The power of personalized nutrition lies in being able to conduct clinical research on healthy people while capturing metabolic markers sensitive to the impact of environmental and metabolic... Show moreThe power of personalized nutrition lies in being able to conduct clinical research on healthy people while capturing metabolic markers sensitive to the impact of environmental and metabolic stressors (e.g. diet, changing sex hormones and the menstrual cycle). Using clinical biomarkers, metabolomics, and diet interventions with intake analyses, we demonstrated the metabolic impact of vegan and animal diet interventions using fasting plasma analysis after 48 hours and using postprandial plasma analysis after meals and snacks. Sexually dimorphic responses were differentiated using proteomics and pathway analyses in two larger, sex-balanced cohorts. Finally, clinical biomarker and metabolomics analyses identified metabolic subtypes across menstrual cycle phases. Although challenges with integrating –omics technology and nutrition remain, the fundamental information generated from these research studies may provide a foundation for future novel personalized nutrition strategies. Show less
The aim of this thesis is to expedite and ensure the systematic accuracy of clearance scaling from adults to paediatric patients, with a special focus on drugs undergoing hepatic metabolism. A... Show moreThe aim of this thesis is to expedite and ensure the systematic accuracy of clearance scaling from adults to paediatric patients, with a special focus on drugs undergoing hepatic metabolism. A physiologically-based pharmacokinetic simulation workflow was developed to unravel the conditions for accurate scaling of drug clearance from adults to children as young as term neonates of one day for various methods. This disproved the belief that a universal allometric exponent can scale size-related changes in clearance across the paediatric age range, and showed that isoenzyme maturation and drug properties, especially extraction ratio and drug binding to alpha-1-acid glycoprotein, should be accounted for when scaling clearance to young children. Based on these results, a clearance scaling decision tree is proposed, which allows pharmacologists for the first-time to select scaling method(s) that require a minimum but still sufficient amount of information to accurately scale clearance of drugs with known properties to a desired paediatric age-range. Moreover, an analysis framework is provided to assess the feasibility and clinical trial requirements for the estimation of PBPK parameters using population pharmacokinetic modelling, which has the potential to expedite development of PBPK models for understudied paediatric subpopulations. Show less
The research described in this thesis focused on identifying novel drug targets and synergistic combinations for triple-negative breast cancer (TNBC), a virulent subtype of breast cancer with a... Show moreThe research described in this thesis focused on identifying novel drug targets and synergistic combinations for triple-negative breast cancer (TNBC), a virulent subtype of breast cancer with a dismal prognosis and limited therapeutic options. In particular, the work centred on reversing resistance of TNBC cells to EGFR inhibitors. High-throughput kinase inhibitor library-based screens were utilised to evaluate the potential of novel targeted agents in a panel of TNBC cell lines and subsequently identify TNBC-specific genetic dependencies using siRNA-based screening. The signal transduction pathways perturbed by drug treatment were delineated and subsequently scrutinised using transcriptomic profiling and western blotting. The impact of drug treatment or gene silencing on cell death, proliferation, cell cycle progression and migration was assessed simultaneously. This work demonstrated that TNBC cell lines resistant to both MEK and Akt inhibitors are sensitive to disruption of CDK function. Additionally, it revealed that novel CDK inhibitors with strong activity against P-TEFb/CDK9 are highly effective against TNBC cells as single agents and in combination with multiple targeted therapies. These agents provoked profound down-regulation of multiple oncogenic pro-proliferative pathways, the silencing of which was detrimental to TNBC cell proliferation, thus defining several genes as potential future drug targets. Show less
Growth and development affect the metabolism of drugs administered to neonates, infants, and children. Research in this thesis focused on the metabolism by cytochrome P450 (CYP) 3A enzymes, aiming... Show moreGrowth and development affect the metabolism of drugs administered to neonates, infants, and children. Research in this thesis focused on the metabolism by cytochrome P450 (CYP) 3A enzymes, aiming to predict CYP3A-mediated clearance in neonates, infants, and children, by development of pediatric (physiological) population pharmacokinetic models.CYP3A-mediated systemic metabolism of midazolam in critically ill pediatric patients was found to be impacted by body weight, critical illness, and inflammation. The developed model was subsequently found to accurately predict clearance in postoperative children or critically ill patients. Furthermore, advanced physiological modelling methods were applied to distinguish between first-pass and systemic CYP3A-mediated metabolism to elucidate the role of intestinal and hepatic CYP3A in neonates and children covering the whole pediatric age range. Lastly, it was described when a pediatric covariate function for CYP3A-mediated midazolam clearance could be applied to scale plasma clearance of other CYP3A substrates in the pediatric population.This work will significantly improve CYP3A-mediated clearance predictions in neonates, infants, and children, which will ultimately lead to rational support for pediatric doses of CYP3A substrates in first-in-child studies during drug development and for pediatric dose recommendations for CYP3A substrates in clinical practice. Show less
