京都大学 / 0048 / 新制・課程博士 / 博士(社会健康医学) / 甲第22384号 / 社医博第106号 / 新制||社||医11(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 妹尾 浩, 教授 古川 壽亮, 教授 中川 一路 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13400号 / 論医博第2224号 / 新制||医||1051(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤田 恭之, 教授 松田 文彦, 教授 柳田 素子 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Tuberculosis (TB) is the leading cause of death due to a single infectious disease and remains a major threat to global public health. The increasing emergence of multi-drug resistance to current anti-TB drugs, exacerbated by the long treatment duration, highlights the need for new effective treatments or strategies to shorten the treatment duration, improve patient adherence and curb the alarming rates of resistance. A key challenge to current strategies employed in the development of anti-TB drugs is the complexity in TB disease pathology which presents as a wide spectrum of lesions in patients presenting with the disease. These lesions occur at different anatomical loci in the same individual and at different stages as the disease progresses. In addition, the interaction between the causative agent, Mycobacterium tuberculosis, and its obligate human host induces physiologic and metabolic changes in the infecting bacillus that are specific to each lesion compartment, and dynamic. This is likely to influence M. tuberculosis susceptibility to antibiotic treatment and, consequently, affect treatment duration and possibly the development of drug resistance. A major limitation in current strategies to address this problem is translation of in vitro compound potency to in vivo efficacy. To reach the target site, a drug must first distribute and accumulate in the lesion microenvironments where bacteria reside: the macrophage host cell and the caseum. This thesis focused on the development of an in vitro infection model that could be used to predict drug penetration into M. tuberculosis-infected macrophages. Of particular interest was the extent to which host intracellular drug concentrations translate into effective antimycobacterial activity. To this end, the thesis comprised three key aspects: (i) characterization of physicochemical properties, antimycobacterial activities and M. tuberculosis-mediated metabolism of selected antiTB compounds; (ii) determination of intracellular drug permeation in resting, activated and foamy macrophages; and (iii) determination of the correlation (or not) between intracellular drug concentration and effective M. tuberculosis growth inhibition. The highly lipophilic natural product, fusidic acid (FA), its known human metabolite, 3-ketofusidic (3-ketoFA or GKFA37), and two C-3 alkyl esters (GKFA16 and GKFA17) as FA prodrugs were utilized in the study. In addition, another chemical class, the less lipophilic benzoxazole-based oxime derivatives were also investigated. Moxifloxacin (MXF), levofloxacin (LVF), bedaquiline (BDQ), rifampicin (RIF) and clofazimine (CFZ) were included for reference as known anti-TB drugs with varying lipophilicities. In chapter 2, FA and derivatives showed potent antimycobacterial activity (~1 µM) with selectivity indices (SI) >20 against the THP-1 macrophage cell line. Predicted artificial membrane permeability assay (PAMPA) results suggested that FA and derivatives would readily permeate the cell membrane. M. tuberculosis metabolized the C-3 alkyl-ester prodrug GKFA17 to form both FA and 3-ketoFA, with complete hydrolysis of the prodrug. FA was metabolized to 3-ketoFA, but the low levels of the metabolite suggested that another unidentified metabolite, presumed to be 3-epifusidic acid (3-epiFA), was formed. In vitro assays revealed that the potent benzoxazole-based oxime carbamates (PMN1-201, PMN1-136 and PMN2-09) were rapidly hydrolyzed by M. tuberculosis and were also susceptible to spontaneous degradation in media, forming the poorly active corresponding free oximes (PMN1-199, PMN1-135 and PMN1-157). In chapter 3, the in vitro macrophage drug uptake assay showed that FA C-3 alkyl prodrugs, GKFA16 and GKFA17, accumulated in significantly higher amounts in resting macrophages in