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 ...... 55
Appendix A ...... 60
A1: Gametocyte induction ...... 60
A1.1 Equipment and Materials ...... 60 A1.2 Reagents ...... 60 A1.3 Procedure (Modified from Fivelman et al., 2007; Lucantoni et al., 2013) ..... 62
A.2 Flow cytometry method to determine gametocyte drug inhibition ...... 64
A2.1 Materials and Equipment ...... 64 A2.2 Reagents ...... 65 A2.3 Procedure (Modified from Wang et al., 2014) ...... 66 A2.4 References ...... 71
Appendix B: Drug-Drug interaction isobolograms for each lab strain ...... 72
vii
LIST OF FIGURES
Figure 1-1. Life-cycle of Plasmodium falciparum...... 6
Figure 1-2. Methods flow-chart for asexual and sexual drug interactions ...... 11
Figure 4-1. Dot plots of control and fluorescent parasites...... 67
Figure 5-1. Isobolograms of asexual and sexual parasite strains...... 72
viii
LIST OF TABLES
Table 1-1. Comparison between P. falciparum and P. vivax malaria parasites ...... 7
Table 1-2. Resistance markers of drugs used in this study...... 10
Table 2-1. Structural classification of antimalarials used in this study...... 25
Table 2-2. Summary of available artemisinin combination therapies ...... 26
Table 2-3. Pharmacokinetics of drugs used in this study...... 32
Table 2-4. Ratios used for asexual TFQ- ACT-partner drug interactions ...... 33
Table 2-5. Drug susceptibilities of asexual parasites ...... 35
Table 2-6. Median TFQ inhibitory concentrations by gametocyte stage ...... 35
Table 2-7. Drug susceptibilites of 3D7αtubIIGFP gametocytes ...... 36
Table 2-8. Summary of TFQ-ACT- partner drug interactions...... 37
ix
LIST OF ABBREVIATIONS
Acronyms of Antimalarial drugs
ACT Artemisinin Combination Therapy
AMQ Amodiaquine
ART Artemisinin
CQ Chloroquine
DHA Dihydroartemisinin
LMF Lumefantrine
MFQ Mefloquine
NQ Naphthoquine
PMQ Primaquine
PND Pyronaridine
PPQ Piperaquine
TFQ Tafenoquine
Acronyms of Materials and Methods
MCM Malaria Complete Medium
G6PD Glucose-6-Phosphate Dehydrogenase
GFP Green Fluorescent Protein
CM Conditioned medium
IC50 Median inhibitory drug concentration
FIC Fractional Inhibitory Concentration
x
ACKNOWLEDGEMENTS
Many thanks to my advisor, Dr. Liwang Cui for his guidance and invested resources in me whilst in his lab. I am grateful to my committee members Dr. Cristina Rosa, Dr. Jason L.
Rasgon and Dr. Kelli Hoover for their generous time and advice. I am grateful to all Cui lab members for helpful discussions and to Dr. Mynthia Cabrera Goss for her mentorship. To Dr.
Gary W. Felton without whose leadership the Dept. of Entomology would not be the same, I am gratified. I am indebted to my parents for their continued support and patience with me being thousands of miles away from home to obtain a good education. I also wish to thank my house- mates, Jennifer T. Yang and Meredith T. Hanlon for always making our grad student home a fun and comfortable environment to live. I acknowledge my boyfriend, Troy F. Carl, and all my friends for their continued love and support. Lastly, this work would not be possible without the
National Institutes of Health’s (fund U19AIO89672) financial support.
1
Chapter 1
An introduction to human malaria, Plasmodium spp.
1.1 Introduction
Malaria, in addition to other infectious diseases like tuberculosis and HIV, is one of the top ten causes of death in low-income countries (WHO, 2014). The name, derived from Italian mal aria or ‘bad air’ came about due to previous beliefs that its cause was air-borne, rather than vector-borne (Wahlgren and Bejarano, 1999). Malaria parasites are single cell eukaryotes or protozoans (not to be confused by popular media as viruses or bacteria) and are spread by female anopheles mosquitos as a by-product of blood feeding behavior (Nilsson et al., 2015). Mosquitoes find their hosts using odorant receptors sensitive to carbon dioxide in exhaled air (Turner et al.,
2011; Pellegrino et al., 2011).