Discovery and development of Central Nervous System (CNS) drugs is hampered by high attrition rates. One of the reasons is the lack of blood-based biomarkers that represent the interaction between... Show moreDiscovery and development of Central Nervous System (CNS) drugs is hampered by high attrition rates. One of the reasons is the lack of blood-based biomarkers that represent the interaction between the drug and the neurological systems of interest. Here we present a systems-pharmacology approach that combines a multi-biomarker approach (e.g. metabolomics) with pharmacokinetic/pharmacodynamic (PK/PD) modeling to reveal quantitative pharmacological characteristics that are relevant to dopaminergic drug action. Moreover, we set out to identify biomarkers that can be obtained from the blood as non-invasive sampling site. In the first section of this thesis the methodology is introduced in the context of translational CNS drug development. Moreover, a systematic search is performed to available biomarkers of dopaminergic drug action. Then, in the second part, the multi-biomarker PK/PD approach is applied to biomarkers from the neuroendocrine system as connection between brain and blood. In the third section, the methodology is developed using the simultaneous, time-resolved metabolomics response in brain extracellular fluid and plasma. By applying multi-biomarker PK/PD modeling we revealed quantitative pharmacological characteristics of dopaminergic drugs with regard to multiple biological processes. Moreover, we identified potential blood-based biomarkers of dopaminergic effect in the brain. Show less
In this thesis, we used genetically engineered mouse models to identify genes and pathways that are involved in ILC formation and in the development of resistance to FGFR-targeted therapy. These... Show moreIn this thesis, we used genetically engineered mouse models to identify genes and pathways that are involved in ILC formation and in the development of resistance to FGFR-targeted therapy. These mice carry conditional alleles of Cdh1, which result in the inactivation of the cell-adhesion molecule E-cadherin when Cre-recombinase is expressed. As mice with mammary-specific inactivation of E-cadherin alone were not prone to develop mammary tumors, they were used to investigate the contribution of additional genetic mutations to the development of ILCs using different genetic approaches. Firstly, we used non-germline modeling to study the role of PI3K-AKT signaling in the development of ILCs by performing intraductal injections of high-titer lentiviruses. Secondly, we employed a Sleeping Beauty (SB)-based insertional mutagenesis screen in conditional Cdh1 knockout mice to identify novel genes and pathways involved in the development of ILCs. We show that active transposon mutagenesis drives ILC formation and analysis of common insertion sites in SB-induced tumors identified a mutually exclusive group of four genes, of which three are frequently aberrated in human ILCs. Lastly, we used active mobilization of transposons in transplanted mouse ILCs to identify genes involved in acquiring resistance to the FGFR inhibitor AZD4547. Show less
In summary, the collective results described in this thesis show that nanoparticulate vaccines can be delivered intradermally by coated and hollow microneedles and evoke antigen-specific immune... Show moreIn summary, the collective results described in this thesis show that nanoparticulate vaccines can be delivered intradermally by coated and hollow microneedles and evoke antigen-specific immune responses. The choice of both the nanoparticles and the microneedle(s) could have important influences on the immune responses. Microneedle arrays coated with antigen loaded and lipid bilayer fused mesoporous silica nanoparticles (MSNs) could be a promising system for convenient and fast intradermal delivery of protein antigen, although our results indicate that the system needs to be improved in order to obtain optimal immune responses. Moreover, antigen and adjuvant loaded nanoparticles can increase IgG2a (Th1) and CD8+ responses after intradermal delivery by hollow microneedles. This effect depends on the type and the physicochemical characteristics of the nanoparticles, in which smaller size and controlled release properties of antigen and adjuvant were found to correlate with the stronger effect. Finally, the combination of separate antigen loaded and adjuvant loaded nanoparticles may be as efficient as the antigen and adjuvant co-encapsulated nanoparticles for modification of the immune responses following intradermal immunization. Show less