comparison to FA and GKFA37. Accumulation of MXF was comparable to the least accumulated FA derivative, GKFA37, and showed steady state intracellular concentrations over a 6-day period. While GKFA16 and GKFA17 showed continued increasing accumulation, intracellular concentrations of FA and GKFA37 decreased after 48 hours, suggesting a likely susceptibility to macrophage efflux. In infected macrophages, the presence of intracellular bacteria or increasing bacterial burden did not affect the host cell ability to accumulate the drugs. FA and derivatives exhibited bacteriostatic inhibition of intracellular mycobacterial growth. MXF showed a potent bactericidal effect, reducing intracellular bacterial counts significantly at 10x MIC, with complete sterilization at 50x MIC even though MXF accumulation was significantly less than that of FA alkyl esters. These results suggested that both the inherent activity of a compound and ability to accumulate within host cells drive cellular efficacy. Given that the C-3 alkyl ester prodrugs accumulated at significantly higher concentrations than FA and GKFA37, this demonstrates the limitations of this assay in ascertaining the impact of intracellular concentration on drug efficacy for bacteriostatic drugs while highlighting its ability to correlate drug penetration and intracellular activity for cidal drugs. The prodrug GKFA17 was shown to undergo metabolism in resting host cells and during infection to form FA and then 3-ketoFA. Therefore, the prodrug strategy could be used to increase intracellular exposure of FA as GKFA17 showed superior macrophage accumulation. Benzoxazole-based oxime carbamates and their corresponding free oximes failed to accumulate in host macrophages and this was corroborated by their failure to control host cell bacterial growth despite the potent in vitro activity against M. tuberculosis of the carbamates, suggesting that they are poorly permeable. Chapter 4 investigated drug permeation in different macrophage phenotypes known to exist in the granuloma during TB disease, including foamy and activated macrophages. The activation state of the host cell did not affect the ability to accumulate anti-TB drugs such as RIF and BDQ. However, FA and its prodrug GKFA17 were significantly reduced in M1 activated macrophages. Despite the significantly reduced intracellular concentration, activated macrophages treated with FA and derivatives showed superior intracellular M. tuberculosis growth inhibition, suggesting that macrophage activation potentiates the activity of these compounds. In order to assess the effect of foamy macrophage lipid bodies (LBs) on drug uptake and intracellular localization, oleic acid-induced foamy macrophages were treated with selected antiTB drugs and experimental compounds. FA and derivatives showed early increased accumulation in foamy cells compared to resting macrophages, while MXF, BDQ and RIF levels were not significantly changed. Intracellular:extracellular (I/E) ratios increased with increase in lipophilicity, with FA C-3 alkyl prodrugs exhibiting the highest I/E ratios of >100. Despite exhibiting increased foamy macrophage concentrations, FA and derivatives exhibited a similar reduction (bacteriostatic) in bacterial counts in both resting and foamy macrophages. The intracellular activity of RIF was also not affected by presence of LBsin foamy macrophages. BDQ, LVF and MXF, however, showed reduced intracellular efficacy against M. tuberculosis in foamy macrophages compared to resting macrophages, suggesting a role for LBs to impact intracellular drug distribution. In conclusion, this thesis demonstrates the potential utility in combining advanced analytical methods and an in vitro infection model to determine cellular drug permeation profiles that might be applied to prioritize compounds and combinations optimized for distribution to target bacterial populations. This will facilitate well-informed decision-making processes in progression of lead compounds in pre-clinical development and, therefore, may offer the potential to reduce high rates of attrition of compounds which enter clinical phase of development.