The disease is reported to have killed approximately 584,000 in the year 2013 according to the most recent World Malaria report (WHO, 2014). Anopheles mosquitos (Diptera: Culicidae) are widespread worldwide with Anopheles gambiae, An. funestus and An. arabiensis being co- dominant in Africa, where most of the malaria fatalities occur. In S. E. Asia, An. dirus is the most prevalent amidst a larger selection of vectors compared to the few in Africa. An. darlingi is prevalent in S. America, An. Albimanus in Central America, and An. fluviatilis and An. stephensi in India/Western Asia (Sinka et al., 2012). Further research is necessary to determine whether these and all other species identified in the field are competent malaria vectors.
There are five known Plasmodium (Haemosporida: Plasmodiidae) species of malaria that infect humans, namely Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi,
2 Plasmodium vivax and Plasmodium falciparum, with the latter two being the most prevalent (Li et al., 2004; Moreno and Joyner, 2015; Biamonte et al., 2013 WHO, 2014). P. vivax is the most wide-spread malaria owing to its lower temperature threshold for sporogony and subsequent parasite development within a mosquito of 16°C, compared to 20°C for P. falciparum, making it withstand temperate, in additional to tropical climate provided that by chance it comes into contact with a competent vector (Weihe et al., 1991; WHO, 2015). All human malarias with the exception of P. malariae which has a 72 hr life- cycle have a 48 hr life-cycle (Moreno and
Joyner, 2015). P. knowlesi is predominantly found in macaque monkeys with a few human infections a year (Lee et al., 2001). P. ovale is mostly found in West Africa and is often misdiagnosed as P. vivax (Roucher et al., 2014; Mueller et al., 2007). P. vivax and P. ovale, unlike P. falciparum have an advantage of having a dormant liver stage that can cause recrudescence or relapses after a few months or years after initial treatment of a malaria infection
(Li et al., 2014; Leroy et al., 2014). This is not only detrimental to people in P. vivax endemic areas, but also for tourists, expats and military personnel who may not know that they harbor dormant malaria until a relapse in their home countries that may be difficult to diagnose.
When infected with P. falciparum malaria, fevers and joint pain are common symptoms, and if left untreated, severe cerebral malaria may develop as well as multi-organ failure
(Biamonte et al., 2013). Due to their underdeveloped immune system, children under 5 years of age account for >75% of malaria deaths and are prone to malaria-related complications like hypoglycemia and anemia, and these can often be fatal if left untreated (WHO, 2014). Rapid diagnostic tests of malaria include a finger prick followed by a thick blood smear that is stained and viewed by microscopy, as well as the more sensitive Real-Time Nucleic Acid Sequence-
Based Amplification to detect sub-microscopic levels of malaria (Schneider et al., 2004).
Wide-spread control of malaria has included the use of pyrethroid- based insecticide treated mosquito nets, indoor residual spraying, draining of all possible larval sites to target the
3 female Anopheles vector, although this has been hindered by the presence of vectors that bite earlier and outdoors (Russell et al., 2011; Mwangangi et al., 2013; Strode et al., 2014; Yohannes and Boelee, 2012), thereby evading any contact with any indoor vector-control methods. To manage the malaria parasite, mass drug administration and chemoprophylaxis for vulnerable populations, such as pregnant women have been successfully carried out (Seidlein and
Greenwood, 2003; Tesfazghi et al., 2015; Steketee, 2014). Other control strategies in development are whole organism and partial protein-in-adjuvant vaccines, use of transgenic mosquitoes, photonic fences to target females using their wing beat frequencies and fungal treatment, among others (Moreno and Joyner 2015; Wells et al; 2015; Heinig and Thomas, 2015;
Marsden et al., 2013; Hughes et al., 2014; Hyde et al., 2014). A multi-faceted target of both vector and parasite is essential if we are to further reduce malaria prevalence.
