Advancing the Zebrafish Embryo Developmental Toxicity Assay (ZEDTA) towards a sensitive screening assay. 01/11/2020 - 31/10/2023

Abstract

Within Europe, alternative methods for toxicity assessment of xenobiotics become very important. Several pharmaceutical, agrochemical and cosmetic companies are currently using the zebrafish embryo as an alternative for animal testing to screen new compounds for developmental toxicity. However, false negative and false positive results are reported in the Zebrafish Embryo Developmental Toxicity Assay (ZEDTA) for known mammalian teratogens and non-teratogens, respectively. For safety, false negative results are more critical, as teratogens may be missed. This project aims to further refine the ZEDTA in order to increase the sensitivity of the assay and as such better predict birth defects caused by using drugs during the 1st trimester of pregnancy. In order to achieve this goal, first the number of evaluated morphological endpoints will be extended by including skeletal staining in the ZEDTA. Second, due to the intrinsically low biotransformation capacity of zebrafish embryos, a metabolic activation system will be developed in order to expose the zebrafish embryos not only to the parent compound but also to its potential teratogenic metabolite(s). Finally, additional morphological endpoints will be programmed in image analysis software that will be combined with an automated handling and imaging system. As such, this project will increase the sensitivity and throughput of the ZEDTA, resulting in better, faster and laboratory animal-free screening of teratogenic drugs.

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I-PREDICT: Innovative Physiology-based pharmacokinetic model to pREdict Drug exposure In neonates undergoing Cooling Therapy. 01/01/2020 - 31/12/2023

Abstract

Drug disposition in neonates depends on maturational and non-maturational covariates. We hypothesize that cooling, a standard therapy for asphyxiated neonates with hypoxic-ischemic encephalopathy, is such a non-maturational covariate. The main goal of the project is to develop an innovative physiology-based pharmacokinetic (PBPK) framework to describe and predict the impact of cooling on drug disposition in asphyxiated neonates. PBPK models combine drug-specific data with physiological data of a population of interest to guide drug dosing. The effect of cooling on drug metabolism is hereby defined as a knowledge gap. Using a multidisciplinary approach, in vitro, animal experimental and human data will be collected for development of the model. First, the impact of temperature on expression and function of drug metabolizing enzymes (DME) and drug transporters (DT) will be assessed in vitro on human and porcine hepatocytes. Second, the utility of endogenous biomarkers reflecting DME and DT activity during cooling is investigated. Third, the results will be used to build PBPK models for selected drugs. Finally, in vivo PK data in human asphyxiated neonates undergoing cooling and in an experimental in vivo setting of neonatal minipigs will be used to evaluate the models. The outcome will be drug dose prediction and drug development in neonates undergoing cooling. The framework may also be applied to develop temperature driven PBPK-models in other special populations.

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Bioactivation of human proteratogens in the zebrafish embryo model, a potential alternative test for development toxicity assessment. 01/01/2018 - 31/12/2021

Abstract

Within Europe, alternative methods for toxicity assessment of xenobiotics become very important. For developmental toxicity, the mouse Embryonic Stem Cell test and the rat Whole Embryo Culture are commonly used, but assays in non-mammalian whole organisms are currently proposed as they more fully represent the complexity of early development. The zebrafish (Danio rerio) is one of the preferred species due to their small size, high fecundity and rapid ex utero development. However, knowledge on the ontogeny of biotransformation enzymes in this species remains scarce. This is a pivotal information, especially for proteratogens that require bioactivation to exert their teratogenic potential. The cytochrome P450 enzymes (CYPs) represent the most important enzyme family in this oxidative process and are also important for the (de) activation of endogenous molecules that regulate normal embryonic development. The aim of this research project is to assess the bioactivation capacity of zebrafish embryos by using molecular techniques that target specific CYP isoenzymes. As such, this project will show whether zebrafish embryos can bioactivate, and thus detect, proteratogens or not, which is key information when considering this alternative assay for developmental toxicity testing.

