Juvenile Göttingen minipigs as pediatric safety testing model for antisense oligonucleotides (ASOs) - Allan Valenzuela (24/11/2023)
- 24 november 2023, 16u
- CDE, gebouw O (D.O.05)
- Promotoren: Prof. Steven Van Cruchten en Dr. Miriam Ayuso Hernando
Antisense oligonucleotides (ASOs) belong to a large group of nucleic acid-based therapeutics that utilize synthetic oligonucleotides to modulate RNA translation. As ASOs need to hybridize to their complementary target RNA, nonhuman primates (NHPs) are the preferred non-rodent model due to their close genetic homology and pharmacokinetics with humans. However, the adult Göttingen Minipig has been recognized as a suitable alternative to NHPs in the safety testing of ASOs, as it showed similar pharmacokinetic, pharmacodynamic, and safety profiles in a previous study. Since ASOs are usually indicated to rare genetic conditions that could start early in life, and as the rapid growth and development of the pediatric population can influence the pharmacokinetics/pharmacodynamics of therapeutic agents, leading to potential toxicities, extending the previous work to the juvenile minipigs was deemed necessary. In this thesis, we evaluated the juvenile Göttingen Minipig as a pediatric safety testing model for ASOs. As such, we assessed potential differences in exposure/toxicity and pharmacologic effect of a model ASO (RTR5001) in the juvenile Göttingen Minipig in an 8-week repeat-dose toxicity study (weekly subcutaneous dosing starting at postnatal day 1). Accordingly, the model ASO that had been previously characterized in adult Göttingen Minipigs and NHPs showed comparable clinical chemistry and toxicity profiles in the juvenile minipigs. However, differences in plasma and tissue exposures, and pharmacologic activity were observed compared to the adult data. The ontogeny evaluation of the key nucleases responsible for ASO metabolism and pharmacologic activity revealed a differential nuclease expression and activity, which could affect the metabolic pathway and pharmacologic effect of ASOs in different tissues and age groups. Likewise, to further understand the species translatability of ASO-induced thrombocytopenia, in vitro platelet activity and aggregometry assays were performed using a panel of tool ASOs with different sequences and modifications. Our data on direct platelet activation and aggregation by ASOs in adult minipig samples are remarkably comparable to human data. Moreover, phosphorothioated ASOs bind to platelet collagen receptor glycoprotein VI (GPVI) and directly activate minipig platelets in vitro, mirroring the findings in human samples. The differential abundance of GPVI (and platelet factor 4) in minipigs provides insight into the influence of ontogeny in potential ASO-induced thrombocytopenia in pediatrics. Therefore, the body of data from this thesis is fundamental for selecting the juvenile Göttingen Minipig in assessing safety concerns for ASOs intended for the human pediatric population.
Mas-related G protein-coupled receptors: activations, interactions, and their role in inflammation – Rohit Arora (9/11/2023)
- 9 november 2023, 15:15u
- Promotoren: Prof. Xaveer Van Ostade en Prof. Alain Labro
The largest family of membrane receptors, known as G protein-coupled receptors (GPCRs), are essential to cellular signaling and regulate physiological processes. Presently, ~35%–40% of US FDA-approved medications target GPCRs. A subfamily of GPCRs, Mas-related G protein-coupled receptors (MRGPRs), which belong to the δ-group of the rhodopsin-like GPCRs, was discovered two decades ago. MRGPRs are expressed by small non-myelinated sensory neurons of the dorsal root ganglia and trigeminal ganglia, mast cells, neutrophils, and macrophages and are known to play a role in itch, pain, and pseudo-allergic drug reactions. Moreover, MRGPRs have been identified as mediators in the renin-angiotensin system and cardiovascular biology. In addition, literature suggests that MRGPRs are also involved in inflammatory processes. Despite the fact that humans express eight MRGPRs (MRGPRD to G and X1-X4), information about their activation, signaling pathways, and role in inflammation is insufficient, and most of them are still classified as orphans. Since MRGPRs are involved in itch, pain, and inflammation, which are important physiological processes, the goal of this PhD was to: i) examine the role of MRGPRs in inflammation biology; ii) decipher the activation mechanism of MRGPRs; and iii) elucidate the oligomeric interaction of MRGPRs.
