Introduction: Pharmacokinetic variability in response to pharmacotherapy contribute to adverse drug reactions, drug-drug interaction and therapeutic failure seen in clinical practice. Poor therapeutic response to medication has been attributed to inter-individual and interethnic variability in cytochrome P450 (CYP450)-dependent metabolism and altered drug absorption via expressed transport channels such as P-glycoprotein (P-gp). An individualised approach in therapeutic management would be beneficial in a South-African population considering the country’s large genetic diversity. A single time point, non-invasive capillary sampling, combined with a low dose probe drug cocktail, to simultaneously quantify in vivo drug and metabolite concentrations, would enhance the feasibility and cost-effectiveness of routine phenotyping in clinical practice and guide personalised prescribing to individual patients. A recent development in dried blood spot sampling is the Mitra™ device, using Volumetric Absorptive Micro Sampling (VAMS™) technology to collect an accurate volume (10-30 µL) of whole blood onto a hydrophilic polymeric tip as an alternative to plasma sampling. Small volume blood sampling however presents bioanalytical challenges in terms of the reproducibility and sensitivity of the quantitative method and the agreement between quantitative measurement from a dried blood spot (DBS) and that from plasma sampling. The physicochemical diversity of the structurally related aromatic probe drugs, used together in a drug cocktail, further require optimised analytical procedures for simultaneous quantification. Phenotyping cocktails are compounded from commercially available dosage forms and introduce challenges with regards to dosage homogeneity, chemical interference or degradation and possible incompatibilities of drugs when used in combination. Aim and objectives: The purpose of this study was to compound the validated “Geneva phenotyping drug cocktail”, from available API sources and develop a validated, targeted, analytical LC-MS/MS method to quantify the seven probe drugs and six respective metabolites in dried blood spots when using the Mitra™ volumetric absorptive micro-sampling device for blood collection. The aim was to assess inter-method agreement of the measured probe drug and metabolite concentrations between the low sample volume, from a dried blood spot, and conventional plasma sampling. Methods: An Agilent binary series LC system coupled to a Sciex 4000 QTRAP triple quadrupole tandem mass spectrometer was used for method optimisation and validation. Targeted LC-MS/MS methods, in both negative and positive ESI mode, were validated according to ICH guidelines for matrix effects, recovery, linearity, limits of quantitation and detection, carry-over, inter and intraday precision and accuracy and analyte stability. The selectivity of the structurally related ionisable analytes was compared between a Kinetex C18 and Kinetex Biphenyl column and the influence of changes in the analytical conditions (involving mobile phase pH and solvent mixture composition as well as the solvent type) studied. An initial assessment of statistical in vitro agreement between plasma and DBS sampling were carried out. USP assays were performed to determine the weight and content uniformity of the compounded phenotyping cocktail containing six of the seven probe drugs. Content uniformity was evaluated with an Acquity UPLC system coupled to a Synapt G2 QTOF mass spectrometer. Results and discussion: A biphenyl stationary phase in combination with methanol as the organic eluent, provided improved resolution and analyte selectivity of the structurally related aromatic compounds. Results from the robustness experiment further confirmed the importance of controlling analytical conditions to ensure reproducibility and reliability of the quantitative method. Separation selectivity and higher throughput were prioritised over optimised ionisation efficiency, although the sensitivity of the analytical method for individual analytes were still within the expected in vivo concentration ranges to infer metabolic and transport phenotypes. This study successfully validated the use of DBS, collected with the volumetrically controlled absorptive microsampling device Mitra™, to measure expected probe drug and metabolite concentrations using the “Geneva phenotyping cocktail”. The validated method met all the required standards accepted in bioanalytical chemistry for specificity, sensitivity, linearity, accuracy, precision, carry-over and stability. From the initial in vitro assessment of agreement, it was concluded that blood cell distribution kinetics are regulated by the blood-to-plasma concentration ratio and time dependent equilibrium between different blood compartments, the physicochemical properties of the analytes, temperature during extraction, analyte concentration and stability. A conclusive confounding factor was the extent to which the extraction procedure liberated bound drug from either plasma proteins or erythrocytes. It was further concluded that the compounded low dose phenotyping cocktail capsules could be used successfully to assess inter-method agreement of drug-based metabolic ratios and drug transport between plasma and DBS collected with the Mitra™ device. Conclusion: To our knowledge, this is the first DBS validation study using the Mitra™ device for the purpose of simultaneous phenotyping of the in vivo P-gp transport and CYP450 metabolic activity of the CYP1A2, -2B6, -2C9, -2C19, -2D6 and -3A4 enzymes and activity. / Thesis (PhD)--University of Pretoria, 2019. / National Research Foundation / Pharmacology / PhD (Pharmacology) / Unrestricted

