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Screening of compounds that alter sleep–wake state in zebrafish
S40
Abstracts / Toxicology Letters 229S (2014) S40–S252
P1: Alternative animal models P-1.1 Utility of the HRNTM (hepatic cytochrome P450 reductase null) mice for investigating mechanisms of liver toxicity of carboxylic-acid-containing drugs James Akingbasote 1,∗ , Alison Foster 2 , Sunil Sarda 3 , Huw Jones 4 , Ian Wilson 5 , Gerry Kenna 6 1 Institute of Translational Medicine, University of Liverpool, Liverpool, Merseyside, UK, 2 Translational Safety, Drug Safety and Metabolism, AstraZeneca, Alderley Park, Cheshire, UK, 3 Discovery Sciences, AstraZeneca, Alderley Park, Cheshire, UK, 4 Pathological Sciences, Drug Safety and Metabolism, AstraZeneca, Alderley Park, Cheshire, UK, 5 Department of Surgery and Cancer, Imperial College, London, UK, 6 Safety Science Consultant, Cheshire, UK
Many carboxylic-acid-containing drugs cause liver injury in humans. Examples include fenclozic acid, which was withdrawn due to jaundice observed in clinical trials, and diclofenac, which remains widely prescribed despite being associated with liver damage. To explore whether metabolic bioactivation mediated by cytochrome P450 (CYP) or UDP-dependent gluronyltransferase (UGT) enzymes could play a role in these toxicities, covalent binding (CVB) assays were performed using liver microsomal incubations from wild-type and hepatic cytochrome P450 reductase null (HRNTM ) mice, which are deficient in hepatic CYP activity. Additionally, wild-type and HRNTM mice were administered fenclozic acid and diclofenac orally for 7 days and the effect on clinical chemistry biomarkers and liver pathology investigated. High levels of CYP-mediated CVB of [14 C]-fenclozic acid and [14 C]-diclofenac were observed in wild-type microsomes, but not in HRNTM microsomes. No UDPGA-mediated CVB was detected in microsomes incubated with [14 C]-fenclozic acid. Exposure to fenclozic acid (100 mg/kg) for 7 days resulted in a significant (p < 0.05) increase in plasma alanine amino transferase (ALT) in wild-type but not HRNTM mice. In HRNTM mice liver histopathology changes and markedly elevated ALT, glutamate dehydrogenase and alkaline phosphatase levels were evident. Treatment with diclofenac and fenclozic acid “normalised” the liver clinical chemistry parameters. These data demonstrate that fenclozic acid undergoes CYP but not UGT mediated bioactivation and that HRNTM mice can provide a valuable insight into the role of hepatic CYPs in drug metabolism. However, HRNTM mice are not well suited to investigations of liver toxicity, due to impaired background liver function. http://dx.doi.org/10.1016/j.toxlet.2014.06.180
P-1.2 Screening of compounds that alter sleep–wake state in zebrafish Olaia Holgado 1 , Arantza Muriana 1 , Anne Dekeyne 2 , Caroline Louis 2 , Ainhoa Alzualde 1,∗ 1
BBD BioPhenix SL, Donostia, San Sebastian, Spain, 2 Unité de Recherche de Découverte en Neurosciences, Institut de Recherches Servier, Croissy-sur-Seine, France The zebrafish is emerging as an alternative to mammalian animal models. Although it is more phylogenetically distant from humans than mammals, zebrafish are vertebrate that display other advantageous characteristics including embryo trans-
parency, which allows direct visualization and evaluation of internal organs; or small size, which allows to manipulate them easily and to use 96-well plates to array embryos. The model is ideal to be used as a predictive tool in Drug Discovery, being able in a high extent to compare the results with those obtained in mammals. Increasing knowledge in zebrafish gives more support to this alternative model. Physiological, biochemical and behavioral daily rhythms are regulated by circadian clock. Apart from having many biological advantages, zebrafish are diurnal and present highly conserved circadian and sleep regulation systems, so they have been postulated also as a good model for circadian rhythm studies. Behavioral profiling in zebrafish reveals a conserved vertebrate neuropharmacology and identifies regulators of rest/wake states. For screening purposes, working with zebrafish larvae has many advantages in terms of rapidity and throughput. The main objective of this study was to set up a protocol to detect compounds with potential to alter rest/wake states in zebrafish. For this purpose, zebrafish larvae were exposed to Melatonin and other reference drugs as Barbiturates and Benzodiazepines. Diurnal and nocturnal locomotor activity and responsiveness of larvae are the main endpoints related to sleep states. This study supports the use of zebrafish in order to detect compounds that alter the sleep–wake states. http://dx.doi.org/10.1016/j.toxlet.2014.06.181
