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Plobsheim, France

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. Source


Grant
Agency: Cordis | 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.


Muller C.,CNRS Chemistry of Complex Matter | Pekthong D.,KaLy Cell | Pekthong D.,Naresuan University | Alexandre E.,KaLy Cell | And 5 more authors.
Combinatorial Chemistry and High Throughput Screening | Year: 2015

In this paper we report quantitative structure-activity models linking in vivo Drug-Induced Liver Injury (DILI) of organic molecules with some parameters both measured experimentally in vitro and calculated theoretically from the molecular structure. At the first step, a small database containing information of DILI in humans was created and annotated by experimentally observed information concerning hepatotoxic effects. Thus, for each compound a binary annotation "yes/no" was applied to DILI and seven endpoints causing different liver pathologies in humans: Cholestasis (CH), Oxidative Stress (OS), Mitochondrial injury (MT), Cirrhosis and Steatosis (CS), Hepatitis (HS), Hepatocellular (HC), and Reactive Metabolite (RM). Different machine-learning methods were used to build classification models linking DILI with molecular structure: Support Vector Machines, Artificial Neural Networks and Random Forests. Three types of models were developed: (i) involving molecular descriptors calculated directly from chemical structure, (ii) involving selected endpoints as "biological" descriptors, and (iii) involving both types of descriptors. It has been found that the models based solely on molecular descriptors have much weaker prediction performance than those involving in vivo measured endpoints. Taking into account difficulties in obtaining of in vivo data, at the validation stage we used instead five endpoints (CH, CS, HC, MT and OS) measured in vitro in human hepatocyte cultures. The models involving either some of experimental in vitro endpoints or their combination with theoretically calculated ones correctly predict DILI for 9 out of 10 reference compounds of the external test set. This opens an interesting perspective to use for DILI predictions a combination of theoretically calculated parameters and measured in vitro biological data. © 2015 Bentham Science Publishers. Source


Alexandre E.,KaLy Cell | Baze A.,KaLy Cell | Parmentier C.,KaLy Cell | Desbans C.,KaLy Cell | And 9 more authors.
Xenobiotica | Year: 2012

Rationale: The aim of the present study was to assess the stability of cryopreserved human hepatocytes over 5 years and to explore experimental condition-related variables such as seeding density, culture matrix and medium, start and duration of treatment that could potentially affect the quality of cultures and their response to cytochrome P450 (CYP) inducers. Results: 63/125 batches of cryopreserved human hepatocytes were plateable after thawing. Of those, 17 batches showed reproducible recovery, viability and plateability (less than 5% intra-batch variability) up to 5 years. When cultured in collagen home-coated 48-well plates at a seeding density allowing 70% confluence, cryopreserved human hepatocytes display activities equivalent to fresh counterparts. Their response to CYP inducers is maximal and equivalent to fresh counterpart for an incubation of 72 h starting at Day 2 or Day 3 after plating when cultured in modified Hepatocyte Maintenance Medium (HMM). The number of cryopreserved human hepatocytes can be further reduced by using a cocktail of CYP substrates for the assessment of their inducibility. Conclusions: Experimental condition-related variables, such as seeding density, culture matrix and medium, start and duration of treatment, affecting the response of plateable thawed cryopreserved human hepatocytes to cytochrome P450 inducers can be reduced by optimizing critical steps of the protocols. © 2012 Informa UK, Ltd. Source


Vidal I.,EA 3921 | Vidal I.,University of Geneva | Blanchard N.,KaLy Cell | Chenard-Neu M.-P.,Service danatomo pathologie | And 5 more authors.
Cell Transplantation | Year: 2014

We recently found that rat hepatocyte transplantation was efficient (liver repopulation: 2.4%) in a sublethal nude mouse model (less than 33% mortality) of repeated liver injury generated using Jo2, a mouse-specific anti-Fas antibody, at sublethal dose of 250 μg/kg for 3 weeks. Genomic analysis of the livers revealed cell cycle blockade and an antiproliferative status of circadian genes, suggesting a selective advantage. By contrast, in the present study, freshly isolated human hepatocyte transplantation performed in the same mouse model resulted in implantation of less than 6,000 cells per liver (about 0.006% repopulation) in all animals. Genomic analysis of nude mouse livers revealed a lack of P21 upregulation, while a signature of stimulation of liver regeneration was observed, including upregulation of early response genes and upregulation of circadian genes. When we translated this sublethal model to a lethal model (65% mortality) by increasing the Jo2 repeated doses to 375 μg/kg, human hepatocyte engraftment was still very low; however, animal mortality was corrected by transplantation (only 20% mortality). Genomic findings in livers from the mice of the lethal Jo2 transplanted group were similar to those of the sublethal Jo2 transplanted group, that is, no selective advantage genomic signature and signature of mouse liver regeneration. In conclusion, transplanted human hepatocytes acted as if they modified nude mouse liver responses to Jo2 by stimulating liver regeneration, leading to an increased survival rate. © 2014 Cognizant Comm. Corp. Source

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