds causing cholestasis via altered bile acid (BA) homeostasis (Chatterjee et al., 2014). In the present study, we assessed whether this model performs equally well with freshly-isolated and cryopreserved hepatocytes. Methods: We exposed sandwich cultures from rat hepatocytes before and after cryopreservation to the cholestatic compounds, cyclosporin A (CsA) and troglitazone (Tro), in the presence and in the absence of a BA mixture. At the end of the incubations, the capability of the hepatocytes to produce urea was measured to determine changes in the drug-induced cholestasis index (DICI). Results: The mean (±SEM) urea production was significantly higher in sandwich cultures from freshly-isolated hepatocytes (27.88 (±0.96) nmol/cm2), compared to cultures from cryopreserved hepatocytes (22.86 (±1.91) nmolurea/cm2). However, after normalization for confluence rate (based on light microscopic image analysis), it appeared that the urea production was similar for all the batches of SCRH. The mean (±SEM) DICI values for CsA 10μM and Tro 75μM were 0.89 (±0.03) and 0.93 (±0.03), respectively. Higher concentrations, CsA (≥15μM) and Tro (≥100μM), elicited a significant decrease in urea production when incubated in the presence of a BA mixture compared to the compound alone. This was the case for all the batches of SCRH, irrespective of cryopreservation history. Discussion: In conclusion, no significant differences were seen when the previously described in vitro cholestasis model was applied in SCRH before or after cryopreservation. This study demonstrates the robustness of the model, which implies that it can be used with SCRH obtained from both freshly-isolated and cryopreserved hepatocytes. © 2015 Elsevier Inc.


Mueller S.O.,Merck KGaA | Mueller S.O.,University of Kaiserslautern | Guillouzo A.,University of Rennes 1 | Hewitt P.G.,Merck KGaA | And 2 more authors.
Toxicology in Vitro | Year: 2015

The overall aim of Predict-IV (EU-funded collaborative project #202222) was to develop improved testing strategies for drug safety in the late discovery phase. One major focus was the prediction of hepatotoxicity as liver remains one of the major organ leading to failure in drug development, drug withdrawal and has a poor predictivity from animal experiments. In this overview we describe the use and applicability of the three cell models employed, i.e., primary rat hepatocytes, primary human hepatocytes and the human HepaRG cell line, using four model compounds, chlorpromazine, ibuprofen, cyclosporine A and amiodarone. This overview described the data generated on mode of action of liver toxicity after long-term repeat-dosing. Moreover we have quantified parent compound and its distribution in various in vitro compartments, which allowed us to develop biokinetic models where we could derive real exposure concentrations in vitro. In conclusion, the complex data set enables quantitative measurements that proved the concept that we can define human relevant free and toxic exposure levels in vitro. Further compounds have to be analyzed in a broader concentration range to fully exploit these promising results for improved prediction of hepatotoxicity and hazard assessment for humans. © 2015 Elsevier Ltd.


den Braver-Sewradj S.P.,VU University Amsterdam | den Braver M.W.,VU University Amsterdam | Vermeulen N.P.E.,VU University Amsterdam | Commandeur J.N.M.,VU University Amsterdam | And 3 more authors.
Toxicology in Vitro | Year: 2016

Cytochrome P450s (CYPs), UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) are the most important enzymes for metabolic clearance. Characterization of phase I and phase II metabolism of a given drug in cellular models is therefore important for an adequate interpretation of the role of drug metabolism in toxicity. We investigated phase I (CYP) and phase II (UGT and SULT) metabolism of three drugs related to drug-induced liver injury (DILI), namely acetaminophen (APAP), diclofenac (DF) and tolcapone (TC), in cryopreserved primary human hepatocytes from 5 donors in suspension and monolayer. The general phase II substrate 7-hydroxycoumarin (7-HC) was included for comparison. Our results show that the decrease in CYP, UGT and SULT activity after plating is substrate dependent. As a consequence the phase I/phase II metabolism ratio is significantly affected, with a shift in monolayer towards phase I metabolism for TC and towards phase II metabolism for APAP and DF. Inter-donor variability in drug metabolism is significant, especially in sulfation of 7-HC or APAP. As CYP, UGT and SULT metabolism may lead to bioactivation and/or detoxification of drugs, a changed ratio in phase I/phase II metabolism may have important consequences for metabolism-related toxicity. © 2016 Elsevier Ltd.


