The Pennsylvania State University The Graduate School College of Agricultural Sciences IN VITRO DRUG INTERACTIONS BETWEEN TAFENOQUINE AND CURRENT ANTIMALARIALS IN PLASMODIUM FALCIPARUM PARASITES A Thesis in Entomology by Karen Kemirembe 2015 Karen Kemirembe Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science December 2015 The thesis of Karen Kemirembe was reviewed and approved* by the following: Liwang Cui Professor of Entomology Thesis Adviser Kelli Hoover Professor of Entomology Jason L. Rasgon Associate Professor of Entomology and Disease Epidemiology Cristina Rosa Associate Professor of Plant Virology Gary W. Felton Professor of Entomology Head of the Department of Entomology *Signatures are on file in the Graduate School iii ABSTRACT Malaria, caused by Plasmodium spp. parasites, is one of the top ten causes of death in low-income tropical/sub-tropical countries. There are five species of human malaria but of these, Plasmodium falciparum and Plasmodium vivax are the most prevalent. The 2014 World Malaria Report estimates that about half of all countries with ongoing malaria transmission are co- endemic for P. vivax and P. falciparum malaria. Special features of P. vivax and P. falciparum respectively are that P. vivax has a latent liver stage that can be re-activated 6 months to several years after initial clearance of an infection with antimalarial drugs, and P. falciparum, although lacking in a dormant liver stage, has the slowest growing infectious sexual stage (gametocytes) of the human malarias. In addition, P. falciparum has prolonged gametocyte clearance within an infected patient due to the inefficacy of current antimalarials at this stage, posing an increased risk of transmission from infected humans to female mosquito vectors that carry malaria. Currently, an 8-aminoquinoline class drug, primaquine (PMQ) is the only antimalarial licensed to target the liver and gametocyte stages of these two malaria species; the first- line treatments, termed artemisinin combination therapy (ACT) preferentially target the asexual stages in a human host that cause the majority of clinical symptoms of the disease. To address this drug shortage, GlaxoSmithKline and the Walter Reed Army Institute of Research have developed an 8- aminoquinoline 5-phenoxyl PMQ derivative, WR 238605/SB-252263, herein referred to as tafenoquine (TFQ). TFQ is currently in late-stage clinical development for the radical cure of liver stage, asexual and sexual stage P. vivax, administered as a single 300 mg dose following a 3- day chloroquine (CQ) regimen to patients pre-screened for a >70% glucose 6-phosphate- dehydrogenase (G6PD) activity in order to minimize severe hemolytic anemia from oxidative stress, a side effect of the drug in deficient individuals. iv On account of 1) a high prevalence of mixed P. vivax and P. falciparum co-infections observed in field clinical trials, 2) possible misdiagnosis of P. falciparum as P. vivax and 3) widespread and emerging CQ resistance in P. falciparum and P. vivax respectively, leading to a shift in antimalarial use from CQ to ACT, studying the drug combination interactions of TFQ with ACT for both P. falciparum and P. vivax parasites is especially important in order to assess the best drug combinations of antimalarial regimens; 3 days of ACT for asexual stages, followed by a single dose of TFQ, that will achieve enhanced treatment efficacy, or synergy. Since there is currently no continuous in vitro culture protocol for P. vivax, this thesis focusses on TFQ’s effect on P. falciparum when given post ACTs for a P. vivax infection. Although TFQ is being developed primarily for P. vivax, it is important to note that studies in P. falciparum have shown causal prophylactic, schizonticidal and gametocytocidal activity of TFQ, suggesting possible off-target inhibition of P. falciparum, should the drug be deployed for P. vivax. In order to assess how TFQ interacts with current ACT antimalarials in vitro, both asexual and sexual stage parasite replication was assessed in the presence of a single drug or combined ACT- partner drugs with TFQ. A SYBR Green I fluorescent dye was used to quantify asexual stage replication whereas a flow cytometry based method was used to quantify the drug inhibition of transgenic parasites expressing green fluorescent protein (GFP) in sexual stage parasites. Fractional inhibitory indices were calculated from growth inhibition curves of single or combined drugs at fixed ratios and used to determine synergistic, additive or antagonistic drug interactions of parasite strains with differing genetic backgrounds. In general, synergism, whereby a given drug in combination with TFQ is more potent than when used alone would be the desired result. Five sensitive or resistant parasite strains to either CQ or Artemisinin (ART) were tested against a panel of long-lasting six ACT component drugs namely amodiaquine (AMQ), lumefantrine (LMF), mefloquine (MFQ), naphthoquine (NQ), piperaquine (PPQ) and v pyronaridine (PND) using a fixed- ratio method based on published Cmax values to mimic in vivo human pharmacokinetics. The short-lived ART and its derivatives were excluded from the experiment on the basis that TFQ peak plasma levels are achieved after their elimination in vivo. Results showed mostly synergistic relationships in all strains at the asexual stage, regardless of CQ or ART sensitivity. Some gametocyte interactions were however found to be antagonistic. Here for the first time, TFQ interactions with ACTs have been investigated. Taken together, TFQ appears to have a positive effect on P. falciparum parasite inhibition, at least in vitro, and patients with mixed malaria infection, will likely benefit from taking TFQ in addition to the standard ACTs. Each malaria-infected region will therefore have to select an ACT-TFQ pair that will likely give the most effective treatment in patients. In vivo drug interaction work in humanized mice with varying G6PD activity as well as clinical drug interaction trials in humans are a necessary follow-up to these claims since host factors such as hematocrit, gender, immunity, drug activation as well as diet might alter the results of drug-drug interaction studies. This work portrays the urgent need for more in vitro studies to perform interaction analyses on both sexual and asexual parasites, not just the former, as the interactions appear to be stage-dependent; there is currently only one publication that includes the sexual stages, perhaps due to previous difficulty in obtaining these in sufficient amounts compared to their asexual stage counterparts. The flow cytometry based method used here, as has been previously shown, is a reproducible way to do this in vitro, although different methods may have to be applied to field parasite isolates. Additionally, the disagreement between results obtained here using ratios based on in vivo plasma concentrations and those reported previously for CQ-TFQ interactions supports the notion of a switch from the more common use of fixed ratios based on in vitro drug inhibitory concentrations to physiologically relevant fixed drug ratios. vi TABLE OF CONTENTS List of Figures .......................................................................................................................... vii List of Tables ........................................................................................................................... viii List of Abbreviations ............................................................................................................... ix Acknowledgements .................................................................................................................. x Chapter 1 An introduction to human malaria, Plasmodium spp. ............................................ 1 1.1 Introduction ................................................................................................................ 1 1.2 P. falciparum and P. vivax life cycle and implications for chemotherapy ................. 4 1.3 Thesis aim, objectives and rationale .......................................................................... 9 1.4 References .................................................................................................................. 12 Chapter 2 Tafenoquine Drug Combinations in Asexual and Sexual parasites ....................... 21 2.1 Introduction ................................................................................................................ 21 2.2 Materials and methods ............................................................................................... 27 2.3 Results ........................................................................................................................ 33 2.4 Discussion .................................................................................................................. 38 2.5 Conclusions ................................................................................................................ 43 Chapter 3 Conclusions and future directions .......................................................................... 50 3.1 Summary of findings and relevance ........................................................................... 50 3.2 Perspectives on malaria control ................................................................................. 52 3.3 References .................................................................................................................