Unfortunately, there have been issues with the control of malaria in the last 50 years due to the evolution of resistance to both insecticides used against the vector, and to antimalarial drugs used against the blood stage parasite in infected humans. There is wide-spread resistance to chloroquine, as well as to sulfadoxine- pyrimethamine, partial mefloquine (MFQ) resistance along the Thai-Cambodia border and emerging resistance to the first- line antimalarial treatment artemisinin (ART) in 6 countries in S. E Asia due to either non-synonymous point mutations of particular genes like Pfcrt in chloroquine and Pfkelch13 mutations in ART resistance or increased copy numbers like in the Pfmdr1gene in the case of MFQ (Ariey et al., 2014; Ranson et al., 2011;
White, 2004; Phyo et al., 2012; Price et al., 2004; Sidhu et al., 2002). Currently, chemotherapy of antimalarial drugs is employed in a co-formulated combination to reduce the evolution of resistance to one drug, with artemisinin combination therapy (ACT) being the first line treatment for malaria. ACTs consist of a fast-acting, short half-life and artemisinin-based component and a longer lasting partner drug to eliminate those parasites that are left-over from the initial killing
(Eastman and Fidock, 2009). In addition to ACT, the WHO recommends prescribing a single
4 0.25mg base/kg dose of the 8-aminoquinoline, primaquine (PMQ), the only licensed gametocytocide, to malaria-infected individuals in areas with emerging artemisinin resistance to curb transmission of gametocytes from infected humans to mosquitoes (WHO, 2014). There is widespread CQ resistance in P. falciparum and ACT is the most used treatment whereas CQ is still efficacious and used commonly against P. vivax in all but 9 countries due to its cheaper price compared to ACT (White, 2013; Price et al., 2014; WHO, 2014). This slow emergence of CQ resistance in P. vivax has been attributed to a shorter duration between peak asexual and gametocyte densities, thus exposing both asexual and sexual parasites to antimalarial drugs when taken doing the symptomatic asexual phase, whereas in P. falciparum., the extended difference in asexual and sexual parasite peak times leads to evasion of an acute febrile response and the release of tumor necrosis factor 2, as well as preferential exposure of only the symptom-causing asexual parasites to antimalarials allowing those asexual parasites that survive drug treatment to differentiate into infectious gametocytes. Furthermore, the reduced parasite numbers in P. vivax versus P. falciparum infections likely plays a role in how many parasites can survive drug treatment, since the evolution of resistance is directly proportional to initial parasite biomass.
Another hypothesis is that since P. vivax gametocytes appear early during infection, transmission may occur successfully before exposure to antimalarial drugs, lessening the likelihood of resistance developing (White, 2004; Mueller et al., 2009; Drakely et al., 2006, Douglas et al.,
2013).
1.2 P. falciparum and P. vivax life cycle and implications for chemotherapy
The malaria life cycle is very complex, with the parasite spending some time both in a human host and in a mosquito. A female Anopheles spp. mosquito, in search of nutrient supplements for egg-laying, bites a person infected with malaria and draws blood containing male
5 and female sexual-stage parasites, named gametocytes. Gametocytes on encountering a change in pH and xanthurenic acid in the mosquito gut are activated in a preparation for gametogenesis where the male and female gametocytes are transformed to gametes by mitosis and meiosis respectively (Delves et al., 2013; Khan et al., 2005). The male gametocyte undergoes three rounds of DNA replication in preparation for the formation of eight motile gametes that each go on to fertilize female gametes, which they encounter through a cell surface adhesion process mediated by Pfs48/45 in a process called exflagellation. Female gametocytes once activated in the mosquito, egress from the red blood cell (RBC), de-repress a lot of silenced transcripts that were under the control of a DOZI gene (Mair et al., 2006). A HAP-2 Arabidopsis plant sterility gene homolog dependent formation of a diploid zygote occurs. The zygote matures into a tetraploid ookinete, meiosis occurs to form to form multi-cellular oocysts that burst to release haploid sporozoites that then travel to the mosquito salivary glands within mosquito hamocoel
(Josling and Llinas, 2015). Mature sporozoites are then passed on from mosquitoes to humans when a female mosquito takes her next blood meal.
Whilst in the human body, sporozoites travel to the liver where they infect hepatocytes and mature into liver schizonts. These schizonts mature in the liver within 7 days and eventually release up to 40,000 merozoites that then initiate the asexual intra-erythrocytic blood stage when they invade red blood cells. In the case of P. vivax, a small proportion of liver parasites become dormant in the liver awaiting activation at a later time point ranging from months to years; these are termed hypnozoites (Mikolaiczak et al., 2015). Within the red blood cells, the malaria parasites replicate asexually from ring stage, progressing through the highly metabolic trophozoite stage, gradually digesting the host red blood cell hemoglobin as they develop over the course of a 48 hour life-cycle with the cycle repeating after a blood-stage schizont bursts to release 8-32 merozoites, and proceeds to invade new red blood cells (Flannery et al., 2013). Due to the rapid replication and exponential growth of these asexual parasites, an immune response is
6 triggered that causes fever and other malaria-induced clinical symptoms like joint pain. Anemia occurs due to the increased rupture of host red blood cells. A small proportion of <10% these haploid asexual blood parasites undergo gametocytogenesis, mediated by the transcription factor,
PfAP2-G (Sinha et al., 2014). P. falciparum gametocytes take 10-12 days to mature within a human host in five distinct stages I-V, and peak in number 7-10 days after the initial asexual stage peak (McKenzie et al., 2006; Talman et al., 2004). Stages II-IV are sequestered in the bone marrow, presumably to avoid clearance by the spleen, and only return to peripheral circulation as mature stage V gametocytes that are then infectious to a mosquito within 2 days (Nilsson et al.,
2015). P. vivax gametocytes on the other hand mature within 3-4 days and reach peak parasitemia simultaneously with asexual parasites and there is no evidence of sequestration
(Mueller et al., 2009; Bousema and Drakely, 2011; White et al., 2008). They are therefore likely to come into contact with antimalarial drugs taken during the symptomatic asexual stage.