Firstly, it was investigated whether β-alanine or alamandine-activated MRGPRD induces interleukin-6 (IL-6) release. It was observed that β-alanine activated MRGPRD-induced IL-6 release via the Gαq/Phospholipase C/NF-kB signaling pathway. Moreover, using IL-6 as a marker for MRGPRD activation, the mechanosensitivity of the MRGPRD and the effect of sterol derivatives, i.e., cholesterol and bile acids, on the activation of MRGPRD were established. Furthermore, it was discovered that the MRGPRD was constitutive (ligand-independent) active. In addition, it was discovered that methyl-β-cyclodextrin, which is known to remove sterols from the plasma membrane, triggered the MRGPRD-mediated IL-6 release.
Secondly, in an effort to deorphanize MRGPRs, it was established that cysteine protease cathepsin S activates MRGPRD and MRGPRF. Lastly, using biophysical and biochemical techniques such as luciferase complementation, bioluminescence resonance energy transfer, and co-immunoprecipitation assays, the heteromeric interactions between MRGPRE and MRGPRF were unambiguously detected.
Overall, in this doctoral thesis, the primary objective was to improve understanding of the involvement of MRGPRs in inflammatory biology, the activation mechanisms of MRGPRs, and the oligomeric interaction of MRGPRs.
Hepatic drug metabolism in pediatrics: investigating the neonatal and juvenile (mini)pig as a translational model - Laura Buyssens (11/10/2023)
- 11 oktober 2023, 17u
- CDE, Aula Fernand Nédée (D.Q.001)
- Promotoren: Prof. Steven Van Cruchten en Prof. Chris Van Ginneken
The pediatric population is one of the most vulnerable age groups within the human population, especially regarding drug development. They comprise approximately 30% of the total human population, but still very little research is conducted in their interest. Although pediatric drug development has been encouraged and efforts are ongoing, knowledge gaps still exist. ADME (i.e., absorption, distribution, metabolism and excretion) properties are particularly understudied possibly resulting in under or overdosing. The neonatal population is especially prone to adverse drug reactions since clinical trials in this group are often perceived as not feasible or unethical. Clinicians are thus required to administer drugs off-label (i.e., without information on safety and efficacy). To overcome this issue, alternatives are explored.
The use of juvenile animal studies, for example, is supported by FDA and EMA to improve our knowledge in the pediatric population. In this regard, the (mini)pig has gained ground as nonclinical species. Newborn piglets share the same size as human neonates and can be assessed in the same way as babies in the neonatal intensive care unit. With regard to hepatic drug metabolism, large similarities have been observed between adult humans and pigs, but data in the human and porcine (preterm) neonatal and juvenile population remain scarce. The goal of this thesis was thus to further characterize hepatic phase I (e.g., cytochrome P450 (CYP)) and phase II (e.g., uridine 5’-diphospho-glucuronosyltransferase (UGT)) enzyme ontogeny in neonatal and juvenile (mini)pigs.
First, we characterized hepatic phase I drug metabolism on protein level in fetal, neonatal, juvenile and adult Göttingen Minipigs. Hepatic CYP protein abundance was investigated in liver microsomes using an LC-MS/MS approach. Various ontogeny profiles were discovered which were in line with observations in man. Sex-related differences were detected with highest expression in female compared to male minipigs. CYP protein abundance was correlated with earlier observed CYP enzyme activity.
Next, the translational potential of the preterm pig model for human neonatal drug metabolism was investigated, as preterm-born neonates are still considered to be “therapeutic orphans” within pediatric drug development. The aim of this study was to examine the ontogeny of CYP3A and UGT enzyme activity in the liver of preterm (90% gestation, gestational day 105 – 107) and term-born (100% gestation, gestational day 115 – 117) domestic piglets. In addition, the effect of chronological (i.e., gestational age, and postconceptional age) and postnatal age (i.e., birth effect) on the onset of enzyme activity was examined. UGT activity showed a significant postnatal increase in both preterm and term-born piglets from birth onwards. CYP3A enzyme activity was only detected in preterm-born piglets at postnatal day 26 while a gradual increase was observed in term-born piglets from postnatal day 11 onwards. In both groups, enzyme activity was lower in the preterm compared to the term group, suggesting that postconceptional age rather than postnatal age is affecting CYP3A and UGT enzyme ontogeny in the pig.