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Qualification of the juvenile minipig for pediatric safety testing of single stranded oligonucleotides (SSOs). 15/07/2020 - 14/07/2021

Abstract

Single stranded oligonucleotides (SSOs) are a new class of drugs that are used to inhibit the expression of specific proteins by hybridizing to specific mRNAs, e.g. in cancer cells. For adult indications, non-human primates (NHPs) are often considered to be the non-rodent species of choice for the safety assessment of SSOs, but recently the Göttingen minipig has been proven to be a good alternative. This animal model also has several ethical and practical advantages over NHPs for pediatric safety testing of SSOs, but the juvenile Göttingen minipig has not yet been characterized for this purpose. In the adult population, SSOs distribute from the circulation into tissues within minutes to a few hours after systemic administration. The aim of this project is to investigate these processes in juvenile Göttingen minipigs. These data will be very useful for the interpretation of pediatric safety data with new SSOs.

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Advancing the zebrafish embryo developmental toxicity assay from screening to regulatory testing. 01/11/2019 - 31/10/2020

Abstract

Within Europe, alternative methods for toxicity assessment of xenobiotics become very important. Several pharmaceutical, agrochemical and cosmetic companies are currently using the zebrafish embryo as an alternative for animal testing to screen new compounds for developmental toxicity. However, false negative and false positive results are reported in the Zebrafish Embryo Developmental Toxicity Assay (ZEDTA) for known mammalian teratogens and non-teratogens, respectively. From a safety point of view, false negative results are more critical because teratogens may be missed by the assay. This project aims to further refine the ZEDTA protocol in order to increase the sensitivity of the assay and to advance the ZEDTA from screening towards potential regulatory acceptance. In order to achieve this goal, first the number of evaluated morphological endpoints will be extended by including skeletal staining in the ZEDTA. Second, due to the intrinsically low biotransformation capacity of zebrafish embryos, a metabolic activation system will be developed in order to expose the zebrafish embryos not only to the parent compound but also to its potential teratogenic metabolite(s). Finally, additional morphological endpoints will be programmed in image analysis software that will be combined with an automated handling and imaging system. As such, this project will increase the sensitivity and throughput of the ZEDTA for potential regulatory use.

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Assessment of skeletal endpoints and exposure in an extended Zebrafish Developmental Toxicity Assay (ZEDTA) using mammalian teratogens. 10/04/2018 - 09/04/2020

Abstract

The number of morphological endpoints in the ZEDTA is much more limited compared to the in vivo developmental toxicity studies. A survey within the laboratories of the proposers pointed out that especially the skeletal endpoints are limited in the ZEDTA. A literature review showed false negative results in the ZEDTA for compounds that showed skeletal abnormalities in mammalian studies. The aim of this project is to assess whether extension of the morphological parameters and evaluation period in the ZEDTA after exposure to mammalian skeletal teratogens during organogenesis increases the concordance of the ZEDTA with the mammalian in vivo developmental toxicity studies.

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Reaction phenotyping and CRISPR/Cas9 genome editing of xenobiotic CYP isoenzymes in the zebrafish embryo: effects on bioactivation of prodrugs and normal functioning. 01/10/2016 - 31/12/2017

Abstract

Within Europe, alternative in vitro methods for toxicity assessment of xenobiotics become very important. For developmental toxicity, the mouse Embryonic Stem Cell test and the rat Whole Embryo Culture are commonly used, but assays in non-mammalian species, as a whole organism, are currently proposed as they more fully represent the complexity of early development. The zebrafish (Danio rerio) is one of the preferred species due to their small size, high fecundity and rapid ex utero development. However, knowledge on the ontogeny of biotransformation enzymes in this species remains scarce. This is pivotal information, especially for proteratogens that require bioactivation to exert their teratogenic potential. The cytochrome P450 enzymes (CYPs) represent the most important enzyme family in this oxidative process and are also important for the (de)activation of endogenous molecules that regulate normal embryonic development. The identification and developmental expression of CYP genes in the zebrafish have been reported but the translation to functional enzymes, i.e. activity of the CYPs, remains to be elucidated despite attempts of several research groups. Indeed, very low CYP1A activity has been detected in microsomes from whole embryo homogenates but whether the in vivo tissue-specific concentration of these metabolites is insufficient to cause a teratogenic effect, could not be concluded. The ultimate proof of the bioactivation capacity of zebrafish embryos is the presence of malformations after exposure of zebrafish embryos to known proteratogens. However, in that particular scenario one needs to be sure that only the metabolite(s) of the prodrug is teratogenic and not the compound itself. Therefore, the aim of this research project is to assess the bioactivation capacity of zebrafish embryos by using molecular techniques that target specific CYP isoenzymes. In that way the role of these CYPs in normal embryonic development and their function in bioactivation of proteratogens will be unraveled.