Fig 1-1 Life cycle of Plasmodium falciparum. Both mosquito and human stages are shown. P. vivax liver hypnozoites are indicated to differentiate the two species. Adapted by permission from
7 Macmillan Publishers Ltd: [Nature Publishing Group] (Michalakis and Renaud, 2009), copyright
(2009). RightsLink license number: 3741480055847. License date: Nov 3rd, 2015.
Table 1-1 Comparison between P. falciparum and P. vivax malaria parasites. Data is from WHO,
2015 and Mueller et al., 2009.
Plasmodium falciparum P. vivax
18ºC sporogony 14ºC sporogony
12 d gametocyte maturation 3-4d gametocyte maturation
Crescent-shaped gametocytes Round gametocytes
Peak gametocyte day 7-11 Peak gametocyte day 1-2
No dormant liver stage parasites Dormant liver stage parasites
Gametocytes more tolerant to ACT Gametocytes killed by ACT
Infects all RBCs Infects reticulocytes
Schüfnner’s dots absent on RBC Schüfnner’s dots present
BOTH REQUIRE HEMOGLOBIN DIGESTION SO SIMILAR HEME-MEDIATED
DRUG TARGETS
Owing to the fact that malaria symptoms are initiated at the asexual intra-erythrocytic blood stage, most antimalarial drugs on the market target this stage, with most drugs depending on the hemoglobin degradation pathway for their action. Gametocytes minimize their hemoglobin digestion machinery past stage III so only drugs affecting other organelles beside the digestive vacuole, such as the mitochondria are effective against them (Butcher, 1997). Only one drug, namely primaquine (PMQ) is licensed to kill gametocyte sexual stages. A derivative of PMQ,
8 tafenoquine (TFQ), is active against the afore-mentioned liver stage that occurs in some P. vivax infections (Li et al., 2014). TFQ is the subject of study in this thesis, because it is currently in late stage clinical trials for the radical treatment of P. vivax, and encounters P. falciparum in areas co- endemic for both malaria species. The problem with both PMQ and TFQ is that they cause hemolysis in individuals with a Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency.
Many studies are underway to identify other drug compounds that are safe for humans and possess the transmission blocking benefit of killing sexual stage gametocytes, but work in this area is relatively in its infancy because it wasn’t until recently that methods were developed to readily obtain large numbers of gametocytes in vitro (Fivelman et al., 2007). Numerous studies have since investigated the effect of potential antimalarials that are gametocytocidal
(Delves et al., 2013; Reader et al., 2015; Peatey et al., 2012). The problem currently lies in the fact that due to differences in methodology, very few of these studies have data in agreement with one another (Reader et al., 2015). One thing that is for certain is that the oldest antimalarial, methylene blue, has received renewed interest because of its inhibitory effects at both the asexual and sexual blood stages of the malaria parasite and is currently in clinical trials (Coulibaly et al.,
2015).
As mentioned earlier, the only drug licensed for gametocytocidal clearance is PMQ, an 8- aminoquinoline. It’s derivative, TFQ, another 8-aminoquinoline is in late stage clinical trials for the treatment of P. vivax relapse, but in vitro studies have shown its efficacy in all stages of P. falciparum as well (Shanks et al., 2001; Crockett and Cain, 2007; Brueckner et al., 1998). Due to a high prevalence of mixed infections of P. vivax and P. falciparum, a person taking a TFQ prescription for P. vivax is likely to be co-infected with both parasites (Snounou and White.,
2004; Douglas et al., 2013). My thesis therefore aims to investigate the off-target effects of TFQ on P. falciparum, when taken with ACT or chloroquine treatment. The recommendation is that a person will be given 3 days of CQ or ACT followed by a single dose of 300mg TFQ (Llanos-
9 Cuentas et al., 2013). The hypothesis is that the TFQ will come into contact with the longest lasting ACT partner drug in vivo and will either enhance, maintain or diminish the efficacy of these drugs on P. falciparum parasites. Although TFQ is being developed primarily for P. vivax, this study will focus only on P. falciparum due to the lack of a continuous in vitro culture method for P. vivax parasite proliferation due to the requirement of fresh reticulocytes (Roobsong et al.,
2015). Follow up studies ex-vivo or in vivo will have to be carried out to further verify the results of this study.