Pomponio G.,Instituto Superiore Of Sanita | Pomponio G.,University of Rome La Sapienza | Savary C.C.,University of Rennes 1 | Parmentier C.,KaLy Cell | And 7 more authors.
Toxicology in Vitro | Year: 2015

The limited value of in vitro toxicity data for the in vivo extrapolation has been often attributed to the lack of kinetic data. Here the in vitro kinetics of amiodarone (AMI) and its mono-N-desethyl (MDEA) metabolite was determined and modelled in primary human hepatocytes (PHH) and HepaRG cells, after single and repeated administration of clinically relevant concentrations. AMI bioavailability was influenced by adsorption to the plastic and the presence of protein in the medium (e.g. 10% serum protein reduced the uptake by half in HepaRG cells). The cell uptake was quick (within 3 h), AMI metabolism was efficient and a dynamic equilibrium was reached in about a week after multiple dosing. In HepaRG cells the metabolic clearance was higher than in PHH and increased over time, as well as CYP3A4. The interindividual variability in MDEA production in PHHs was not proportional to the differences in CYP3A4 activities, suggesting the involvement of other CYPs and/or AMI-related CYP inhibition. After repeated treatment AMI showed a slight potential for bioaccumulation, whereas much higher intracellular MDEA levels accumulated over time, especially in the HepaRG cells, associated with occurrence of phospholipidosis.The knowledge of in vitro biokinetics is important to transform an actual in vitro concentration-effect into an in vivo dose-effect relationship by using appropriate modelling, thus improving the in vitro-to-in vivo extrapolation. © 2014 Elsevier Ltd.


Oorts M.,Catholic University of Leuven | Baze A.,KaLy Cell | Bachellier P.,University of Franche Comte | Heyd B.,University of Franche Comte | And 4 more authors.
Toxicology in Vitro | Year: 2016

Drug-induced cholestasis (DIC) is recognized as one of the prime mechanisms for DILI. Hence, earlier detection of drug candidates with cholestatic signature is crucial. Recently, we introduced an in vitro model for DIC and evaluated its performance with several cholestatic drugs. We presently expand on the validation of this model by 14 training compounds (TCs) of the EU-EFPIA IMI project MIP-DILI.Several batches of human hepatocytes in sandwich-culture were qualified for DIC assessment by verifying the bile acid-dependent increase in sensitivity to the toxic effects of cyclosporin A. The cholestatic potential of the TCs was expressed by determining the drug-induced cholestasis index (DICI). A safety margin (SM) was calculated as the ratio of the lowest TC concentration with a DICI ≤ 0.80 to the Cmax,total. Nefazodone, bosentan, perhexiline and troglitazone were flagged for cholestasis (SM < 30). The hepatotoxic (but non-cholestatic) compounds, amiodarone, diclofenac, fialuridine and ximelagatran, and all non-hepatotoxic compounds were cleared as "safe" for DIC. Tolcapone and paracetamol yielded DICI-based SM values equal to or higher than those based on cytotoxicity, thus excluding DIC as a DILI mechanism.This hepatocyte-based in vitro assay provides a unique tool for early and reliable identification of drug candidates with cholestasis risk. © 2016 Elsevier B.V..


Sison-Young R.L.C.,Center for Drug Safety Science | Mitsa D.,Center for Drug Safety Science | Jenkins R.E.,Center for Drug Safety Science | Mottram D.,Center for Drug Safety Science | And 11 more authors.
Toxicological Sciences | Year: 2015

In vitro preclinical models for the assessment of drug-induced liver injury (DILI) are usually based on cryopreserved primary human hepatocytes (cPHH) or human hepatic tumor-derived cell lines; however, it is unclear how well such cell models reflect the normal function of liver cells. The physiological, pharmacological, and toxicological phenotyping of available cell-based systems is necessary in order to decide the testing purpose for which they are fit. We have therefore undertaken a global proteomic analysis of 3 human-derived hepatic cell lines (HepG2, Upcyte, and HepaRG) in comparison with cPHH with a focus on drug metabolizing enzymes and transport proteins (DMETs), as well as Nrf2-regulated proteins. In total, 4946 proteins were identified, of which 2722 proteins were common across all cell models, including 128 DMETs. Approximately 90% reduction in expression of cytochromes P450 was observed in HepG2 and Upcyte cells, and approximately 60% in HepaRG cells relative to cPHH. Drug transporter expression was also lower compared with cPHH with the exception of MRP3 and P-gp (MDR1) which appeared to be significantly expressed in HepaRG cells. In contrast, a high proportion of Nrf2-regulated proteins were more highly expressed in the cell lines compared with cPHH. The proteomic database derived here will provide a rational basis for the context-specific selection of the most appropriate 'hepatocyte-like' cell for the evaluation of particular cellular functions associated with DILI and, at the same time, assist in the construction of a testing paradigm which takes into account the in vivo disposition of a new drug. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.