Personalised medicine describes the practice of tailoring medical care to the individual characteristics of each patient. Fundamental to this practice is the identification of markers associated with differential treatment response. Such markers can be identified through the assessment of treatment effect modification using statistical methods. Randomised controlled trials provide the optimal setting for evaluating differential response to treatment. Due to restrictions regarding sample size, study length and ethics, observational studies are more appropriate in many circumstances, particularly for the identification of markers associated with adverse side-effects and long term response to treatments. However, the analysis of observational data raises some additional challenges. The overall aim of this thesis was to develop statistical methodology for the analysis of observational data, specifically primary care databases, to identify and evaluate markers associated with differential treatment response. Three aspects of the assessment of treatment effect modification in an observational setting were addressed. The first aspect related to the assessment of treatment effect modification on the additive measurement scale which corresponds to a comparison of absolute treatment effects across patient subgroups. Various ways in which this can be assessed in an observational setting were reviewed and a novel measure, the ratio of absolute effects, which can be calculated from certain multiplicative regression models, was proposed. The second aspect regarded the confounding adjustment and it was investigated how the presence of interactions between the moderator and confounders on both treatment receipt and outcome can bias estimates of treatment effect modification if unaccounted for using Monte Carlo simulations. It was determined that the presence of bias differed across different confounding adjustment methods and, in the majority of settings, the bias was reduced when the interactions between the moderator and confounders were accounted for in the confounding adjustment model. Thirdly, it has been proposed that patient data in observational studies be organised into and analysed as series of nested nonrandomised trials. This thesis extended this study design to evaluate predictive markers of differential treatment response and explored the benefits of this methodology for this purpose. It was suggested how absolute treatment effect estimates can be estimated and compared across patient subgroups in this setting. A dataset comprising primary care medical records of adults with rheumatoid arthritis was used throughout this thesis. Interest lay in the identification of characteristics predictive of the onset of type II diabetes associated with steroid (glucocorticoid) therapy. The analysis in this thesis suggested older age may be associated with a higher risk of steroid-associated type II diabetes, but this warrants further investigation. Overall, this thesis demonstrates how observational studies can be analysed such that accurate and meaningful conclusions are made within personalised medicine research.

Analyse intégrative de données génomiques et pharmacologiques pour améliorer la prédiction de la réponse aux thérapies cibléesL'utilisation de thérapies ciblées dans le contexte de la médecine personnalisée du cancer a permis d’améliorer le traitement des patients dans différents types de cancer. Cependant, alors que la décision thérapeutique est basée sur une unique altération moléculaire (par exemple une mutation ou un changement du nombre de copies d’un gène), les tumeurs montrent différents degrés de réponse. Dans cette thèse, nous démontrons que la décision thérapeutique basée sur une unique altération n’est pas optimale et nous proposons un modèle mathématique intégrant des données génomiques et pharmacologiques pour identifier de nouveaux biomarqueurs prédictifs de la réponse thérapeutique. Le modèle a été construit à partir de deux bases de données de lignées cellulaires (the Genomics of Drug Sensitivity in Cancer, GDSC and the Cancer Cell Line Encyclopedia, CCLE) et validé avec des données de lignées et des données cliniques. De plus, nous avons également développé une nouvelle méthode pour améliorer la détection des mutations somatiques à partir de données de séquençage d'exomes complets et proposons un nouvel outil, cmDetect, disponible gratuitement pour la communauté scientifique. / Integrated analysis of genomic and pharmacological data to better predict the response to targeted therapiesThe use of targeted therapies in the context of cancer personalized medicine has shown great improvement of patients’ treatment in different cancer types. However, while the therapeutic decision is based on a single molecular alteration (for example a mutation or a gene copy number change), tumors will show different degrees of response. In this thesis, we demonstrate that a therapeutic decision based on a unique alteration is not optimal and we propose a mathematical model integrating genomic and pharmacological data to identify new single predictive biomarkers as well as combinations of biomarkers of therapy response. The model was trained using two public large-scale cell line data sets (the Genomics of Drug Sensitivity in Cancer, GDSC and the Cancer Cell Line Encyclopedia, CCLE) and validated with cell line and clinical data. Additionally, we also developed a new method for improving the detection of somatic mutations using whole exome sequencing data and propose a new tool, cmDetect, freely available to the scientific community.