Finally, the data obtained in this thesis contribute to a better understanding of the biotransformation capacity in the (mini)pig. In general, large similarities with human pediatric hepatic drug metabolism were found which encourages the use of the pig as nonclinical species for pediatric drug development. However, it needs to be emphasized that nonclinical species selection for juvenile animal studies is not just driven by hepatic drug metabolism, as other factors are also important. Therefore, species selection has to be considered very carefully on a case-by-case basis.
Opportunities for improvement of oocyte quality in metabolically compromised conditions: from discoveries in the well until the development of preconception care strategies in an obese mouse model – Anouk Smits (31/03/2022)
- 31 maart 2022, 17u
- CDE, auditorium O.07
- Promotoren: Prof. Jo Leroy en Prof. Diane De Neubourg
There is a significant negative impact of metabolic health disorders (like obesity) on female fertility, with an important role of reduced oocyte and embryo quality. As a result, more and more research focuses on the possibilities of specific interventions to improve fertility. Such intervention should prevent further damage or should improve and/or recover oocyte quality leading to an optimized oocyte developmental competence and ultimately restored fertility. Can we substantiate these concepts in vitro and which mouse model should be used to investigate the importance of preconception care in vivo?
The consequences of maternal metabolic disorders on oocyte quality are clear. However, it is not known if this metabolically-compromised oocyte can be rescued. An in vitro bovine exposure model was used to obtain metabolically-compromised oocytes. To investigate the impact of post-conception care interventions, the in vitro culture medium was supplemented with anti-apoptotic, antioxidative and mitogenic factors. Results indicated that supportive embryo culture conditions might improve oocyte developmental competence but resulting embryo quality was still aberrant.
Research investigating the impact of obesity on metabolic health (and fertility) often uses the inbred C57BL/6 strain. However, inbred strains are characterized by reduced fertility and might limit translation to human outbred physiology. We showed that control inbred C57BL/6 mice had oocytes with already high cellular stress and significant ultrastructural abnormalities. This deviating profile was only present in outbred Swiss mice after exposure to an obesogenic diet for 13 weeks. These results indicate that Swiss mice are the better choice for research regarding obesity and oocyte quality.
Nowadays, the development of preconception care interventions (PCCI) for obese women to improve their fertility is gaining more and more attention. However, so far there are no clear preconception guidelines. Knowledge on the impact of dietary PCCI on oocyte quality and the necessary duration for this PCCI is lacking. Therefore, the impact of diet normalization or caloric restriction for different time periods on metabolic health and oocyte quality of high fat or high-fat/high-sugar diet-induced obese mice was tested. The impact of the tested PCCI seemed to depend partially on the obesogenic diet used. Overall, the results indicated that switching to an ad libitum control diet for at least four weeks was the most promising approach to improve metabolic health and oocyte quality. However, complete recovery of oocyte quality was not present. These increased fundamental insights can bridge the gap towards clear preconception guidelines for obese women planning for pregnancy.
Assessment of the effects of cryostorage on pre-antral follicle survival and preservation of intercellular connections - Anniek Bus (20/12/2021)
- 20 december 2021, 17u
- CDE, Promotiezaal (Q.002)
- Promotoren: Prof. Peter Bols en Prof. Jo Leroy
Nowadays, an emerging need for (human) fertility preservation (FP) strategies is present, in particular when young girls and woman are confronted with cancer treatment. In addition, the preservation of genetic material from endangered animal species or animals with important genetic traits will also benefit from the development of alternative FP strategies. The strategies in this thesis focus on the use of pre-antral follicles (PAF) as, for prepubertal girls and women whose cancer treatment cannot be postponed, there are no other options. As PAFs account for the vast majority of follicles in the ovarian cortex, they represent an untapped potential, which could be cultivated for reproduction, preservation or research purposes. Vitrification is the most successful preservation method for gametes and embryos. However, a negative consequence of this strategy is the increased probability of injuries caused by exposure of cells to other non-physiological conditions that may impair their further development. Research performed on effects of vitrification on the viability of isolated PAFs remains scarce and protocols still need to be optimized for each specific cell type and species. Due to their small size (30 µm), the isolation and processing of isolated PAFs remains a huge challenge in terms of follicular retrieval and manipulation. Research presented in this thesis shows that the vitrification of isolated PAFs is a promising FP strategy. This is particularly relevant for cancer patients at moderate-to-high risk of ovarian metastasis. Different vitrification techniques including straws, follicle embedding, cell strainers and Cryotop were evaluated for follicle survival and applicability in clinical practice. We however, found that all evaluated techniques, next to their advantages also had their downsides. Several methods were used to assess follicle quality, ranging from gross morphology, simple viability stains as Neutral red and Calcein, immunochemical methods to more complex xenografting procedures. Our main finding is that isolated PAFs can survive the cryopreservation process and maintain their intercellular connections which are crucial for further development. In vitro development of PAFs to the pre-ovulatory stage has not yet been achieved in humans and larger animals. However, in vitro culture systems for PAFs are under development and are expected to become available in the near future. Animal in vitro models such as the bovine that rely on unlimited sources of research material and which are largely free of ethical or moral restrictions, have an important role in contributing to make faster progress in the development of these FP strategies.
