Background: The retail sector is an important channel for increasing access to adequate treatment of fevers in Africa. The objectives of the thesis were to assess the performance of three malaria control programmes targeting private medicine retailers (PMRs) by addressing coverage, utilisation, impact on PMRs' knowledge, practices and implementation processes in Kisii central, Kwale and Bungoma districts of Kenya. Methods: The thesis used mixed methods including retail audits, surrogate client surveys based on post intervention cross sectional surveys in intervention and controls and mapping of outlets in intervention areas. Qualitative methods including record reviews, in-depth interviews and focus group discussions with programme stakeholders were analysed using thematic framework and policy analysis. Results: There was a significant impact on PMR knowledge and practice of an NGO-led participatory training programme in Kisii-central district with 60.5% of trained PMRs selling AQ medicines adequately compared to 2.8% in the untrained ones (OR; 53.5: 95% CI 6.7, 428.3). There was some evidence of a limited impact for the MoH-led participatory training programme in the Kwale district, where 18.8% of trained PMRs sold AQ medicines adequately compared to 2.3% of control PMRs (OR; 9.4: 95% CI 1.1, 83.7). This study was unable to show evidence of impact in the social marketing programme in Bungoma district.

The rapid development of resistance to currently deployed antimalarial drugs has raised the desperate need for new chemotherapies, preferably with novel therapeutic target. This thesis explores the synthesis of novel quinolone compounds and artemisinin derivatives targeting the mitochondrial electron transfer chain (ETC) and the haemoglobin degradation of Plasmodium falciparum respectively. The respiratory chain of the human malaria is an attractive target for antimalarial drugs. It is believed that the collapse of the mitochondrial potential will shut down the metabolism and malaria parasite de novo synthesis of pyrimidines, ultimately leading to the death of parasite. The bc1, (Complex III) inhibitors are being studied by many scientists, with recent studies targeting PfNDH2 due to its potential as a therapeutic target (Humans lack this enzyme in the respiratory chain). Following the hit to lead optimisation of chemical name here CK-2-68 against the PfNDH2 enzyme in the group, a series of quinolones were designed to improve the ClogP and aqueous solubility. Analogues in this series were synthesised in less than six-step. Our strategy for reducing the ClogP of the original series involves the incorporation of heterocycles into the C and 0 rings of the side chain. Work describes in this thesis was principally cover the 2-pyridyl series of compounds. Two of these analogues have ICso values in the nanomolar range versus PfNOH2 enzyme and 307 strain of Plasmodium falciparum. Further in vivo studies showed that these two analogues have notable ED50/ED90 against Plasmodium berghei (NS Strain) following oral administration. A series of 6-substituted quinolone esters and pyrrolidine-fused quinolones were also prepared for targeting bc1 complex. They were tested in vitro to explore their structure-activity relationship (SAR). The 6-substituted quinolone ester were synthesised in four steps employing the Gould-Jacobs method. It was noticed that the 3-ester functionality and its steric size are essential for good activity. The 6- substituted quinolone esters possess moderate antimalarial activity with the lead analogue IC50 of 40.4 nM. The 6-substituted quinolone esters were compared head to head with the 7-series of analogues. In an attempt to enhance solubilities, pyrrolidine-fused quinolones were synthesised using Winterfeldt oxidation. Although the compounds were poorly soluble, the potent in vitro result of one of the analogues underlines the potential of the template for further study. Artemisinin and its semi-synthetic derivatives are the most effective drugs in malarial chemotherapy. Despite of their high therapeutic indices, they have poor bioavailability and short half-lives in general. To improve the aqueous solubility and metabolic stability, a series of semi-synthetic C-10 pyrrole Mannich artemisinin derivatives were prepared in 2 steps from dihydroartemisinin. These analogues have demonstrated nanomolar antimalarial activity against the 3D7 strain and Kl strain of Plasmodium falciparum in vitro with high therapeutic indices. Further in vivo studies showed that three of the analogues have excellent ED50/ED90 indicating their overall in vitro and in vivo drug profiles are superior to those of clinically used artemether and artesunate.

In low income countries the majority of health care is sought in the private sector, often through drug retailers, but little information is available on retail competition and regulation. This thesis addresses this gap, in the context of the market for fever and malaria treatment in rural Tanzania, investigating how market structure, provider conduct, regulation and consumer demand influence treatment outcomes. Data were collected in three districts, where the main treatment providers were public and private health facilities, drug stores and general shops. Following a census of retailers, data on supply were collected from facilities and shops through in-depth interviews, a structured survey, and retail audits. Data on demand were collected through a household survey. Analysis focused on retailers, supplemented by data on facility services and consumer demand, where this was central to an understanding of retailer behaviour. Retailers were an important source of fever/malaria treatment, with the majority of retail antimalarial sales occurring through drug stores. Retail providers increased the accessibility, range and reliability of drug stocks, but several market failures were evident. Market concentration was high, price competition was weak, information on treatment quality was poor, and negative externalities arose from inappropriate drug use. These failures contributed to low antimalarial coverage, use of ineffective antimalarials, under-dosing, and inequitable access to quality care. Government failures were also evident, in the form of poor quality public sector treatment, and inadequately implemented regulation. To optimise the planned introduction of antimalarial combination therapy, public facility care must be improved. However, facility-only provision will not improve treatment for the majority of fever/malaria visits, which are likely to remain to shops. Implications for widening combination therapy provision to the retail sector are outlined, including the selection of appropriate retailers, maintenance of affordable prices, effective communication with consumers and providers, and a constructive role for regulation.