No / Traditional Chinese medicine (TCM) investigates the clinical diagnosis and treatment regularities in a typical schema of personalized medicine, which means that individualized patients with same diseases would obtain distinct diagnosis and optimal treatment from different TCM physicians. This principle has been recognized and adhered by TCM clinical practitioners for thousands of years. However, the underlying mechanisms of TCM personalized medicine are not fully investigated so far and remained unknown. This paper discusses framework of TCM personalized medicine in classic literatures and in real-world clinical settings, and investigates the underlying mechanisms of TCM personalized medicine from the perspectives of network medicine. Based on 246 well-designed outpatient records on insomnia, by evaluating the personal biases of manifestation observation and preferences of herb prescriptions, we noted significant similarities between each herb prescriptions and symptom similarities between each encounters. To investigate the underlying mechanisms of TCM personalized medicine, we constructed a clinical phenotype network (CPN), in which the clinical phenotype entities like symptoms and diagnoses are presented as nodes and the correlation between these entities as links. This CPN is used to investigate the promiscuous boundary of syndromes and the co-occurrence of symptoms. The small-world topological characteristics are noted in the CPN with high clustering structures, which provide insight on the rationality of TCM personalized diagnosis and treatment. The investigation on this network would help us to gain understanding on the underlying mechanism of TCM personalized medicine and would propose a new perspective for the refinement of the TCM individualized clinical skills.

A comprehensive molecular and phenotypic analysis of congenital and childhood cataractRachel L. Gillespie; The University of Manchester, Doctor of Philosophy, 2015Congenital and childhood cataract (CCC) is estimated to affect 3.5-6 per 10,000 children under 16 years in developed countries - a major cause of lifelong visual impairment. It is estimated that 25-50% of CCC cases are caused by genetic mutations. CCC demonstrates extreme heterogeneity with more than 100 associated genes, and may occur as an isolated anomaly of the eye (non-syndromic) or as a manifestation of a multisystem condition (syndromic). Limitations of conventional sequencing technologies have precluded precise genetic diagnosis and limited understanding of the epidemiological basis of the condition. Next generation sequencing (NGS) technologies have revolutionised the approach to the study of human disease. The aim of this research was to conduct a comprehensive molecular and phenotypic analysis of CCC using NGS.A disease-targeted NGS assay was designed to screen, in parallel, 115 genes associated with all forms of CCC. DNA from 36 patients, randomly selected from the study cohort, underwent cataract-targeted NGS. Putative cataract-causing variants were identified in 75% of individuals; 85% of non-syndromic patients and 63% of syndromic CCC patients. Cataract-targeted NGS was able to efficiently delineate disease sub-type and in some cases identified rare syndromic forms of the condition. These findings were envisaged to alter care and management of CCC patients demonstrating the potential clinical utility of the test. In a subset of cases, NGS identified CCC was a manifestation of an inborn error of metabolism. A number of these conditions were eligible for preventative treatment emphasizing the importance of early diagnosis. A strategic approach to the identification of novel recessive causes of CCC was also undertaken. Affected children from seven consanguineous families underwent pre-screening by cataract-targeted NGS to delineate those with mutations in known genes. Mutation negative patients underwent autozygosity-guided whole exome sequencing (WES) analysis. This strategic approach to disease gene discovery led to the identification three novel cataract-causing candidate genes, TRIM8, CCDC13 and GRWD1. It also led to the association of EIF2B2, known to cause adult-onset leukoencephalopathy with vanishing white matter (VWM) disease featuring pre-senile onset cataract, with childhood-onset cataract. This work demonstrated that cataract-targeted NGS offers an efficient and unbiased means of pre-screening, however, causation is difficult to assign to novel disease genes in the absence of experimental evidence. Correspondingly, in vitro analysis of a missense variant in HMX1 demonstrated the deleterious effect of the mutation on protein function. This work confirmed HMX1 as the cause of a rare oculoauricular phenotype and expanded the class of disease-causing mutations in this gene. In conclusion, this study has demonstrated that NGS is effective in the study of CCC and has provided a platform for future studies in to the genetic aetiology of the condition, as well as the molecular mechanisms underlying lens transparency and human development. The work adds to the increasing body of evidence that augurs an era of personalised genomic medicine in ophthalmology that will foresee improved patient outcomes attributable to the implementation of a stratified approach to medicine.

Cancer is a global health issue and is estimated to be the second leading cause of death worldwide. Within cancer treatment, it has become attractive to introduce precision medicine to harness the body’s own immune system to fight cancer. Personalised peptide cancer vaccine can activate the immune system and elicit an immune response by using the patient’s own blood and tumour cells.   The aim of this study was to gather information to better understand the production of individual-specific peptides and the challenges when producing these peptides with focus from a GMP perspective. The methodology for conducting this study and retrieve information for the result was gathered by three different data bases; EMA, European Commission and FDA, and by qualitative semi-structured interviews.   The findings show that these personalised peptides should be manufactured by using solid-phase peptide synthesis, cleavage from the resin, HPLC purification, and final salt exchange. The traditional GMP requirements must be used as a basis to build a pragmatic program. In terms of delivery time, the peptides can be delivered within a few weeks. The main challenges will be to manufacture successful peptides since the peptide sequences vary from patient to patient and due to that fact have different production efficacy, deliver within the set timeframe, and have flexibility in the manufacture process.   Multiple guidelines need to be followed in order to set up a process that consistently delivers the intended product. When producing oncology drugs on-demand it is essential to keep the timeline since the patients are severely ill. However, further studies are needed to determine how the manufacture of personalised peptides could be performed to harmonise with regulatory guidelines.

Background and Objectives: Economic evaluation and decision analysis provide a framework to evaluate incremental costs and effects associated with alternative health interventions. These methods can also be used as a tool to evaluate alternative clinical behaviours or practice patterns. The objective of this thesis was to investigate the impact of current Canadian practices in human epidermal growth factor receptor-2 (HER2) testing to target trastuzumab in early-stage breast cancer (BC). Methods: Project 1: A systematic review of previous trastuzumab and HER2 testing economic analyses was conducted to identify methodological gaps and key lessons. Project 2: A population-level, retrospective cohort was studied to determine HER2 testing and trastuzumab treatment patterns in Ontario early-stage BC patients. Project 3: A cost-utility analysis of alternative test-treat strategies was conducted using a Markov model of BC calibrated to the Canadian setting, and incorporating Project 2 findings. Results: Project 1: Previous economic evaluations demonstrated that HER2 test accuracy and sequencing were key considerations when modelling the cost-effectiveness of trastuzumab treatment. Consideration of local testing and treatment practices was lacking. Project 2: HER2 testing and treatment practice differed from guidelines, where documentation was available. Only 