P-1.3 Analysis of the influence of vehicles on the sensitizing potential of 3 allergenic compounds Marie-Pierre Berrada-Gomez 1,∗ , Hanna Dalhuchyts 1 , Sandy Rattier 1 , Catherine Bidan 1 , Hervé Groux 2 , Pierre-Jacques Ferret 1 1 2
Pierre Fabre Dermo Cosmetique, Toulouse, France, IMMUNOSEARCH, Grasse, France
Purpose: Cutaneous sensitization results from the induction of an immune response following the exposure of the skin to a substance. A compound sensitizing potential depends on its capacity to penetrate the skin. This potential could be reduced or increased by the bioavailability of the substance. Thus, the use of an appropriate vehicle could play a leading role in the bioavailability of the compound in order to decrease the sensitizing risk. Method: The aim of this study was to determine the influence of 5 cosmetic forms (oil-in-water emulsion, cleansing water, oil, water-in-oil emulsion, microemulsion) on the sensitizing potential of 3 allergens. Cinnamaldehyde, isoeugenol and hydroxycitronellal were tested at several concentrations 10%, 5%, 2%, 1%, 0.1% on the Sens-is model. This assay assesses the ability of the test item to specifically induce the gene expression of irritation and sensitization biomarkers in an in vitro 3D model of reconstructed skin. Results and conclusion: The 3 compounds sensitizing potential has been significantly reduced by the microemulsion while it has been significantly increased by the cleansing water. These observations have been less important with the emulsions. On the other hand the oil vehicle has not presented any influence on this potential. Moreover it would be interesting to carry out a study with complex mixtures as perfumes used in cosmetic products and compare the results to those obtained with the Human Repeated Insult Patch Test. The Sens-is model could also provide relevant data to assess the safety of hypoallergenic products. http://dx.doi.org/10.1016/j.toxlet.2014.06.182
Abstracts / Toxicology Letters 229S (2014) S40–S252
P1: Alternative animal models P-1.1 Utility of the HRNTM (hepatic cytochrome P450 reductase null) mice for investigating mechanisms of liver toxicity of carboxylic-acid-containing drugs James Akingbasote 1,∗ , Alison Foster 2 , Sunil Sarda 3 , Huw Jones 4 , Ian Wilson 5 , Gerry Kenna 6 1 Institute of Translational Medicine, University of Liverpool, Liverpool, Merseyside, UK, 2 Translational Safety, Drug Safety and Metabolism, AstraZeneca, Alderley Park, Cheshire, UK, 3 Discovery Sciences, AstraZeneca, Alderley Park, Cheshire, UK, 4 Pathological Sciences, Drug Safety and Metabolism, AstraZeneca, Alderley Park, Cheshire, UK, 5 Department of Surgery and Cancer, Imperial College, London, UK, 6 Safety Science Consultant, Cheshire, UK
Many carboxylic-acid-containing drugs cause liver injury in humans. Examples include fenclozic acid, which was withdrawn due to jaundice observed in clinical trials, and diclofenac, which remains widely prescribed despite being associated with liver damage. To explore whether metabolic bioactivation mediated by cytochrome P450 (CYP) or UDP-dependent gluronyltransferase (UGT) enzymes could play a role in these toxicities, covalent binding (CVB) assays were performed using liver microsomal incubations from wild-type and hepatic cytochrome P450 reductase null (HRNTM ) mice, which are deficient in hepatic CYP activity. Additionally, wild-type and HRNTM mice were administered fenclozic acid and diclofenac orally for 7 days and the effect on clinical chemistry biomarkers and liver pathology investigated. High levels of CYP-mediated CVB of [14 C]-fenclozic acid and [14 C]-diclofenac were observed in wild-type microsomes, but not in HRNTM microsomes. No UDPGA-mediated CVB was detected in microsomes incubated with [14 C]-fenclozic acid. Exposure to fenclozic acid (100 mg/kg) for 7 days resulted in a significant (p < 0.05) increase in plasma alanine amino transferase (ALT) in wild-type but not HRNTM mice. In HRNTM mice liver histopathology changes and markedly elevated ALT, glutamate dehydrogenase and alkaline phosphatase levels were evident. Treatment with diclofenac and fenclozic acid “normalised” the liver clinical chemistry parameters. These data demonstrate that fenclozic acid undergoes CYP but not UGT mediated bioactivation and that HRNTM mice can provide a valuable insight into the role of hepatic CYPs in drug metabolism. However, HRNTM mice are not well suited to investigations of liver toxicity, due to impaired background liver function. http://dx.doi.org/10.1016/j.toxlet.2014.06.180
