L’œdème maculaire est une complication majeure de nombreuses pathologies rétiniennes induisant la cécité. Les traitements actuels ne sont pas curatifs. Ce sont des injections intraoculaires de corticostéroïdes et d'anti-VEGF. Les mécanismes d'action de ces médicaments sont mal connus dans l'œil car ces thérapies ont été initialement développées pour des pathologies non-oculaires (tels que l'asthme et les maladies auto-immunes pour les corticostéroïdes et les cancers pour certains anti-VEGF). De plus, les effets bénéfiques de ces molécules sont contrebalancés par des effets secondaires graves (glaucomes et cataractes pour les corticostéroïdes) et la résistance au traitement. Le projet vise à élucider les mécanismes d'action oculaires des corticostéroïdes et anti-VEGF; dans le but d'identifier de nouvelles cibles thérapeutiques pour le traitement de l’œdème maculaire. Ce travail a porté sur : 1) La compréhension des mécanismes des corticostéroïdes et de leurs récepteurs (le récepteur aux glucocorticoïdes ou GR et le récepteur aux minéralocorticoïdes ou MR) suite à l’injection intravitréene de corticostéroïdes ou d’aldostérone chez le rat. Nous avons ainsi : -1.1. Identifier les protéines partenaires du GR et du MR avant et après traitements par une approche protéomique (immunoprécipitations endogènes suivies d’une analyse par spectrométrie de masse). -1.2. Identification les gènes cibles dont l'expression est modulée par les corticostéroïdes ou l’aldostérone par ARN-sequencing. 2) L'identification des mécanismes d’action des aux anti-VEGFs et leur comparaison avec des molécules de même nature physico-chimique. 3) L’identification des gènes différentiellement exprimés entre la rétine périphérique et la macula (zone d’acuité visuelle susceptible à la formation d’œdèmes) chez le singe (modèle qui possède, comme l’homme, une macula). Ceci afin de comprendre pourquoi la macula est particulièrement sensible. 4) L’analyse et la comparaison de ces différents traitements. Ces études ont permis d’identifier les cofacteurs des récepteurs aux corticostéroïdes, ainsi que la dynamique de leurs interactions avant et après traitement à la corticostérone et à l’aldostérone; ainsi que les gènes dont l’expression est régulée par les corticostéroïdes et les anti-VEGF. L’analyse croisée des données, des analyses bioinformatiques ainsi que l’étude bibliographique des protéines et gènes identifiés nous ont permis d’établir une liste restreinte de cibles potentielles des traitements anti-œdémateux, dans le but final de potentialiser l’effet de ces traitements, trop souvent associés à des effets secondaires qui limitent considérablement leur(s) action(s) bénéfique(s) sur la fonction visuelle dans le traitement de l’œdème maculaire. / Macular Edema is a major complication of numerous retinal pathologies that lead the blindness. The available treatments (intra-ocular injection of corticosteroids and anti-VEGF) do not cure this disease. The ocular mechanisms of action of these drugs are not very well understood. In fact, these molecules have been initially developed for non-ocular pathologies, such as asthma, auto-immune diseases (for corticosteroid) and cancers (for anti-VEGF). In addition, the beneficial effects of these drugs are counteracted by their side effects (cataracts and glaucoma for corticosteroids) as well as resistance to treatment. The project aims at unraveling the ocular mechanisms of action of corticosteroids and anti-VEGF; in order to identify new therapeutic targets for the treatment of macular edema. The project focused on: 1) The understanding of the mechanisms of the corticosteroids and their receptors after an intravitreal injection of Corticosterone or Aldosterone in the rat model. We therefore: 1.1. Identified the partner proteins of the glucocorticoid and mineralocorticoid receptors before and after treatment, using a proteomic approach. 1.2. Identified the target genes which expression is modulated by the corticosteroid using the RNA-Sequencing technique. 2) The identification of the mechanisms of action of anti-VEGF molecules and their comparison with molecules that have similar physico-chemical structure. 3) The identification of differentially expressed genes between the retina and the macula in the monkey model. This aimed at understanding why the macula is particularly sensitive to edemas. 4) The analysis and comparison of these treatments. We were able to identify co-factors for corticosteroids receptors as well as the dynamics of these interactions (before and after treatment), as well as the genes which expression is regulated by the corticosteroids or anti-VEGF. The analysis of the bio-informatic data as well as the bibliographic study of the identified proteins and genes allowed us to establish a list of partiMacularly interesting genes that are potentially therapeutic targets for the treatment of macular edema.