An adverse outcome pathway-based tiered testing strategy for the assessment of thyroid hormone disruption in fish - Evelyn Stinckens (22/09/2020)
- 22 september 2020
- Promotoren: Prof. Dries Knapen, Dr. Hilda Witters en Dr. Lucia Vergauwen
Thyroid hormones (THs) play a crucial role in the regulation of vertebrate development and homeostatic processes. A growing number of environmental pollutants are known to adversely affect the TH system. Disruption of this system is increasingly being recognized as an important endocrine disrupting mode of action that can cause a wide variety of adverse effects.
Major gaps have been identified in the tools available for the hazard and risk assessment of TH disrupting substances. The scientific community is currently challenged with developing new or improved testing approaches to evaluate TH disruption in fish. Therefore, the overall aim of this thesis was the development of a tiered testing strategy for the assessment of TH disruption in fish. We used the Adverse Outcome Pathway (AOP) framework for guiding our work.
First, we constructed AOPs leading from thyroperoxidase (TPO, essential for TH synthesis) and deiodinase (DIO, critical for TH activation) inhibition to impaired swim bladder inflation of zebrafish and fathead minnow through decreased thyroid hormone concentrations, ultimately affecting survival probability and population growth.
Next, we provided additional biological and toxicological data supporting the mechanisms and processes captured in the AOPs. We performed in chemico assays targeting the specific molecular initiating events, DIO and TPO inhibition, and we evaluated the use of the resulting in chemico data for predicting higher level in vivo endpoints. We demonstrated that the combined information of the enzyme inhibition assays can be used as a tool to reliably predict the biological effects on swim bladder inflation with only few outliers. This work thus provides an example of how the AOP framework and associated data generation can address current TH disruption testing challenges in the context of fish early-life stage assays, and fish tests in general.
Finally, we demonstrate how different assays covering essential biological processes along the continuum of the AOP network can be implemented in a tiered screening and testing approach for TH disruption in fish. The addition of the associated assays to existing test guidelines allows to increase their diagnostic value for the assessment of TH disruption. Further expanding the applicability domain of our AOP network would be of great value for screening and testing of TH disrupting compounds.
Drug disposition in the zebrafish embryo and larva: focus on cytochrome P450 activity - Evy Verbueken (16/12/2019)
- 16 december 2019
- Promotoren: Prof. Steven Van Cruchten en Prof. Chris Van Ginneken
The zebrafish embryo is increasingly used as an alternative model to screen new drug candidates and environmental pollutants for developmental toxicity (i.e. teratogenicity). Since the zebrafish is not considered to be a test animal until it reaches the stage of independent feeding, the zebrafish embryo developmental toxicity assay (ZEDTA) fits within the 3Rs (i.e. Replacement, Reduction and Refinement) concept as described within laboratory animal sciences.
The externally developing zebrafish embryo cannot rely on maternal metabolism unlike the mammalian embryo. Hence, the zebrafish embryo gets directly exposed to the parent compound and depends on its own drug–metabolizing capacity for the detoxification or bioactivation of xenobiotics. In this respect, knowledge of the intrinsic biotransformation capacity during zebrafish organogenesis, which coincides with the exposure window of the ZEDTA, is key in order to correctly interpret the outcome of the ZEDTA. However, the overall results of studies described in literature regarding the xenobiotic–metabolizing capacity of zebrafish embryos are contradictory.