The stratum corneum is the outermost skin layer and consists of dead cells embedded in a lipid matrix. The lipid matrix, consisting of ceramides, fatty acids, and cholesterol, is crucial for a... Show moreThe stratum corneum is the outermost skin layer and consists of dead cells embedded in a lipid matrix. The lipid matrix, consisting of ceramides, fatty acids, and cholesterol, is crucial for a proper skin barrier function. In inflammatory skin diseases the lipid composition and ordering is altered contributing to the impaired skin barrier. Vernix caseosa (VC) is the cheesy, white cream that covers the skin of the human fetus. Application of an in house developed synthetic VC enhanced skin barrier repair in mice. Currently, there are no suitable skin models available to study human skin barrier repair after application of a topical formulation. This thesis describes the development of a human skin barrier repair model and evaluates VC based formulations using this model. The results demonstrate that the barrier of this repair model mimics several aspects of inflammatory skin diseases. Additionally, it was shown that the lipid properties in this model were improved when a synthetic VC-based formulation was applied. Based on the outcome, clinical studies were performed. These studies showed that application on a disrupted human skin barrier in vivo enhanced the barrier repair. However, the effects of the formulation are limited when applied on atopic dermatitis skin Show less
This thesis describes the development of improved formulations and alternative delivery strategies for polio vaccination. A new generation of IPV should not only be affordable and safe to... Show moreThis thesis describes the development of improved formulations and alternative delivery strategies for polio vaccination. A new generation of IPV should not only be affordable and safe to produce, but preferably should also induce mucosal immunity, remain stable at unrefrigerated conditions, and be easy to administer. This is also important with regard to stockpiling and outbreak management in the period towards and beyond polio eradication. Solid dosage forms that have improved thermostability, like biodegradable Bioneedles comprising lyophilized IPV or polymer-based oral films, would be favorable to reach remote areas in developing countries for which proper logistics are not available. Replacing the currently used polio vaccines with a thermostable vaccine may yield significant cost savings. Furthermore, vaccination via mucosal routes showed to have important advantages over conventional intramuscular vaccination using needle and syringes. Show less
During the course of drug discovery translational steps are made. The translation from in vitro to in vivo experiments is not as predictive as one would desire, resulting in selection of... Show moreDuring the course of drug discovery translational steps are made. The translation from in vitro to in vivo experiments is not as predictive as one would desire, resulting in selection of inefficacious compounds but also in overlooking of promising drug candidates. This is not different for the mGlu2 receptor for which no drugs are available on the market so far despite enormous drug discovery efforts. Therefore, there is a need to improve the molecular understanding of key in vitro parameters that drive in vivo efficacy. Hence, this thesis focuses on the concepts of target binding kinetics and functional efficacy of both allosteric and orthosteric ligands of the mGlu2 receptor. Show less
In this thesis, a system pharmacology approach, integrating metabolomics, pharmacology and chemical biology, was applied to understand and modulate the endocannabinoid system across different model... Show moreIn this thesis, a system pharmacology approach, integrating metabolomics, pharmacology and chemical biology, was applied to understand and modulate the endocannabinoid system across different model systems (cells, zebrafish, mice and humans). The endocannabinoid system (ECS) and its function in the brain was discovered after identifying THC: the active ingredient of cannabis (marijuana) plant. It is perhaps the most important physiological signalling system involved in establishing and maintaining human health. In disease conditions the ECS system is dysregulated and becomes either highly active or less active. Due to the widespread effects throughout the body, targeting ECS system is believed to hold promise as a treatment target in the field of medicine. In this thesis, the functional role of the ECS was studied in healthy and diseased conditions. In addition, the ECS was modulated using enzyme inhibitors to gain a better understanding in different signalling pathways. Furthermore, to understand the drug pharmacology and whether such new compound inhibitors may modify disease biochemistry; this includes the study of target engagement, downstream effects and the off-target effects inhibitors. Overall, we are convinced that by applying system pharmacology approach to study ECS can lead to develop novel therapeutic biomarkers to treat metabolic or neurological diseases. Show less