88% of equivocal results were confirmed, while 57% of HER2 positive patients received trastuzumab. Project 3: Calibration of the BC model minimised gaps between trial-based survival and expected Canadian survival patterns. Deviations from guidelines in practice suggest that primary testing with fluorescence in situ hybridization (FISH) would produce greater health gains at a reduced cost vs. primary immunohistochemistry with FISH confirmation. This finding was more apparent as the prevalence of HER2 positive disease increased. Introduction of newer in situ hybridisation tests may be cost-effective as well. Conclusions: Practice deviations from guidelines are an important consideration when modelling the cost-effectiveness of trastuzumab therapy. Underlying local disease progression and prevalence can also significantly impact outcomes. / Dissertation / Doctor of Philosophy (PhD)

Yes / Despite the abundant use of polyethylene oxides (PEOs) and their integration as an excipient in numerous pharmaceutical products, there have been no previous reports of applying this important thermoplastic polymer species alone to fused deposition modelling (FDM) 3D printing. In this work, we have investigated the manufacture of oral doses via FDM 3D printing by employing PEOs as a backbone polymer in combination with polyethylene glycol (PEG). Blends of PEO (molecular weight 100 K, 200 K, 300 K, 600 K or 900 K) with PEG 6 K (plasticiser) and a model drug (theophylline) were hot-melt extruded. The resultant filaments were used as a feed for FDM 3D printer to fabricate oral dosage forms (ODFs) with innovative designs. ODFs were designed in a radiator-like geometry with connected paralleled plates and inter-plate spacing of either 0.5, 1, 1.5 or 2 mm. X-ray diffraction patterns of the filaments revealed the presence of two distinctive peaks at 2θ = 7° and 12°, which can be correlated to the diffraction pattern of theophylline crystals. Blends of PEO and PEG yielded filaments of variable mechanically resistance (maximum load at break of 357, 608, 649, 882, 781 N for filament produced with PEO 100 K, 200 K, 300 K, 600 K or 900 K, respectively). Filaments of PEO at a molecular weight of 200–600 K were compatible with FDM 3D printing process. Further increase in PEO molecular weight resulted in elevated shear viscosity (>104 Pa.S) at the printing temperature and hindered material flow during FDM 3D printing process. A minimal spacing (1 mm) between parallel plates of the radiator-like design deemed essential to boost drug release from the structure. This is the first report of utilising this widely used biodegradable polymer species (PEOs and PEG) in FDM 3D printing.

Yes / Appeals have been made for eye care professionals to start prescribing anti-myopia therapies as part of their routine management of myopic children. 1–3 These calls are fuelled by two key considerations. Firstly, that interventions to slow myopia progression have shown success in randomized controlled trials (RCTs) 4–7, and secondly, appreciation that the risk of sight-threatening complications rises dose-dependently with the level of myopia. 8,9 Notwithstanding existing gaps in knowledge regarding the efficacy of current treatments (see below), these considerations argue that myopia control interventions should be widely adopted, and that they should be instigated at an early age – especially in children most at risk – in order to reduce the final level of myopia. Therefore in managing a child with myopia, an eye care professional would have to decide not only which therapy to recommend, but at what age to start treatment. In this review we discuss the future role of genetic prediction in helping clinicians treat myopia. / NIHR Senior Research Fellowship. Grant Number: SRF‐2015‐08‐005