1.3 Thesis aim, objectives and rationale
Aim: To determine if there is an off-target treatment benefit of TFQ against P. falciparum parasites when administered with ACT for P.vivax in areas where mixed infections occur.
Objective 1
To determine asexual stage TFQ-ACT-partner drug combination interactions
Rationale: First line ACT targets asexual stage parasites. ACT consists of a fast-acting and rapidly eliminated artemisinin derivative such as dihydroartemisinin or artemether and a long lasting partner drug, such as the bisquinoline, piperaquine. TFQ, if administered after 3 days of
ACT will encounter the long lasting drug compound in peripheral blood. The resulting TFQ-drug combinations’ ability to inhibit growth and proliferation of asexual parasites will either be enhanced (synergy), maintained (additivity) or diminished (antagonism).
Method: SYBR Green I fluorescent dye is used to stain DNA of the parasite with higher fluorescence corresponding to more parasite survival post-drug treatment (Smilkstein et al.,
2004).
10 Asexual strains used:
- IPC5202 (Cambodia: DHA resistant [Pfkelch13 R539T mutation])
- 7G8 (Brazil: CQ resistant [Pfcrt K76T SVMNT haplotype])
- DD2 (Indo-China: CQ resistant [Pfcrt K76T CVIET haplotype, Pfmdr1 copy number: 4])
- 3D7 (Africa: CQ sensitive)
- HB3 (Honduras: CQ sensitive)
Table 1-2 Resistance markers of drugs used in this study.
DRUG RESISTANCE NOTES REFERENCE
MARKERS
LMF, MFQ Pfmdr1 Multi-drug resistance Sidhu et al., 2006;
amplification, N86Y transporter, influx pump Folarin et al., 2011
CQ Pfcrt K76T Chloroquine resistance Valderramos et al.,
transporter, efflux pump 2010
ART R539T (Pfkelch13) KLHL12 homolog likely Ariey et al., 2014
involved in ubiquitination
Objective 2
To determine sexual stage TFQ-schizonticide combination interactions
Rationale: Most antimalarial drug treatment (ACT) targets the asexual stage of parasites. A single drug, an 8-aminquinoline, PMQ additionally targets both sexual stage gametocyte parasites of P. falciparum, P. vivax and liver stage dormant parasites in P. vivax. TFQ, a PMQ derivative is in late stage clinical trials to carry out PMQ’s role in P. vivax, taken on the third day of antimalarial treatment. Since mixed infections are common in the field, TFQ will encounter P.
11 falciparum gametocytes. Although TFQ is being developed for P. vivax, a desired off-target effect on P. falciparum, either additivity or synergy is desired. In addition, past drug-drug interaction studies have only been conducted in asexual parasites.
Method: A transgenic line of parasite expressing GFP under the α-tubulin II promoter is used to determine cell viability after drug treatment by flow cytometry (Wang et al., 2014).
Gametocyte strain used: 3D7αtubIIGFP [PlasmoDB: PF3D7_042230]
Drugs used for both asexual and sexual drug interactions include the 4-aminoquinolines chloroquine (CQ), amodiaquine (AMQ) and naphthoquine (NQ), the bisquinoline piperaquine
(PPQ), aryl amino-alcohols lumefantrine (LMF) and mefloquine (MFQ) and the mannich base, pyronaridine (PND).
Figure 1-2 Methods flow-chart for asexual and sexual drug interactions.
12 1.4 References
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Langlois, Nimol Khim, Saorin Kim et al. "A molecular marker of artemisinin-resistant
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Biamonte, Marco A., Jutta Wanner, and Karine G. Le Roch. "Recent advances in malaria drug discovery." Bioorganic & medicinal chemistry letters 23, no. 10 (2013): 2829-2843.
Bousema, Teun, and Chris Drakeley. "Epidemiology and infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination." Clinical microbiology reviews 24, no. 2 (2011): 377-410.