Antimalarial drug resistance is a major cause of the increasing burden due to P. falciparum malaria. Artemisinin-based combination therapies (ACTs) are now recognised to be the ideal choice for the first-line treatment of uncomplicated malaria, in order to achieve two beneficial outcomes: improvement of treatment efficacy and delay in the development of drug resistance. However uncertainties remain about the current and future benefits, risks and costs of ACTs and in particular how these outcomes are affected by differences in malaria epidemiology, health care settings, human behaviour and implementation strategies. This thesis seeks to address these uncertainties by creating a comprehensive, dynamic, bio- economic model of malaria transmission and the spread of drug resistance, which incorporates vector factors, human immunity, human behaviour, drug characteristics and costs. Central to the model is a biological model, developed in collaboration with a mathematician, which outputs the proportion of drug resistant infections and the incidence of new and recrudescent infections. Parasite biomass is also tracked in order for human "infectiousness" to be measured and fed-back into the model. Sub-models are used to calculate severe malaria, deaths, costs and cost-effectiveness. Data were obtained to develop and populate the model. This included a community drug usage survey in Cambodia, which was undertaken in order to document the adherence and coverage rates to ACT following the implementation of locally blister-packaged ACT. Coverage was found to be extremely low, and the use of artemisinin derivatives on their own was widespread. However, both of these outcomes were improved by interventions to increase coverage, particularly village malaria volunteers. Application of the model in a low transmission setting suggests that with a 10-year time-frame, switching from monotherapy to an ACT is very cost-effective and results in overall cost savings in a range of scenarios. High coverage rates with an ACT are required to delay the spread of drug resistance if resistance has already arisen to one of the partner drugs. Running the model with data from Cambodia suggests that even in settings with low coverage, the change will be cost-effective and significant benefits are gained from the implementation of the specific delivery interventions. Strategies for optimising the implementation of ACTs are discussed in light of the findings.

Background: With a low proportion of children receiving the first line treatment for suspected malaria, it has been proposed that artemisinin based combination therapy be subsidised in the private sector in order to improve affordability and access. This thesis presents an evaluation of a pilot subsidy mechanism in Western Kenya. Methods: The primary objective was to evaluate the impact of providing subsidized artemether-lumefantrine (AL) through trained retailers, on the coverage of prompt effective anti-malarial treatment for febrile children aged three to 59 months. I used a cluster-randomised, controlled design with nine control and nine intervention sublocations, equally distributed across three districts. Provider, mystery shopper and household cross- sectional surveys were conducted at baseline and one year later. Data were analysed based on cluster-level summaries, comparing control and intervention arms, while adjusting for covariates. On average details of 2,706 children and 564 retail outlets were captured per year. ., Results: Provider survey and mystery shopper data showed that at follow-up a significantly greater percentage of retailers stocked and dispensed AL, and knew that AL was the first line treatment for uncomplicated malaria in the intervention arm compared to the control. Significantly fewer retailers stocked antimalarial monotherapies. Household survey data showed that an average of 29% of children had experienced fever within the previous two weeks.

Purine nucleoside phosphorylase (PNP) and lactate dehydrogenase (LDH) from Plasmodium parasites are two potential targets for novel anti-malarial compounds. Whereas these enzymes have been well-characterised from the Plasmodium falciparum parasite, information on these enzymes from the similarly prevalent species, Plasmodium vivax, is very limited. This hinders the development of effective common anti-malarial compounds.

