PHARMACODYNAMICS OF PYRONARIDINE IN COMBINATION WITH CURRENT ANTIMALARIALS IN A CYTOCIDAL MURINE MALARIA MODEL by GaYoung Lee A thesis submitted to Johns Hopkins University in conformity with the requirement for the degree of Master of Science. Baltimore, Maryland April, 2018 © GaYoung Lee 2018 All right reserved Abstract While there has been a progress in understanding P. falciparum genomics, and exciting discoveries of promising leads in the antimalarial pipeline, the efficacies of existing artemisinin combination therapies are threatened by incorrect dosing and non-compliance with duration of dosing regimen, which might hasten the emergence of resistant parasites. Understanding of the effects of drug interactions on combination therapy with existing antimalarial agents may provide useful information such as their mechanism of action, and influence the development of new drug combination. A traditional way of evaluating antimalarial drug efficacy and combined drug interactions has been relying on in vitro drug sensitivity assays using cultured cells. This method measures the rate of suppression or inhibition of parasite growth during 48 – 72 hours in comparison to untreated controls. However, extrapolating in vitro results from cultured parasites to humans remains challenging as cytocidal activity of antimalarials (reduction in absolute parasite numbers) ii in patients is measured at a high asynchronous parasitemia at close to 1 trillion (per adult). In this thesis, we adapted a transgenic luciferase reporter, P. berghei ANKA to study cytocidal activity of tafenoquine and pyronaridine in mice for combinations of three current antimalarials, artesunate, amodiaquine, and azithromycin at clinically relevant dosage. We initiated each drug treatment at a high parasitemia (~10%) and followed for 30 days. Three metrics of measures we used in characterizing pharmacodynamic properties are 30-day survival, parasite log reduction over 48 hours, and recrudescent parasitemia (i.e., return to initial parasitemia). We adapted the parasite reduction ratio (PRR), or fractional log reduction in parasitemia as the PRR can be analogous to the killing rate, thus serving as a measure to compare the different intrinsic pharmacodynamic between the drugs in question. Pyronaridine when compared to tafenoquine at the same dose exhibited more potent cytocidal activity with approximately 1,000-fold more reduction in the number of parasite over the single 24-hour parasitic life cycle. Tafenoquine synergized with artesunate. In contrast, additive interactions were evident for pyronaridine in combination with artesunate. In addition, while pyronaridine synergized amodiaquine, differential interactions were shown with azithromycin, suggesting that synergy may only be achieved at certain dose ratios. Primary Reader: David Sullivan, M.D. (JHSPH) Secondary Reader: Theresa Shapiro M.D., Ph.D. (JH SOM) iii Acknowledgments The following people had a significant influence on this thesis: David Sullivan and Theresa Shapiro. I’d like to thank Sullivan lab members (past and present): Grace, Kristin, Leah, Rachel, Scott and Tamaki. Special thanks to: Dick, David, Jeremy and Sean for their relentless support and encouragement throughout the period. Finally, I’d like to remember my parents from whom I learn to be kind and courageous. And, my big brother, Matthew H.K who challenges me to appreciate the little things in life and see beautiful things in humble places. iv Contents Acknowledgement iv An introduction to Plasmodium spp. and antimalarial chemotherapy 1 1.1 Malaria ........................................................................................................ 1 1.2 Antimalarial chemotherapy ......................................................................... 7 1.3 Rationale ................................................................................................... 17 1.4 Research Aims .......................................................................................... 21 Methodology 23 Results 28 3.1 The minimum curative single oral administration of tafenoquine is 30 mg/kg ........................................................................................................ 28 3.2 The minimum curative single intraperitoneal administration of pyronaridine is 10 mg/kg .......................................................................... 30 3.3 Pyronaridine exhibits more rapid killing than tafenoquine. ...................... 31 3.4 Checkerboard experiments to examine in vivo drug interactions between tafenoquine or pyronaridine with ACT partner compounds ..................... 33 v 3.4.1 Tafenoquine synergizes with artesunate ........................................ 33 3.4.2 Pyronaridine exhibits additive interactions with artesunate .......... 