Doctor Pharmaceuticae - DPharm / Treatment of the human immunodeficiency virus (HIV) and tuberculosis (TB) infections have become very complicated due to the advent of drug resistance. Drug combinations offer an alternative approach to reducing the emergence of drug resistance. Pharmaceutical co-crystals have provided the pharmaceutical industry with the ability to optimise the physicochemical properties of active pharmaceutical ingredients (APIs) while preserving the biological activity. Pharmaceutical co-crystals are formed between APIs and suitable co-formers that are biologically safe or even a second or third API.

The usage of antimicrobials either as performance enhancers or for prophylactic and therapeutic purposes in food animals, such as chickens, increases the prevalence of antimicrobial drug resistance among enteric bacteria of these animals. This may be transferred to people working with such animals, e.g. abattoir workers, or the products arising from these animals. In this study antimicrobial drug resistance was investigated for selected enteric bacteria from broilers raised on feed supplemented with antimicrobial growth enhancers, and the people who carry out evisceration, washing and packing of intestines in a high throughput poultry abattoir in Gauteng, South Africa. Poultry farms (n=6) were purposively selected on the basis of allowing for sampling of farms from more than one grow out cycle. Broiler carcases (n=100) were randomly selected per farm five minutes after slaughter and sampled by incising caecae from the rest of the gastro-intestinal tract (GIT). The ends of each caecae were tied off to prevent contamination and to enhance the culturing of anaerobic bacteria. In the laboratory, caecal contents were selectively cultured for Clostridium perfringens, Escherichia coli, Enterococcus faecium, E. faecalis, and vancomycin-resistant enterococci (VRE). Salmonella enterica was isolated using pre-enrichment followed by selective culture. The minimum inhibitory concentration (MIC) micro broth dilution test as prescribed by the Clinical and Laboratory Standards Institute USA (CLSI), previously known as National Committee of Clinical Laboratories (NCCL), was used to determine the susceptibility of the isolates to the following antimicrobials: vancomycin, virginiamycin, doxycycline, trimethoprim, sulphamethoxazole, ampicillin, bacitracin, enrofloxacin, erythromycin, fosfomycin, ceftriaxone and nalidixic acid. The same was done on the faeces of 29 abattoir workers exposed to potentially resistant micro-organisms from broilers and 28 persons used as controls, who had not been equally exposed to potentially resistant micro-organisms from broilers. Both of the human populations had not been treated with antimicrobials within three months prior to sampling. Statistical analysis was done by Fisher’s exact test. No salmonellae and VRE on VRE selective agar (Oxoid UK) were cultured. Two Clostridium perfringens, 168 E. coli, 20 E. faecalis and 96 E. faecium isolates from the broiler caecae were cultured. Fifty four (28 and 26) E. coli, 24 (21 and 3) E. faecalis and 12 (2 and 10) E. faeciumfrom humans were cultured. The figures in brackets represent the abattoir workers and human controls respectively. The majority of E. coli isolates from broilers had MIC’s above the cut off point for the antimicrobials tested. Low resistance was observed among broiler enterococci isolates to vancomycin, virginiamycin, trimethoprim and ampicillin. A comparison of the median MIC’s of isolates from abattoir workers (packers) and the control group revealed significant differences in the median MIC’s for the following antimicrobials; E. faecalis: enrofloxacin (p=0.019). E. faecium, trimethoprim (p=0.01), enrofloxacin (p=0.029) and erythromycin (p=0.03). E. coli: trimethoprim (p=0.012) and ampicillin (p=0.036). Use of antimicrobials as feed additives causes resistance among enteric bacteria from broilers. Significant differences between median MIC’s of abattoir workers (packers) and the control group were observed for therapeutics and not growth enhancers. There was a tendency for isolates from abattoir workers to have a higher median MIC and a higher number of resistant isolates as compared to the control group. In spite of the fact that there was a high level of resistance in the enteric commensal bacteria of broiler caecae, an association could not be shown with that of the human enteric bacteria. It could not be concluded that a significant AMR transfer to poultry abattoir workers existed. This notwithstanding, both the control and experimental group, carried levels of resistance among their enteric bacteria that could be described as being high. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2007. / Paraclinical Sciences / unrestricted

The malaria parasite Plasmodium falciparum is responsible for about 500,000 deaths a year and is evolving resistance to the front-line treatment of artemisinin-based combination therapy. Resistance is currently confined to South East Asia, however millions of lives will be at risk if resistance spreads to Africa. Understanding the mechanism of resistance to artemisinins would aid containment strategies to prevent the spread of artemisinin resistance. There is also an urgent need to accelerate drug discovery since drug resistance has already been documented to all existing antimalarials. Here, I report on our efforts to understand the function of the gene k13, the gene with the strongest association with artemisinin resistance, and the potential genetic mechanisms associated with resistance to atovaquone, another widely used antimalarial. To precisely study the transcriptome characteristics of an isogenic k13 dysregulation mutant and wild type strain, I developed a new computational algorithm called Dephasing Identifier (DI) that is capable of identifying the genes dysregulated in cell cycle shifts. DI is designed to solve the problem of pinpointing important patterns in complex genomics data with temporal sequences that cannot be resolved by standard pair-wise comparison methods, by using an innovative method that leverages external reference data for systematic comparisons. In the k13 study, I demonstrated that the algorithm identifies co- regulated gene sets that have consistent annotated functions. The DI algorithm successfully identified aberrantly early DNA replication as the driving process of transcriptome changes in the mutant. To understand genome-wide changes that occurred in a set of atovaquone resistance stains, I analyzed whole genome sequencing data previously generated for a P. falciparum strain that underwent in vitro atovaquone selection to create atovaquone resistant strains. I systematically analyzed the genomes of these strains to search for significant genetic changes associated with atovaquone resistance; and used stringent criteria to identify genes involved in regulating transcription and protein modifications as acquiring non- synonymous mutations. Additionally, copy number variations in plasmepsin genes, a family known to be involved in resistance, were found in the resistant strains. In summary, genomics and transcriptomics technologies can be used to rapidly identify resistance mechanisms allowing for faster adjustment of current containment strategies. Future research on the critical targets identified in this study can aid new drug discovery efforts and novel control strategies.

Magister Pharmaceuticae - MPharm / Malaria is an infectious disease which continues to kill more than one million people every year and the African continent accounts for most of the malaria death worldwide. New classes of medicine to combat malaria are urgently needed due to the surge in resistance of the Plasmodium falciparum (the parasite that causes malaria in humans) to existing antimalarial drugs. One approach to circumvent the problem of P. falciparum resistance to antimalarial drugs could be the discovery of novel compounds with unique scaffolds and possibly new mechanisms of action. Natural products (NP) provide a wide diversity of compounds with unique scaffolds, as such, a library of virtual compounds (VC) designed from natural products with antiplasmodial activities (NAA) can be a worthy starting point.