Philosophiae Doctor - PhD / Solute carrier transporters belonging to the major facilitator family of membrane transporter are increasingly being recognized as a possible mechanism to explain inter-individual variation in drug efficacy and response. Genetic factors are estimated to be responsible for approximately 15-30% of inter-individual variation in drug disposition and response. The aims of this study were to determine the minor allele frequencies of 78 previously identified single nucleotide polymorphisms in the pharmacogenomically relevant SLC22A1-3 and SLCO1B1 genes in the Admixed population of South Africa. Thereafter, to determine whether allele and genotype frequencies for these SNP were different from that reported for other African, Caucasian, and Asian populations. The inferred haplotypes from the genetic information possessed the potential to subsequently be used in future to design and interpret results of pharmacogenomic association studies involving these genes and their substrate drugs. Furthermore, to determine whether the Cape Admixed population harbour novel SNPs in the proximal promoter regions of SLC22A1- 3 and SLCO1B1-3 genes, that encodes hOCT1-3 and hOATP1 and hOATP3, respectively. SNaPshot™ multiplex single base mini-sequencing systems were developed and optimized for each of SLC22A1, SLC22A2, SLC22A3, and SLCO1B1 genes covering the previously identified 78 SNPs. These systems were then used to genotype the alleles of 130 healthy Cape Admixed subjects residing in Cape Town, South Africa. In addition, the proximal promoter regions of the SLC22A1-3 and SLCO1B1-3 genes of 96 of the participants were screened for novel SNPs by direct sequencing. The Cape Admixed subjects investigated displayed a lack of variation and were monomorphic for 78% of the SNPs screened. None of the SLC22A3 SNPs investigated was observed in this study. Sequencing of the proximal promoter regions of the SLC22 and SLCO genes did not reveal any novel SNPs in the 96 Cape Admixed subjects that were screened. This study highlights the fact that African populations do not have the same allele frequencies for SNPs in harmacogenomically relevant genes. Furthermore, the Cape Admixed and other African populations do not share all reduced-function variants of the SLC22A1-3 and SLCO1B1-3 genes with Caucasian and Asian populations. In addition, previously identified novel regulatory variants in SLC22A2 did not exhibit a significant effect on the ability of the promoter to drive transcription. However, it must be noted that these results were observed at 95% confidence interval, and that a 99% confidence interval the significance may increase theoretically. Additionally, it should be noted that more intensive studies are required to determine the potential effect these novel variants may well cause. This study lays the foundation for the design and interpretation of future pharmacogenomic association studies between the variant alleles of the SLC22A and SLCO genes in the Cape Admixed population, as well as optimizations for future expression, and more importantly, drug transport assays with respect to drug disposition and efficacy. / National Research Foundation (NRF) and the Medical Research Council (MRC)

The Argus Commission identified three major federal priorities related to health care, including the precision medicine initiative, the Cancer Moonshot and the opioid abuse epidemic. Current activities at the federal level were summarized and an analysis of activities within the profession, and academic pharmacy specifically, was prepared. The implications for pharmacy education, research and practice are compelling in all three areas. Recommendations, suggestions and two policy statements aim to optimize the attention to these priorities by the academy. Further, aligning the AACP Strategic Engagement agenda with the opportunities and threats acknowledged in the analysis is essential.