The present thesis focuses on the pharmacological concept of drug-target interaction, which dates back to the beginning of modern pharmacology. A traditional equilibrium metrics-based... Show moreThe present thesis focuses on the pharmacological concept of drug-target interaction, which dates back to the beginning of modern pharmacology. A traditional equilibrium metrics-based rationale (i.e. optimization of drug affinity leads to better efficacy and safety) is unable to prevent current high attrition rates in the early phase of drug discovery. In the past decade drug-target binding kinetics (i.e. association and dissociation rate constants, residence time) has been gaining more and more attention, which constitutes a paradigm shift to better predict parameters of drug efficacy and safety. We decided to investigate binding kinetics of G protein-coupled receptors (GPCR), since GPCR are involved in various critical physiological and pharmacological functions, being the target of about 30% of all drugs on the market. Both the human cannabinoid receptor 1 and the human adenosine A1 and A3 receptors were chosen as prototypical GPCR as well as potential drug targets. The binding kinetics investigations described in this thesis provide a better and multi-faceted understanding of drug-target interactions and offer suggestions for the design of better ligands with an appropriate kinetic profile, new technologies for rapid kinetic assessment, and ultimately suitable evaluation schemes for a better translation towards effective and safe drugs. Show less
Traditional drug discovery approaches have been hampered by (in vitro) cell-culture models that poorly represent the situation in the human body. Principally, cells grow in the body in a three... Show moreTraditional drug discovery approaches have been hampered by (in vitro) cell-culture models that poorly represent the situation in the human body. Principally, cells grow in the body in a three-dimensional (3D) environment that cannot generally be captured using cell culture methods. For this reason, cell-culture models have been developed where cells grow in a 3D-environment, which allows them to form structures that are more comparable to tissue in the body. However, the full complexity of these advanced cell-culture models can only be fully used for routine drug testing if the cell culture model can be used on a large scale (also termed high-throughput screening or HTS), and if the readout can capture all of the biological complexity reflected by the 3D-cultured cells (high-content screening or HCS). Due to these technological limitations, 3D cellular models are not yet routinely applied in drug and drug-target discovery. This thesis describes the development of fully-scalable 3D cell-culture screening platforms in the context of cancer and polycystic kidney disease. Show less
Drug-target binding kinetics determine the time course of the central event in pharmacotherapy: Drug-target interaction. However, the time course of a drug effect is also influenced by many... Show moreDrug-target binding kinetics determine the time course of the central event in pharmacotherapy: Drug-target interaction. However, the time course of a drug effect is also influenced by many other physiological processes such as the metabolism and excretion of a drug and the transduction of the relevant biological signals. In this study, we investigate when target binding kinetics are determining the time course of drug effect and generate understanding into the relation between the parameter values and the rate-limiting step in the duration of a drug effect. Show less
There is an urgent need for more physiologically relevant cell culture methods to guide compound selection in pre-clinical stages of the drug development pipeline. This thesis describes the... Show moreThere is an urgent need for more physiologically relevant cell culture methods to guide compound selection in pre-clinical stages of the drug development pipeline. This thesis describes the development of the OrganoPlate, a microfluidic platform that enables enhanced physiology in cell culture models by combining 3D cell culture, co-culture and perfusion flow, whilst maintaining ease of use, compatibility and throughput. Phaseguides are capillary pressure barriers that enable microfluidic liquid routing and patterning without the use of membrane or other physical barriers. This technology was further developed to enable complex liquid routing using only a standard pipette Phaseguide technology was implemented for gel patterning in a dedicated 3D cell culture device embedded in a standard 384 wells plate. Each plate contains up to 96 microfluidic networks that enable perfusion culture of extracellular matrix embedded tissues and perfused epithelial or endothelial tubules. The standard dimensions and high quality optical readout allows interrogation of these tissues using high content readers as well as other standard readout equipment. The platform has been used for the culture of a variety of tissue types and disease models by the authors, but has also been adopted by expert and non-expert users across the field. Show less
