Pharmacology and pharmacodynamics of selected antimalarials against P. falciparum gametocytes

Abstract

Malaria is a vector-borne disease that is still responsible for high human morbidity and mortality. Of the five Plasmodium species that can cause malaria in humans, Plasmodium falciparum is regarded the most virulent species. The most fundamental component of sustained control and eradication efforts is the development of effective drugs for malaria treatment and prophylaxis. Plasmodium falciparum’s sexual stages (gametocytes) are not associated with malarial pathogenesis or the clinical symptoms, but they are responsible for the transmission of the disease from human hosts to mosquitos. As such, the development of gametocytocidal interventions that targets the transmission stage to break the disease’s lifecycle forms the basis of efforts towards malaria elimination and eradication. However, despite the importance of this developmental stage, the biology and pharmacology of gametocytes are still very poorly understood. This thesis has set out to gain a better understanding of the identity of gametocyte-active antimalarials and a deeper understanding of the mechanisms underpinning the activity. Using a newly generated luciferase-reporting transgenic line, pharmacodynamic gametocyte studies could be performed to help characterise the activity of selected known reference antimalarials, new potential gametocyte inhibitors in pre-clinical development as well as newly developed fully synthetic compounds designed against the sexual stages. This novel assay revealed that the efficacy of active tested compounds is highly stage-specific. Of all the tested reference antimalarial drugs, MB and DHA were the most potent antimalarial across all gametocyte stages and importantly they were active at clinically relevant levels. These observations were progressed further, developing a time- dependent killing assay that was performed with different concentrations of targeted drug over discrete time intervals to determine the drug’s kill rate. These parameters were then used to simulate the PK/PD relationship of the drug in order to estimate gametocyte clearance profiles during the human treatment period (Chapter 3 and 4). A main focus of the thesis was conducted to better understand the mechanism of drug activity of the 8-aminoquinolines against gametocytes. The ability of a series of 8-aminoquinolines (primaquine as the parent drug, synthesised metabolites in chapter 5, three novel analogues and tafenoquine in (chapter 6) to interact with CYP2D6 was tested by measuring their ability to specifically inhibit the metabolism of fluorescently-tagged tracer substrate by recombinant human CYP 2D6. Reaction products from the CYP metabolites and HLM were then used to test firstly their ability to kill gametocytes, and then to establish their ability to generate hydrogen peroxides and finally measure their haemolytic toxicity. At 10 µM, primaquine CYP metabolites showed activity against the gametocytes that was higher than that of the parent drugs, with the exception of tafenoquine which, interestingly, demonstrated good activity and haemolytic toxicity as a parent drug. These analyses are presented and discussed in the context of strategies that aim at the discovery and development of new transmission-reducing antimalarial drugs.