Ces dernières années, les thérapies ciblées pour le traitement du cancer, ont été introduites. Cependant, un médicament fonctionnant chez un patient peut ne pas fonctionner chez un autre. Pour éviter l'administration de traitements inefficaces, des méthodes capables de prédire les patients qui répondront à un médicament donné doivent être mises au point.Il n'est actuellement pas possible de prédire l'efficacité de la grande majorité des médicaments anticancéreux. L’apprentissage automatique (AA) est un outil particulièrement prometteur pour la médecine personnalisée. L’AA est un champ d’étude de l'intelligence artificielle ; elle concerne la mise au point et l'application d'algorithmes informatiques qui s'améliorent avec l'expérience. Dans ce cas, l'algorithme d’AA apprendra à faire la distinction entre les tumeurs sensibles et résistantes en fonction de plusieurs gènes au lieu d'un seul gène. Cette étude se concentre sur l'application de différentes approches de l’AA pour prédire la réponse à des médicaments anticancéreux des tumeurs et générer des modèles précis, biologiquement pertinentes et faciles à expliquer. / In recent years, targeted drugs for the treatment of cancer have been introduced. However, a drug that works in one patient may not work in another patient. To avoid the administration of ineffective treatments, methods that predict which patients will respond to a particular drug must be developed.Unfortunately, it is not currently possible to predict the effectiveness of most anticancer drugs. Machine learning (ML) is a particularly promising approach for personalized medicine. ML is a form of artificial intelligence; it concerns the development and application of computer algorithms that improve with experience. In this case, ML algorithm will learn to distinguish between sensitive and non-sensitive tumours based on multiple genes instead of a single gene. Our study focuses on applying different approaches of ML to predict drug response of tumours to anticancer drugs and generate models which have good accuracy, as well as are biologically relevant and easy to be explained.

Indiana University-Purdue University Indianapolis (IUPUI) / Adverse drug events (ADEs) are injuries resulting from drug-related medical interventions. ADEs can be either induced by a single drug or a drug-drug interaction (DDI). In order to prevent unnecessary ADEs, many regulatory agencies in public health maintain pharmacovigilance databases for detecting novel drug-ADE associations. However, pharmacovigilance databases usually contain a significant portion of false associations due to their nature structure (i.e. false drug-ADE associations caused by co-medications). Besides pharmacovigilance studies, the risks of ADEs can be minimized by understating their mechanisms, which include abnormal pharmacokinetics/pharmacodynamics due to genetic factors and synergistic effects between drugs. During the past decade, pharmacogenomics studies have successfully identified several predictive markers to reduce ADE risks. While, pharmacogenomics studies are usually limited by the sample size and budget. In this dissertation, we develop statistical methods for pharmacovigilance and pharmacogenomics studies. Firstly, we propose an empirical Bayes mixture model to identify significant drug-ADE associations. The proposed approach can be used for both signal generation and ranking. Following this approach, the portion of false associations from the detected signals can be well controlled. Secondly, we propose a mixture dose response model to investigate the functional relationship between increased dimensionality of drug combinations and the ADE risks. Moreover, this approach can be used to identify high-dimensional drug combinations that are associated with escalated ADE risks at a significantly low local false discovery rates. Finally, we proposed a cost-efficient design for pharmacogenomics studies. In order to pursue a further cost-efficiency, the proposed design involves both DNA pooling and two-stage design approach. Compared to traditional design, the cost under the proposed design will be reduced dramatically with an acceptable compromise on statistical power. The proposed methods are examined by extensive simulation studies. Furthermore, the proposed methods to analyze pharmacovigilance databases are applied to the FDA’s Adverse Reporting System database and a local electronic medical record (EMR) database. For different scenarios of pharmacogenomics study, optimized designs to detect a functioning rare allele are given as well.

Cytochrome P450 (CYP) enzymes play a crucial role in the human body. These enzymes are responsible for the synthesis of steroid hormones and cholesterol, as well as the metabolism of external substances such as medications. While more than 50 CYP enzymes have been identified, just 6 are credited with metabolizing most drugs. Of note is CYP 3A4, which metabolizes ~34% of medications that use the CYP enzyme system. CYP enzymes are polymorphic, meaning there are different versions of the same enzyme; therefore there is variability from individual to individual in their ability to metabolize medications. In the oncology field tyrosine kinase has been identified as an important target controlling cell regulatory functions and proliferation. Thus, tyrosine kinase inhibitors have become widely used for a variety of malignancies. Many of these tyrosine kinase inhibitors rely on CYP 3A4 for metabolism and are subject to variable toxicities based on an individual patient’s genome. A 62-year-old female was diagnosed with Renal Cell Carcinoma (RCC). After undergoing a left nephrectomy, a surveillance scan 21 months after diagnosis was concerning for metastatic disease, which was then confirmed through biopsy. The patient was started on sunitinib 50 mg on days 1-28 of a six-week cycle for metastatic RCC. The patient suffered from Grade 3 myelosupression/mucositis within two weeks of the initiation of therapy. The early onset and severity of the toxicity lead to CYP 3A4 pharmacogenetic testing. She was subsequently found to have a 3A4 polymorphism (*1/*28). The dose of sunitinib was reduced to 25 mg followed by a further reduction to 12.5 mg due to toxicity. Eighteen months after starting sunitinib, a CT scan showed disease progression and therapy was changed to pazopanib. Due to her 3A4 polymorphism, the starting pazopanib dose was empirically reduced by 50% and was started at 400 mg/daily. Pazopanib was held for episodes of severe diarrhea and was further reduced to 200 mg/daily. Nine months after starting pazopanib, new imaging showed lesions in the patient’s liver, confirming disease progression. The patient was subsequently started on nivolumab but quickly progressed. She was then started on cabozantinib at a dose reduced 20 mg/daily. This initial dosing was tolerated well by the patient, so a decision was made to alternate between 20 and 40 mg/daily to increase to an average of 30 mg/daily. She is currently tolerating the 30 mg/daily and continues treatment for metastatic RCC. As described above, CYP 3A4 polymorphisms can result in severe toxicities that present earlier in the treatment course than traditionally expected. Moving forward there may be a role in testing for these polymorphisms to determine an individual’s optimal dose before initiating therapy with tyrosine kinase inhibitors. The advantage of performing such testing would be to limit the severity of toxicities experienced by this patient population, while retaining the overall benefit of these medications.