P-1.2 Screening of compounds that alter sleep–wake state in zebrafish Olaia Holgado 1 , Arantza Muriana 1 , Anne Dekeyne 2 , Caroline Louis 2 , Ainhoa Alzualde 1,∗ 1
BBD BioPhenix SL, Donostia, San Sebastian, Spain, 2 Unité de Recherche de Découverte en Neurosciences, Institut de Recherches Servier, Croissy-sur-Seine, France The zebrafish is emerging as an alternative to mammalian animal models. Although it is more phylogenetically distant from humans than mammals, zebrafish are vertebrate that display other advantageous characteristics including embryo trans-
parency, which allows direct visualization and evaluation of internal organs; or small size, which allows to manipulate them easily and to use 96-well plates to array embryos. The model is ideal to be used as a predictive tool in Drug Discovery, being able in a high extent to compare the results with those obtained in mammals. Increasing knowledge in zebrafish gives more support to this alternative model. Physiological, biochemical and behavioral daily rhythms are regulated by circadian clock. Apart from having many biological advantages, zebrafish are diurnal and present highly conserved circadian and sleep regulation systems, so they have been postulated also as a good model for circadian rhythm studies. Behavioral profiling in zebrafish reveals a conserved vertebrate neuropharmacology and identifies regulators of rest/wake states. For screening purposes, working with zebrafish larvae has many advantages in terms of rapidity and throughput. The main objective of this study was to set up a protocol to detect compounds with potential to alter rest/wake states in zebrafish. For this purpose, zebrafish larvae were exposed to Melatonin and other reference drugs as Barbiturates and Benzodiazepines. Diurnal and nocturnal locomotor activity and responsiveness of larvae are the main endpoints related to sleep states. This study supports the use of zebrafish in order to detect compounds that alter the sleep–wake states. http://dx.doi.org/10.1016/j.toxlet.2014.06.181
P-1.3 Analysis of the influence of vehicles on the sensitizing potential of 3 allergenic compounds Marie-Pierre Berrada-Gomez 1,∗ , Hanna Dalhuchyts 1 , Sandy Rattier 1 , Catherine Bidan 1 , Hervé Groux 2 , Pierre-Jacques Ferret 1 1 2
Pierre Fabre Dermo Cosmetique, Toulouse, France, IMMUNOSEARCH, Grasse, France
Purpose: Cutaneous sensitization results from the induction of an immune response following the exposure of the skin to a substance. A compound sensitizing potential depends on its capacity to penetrate the skin. This potential could be reduced or increased by the bioavailability of the substance. Thus, the use of an appropriate vehicle could play a leading role in the bioavailability of the compound in order to decrease the sensitizing risk. Method: The aim of this study was to determine the influence of 5 cosmetic forms (oil-in-water emulsion, cleansing water, oil, water-in-oil emulsion, microemulsion) on the sensitizing potential of 3 allergens. Cinnamaldehyde, isoeugenol and hydroxycitronellal were tested at several concentrations 10%, 5%, 2%, 1%, 0.1% on the Sens-is model. This assay assesses the ability of the test item to specifically induce the gene expression of irritation and sensitization biomarkers in an in vitro 3D model of reconstructed skin. Results and conclusion: The 3 compounds sensitizing potential has been significantly reduced by the microemulsion while it has been significantly increased by the cleansing water. These observations have been less important with the emulsions. On the other hand the oil vehicle has not presented any influence on this potential. Moreover it would be interesting to carry out a study with complex mixtures as perfumes used in cosmetic products and compare the results to those obtained with the Human Repeated Insult Patch Test. The Sens-is model could also provide relevant data to assess the safety of hypoallergenic products. http://dx.doi.org/10.1016/j.toxlet.2014.06.182