Brueckner, Ralf P., Kenneth C. Lasseter, Emil T. Lin, and Brian G. Schuster. "First-time- in-humans safety and pharmacokinetics of WR 238605, a new antimalarial." The American journal of tropical medicine and hygiene 58, no. 5 (1998): 645-649.
Butcher, G. A. "Antimalarial drugs and the mosquito transmission of
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Crockett, Maryanne, and Kevin C. Kain. "Tafenoquine: a promising new antimalarial agent." (2007): 705-715.
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13 Douglas, Nicholas M., Julie A. Simpson, Aung Pyae Phyo, Hadjar Siswantoro, Armedy
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21
Chapter 2
Tafenoquine Drug Combinations in Asexual and Sexual parasites
*Status: In prep for submission to Antimicrobial Agents and Chemotherapy
2.1 Introduction
The current first- line treatment for Plasmodium malaria parasites is artemisinin combination therapy (ACT), comprising a co-formulated fixed-dose tablet of a fast-killing and rapidly eliminated artemisinin (ART) derivative, combined with a slower-acting partner drug with a longer elimination half-life in humans [1]. The majority of ACTs however, are highly effective against the asexual blood parasites that cause clinical symptoms, but ineffective against the infectious sexual stage gametocytes in P. falciparum and the dormant liver stage parasites, namely hypnozoites of Plasmodium vivax [2]. To address this, the World Health Organization recommends prescribing a 0.25mg base dose/kg of the only licensed gametocytocidal and liver schizonticidal drug, primaquine (PMQ), an 8-aminoquinoline, following either chloroquine (CQ) or ACT to suppress human to mosquito transmission and P. vivax relapses respectively in areas of emerging P. vivax CQ resistance and emerging P. falciparum ART resistance [3]. Owing to the shortage of antimalarial therapies with similar parasite stage targets as PMQ, the Walter Reed
Army Institute of Research in collaboration with GlaxoSmithKline began testing the antimalarial efficacy of a 5-phenoxyl PMQ derivative, tafenoquine (TFQ), also denoted as WR 238605/SB-
252263. [4]. Compared to PMQ, TFQ has a longer elimination half- life of 2 weeks compared to the 6-8 hours of PMQ, appears to have better bioavailability and enhanced hypnozoite suppression [5, 6]. TFQ, is currently in late stage clinical trials for the radical cure of P. vivax [5]
22 as a single 300mg dose after either 3 days of chloroquine, or ACT, a drug regimen similar to the current one of PMQ for P. vivax (Unpublished data: Clinical Trials.gov identifier: NCT01376167 and NCT02184637 respectively). A completed clinical trial showed a higher protection against P. vivax relapse for a CQ+TFQ regimen compared to CQ alone [7].
TFQ’s mechanism of action is unknown but studies in Leishmania spp. and Trypanosoma spp. have yielded possible hypotheses that it acts by inducing mitochondrial dysfunction [8, 9].
Studies in Plasmodium are yet to reveal the link between mitochondrial function and TFQ. Both
TFQ and PMQ have the disadvantage of causing severe hemolytic anemia in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency [7]. Screening for this common X-linked genetic disorder must be carried out prior to dosing with PMQ or TFQ for transmission blocking and/or killing dormant liver stage parasites. The classic Fluorescent Spot Test and the recently developed CareStart (AccessBio, Somerset, NJ) G6PD kit are two effective rapid diagnostic tests for this, although test results must be taken with caution since heterozygous women carriers may show false negative results [10].
Should TFQ be deployed for P. vivax radical treatment in a P. falciparum and P. vivax co-endemic zone, knowledge of drug-drug interactions between either ACT or CQ in the presence of TFQ will be essential, since drug-drug interactions within the human host could enhance or reduce the efficacy of all the drugs involved. In order to study this, interactions in P. vivax would have to be investigated ex-vivo or in-vivo since continuous P. vivax in vitro culture is challenging and unstable, requires a constant supply of reticulocytes and yields very low parasitemia [11, 12].
This study therefore focused on only P. falciparum in vitro drug-drug interactions with ACT- partner drugs and since mixed P. falciparum and P. vivax malaria infections are extremely common in co-endemic countries, the goal was to determine off-target benefits/limitations of
TFQ dosage to P. falciparum parasite inhibition.