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Background: Major depressive disorder (MDD) is commonly treated with antidepressants, but many patients undergo trial-and-error to find the medication best suited for them. Pharmacogenomic (PGx) testing was developed to provide prescribing guidance for a variety of medications including antidepressants. PGx testing is not yet part of standard depression care in Canada, but clinical implementation efforts are ongoing. Patient perspectives, as well as the views of people with professional expertise about this technology, are critical to health technology assessment (HTA) and policy decisions, and can inform implementation. Methods: This dissertation produces actionable patient and key informant (i.e., clinician, scientists, policy actor, industry member) perspectives evidence for future HTA and policy- making through three independent studies: a systematic review and qualitative meta-synthesis of patient experiences of treatment-resistant depression (TRD), and two qualitative description studies about patient and key informant perspectives on PGx testing to guide antidepressant prescribing. Results: Trial-and-error of medications often leaves patients feeling hopeless but desperate to get better. Patients and key informants both felt the main benefit of PGx testing is its potential to reduce the time between diagnosis and successful treatment. The main findings of this research were concrete suggestions for how PGx testing may be integrated in the health system. Participants preferred this technology be deployed in primary care, with patients with TRD identified as a possible priority population. The ideal PGx test would incorporate genetic variants common in non-white populations to ensure equity of health outcomes, and mechanisms to facilitate equity of access (eg., public funding) were considered important. Conclusions: Patients and key informants both described benefits of PGx testing to guide antidepressant prescribing. Patients typically raised more concerns than key informants. Future research is needed around some of the ethical and social issues related to PGx testing, as well as to address feasibility questions. / Dissertation / Doctor of Philosophy (PhD) / Pharmacogenomic (PGx) testing may facilitate targeted prescribing of antidepressants by identifying genetic factors that play a role in how a person responds to these drugs. However, this technology is not yet widely or systematically used in clinical care. This dissertation focuses on how PGx testing might be implemented in primary care in Ontario, Canada. It examines which patients this type of genetic testing may be most appropriate for, and collects and analyzes the views of patients with depression and experts on PGx testing about this technology. The main benefit of PGx testing was its potential to help people get better faster, and primary care was uniformly considered an appropriate care setting for the use of this technology. There were some concerns that people may not fully understand the limitations of PGx testing. Future research is needed to understand how this technology might fit into clinician workflows across the healthcare system.