Hence, the main goal of this doctoral project was to characterize drug disposition in zebrafish during organogenesis with a main focus on cytochrome P450 (CYP)–mediated metabolism since the latter enzymes are responsible for the oxidation of the majority of marketed drugs. To this end, the thesis investigates the ontogeny of CYP enzymes on mRNA as well as on activity level, and to a lesser extent also of the expression levels of two major phase II enzymes and a drug transporter, i.e. abcb4, at different time–points during zebrafish organogenesis and beyond.
This project mainly showed that CYP–mediated biotransformation of xenobiotics appears to be immature during a major part of the ZEDTA exposure window (i.e. 4–120 h post–fertilization (hpf)). Moreover, the mRNA expression levels of the phase II enzymes and abcb4 reached maximum expression levels by the end of zebrafish organogenesis. These findings can have a profound impact on the predictivity of the ZEDTA for human safety assessment in the drug development process, especially in case of proteratogenic compounds that require bioactivation to exert their teratogenic potential.
A solution to overcome the immature biotransformation capacity of zebrafish embryos is to co–incubate the ZEDTA with a human–derived external metabolic activating system (MAS), such as human liver microsomes, during the entire exposure window of the ZEDTA. However, the co–incubation method with the external MAS needs to be further optimized and validated before it can be used in regulatory developmental toxicity testing.
Advancing the zebrafish embryo test for estrogen disruptor screening - Ellen Michiels (06/12/2019)
- 6 december 2019
- Promotoren: Prof. Dries Knapen en Prof. Steven Van Cruchten
The presence of endocrine disrupting compounds (EDCs) in the environment is a major concern. EDCs directly interact with the endocrine system, leading to important adverse effects in humans and wildlife (e.g, related to reproduction, energy metabolism and growth, behaviour, etc.). To identify these compounds, several regulatory strategies and frameworks have been developed in many parts of the world. Various assays are available to assess the endocrine disrupting potential of compounds, and several methods are used to administer the compounds. Within the context of non-mammalian assays, fish are often used as model organisms.
Almost all current fish assays use adult animals, but there is increasing public pressure to reduce or even avoid the use of animal testing. Therefore, significant efforts are being made to develop new, alternative testing methods as well as to optimize existing methods for the assessment of endocrine disrupting compounds. The development of a fish embryo test for the detection of EDCs may offer a particular advantage in this context, since fish embryos are not considered test animals until the stage of free feeding while they do offer the biological relevance gained from using a model organism with an intact endocrine system.
Assays using fish and fish embryos are mostly performed via aquatic exposure. However, many EDCs are hydrophobic compounds. Aquatic exposure may therefore not always be the most biologically relevant exposure method. In addition, the use of hydrophobic compounds in an aquatic exposure set-up brings several practical challenges as well. In the present work, we used an existing fish embryo test guideline as a basis for the development of an alternative test system for the assessment of EDCs.
Specifically, the Fish Embryo Acute Toxicity (FET) Test (OECD Test Guideline 236) was supplemented with a number of specific endpoints relevant to the screening and testing of estrogen disrupting compounds. In addition, we suggest a combination of aquatic exposure (i.e, as currently required in the FET test) and administration of the compound under consideration via micro-injection. Micro-injection into the yolk of the embryo can possibly mimic maternal transfer, and it helps to solve a number of practical challenges that are associated with the use of hydrophobic compounds in aquatic exposure scenarios. Overall, we developed a new approach allowing us to advance the zebrafish embryo test for use in the assessment of estrogen disrupting compounds.
Maternal metabolic disorders and fertility: importance of lipotoxicity on oocyte, sperm and embryo - Karolien Desmet (19/11/2019)
- 19 november 2019
- Promotoren: Prof. Jo Leroy en Prof. Peter Bols
Lipolysis-associated maternal metabolic disorders, like obesity and diabetes type II in women and negative energy balance in dairy cows, are a major risk factor for subfertility. These altered metabolic states are reflected in the follicular and oviductal micro-environment with emphasis on elevated concentrations of non-esterified fatty acids (NEFAs). Although information is available about the toxic effects of elevated NEFA concentrations on oocyte and embryo developmental competence, there remains a lack of information regarding the underlying mechanisms. In this dissertation, the bovine model was used to study the relation between lipolysis-linked maternal metabolic disorders and fertility.