In recent decades, the use of a systems-based view of life has provided key insight into fundamental processes with respect to biology. In life sciences, important paradigm shifts are the way in... Show moreIn recent decades, the use of a systems-based view of life has provided key insight into fundamental processes with respect to biology. In life sciences, important paradigm shifts are the way in which we approach health and disease. Although modern medicine has traditionally emphasized pathology and acute conditions, our current understanding is that different interventions are needed for treating and preventing chronic disease. To design better interventions, new diagnostic tools are urgently needed in order to create new opportunities for achieving personalized health and medicine. The focus in diagnosis is, therefore, shifting from measuring single biomarkers such as glucose and cholesterol to creating complex maps of the dynamic patterns underlying regulatory processes. Moreover, the notion of "health" is viewed in a holistic context using biochemistry as a basis and then expanding this basis to include the psycho-social environment, including the individual's worldview. In the field of diagnostics, a highly promising new tool has recently emerged based on ultra-weak photon emission from biological systems, including all living cells. The aim of this thesis was to explore the applications of UPE and correlate it with biochemistry in order to obtain a deeper understanding of the processes that occur in living systems. Show less
The traditional medical treatment paradigm focuses on prescribing one drug to treat all patients with a specific disease or condition, so called ‘one-size-fits-all’. However, it has been shown... Show moreThe traditional medical treatment paradigm focuses on prescribing one drug to treat all patients with a specific disease or condition, so called ‘one-size-fits-all’. However, it has been shown increasingly that differences between persons, such as in lifestyle or genes, can change both the course of a disease and effect of a drug. In order to adapt medical treatment and drug development to that, a concept know as precision medicine, it is essential to study which and how genetic differences affect drug response. This thesis describes the study of the influences of genetic variation on a specific class of drug targets, the G protein-coupled receptors (GPCRs).Altogether a novel cellular approach towards studying genetic effects on GPCR function has been explored and detailed throughout this thesis. Several GPCRs and different types of genetic variations were investigated, demonstrating together that personal cell lines in combination with label-free technology are an appropriate tool to enable GPCR pharmacogenetic studies. Incorporating aspects such as genetic variation in drug targets, representative model systems and appropriate assay technology are important factors for advancing GPCR drug discovery. The data presented in this thesis contributes towards the progress of applying precision medicine concepts to this class of drug targets. Show less
In previous studies at our laboratory it was demonstrated that drug exposure of HepG2 cells can lead to an altered TNFα-induced NF-κB oscillatory phenotype, concurrent with a synergistically... Show moreIn previous studies at our laboratory it was demonstrated that drug exposure of HepG2 cells can lead to an altered TNFα-induced NF-κB oscillatory phenotype, concurrent with a synergistically increased sensitivity for TNFα-induced apoptosis. We have also shown that synergistic drug/ TNFα-induced cell death is dependent on concurrent cellular stress responses. To monitor these stress responses, we have developed a fluorescent protein stress response reporter platform, feasible for high throughput approaches. In this thesis, we focus on an in vitro HepG2 cell model, in which the addition of TNFα reflects inflammatory stress. We studied inflammatory signaling, both for use in improved in vitro testing approaches and to gain mechanistic understanding of TNFα-induced signaling in hepatocytes. Show less
According to the amyloid cascade hypothesis, accumulation of beta-amyloid (Aβ) peptides initiates the pathological cascade in Alzheimer's disease (AD). Early in the disease process, before clinical... Show moreAccording to the amyloid cascade hypothesis, accumulation of beta-amyloid (Aβ) peptides initiates the pathological cascade in Alzheimer's disease (AD). Early in the disease process, before clinical symptoms, an increase in Aβ concentrations leads to formation of toxic Aβ oligomers. These oligomers drive the neurodegeneration in AD brain. An important therapeutic strategy is to lower Aβ concentration in the CNS. Theoretically, this can prevent all subsequent pathological processes. Aβ is the final product of sequential proteolytic cleavages of the precursor protein APP. The drug effects on the individual pathways of APP processing are hard to predict, because these are regulated by a complex biochemical network. In this research, a 'systems pharmacology' approach was applied, integrating available knowledge of biology and pharmacology of system reactions into mathematical models. The APP-system-pharmacology-model provides important information about the APP processing pathways: (i) Aβ production inhibition leads to a relatively greater decrease in Aβ oligomers compared to monomers (ii) dissociation of oligomers contributes to the drug effect; (iii) Aβ42 is the major Aβ variant that contributes to the oligomer pool; (iv) inhibition of the enzyme GS stimulates alternative processing of APP by feedback. The APP-system-pharmacology-model can be of value in development of therapeutic interventions for AD. Show less