Chatterjee S.,Catholic University of Leuven | Richert L.,KaLy Cell | Augustijns P.,Catholic University of Leuven | Annaert P.,Catholic University of Leuven
Toxicology and Applied Pharmacology | Year: 2014

Early detection of drug-induced cholestasis remains a challenge during drug development. We have developed and validated a biorelevant sandwich-cultured hepatocytes- (SCH) based model that can identify compounds causing cholestasis by altering bile acid disposition. Human and rat SCH were exposed (24-48. h) to known cholestatic and/or hepatotoxic compounds, in the presence or in the absence of a concentrated mixture of bile acids (BAs). Urea assay was used to assess (compromised) hepatocyte functionality at the end of the incubations. The cholestatic potential of the compounds was expressed by calculating a drug-induced cholestasis index (DICI), reflecting the relative residual urea formation by hepatocytes co-incubated with BAs and test compound as compared to hepatocytes treated with test compound alone. Compounds with clinical reports of cholestasis, including cyclosporin A, troglitazone, chlorpromazine, bosentan, ticlopidine, ritonavir, and midecamycin showed enhanced toxicity in the presence of BAs (DICI. ≤. 0.8) for at least one of the tested concentrations. In contrast, the in vitro toxicity of compounds causing hepatotoxicity by other mechanisms (including diclofenac, valproic acid, amiodarone and acetaminophen), remained unchanged in the presence of BAs. A safety margin (SM) for drug-induced cholestasis was calculated as the ratio of lowest in vitro concentration for which was DICI. ≤. 0.8, to the reported mean peak therapeutic plasma concentration. SM values obtained in human SCH correlated well with reported % incidence of clinical drug-induced cholestasis, while no correlation was observed in rat SCH. This in vitro model enables early identification of drug candidates causing cholestasis by disturbed BA handling. © 2013 Elsevier Inc.


Grant
Agency: European Commission | Branch: FP7 | Program: CP-IP | Phase: HEALTH-2007-1.3-1 | Award Amount: 16.43M | Year: 2008

The overall aim of Predict-IV is to develop strategies to improve the assessment of drug safety in the early stage of development and late discovery phase, by an intelligent combination of non animal-based test systems, cell biology, mechanistic toxicology and in-silico modelling, in a rapid and cost effective manner. A better prediction of the safety of an investigational compound in early development will be delivered. Margins-of-safety will be deduced and the data generated by the proposed approach may also identify early biomarkers of human toxicity for pharmaceuticals. The results obtained in Predict-IV will enable pharmaceutical companies to create a tailored testing strategy for early drug safety. The project will integrate new developments to improve and optimize cell culture models for toxicity testing and to characterize the dynamics and kinetics of cellular responses to toxic effects in vitro. The target organs most frequently affected by drug toxicity will be taken into account, namely liver and kidney. Moreover, predictive models for neurotoxicty are scarce and will be developed. For each target organ the most appropriate cell model will be used. The approach will be evaluated using a panel of drugs with well described toxicities and kinetics in animals and partly also in humans. This approach will be highly advantageous as it will allow a direct comparison between the in vivo to the in vitro data. A parallel analysis of several dynamic and kinetic models with a broad spectrum of endpoints should allow for the identification of several relevant biomarkers of toxicity. Inter-individual susceptibilities will be taken into account by integrating the polymorphisms of the major drug metabolizing enzymes and correlating the observed effects in the human cell models with their genotype. Environmental influences on cellular toxicity to these compounds will also be evaluated using hypoxic stress as a relevant test model.


Gerin B.,UCB Pharma | Dell'aiera S.,UCB Pharma | Richert L.,Kaly Cell | Smith S.,UCB Pharma | Chanteux H.,UCB Pharma
Xenobiotica | Year: 2013

1. A fast, straightforward and cost-effective assay was validated for the assessment of CYP induction in cryopreserved human hepatocytes cultured in 48-well plates. The cocktail strategy (in situ incubation) was used to assess the induction of CYP1A2, CYP2B6, CYP2C9 and CYP3A4 by using the recommended probe substrate, i.e. phenacetin, bupropion, diclofenac and midazolam, respectively. 2. Cryopreserved human hepatocytes were treated for 72 h with prototypical reference inducers, β-naphthoflavone (25 μM), phenobarbital (500 μM) and rifampicin (10 μM) as positive controls for CYP induction. The use of a cocktail strategy has been validated and compared to the classical approach (single incubation). The need of using phase II inhibitor (salicylamide) in CYP induction assay was also investigated. 3. By using three different batches of cryopreserved human hepatocytes and our conditions of incubations, we showed that there was no relevant drug-drug interaction using the cocktail strategy. The same conclusions were observed when a broad range of enzyme activity has to be assessed (wide range of reference inducers, i.e. EC50-Emax experiment). In addition, the interassay reproducibility assessment showed that the day-to-day variability was minimal. 4. In summary, the study showed that the conditions used (probe substrates, concentration of probe substrate and time of incubation) for the cocktail approach were appropriate for investigations of CYP induction potential of new chemical entities. In addition, it was also clear that the use of salicylamide in the incubation media was not mandatory and could generate drug-drug interactions. For this reason, we recommend to not use salicylamide in CYP induction assay. © 2013 Informa UK, Ltd.

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