The mitochondria of malaria have big potential to be explored as a drug targeting site. This is due to the differences in the composition of the Plasmodium respiratory complex compared with the human host. The Plasmodium respiratory chain consists of 2 unique dehydrogenases PfNDH2 and MQO which are only encoded in prokaryotic cells. Apart from these two, thy Plasmodium bc1 complex has low amino acid similarity when compared to the bc1 cQmplex of other eukaryotes. Currently the only approved antimalarial drug targeting Plasmodium mitochondria is atovaquone. This drug is used in combination with proguanil (Malarone™) and is widely used for the curative and prophylactic treatment of malaria. Atovaquone, a 2-hydroxynaphthoquinone, is a competitive inhibitor of the quinol oxidation (Qo) site of the mitochondrial cytochrome bc] complex. Inhibition of this enzyme results in the collapse of the mitochondrial membrane potential and subsequent parasite death. However, atovaquone resistance developed very soon after its implementation as malaria chemotherapy. Previous studies have established that the resistance of the parasite is the result of mutations in the bc1 Q0. To date we are the first group to elucidate the genotype and biochemical characterisation of the atovaquone resistant P. Jalciparum TM90C2B. The GeXP multiplex quantitative PCR (qPCR) revealed that a number of genes encoding energy metabolism proteins and genes associated with redox control are upregulated in the resistant parasite compared to the atovaquone sensitive strain 3 D7. This has been supported by automated sequencing results which indicate the presence of a single point mutation in the DNA sequence of TM90C2B cytochrome b which resulted in the substitution of tyrosine by serine at position 268. The drug sensitivity assays and kinetic studies were conducted in order to understand the phenotypic consequences of this mutation. As expected, TM90C2B strain was resistant to almost all electron transport chain inhibitors. The enzymological characterisation of TM90-C2B bc] complex (steady-state decylubiquinol:cytochrome oxidoreductase assay) showed that enzyme turnover was approximately 50% of the atovaquone-sensitive strain with a threefold increase in Krn for decylubiquinol (3D7 : Vrnax = 97.4 ± 5.1 nmol cyt c reduced/min/mg protein, Krn = 5.5± 1.1 mM dQH2, ICSD 6 nM, K; = 0.6 nM; TM90C2B : Vrnax = 60.2 ± 3.2 nmol cyt C reduced/min/mg protein, Krn = 18.5± 2.6 mM dQH2, leSD 600 nM, K; = 162 nM). The other mitochondrial respiratory complexes' specific activities were also measured using samples prepared from Percol® fractionation. Apart from Complex II, we also managed to demonstrate the specific activities of the other complexes'. The PfNDH2 and bCI complex proteins ofTM90C2B strain had 50% less activity than the 3D7 strain. This was supported by data from western blot analysis showing a decrease in PfNDH2 and ISP proteins content of the TM90C2B compared to the 3D7 strain. In addition to this study, we also characterised the Plasmodium Jalciparum mitochondrial malate quinone oxidoreductase (MQO). MQO is involved in the electron transport chain, oxidising malate to oxaloacetate in the oxidative arm of Plasmodium tricarboxylic acid cycle (TCA cycle). This enzyme contains a flavin cofactor which donates electrons to cytochrome bCI complexvia reduction ofubiquinone to ubiquinoL The bioinformatic analysis data indicates that PfMQO is a membrane-bound mitochondrial protein with only one transmembrane domain. The PfMQO gene was successfully amplified from genomic DNA of 3D7 P. falciparum and cloned into 2 expression vectors pET-15b and pUCI9. The presence and orientation of the gene in the respective vector were confirmed by restriction enzyme analysis and automated sequencing. The putative PfMQO gene (1566 bp) was successfully amplified and predicted to produce an approximately 60-kDa protein. Only when the PfMQO gene was cloned into pET15b was overexpression of the recombinant protein achieved. The recombinant PfMQO was purifiable under denaturing conditions due to its insolubility and formation of inclusion bodies. This protein is inactive and appears to be improperly folded thus producing the inconsistent results of the kinetic analyses. In conclusion, our study provides new insights into the understanding of the moleCular and biochemical regulation of atovaquone resistant parasites through a mutation in the bc1 complex. The partial characterisation of PfMQO is a starting point for the development of a new drug target in Plasmodium mitochondria by taking into account the uniqueness of this enzyme which is not found in the human host.

In recent years investigation of the biological mode of action of the antimalarial compound atovaquone has validated the Plasmodium falciparum bC1 protein complex as a target for the development of novel antimalarials. This project is concerned with the design, synthesis and development of quinolone based antimalarial compounds targeting the Qo site of the Plasmodium falciparum bC1 protein complex. -......, ..•. : - A small array of quinolone-based compounds, substituted at the 3 and 7 positions, has been developed utilising the Gould-Jacobs methodology. The synthesised compounds express activity as low as 0.46 nM versus Plasmodium falciparum malaria parasites in vitro. Activity at the bC1 protein complex has been confirmed and selectivity for the parasite protein over the mammalian equivalent has been demonstrated. Poor solubility has been highlighted as a potential issue; however steps have been taken towards the ,/ development of carbamate prod rugs with encouraging results. Docking studies performed in silico, using the crystallised structure of yeast bC1 protein, demonstrate a key role for residues His181 and Glu272 in the recognition of high potency inhibitors and have aided in the rationalisation of the observed biological activities. The orientation of the quinolone group in the active site and interaction of the inhibitors with the hydrophobic channel leading to the active site have both been shown to be of significance for biological potency. Computer lead design of a new chemotype has resulted in the development of a second array of quinolone based compounds synthesised by a divergent method based on the Huisqen 1,3-dipolar cycloaddition. Docking studies predicted the synthesised compounds to be potent inhibitors of Plasmodium falciparum bC1. However, in vitro results for the array proved disappointing, an outcome attributed to a failure to account for membrane permeability and the physical location of the targeted bC1 protein complex.

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