The lack of access to effective diagnosis and treatment of drug-resistant tuberculosis (DR-TB) remains a persistent global challenge. Human rights arguments for access to treatment mostly focus on the right to health. However, a key challenge in access to effective diagnosis and treatment is the glaring absence of scientific research in neglected diseases such as TB. This thesis sets out to elaborate the right to enjoy the benefits of scientific progress and explore its potential to increase scientific research in DR-TB and consequently enhance access to effective diagnosis and treatment in South Africa. This research project was conducted using three interrelated sub-studies; a legal analysis sub-study which examines the current conceptualisation of the REBSP in international law; a policy analysis sub-study which interrogates South Africa’s legal and policy efforts towards the realisation of the REBSP and access to diagnosis and treatment for DR-TB; and a qualitative sub-study which explores the South African context regarding research and development (R&D) in general, and in DR-TB in particular. The qualitative sub-study included 17 stakeholders who are active in TB R&D, advocacy and policy work, from human rights and research institutions, government agencies, civil society organisations, and donor agencies. This thesis finds that the REBSP essentially ensures two things, namely the production of science and access to the benefits of scientific progress. However, most countries including South Africa have systems, policies and resources aimed at advancing the production of science but lack similar systems, policies and resources to purposely ensure the enjoyment of the benefits from scientific progress. Internationally, there is no clear guidance on the interpretation of the REBSP, making it difficult for states to domesticate it in their national policies and framework laws. A General Comment by a UN human rights monitoring body is therefore urgently needed to secure global consensus on the interpretation of the REBSP. In the meanwhile, South Africa can still draw inspiration for the REBSP and together with the right to health, use it to advance access to DR-TB diagnosis and treatment alongside many other interventions. To enable better access to effective diagnosis and treatment of DR-TB, this thesis recommends that South Africa i) develops systems that would make scientific progress and results accessible, and affordable; ii) removes system and regulatory barriers that hinder the conduct of research or that delay registration of new drugs; iii) monitors and regulates the conduct of third parties and prevent them from exploiting communities; iv) encourages pharmaceutical companies to provide free access to successful treatment and tools in communities where trials are conducted; and v) mobilises financial and technical resources and allocates them to DR-TB researchfrom drug discovery through to implementation science.

Charles University Faculty of Pharmacy in Hradci Králové Departement of Pharmacology & Toxicology Student: Magdalena Moriová Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on the role of selected cytochrome P450 isoforms in cytostatic resistance at apoptosis level Cytostatic resistance is one of the most problematic obstacles in oncological treatment. Beside pharmacodynamic mechanisms, pharmacokinetic factors play an important role in drug resistance as well. Enzymatic transformation of active substance to inactive metabolite in tumor cells probably belongs to these mechanisms, however, evidences concerning the relevance of this phenomenon are predominantly either indirect and/or affected by interference elements. Using comparative experiments with HepG2 cell lines with/without CYP3A4 overexpression, we focused on the evaluation of the role of this clinically important enzyme in the resistance against docetaxel. Methodologically, it was the assessment of apoptosis induction (activation of caspases 3/7, 8 and 9) using commercial luminescent kits. Our results suggest significant participation of CYP3A4 enzyme on the reduction of docetaxel anticancer efficacy after 48 h from treatment, whereas this effect was not recorded in earlier intervals. These findings perfectly correlate...

Background: There is a dearth of information regarding prevalence of extensively drugresistant tuberculosis (XDR-TB) in Africa. Although countries in Africa conduct national tuberculosis surveys on a regular basis, this information has not been systematically reviewed to ascertain the overall prevalence of XDR-TB in Africa. Methods: The study aimed to perform a systematic review and meta-analysis of the prevalence and factors associated with prevalence of pulmonary XDR-TB among adults in Africa. Eligible studies, published between 2006 and 2018, were sourced from various electronic databases including PubMed, Scopus, and Web of Science. Meta-analysis was performed using STATA (version 14.2) statistical software. The protocol of this review was registered with PROSPERO, reg No CRD42018117037. Result: A total of 6242 records were retrieved. Forty-eight studies were screened for eligibility and seven, which varied in terms of country setting and study design, were included. The prevalence of XDR-TB is 4% (95%CI 2-7) among participants tested for second-line anti-TB drug resistance, and 3% (95%1-6) among participants with drug resistant TB. The prevalence of XDR-TB was 7% (95%CI 1-18) among participants with MDR-TB. A few studies reported on the factors associated with the prevalence of XDR-TB. Discussion: The reported prevalence of XDR-TB among participants tested for second-line anti-TB drug resistance is low compared to WHO estimates. The systematic review underscores a dearth of studies depicting the reality regarding the prevalence of XDR-TB in Africa. Policymakers and stakeholders interested in drug-resistant TB should apply prudence when considering XDR-TB prevalence reported for Africa.