36 3.4.3 Pyronaridine synergizes with amodiaquine ................................... 39 3.4.4 Pyronaridine exhibits differential interactions with azithromycin..42 Discussion 46 References 53 Curriculum Vitae 69 vi List of Tables 1.2 Chemical structures of antimalarial drugs used in study……………………16 1.3 In vitro and in vivo drug interaions between antimalarial agents on Plasmodium spp. ............................................................................................ 21 2.1 Mouse doses and human equivalents of antimalarial drugs ........................... 27 3.1.1 Curative dose of tafenoquine, and measure of parasite log reduction on P. berghei ANKA infection ............................................................................... 30 3.2.1 Curative dose of pyronaridine, and measure of parasite log reduction on P. berghei ANKA infection ............................................................................... 30 3.3.1 Measure of parasite log reduction of tafenoquine vs. pyronaridine on P. berghei ANKA infection ............................................................................... 32 3.4.1.1 Curative dose and measure of parasite log reduction of tafenoquine in combination with artesunate ......................................................................... 36 3.4.2.1 Curative dose and measure of parasite log reduction of pyronaridine in combination with artesunate .......................................................................... 38 vii 3.4.3.1 Curative dose and measure of parasite log reduction of pyronaridine in combination with amodiaquine ..................................................................... 41 3.4.4.1 Curative dose and measure of parasite log reduction of pyronaridine in combination with azithromycin ..................................................................... 44 viii List of Figures 1.1 Plasmodium Life Cycle ................................................................................... 2 1.2 Possible drug interactions illustrated in isobolograms .................................. 18 3.1.1 Curative efficacy of tafenoquine on P. berghei ANKA infection ................. 29 3.2.1 Curative efficacy of pyronaridine on P. berghei ANKA infection ............... 31 3.3.1 Comparing curative efficacy of pyronaridine vs. tafenoquine ...................... 32 3.4.1.1 Checkerboard and isobolograms showing in vivo interactions between tafenoquine and artesunate ............................................................................ 34 3.4.1.2 Curative efficacy of tafenoquine in combination with artesunate ................. 35 3.4.2.1 Curative efficacy of pyronaridine in combination with artesunate ............... 38 3.4.2.2 Checkerboard and isobolograms showing in vivo interactions between pyronaridine and artesunate ........................................................................... 39 3.4.3.1 Curative efficacy of pyronaridine in combination with amodiaquin ............. 40 3.4.3.2 Checkerboard and isobolograms showing in vivo interactions between pyronaridine and amodiaquine ...................................................................... 41 ix 3.4.4.1 Curative efficacy of pyronaridine in combination with azithromycin ........... 43 3.4.4.2 Checkerboard and isobolograms in vivo interactions between pyronaridine and azithromycin ........................................................................................... 45 x Chapter 1 An introduction to Plasmodium spp. and antimalarial chemotherapy 1.1 Malaria Malaria, a mosquito-borne, acute febrile illness caused by a protozoan parasites of the genus Plasmodium, remains one of the life-threatening infectious diseases with 216 million cases and 445,000 deaths(1). In humans, five different Plasmodium species, namely P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi can cause malaria through the bites of infected female mosquitoes of the genus Anopheles. While other Plasmodium species remain important causes of significant disease, including severe illness, P. falciparum and P. vivax pose the greatest public health challenge globally. Infection with P. falciparum is most 1 1.1 MALARIA prevalent on the African continent where more than two thirds (70%) of all fatal malaria cases occur in children under age 5. P. vivax has a wider geographical distribution, covering most countries outside of sub-Saharan Africa owing to its ability to develop in the Anopheles mosquito at lower temperatures (below 20C) and survive at higher altitudes. In case of P. vivax and P. ovale malaria, parasites have an advantage of having a dormant liver-resident hypnozoite stage, producing no clinical manifestation, but causing recrudescence or relapse months or years