Abstract Novel genetic aetiologies for epileptic encephalopathies and movement disorders have been discovered by using next-generation sequencing methods. The phenotypic and genotypic variability in these conditions is very wide. The aim of this study was to discover novel genetic causes and phenotypes of childhood-onset drug-resistant epilepsy and epileptic or developmental encephalopathies that occur separately or together with movement disorders, and familial movement disorders. Furthermore, the use of whole-exome sequencing (WES) as a diagnostic tool in clinical practice was evaluated. Altogether, 12 children with undefined aetiology, who fulfilled the inclusion criteria, were included in the study. GABRG2 gene was identified as a genetic cause of epileptic encephalopathies. Novel GABRG2-associated phenotypes included progressive neurodegeneration, epilepsy in infancy with migrating focal seizures, and autism spectrum disorder. New pathogenic variants, GABRG2 p.P282T and p.S306F, were discovered. The pathogenic NACC1 variant caused focal epilepsy, developmental disability, bilateral cataracts, and dysautonomia. The novel phenotype associated with the NACC1 p.R298W variant included hyperkinetic movement disorder. SAMD9L was found to be the genetic cause for the familial movement disorder. The phenotype associated with the novel SAMD9L p.I891T variant was very variable. Neuroradiological findings included cerebellar atrophy and periventricular white matter changes. After publication of these results, SAMD9L was reported to be one of the most common genetic aetiologies of childhood bone marrow failure and myelodysplastic syndrome. The pathogenic homozygous MTR variant was found to cause early-onset epileptic encephalopathy that occurred together with movement disorder and haematological disturbances. Drug resistant seizures responded to cofactor and vitamin treatments. Whole-exome sequencing for 10 patients with drug-resistant epilepsy or epileptic or developmental encephalopathy provided a genetic diagnosis for two patients (20%). This study confirmed that, for epileptic encephalopathies and movement disorders in which the genetic causes and phenotypes are heterogeneous and sometimes treatable, WES is a useful tool for diagnostics and in the search for novel aetiologies, which might turn out to be more common than expected. / Tiivistelmä Uusien sekvensointimenetelmien käyttöönotto on mahdollistanut epileptisten enkefalopatioiden ja liikehäiriöiden uusien geneettisten syiden löytymisen. Näissä sairausryhmissä geenien ja ilmiasujen vaihtelevuus on suurta. Tutkimuksen tarkoituksena oli löytää uusia geneettisiä syitä ja ilmiasuja lapsuusiällä alkavissa vaikeahoitoisissa epilepsioissa ja epileptisissä tai kehityksellisissä joko itsenäisesti tai yhdessä liikehäiriön kanssa esiintyvissä enkefalopatioissa sekä perheittäin esiintyvissä liikehäiriöissä. Lisäksi selvitettiin eksomisekvensoinnin käyttökelpoisuutta kliinisessä diagnostiikassa näiden potilasryhmien kohdalla. Tutkimukseen osallistui yhteensä 12 sisäänottokriteerit täyttävää lasta, joiden sairauden syy oli jäänyt tuntemattomaksi. GABRG2-geenin mutaatiot aiheuttivat epileptisiä enkefalopatioita, joiden uutena ilmiasuna oli etenevä taudinkuva, johon liittyivät aivojen rappeutuminen, migroiva imeväisiän paikallisalkuinen epilepsia sekä autismikirjon häiriö. Tutkimuksessa löydettiin uusia GABRG2-mutaatioita: p.P282T ja p.S306F. NACC1-geenin mutaatio aiheutti epilepsian, kehitysvammaisuuden, molemminpuolisen kaihin ja autonomisen hermoston toiminnan häiriön. Hyperkineettinen liikehäiriö oli uusi NACC1 p.R298W -mutaatioon liittyvä ilmiasu. SAMD9L-geenin mutaatio aiheutti perheessä esiintyvän liikehäiriön. Neurologinen ja hematologinen ilmiasu olivat hyvin vaihtelevia. Aivojen kuvantamislöydöksiin sisältyi pikkuaivojen rappeutumista ja valkoisen aivoaineen muutoksia aivokammioiden ympärillä. Näiden tutkimustulosten julkaisemisen jälkeen SAMD9L-geenin mutaatioiden on todettu olevan yksi yleisimmistä perinnöllisistä luuytimen vajaatoiminnan ja myelodysplasian syistä. Homotsygoottinen MTR-geenin mutaatio aiheutti varhain alkaneen epileptisen enkefalopatian, liikehäiriön ja hematologisen häiriön. Kofaktori- ja vitamiini hoidot vähensivät epileptisiä kohtauksia, joihin tavanomainen lääkitys ei tehonnut. Geneettiset syyt ja ilmiasut ovat epileptisissä enkefalopatioissa ja liikehäiriöissä hyvin vaihtelevia, ja osaan on olemassa spesifi hoito. Eksomisekvensointi on käyttökelpoinen diagnostiikan ja uusien geneettisten syiden etsimisen apuna. Tässä tutkimuksessa eksomisekvensoinnin avulla kymmenestä potilaasta kahdelle (20%) saatiin varmistettua geneettinen diagnoosi.