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Construction and activity of recombinant cythochrome P450 enzymes as an in vitro system for reaction-phenotyping of drug candidates in the Göttingen minipig. 18/07/2016 - 31/12/2017

Abstract

During drug development, pharmaceutical companies are required to characterize all routes of clearance (drug elimination) in man. Although the characterization of the metabolic profile of a drug is only required prior to the start of Phase 3 clinical trials, earlier information is very useful for the species selection in safety studies. Therefore, in vitro drug metabolism studies in several species are often performed early during drug development. Liver microsomes or the S9 fraction are the most common tools for screening of effects of a new drug on the CYP450 pathways, although liver slices or isolated hepatocytes give a more complete picture. To unravel the specific isoenzymes involved in the metabolism of drugs, recombinantly expressed cDNA of several CYP isoforms is used and commercially available for a number of species, including man, mouse, rat, dog and monkey. For the minipig no recombinant CYPs are commercially available. This species is, however, increasingly used in juvenile and general toxicity studies. Therefore, the aim of our project is to develop recombinant CYP enzymes of the minipig and compare their activity with adult minipig liver microsomes and in vivo pharmacokinetic (PK) data. This minipig recombinant system can be very valuable for pharmaceutical companies for species selection in non-clinical studies.

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Ontogeny of cytochrome P450 activity in the zebrafish: an in vitro investigation. 15/07/2015 - 14/07/2016

Abstract

The zebrafish is increasingly used for toxicological purposes. However, knowledge on the biotransformation processes in this species is scarce, especially in zebrafish embryos which are increasingly used as an alternative model for teratogenicity screening of pharmaceuticals. In this project, we will assess the intrinsic activity of cytochrome P450 (CYPs) in adult male and female zebrafish and during embryonic development. This project will provide information on possible gender differences in CYP activity in the adult zebrafish and also on the capacity of zebrafish embryos to metabolize xenobiotics.

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Development of a zebrafish embryo test for environmental risk assessment of pharmaceuticals with estrogenic properties. 01/01/2015 - 31/12/2018

Abstract

Pharmaceutical companies are obligated to perform an environmental risk assessment for each new drug that they launch on the market. The mandatory tests for potential endocrine disrupting compounds require a lot of time and laboratory animals. Therefore, the purpose of this study is to develop a zebrafish embryo test, which is not considered as animal test, that is capable of quickly detecting estrogenic properties of pharmaceuticals.

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Influence of temperature on bioactivation and embryotoxicity in a modified Zebrafish developmental toxicity assay. 01/10/2014 - 30/09/2018

Abstract

The zebrafish embryo is already being used for teratogenicity testing, i.e. in the Zebrafish Developmental Toxicity Assay (ZEDTA). However, in vitro data of our research group and in vivo data from other labs indicate that the zebrafish embryo shows no or low bioactivity. As such proteratogens, i.e. compounds that require bioactivation to exert their teratogenic potential, may be missed in the ZEDTA and lead to false negative results. Currently, rat exogenous metabolic activation system (MAS) is used to optimize the ZEDTA but it is controversial as it causes embryotoxicity by itself and its metabolic profile may be different than in man. The temperature of the co-incubation system may contribute to the observed embryotoxicity. Zebrafish embryos develop optimally between 26,5-28,5°C and do not tolerate the physiological temperature of rat MAS (38-39°C). Therefore, 32°C is often used in the coincubation system as a compromise. However, very recently we assessed temperature effects on zebrafish embryonic development and observed that 32,5°C causes embryotoxicty. Therefore, the aim of this research project is to develop a novel coincubation system (MAS) that functions at a non-embryotoxic temperature. Instead of rat MAS, we will evaluate in a first phase the bioactivity and embryotoxicity of human MAS (to obtain a human relevant metabolic profile) at different temperatures and of zebrafish MAS at 28,5°C (physiological condition for embryonic development). Regarding bioactivity, we will focus on xenobiotic metabolizing CYP families (i.e. CYP 1-3) and limit ourselves to fluorogenic substrates that are relevant to man and covering approx. 95% of human xenobiotic metabolism. Metabolite concentrations will be determined in the supernatans over-time by detection of the fluorescent signal. In a second phase we will evaluate biotransformation in the ZEDTA in presence and absence (controls) of hMAS and zMAS. This will be achieved by exposing early zebrafish embryos to the same substrates of the first phase. For the co-incubation, we will select the optimal temperature from the first phase. Metabolite concentrations will be determined over-time in the embryo and in the supernatans by detection of the fluorescent signal.