We showed that elevated NEFA concentrations during either in vitro oocyte maturation or early embryo development impact the resultant blastocysts’ transcriptomic and epigenetic profiles. This disturbance in epigenetic reprogramming may render the embryo susceptible to changes during further development. We observed that good quality blastocysts produced from metabolically compromised oocytes have defective post-hatching development after embryo transfer to a recipient cow. These embryos were retarded in growth, exhibited disturbed metabolism and transcriptomic profile, and displayed a reduced capacity to signal its presence to the mother.
Also the oviductal environment plays a vital role in the functionality of and interaction between the oocyte and sperm cells. We showed that although sperm functionality was affected under lipolytic conditions, adequate numbers of viable sperm cells retained their fertilizing ability. In contrast, the fertilization process was affected as well as subsequent embryo development.
Metabolic disorders and sub-/infertility are both increasing in men and women. The combination of having both obese parents on fecundity has received minimal attention although most couples share lifestyle and thus obesity in both persons is more likely to occur. We investigated if metabolic conditions of couples seeking assisted reproductive services in an IVF-clinic could be linked to embryo quality and metabolism. Aberrations in follicular fluid, female and male waist circumference had an effect on embryo quality and metabolism. Combining BMI of both parents led to an association between parental BMI and embryo metabolism. Only male BMI was associated with pregnancy outcome.
We concluded that lipolytic conditions cause subfertility through impairment of oocyte maturation, fertilization and embryo development. We also substantiated an effect of the parents’ metabolic health on quality and metabolism of their embryos in a human setting. In characterizing these effects, we may increase the awareness of the importance of parental metabolic health in relation to fertility and carryover effects persisting in the offspring.
The use of zebrafish feeding trials for the safety assessment of genetically modified crops - Isabelle Gabriëls (06/06/2019)
- 6 juni 2019
- Promotoren: Prof. Dries Knapen, Prof. Marc De Loose en Dr. Lucia Vergauwen
In the EU, a mandatory part of the safety assessment of genetically modified crops (GMOs) consists of a 90-day rodent feeding trial. As a relevant addition to the current assessment strategy, our aim was to develop a complementary zebrafish feeding trial specifically designed to address a number of shortcomings of existing test methods, using maize as a case study.
First, to ensure the use of nutritionally balanced experimental diets, we investigated whether substitution of a non-GM maize into the zebrafish diet causes component-related effects. High dietary maize percentages resulted in an overall disturbed uptake and processing of carbohydrates, influencing processes such as growth rate, and affected mRNA transcriptional liver patterns of genes involved in key metabolic processes. Balancing the physiological tolerance of the fish and achieving a toxicologically relevant dose of the GMO, 15% was selected as the optimal maize substitution level to be used for all experiments.
Next, a subchronic zebrafish feeding trial was developed for the toxicological safety assessment of GMOs. Endpoints included general health parameters, effects on energy metabolism, and reproductive effects including transgenerational effects. As a key aspect of our experimental design, we investigated the importance of different non-GM comparators. Our results highlight the importance of distinguishing between potential effects caused by the transformation process (i.e., the process of genetically modifying the plant) and those caused by the presence of the transgenic event itself. Indeed, most differences (in general health endpoints as well as transcriptional liver profiles) were identified between the conventional wild type control (i.e., the original genetic starting material) and null segregant (i.e., the plant progeny created during the transformation process but lacking the transgenic event) on the one hand, and between wild type and GM maize on the other hand, while differences between GM maize and null segregant were limited. To assess the toxicological relevance of biological responses, we further optimized a method allowing the interpretation of results relative to the natural response variation, i.e., the normal biological range in endpoint responses of the fish. We established natural response variation datasets for various zebrafish endpoints and discussed equivalence testing as a conceptual approach using examples originating from the subchronic/transgenerational feeding trial.
Overall, this work forms a strong foundation for advancing zebrafish feeding trials and, to facilitate the development of further strategies, main conclusions and recommendations have been summarized in a Guidance Document for the safety evaluation of GM crops using the zebrafish model.