23 Few in vitro studies pertaining to TFQ drug-drug interactions have been performed. One study reports either antagonism or additivity of TFQ when combined with CQ or AQ respectively, whereas another conflicting study reports synergism when combined with CQ [13,
14]. Another study reports synergism when TFQ is combined with Artemisinin at a single 1: 1 ratio [15]. Neither of these tested drug-drug interactions against the full panel of ACT- partner drugs and neither of them tested these interactions in sexual stage gametocytes. Drug-drug interactions of these are expected to differ since asexual parasites are sometimes as much as 2000 fold more susceptible to most antimalarials especially the first- line ACTs, compared to their sexual gametocyte-stage parasite counterparts [16]. With regard to PMQ, a recently published study investigated the effects of PMQ-ACT partner drugs in vitro on asexual stage and sexual stage gametocyte P. falciparum malaria parasites, reporting mostly synergistic drug-drug interactions [16]. The study focused on only the longer lasting ACT partner drugs since it was assumed that ART derivatives did not stay in the blood stream long enough to greatly impact the drug-drug interactions.
Here, for the first time, the in vitro interactions between TFQ and six ACT schizonticides, as well as chloroquine in asexual and sexual stage P. falciparum are investigated.
Using a SYBR Green I method for asexual stage and flow cytometry based methods for gametocytes, fractional inhibitory concentrations and isobolograms derived from pharmacologically relevant fixed ratios are used to determine decreased (antagonistic) or enhanced (synergistic) drug efficacy of ACT-partner drugs in the presence of TFQ. Only the long lasting ACT-partner drugs are used here, namely the 4-aminoquinolines amodiaquine (AMQ) and naphthoquine (NQ), the bisquinoline piperaquine (PPQ), aryl amino-alcohols lumefantrine (LMF) and mefloquine (MFQ) and the mannich base, pyronaridine (PND) because TFQ and ART derivatives have durations to reach plasma concentrations (Tmax) of 15 hours and <1.8hours respectively, therefore are less likely to interact in vivo [17–19]. These correspond to current
24 ACTs namely artesunate- amodiaquine, artemisinin- naphthoquine, dihydroartemisinin- piperaquine, artemether- lumefantrine, artesunate- mefloquine and artesunate- pyronaridine, all of which are used in different malaria endemic regions (see Table 2-2, Table 2-3) [1, 20]. The 4- aminoquinoline chloroquine (CQ) is included as well because in areas where P. vivax has not yet developed, CQ is still the first- line treatment prior to a PMQ dosage [2]. Drug-drug combination ratios were chosen to reflect both peak blood plasma concentrations (Cmax) of TFQ and ACT- partner drugs, as well as the in vitro median inhibitory drug concentrations (IC50s) of each individual drug to ensure that proper drug-response curves were obtained (see Tables 2-3 and 2-
4). Parasites with different genetic backgrounds and differential drug susceptibilities to both CQ
(Pfcrt K76T mutation) and the ART metabolite dihydroartemisinin (PfKelch13 R539T mutation) are used to assess whether genetic factors might affect the observed asexual stage drug-drug interactions. 3D7αtubIIGFP parasites expressing GFP under the gametocyte-specific α - tubulin II gene promoter are used for the gametocyte drug interaction assays [21]. Differential TFQ drug interactions between asexual and sexual stage parasites are reported.
Overall, our data sheds some light upon TFQ interactions with ACT partner drugs in vitro for P. falciparum and re-emphasizes the need for future interaction studies to include gametocyte stages to test pharmacologically-relevant drug concentrations in new antimalarial combinations.
Follow-up in vivo experiments, either using humanized mice/humans infected with P. vivax/ P. falciparum with varying G6PD activity will be necessary to verify these results.
25 Table 2-1. Structural classification of antimalarial drugs used in study. Canonical SMILES were obtained from PubChem, and structures drawn using ChemDraw 3D Pro14.0 software.