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Equipment for high-speed refrigerated, preparative ultracentrifugation, automated gradient formation and fraction collection and analysis. 19/05/2014 - 31/12/2018

Abstract

This project represents a formal research agreement between UA and on the other hand the Hercules Foundation. UA provides the Hercules Foundation research results mentioned in the title of the project under the conditions as stipulated in this contract.

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Pathways to subfertility: elevated non-esterified fatty acid concentrations as the causative link between maternal metabolic disorders and reduced embryo quality. 01/01/2014 - 31/12/2017

Abstract

This research project concentrates on the effect of oocyte maturation, fertilization and embryo culture under high NEFA conditions on fertilization rate, DNA transcription and methylation patterns in Day 7 embryos.

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Optimalisation of the biotransformation in the Zebrafish Teratogenicity Test. 01/01/2014 - 30/09/2014

Abstract

Zebrafish (Danio rerio) are commonly used in drug development because of its advantages. As zebrafish are small, only little compound is needed compared to mammals. Furthermore, the expenses for the maintenance of the fish stock are limited and the fish show a high fecundity throughout the year. Moreover, the externally fertilised eggs develop in a similar way as higher vertebrate species, including humans. Because of these advantages, zebrafish are nowadays being used for screening new drugs for their teratogenic potential in the Zebrafish Teratogenicity Test (ZTT). However, optimal study conditions aren't available, indicating the need for further optimalisation of the test before it can be used as a valid screening model. Therefore, we will investigate the use of human- and zebrafish-derived liver microsomes in order to optimize the biotransformation of compounds.

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Optimization of biotransformation in the Zebrafish TeratogenicityTest. 01/10/2013 - 30/09/2014

Abstract

The aim of this research project is to develop a new exogenous metabolic activation system (MAS) to optimize the biotransformation of the ZET. To achieve this goal we will first evaluate and compare the biotransformation and embryotoxic potential of human MAS with induced zebrafish MAS (izMAS) by exposure to different substrates. We will focus on xenobiotic metabolizing CYP families (i.e. CYP 1-3) and limit ourselves to substrates that are relevant to man and covering approx. 95% of human xenobiotic metabolism. Parent compound and metabolites will be determined in the supernatans over-time by LC-MS.

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Ontogeny of CYPs and drug transporters in the gastrointestinal tract of zebrafish. 16/09/2012 - 15/07/2015

Abstract

This project represents a formal research agreement between UA and on the other hand EU. UA provides EU research results mentioned in the title of the project under the conditions as stipulated in this contract.

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Ontogeny of drug transporters and metabolizing enzymes in the minipig: a genomic approach 01/01/2012 - 31/12/2012

Abstract

Drug transporters and metabolizing enzymes play a pivotal role in the pharmacokinetics of most drugs. The ATP-binding cassette (ABC) drug efflux transporters and cytochrome P450 isoenzymes (CYP) are the best known of these two groups, respectively, but numerous other subgroups are involved in the absorption, distribution, metabolism and elimination of drugs. The presence and activity of these systems in hepatocytes and enterocytes influence oral bioavailability and as such they may determine the clinical efficacy but also the toxicity of orally ingested drugs. Although many of these pharmaceuticals are also used in (very young) children and differences in oral bioavailability are common compared to adults, studies on the ontogeny of drug transporters and metabolizing enzymes are scarce. This information is also pivotal in juvenile animals as, from a safety perspective, toxicity studies in one or more species may be required prior to the start of clinical trials in children. Knowledge on the ontogeny of these enzymes and transporters in different organ systems is building up in the rat, but in non-rodent species data are lacking. As the minipig is the most commonly used non-rodent species in juvenile toxicity studies besides the dog, information on the ontogeny of drug efflux transporters and metabolizing enzymes is pivotal in the interpretation of these studies. Therefore, the aim of this project is to verify the gene expression levels of different drug transporters and metabolizing enzymes in the liver and small intestine of fetal, neonatal, suckling and weanling minipigs in order to cover a pediatric population of < 2 years of age.

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