Η μεθυλοτρανσφεράση της θειοπουρίνης (TPMT) είναι ένα ένζυμο του κυτταροπλάσματος, που καταλύει τη μεθυλίωση των θειοπουρινικών φαρμάκων, όπως η 6-μερκαπτοπουρίνη (6-MP), η οποία έχει καθιερωθεί στη θεραπεία συντήρησης παιδιών με ΟΛΛ. Ένα πολυμορφικό στοιχείο (VNTR) στον υποκινητή του γονιδίου ΤΡΜΤ, φαίνεται να επηρεάζει τα επίπεδα δραστικότητας του ενζύμου που παράγεται. Πρόκειται για διαδοχικές επαναλήψεις τριών διαφορετικών αλληλουχιών (A, B και C), ο αριθμός των οποίων κυμαίνεται από τρεις έως εννέα. Η περιοχή VNTR είναι πλούσια σε επαναλήψεις των βάσεων GC, οι οποίες αποτελούν πιθανές θέσεις πρόσδεσης μεταγραφικών παραγόντων. Μερικοί ασθενείς, οι οποίοι ακολουθούν θεραπεία με 6-ΜΡ, δεν είναι ανθεκτικοί στην καθιερωμένη δόση του φαρμάκου και παρουσιάζουν μυελοτοξικότητα ως αποτέλεσμα της ανεπάρκειας του ενζύμου TPMT. Στην παρούσα εργασία πραγματοποιήθηκε μία εκτεταμένη μελέτη της περιοχής VNTR, με απώτερο σκοπό την αξιολόγησή της ως πιθανό φαρμακογονιδιωματικό δείκτη, που να σχετίζεται με την πρόβλεψη ανεπιθύμητων παρενεργειών σε ασθενείς με ΟΛΛ, οι οποίοι λαμβάνουν 6-ΜΡ. Αρχικά, προσδιορίστηκε η συχνότητα των διαφορετικών VNTR αλληλομόρφων στον ελληνικό πληθυσμό, σε ένα σύνολο υγιών ατόμων, με σκοπό να αντιπαραβάλλουμε τις συχνότητες αυτές με δεδομένα από άλλες πληθυσμιακές ομάδες. Τα αποτελέσματά που προέκυψαν συμφωνούν με τα αντίστοιχα στοιχεία που υπάρχουν για άλλους πληθυσμούς. Οι μέθοδοι γονοτύπησης που εφαρμόσθηκαν περιλαμβάνουν την αντίδραση της PCR και τον προσδιορισμό αλληλουχίας κατά Sanger. Στη συνέχεια μελετήθηκε η επίδραση συγκεκριμένων μοτίβων VNTR στον υποκινητή του γονιδίου υγιών ατόμων, στη μεταγραφή του γονιδίου, καθώς και στην έκφραση του ενζύμου ΤΡΜΤ. Η ικανότητα μεταγραφής εκτιμήθηκε με τη μέθοδο της ποσοτικής Real Time PCR, ενώ τα επίπεδα έκφρασης του ενζύμου προσδιορίστηκαν μέσω της αντίδρασης χημειοφωταύγειας, με χρήση κατάλληλων αντισωμάτων. Ο μικρός αριθμός δειγμάτων δεν επέτρεψε την εξαγωγή ασφαλών συμπερασμάτων για το ρόλο της VNTR περιοχής, στην μεταγραφή του γονιδίου ΤΡΜΤ. Σύμφωνα με τα αποτελέσματά μας, διακρίνεται μία τάση συσχέτισης, η οποία όμως απαιτεί περαιτέρω διερεύνηση. / Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyzes S-methylation of thiopurine drugs, such as 6-mercaptopurine (6-MP), which is typically used as maintenance therapy in pediatric acute lymphoblastic leukemia (ALL). A variable number of tandem repeat (VNTR) within the TPMT promoter has been reported to “modulate” levels of this enzyme activity. The VNTR region architecture is defined by three types of repeats (A, B and C) rang¬ing from three to nine (VNTR*3 to VNTR*9). These repeats are GC rich, and are putative binding sites of various transcription factors. Some patients do not tolerate standard doses of 6-MP and develop myelotoxicity as a consequence of inherited TPMT deficiency. In this study, we performed an extensive analysis of VNTR region, in order to evaluate it as a putative pharmacogenomic marker to predict 6-MP toxicity, in ALL patients. The frequency of TPMT alleles bearing different VNTR architectures was investigated in the Hellenic population. The results of this study were consistent with data, already existed, from other populations. The number and type of tandem repeats were determined by PCR amplification followed by direct re-sequencing. Moreover, we explored the influence of specific VNTR motifs, within the TPMT promoter of healthy individuals, on transcription and expression of TPMT gene. Quantitative Real-Time PCR was performed in order to investigate the influence of VNTR architecture on TPMT gene transcription. The expression levels of TPMT enzyme were estimated by chemiluminescence reaction, using appropriate antibodies. The role of VNTR region on TPMT gene transcription was not clarified through this study, owing to small cohort of samples included. A statistical trend of association has been observed, requiring, however, further investigation.