Antimalarial Classification Chemical structure
Chloroquine 4-aminoquinoline
Amodiaquine 4-aminoquinolines
Naphthoquine
Piperaquine Bis-4-
aminoquinoline
Mefloquine Aryl amino-
alcohols
Lumefantrine/
Benflumetol
Pyronaridine Aza-acridine
mannich base
26
Tafenoquine 8-aminoquinoline
Table 2-2 Summary of available artemisinin combination therapies (ACTs). List is a compilation from [1, 3, 20, 22]. N/A represents drugs manufactured in China but data on world usage is missing. EM, WP, SEA and LA stand for Eastern Mediterranean, Western Pacific, S. East Asia and Latin America respectively. Artemisinin/derivative- Brand names World Region in Use partner antimalarial co- formulations (ACT) Artesunate- amodiaquine Coarsucam®, ASAQ-Winthrop®, Co- Africa and EM Artusan®, MalmedFD® Artemisinin- naphthoquine Arco® Africa and WP Artemether- lumefantrine Coartem®, Riamet®, Faverid®, Amatem®, Africa, EM, WP, Lonart®, AL®, Artemine®, Fantem®, LA,SEA Artefan®, Lumartem®, Lumet® Artesunate-mefloquine ASMQ®, Artequin®, Mefliam Plus® Africa, WP, LA, SEA Artesunate- pyronaridine Pyramax® N/A (China) Dihydroartemisinin- Duocotecxin®, Eurartekin®, Eurartesim®, Africa, SEA,WP piperaquine Combimal®, P-Alaxin® Artemisinin- piperaquine Artequick® N/A (China)
27 2.2 Materials and methods
Chemical reagents
Parasite culture medium starters RPMI 1640 and Albumax II were purchased from Gibco
Life Technologies (Grand Island, NY, USA). Antimalarials for drug susceptibility assays namely chloroquine diphosphate (CQ) [Molecular Weight (MW): 515.86], amodiaquine dihydrochloride dihydrate (AMQ) [MW: 464.81], mefloquine hydrochloride (MFQ) [MW: 414.77] and tafenoquine succinate (TFQ) [MW: 581.58] were purchased from Sigma-Aldrich (St. Louis,
MO). Piperaquine tetraphosphate (PPQ) [MW: 999.55] was kindly provided by Chongqing
Kangle Pharmaceuticals (Chongqing, China). Naphthoquine phosphate (NQ) [MW: 605.94], pyronaridine tetraphosphate (PND) [MW: 910.03] and lumefantrine (LMF) [MW: 528.94] were kindly provided by Kunming Pharmaceuticals (Yunnan, China). PND, AMQ, NQ and CQ were dissolved in distilled water to 20mM stock concentrations. PPQ was dissolved in 90% methanol +
10% 1M HCl to 10mM [23]. TFQ, MFQ, and LMF were dissolved in 100% dimethyl sulfoxide
(DMSO: Alfa-Aesar, Ward Hill, MA) to stock concentrations of 40mM, 20mM and 40mM respectively. Cellulose acetate or nylon 0.2um membrane syringe filters (VWR International,
Radnor, PA) were used to sterilize water and DMSO dissolved drugs respectively. All drug stocks were stored at -80°C until ready for use. Chloroquine stocks were protected from light. Working drug concentrations ranging from 20mM to 100nM were freshly prepared in malaria complete medium (MCM: see In vitro parasite culture below) the same day of drug inhibition assay set up.
SYBR Green I PCR Master Mix for asexual parasite growth inhibition assays was purchased from Invitrogen (Eugene, OR, USA). Giemsa azure eosin blue for parasite staining was purchased from Fluka Chemical Corp. (Ronkonkoma, NY). Percoll for density gradient centrifugation was purchased from Sigma.
28 Parasite culture
P. falciparum laboratory- adapted strains of varying genetic backgrounds 3D7 (Africa:
CQ sensitive), HB3 (Honduras: CQ sensitive), 7G8 (Brazil: CQ resistant Pfcrt K76T), DD2
(Indo-China: CQ resistant Pfcrt K76T) and IPC5202 (Cambodia: ART resistant Pfkelch13:
R539T) were obtained from MR4 (Manassas, VA) and maintained at 37°C under an atmosphere of 90% N2, 5%O2, and 5% CO2 in asexual malaria complete medium (MCM) containing RPMI
1640, 25 mM NaHCO3, 25 mM HEPES (pH 7.4), 11 mM glucose, 0.367 mM hypoxanthine and
5 µg/L gentamycin supplemented with 0.5% Albumax II [24, 25]. Asexual MCM was changed daily and percentage parasitemia maintained below 6.5%, at 2.5% hematocrit in O+ human whole blood (Biological Specialty, Colmar, PA).
A transgenic 3D7αtubIIGFP parasite strain with green fluorescent protein expression under the α- tubulin II promoter was kindly provided by Jun Miao, and sexual stage gametocytes induced from it as previously described in [26] with a few alterations. Heparin sodium salt
(Sigma-Aldrich, St. Louis, MO) was used instead of n-Acetyl Glucosamine to inhibit asexual parasite proliferation in gametocyte cultures and late stage II gametocytes were purified by a
75%/35% percoll gradient (pH 7.4) on day 4 post-gametocyte induction [27]. Gametocytes were maintained in gametocyte MCM that unlike asexual MCM contained RPMI 1640 supplemented with 0.25% Albumax II + 5% heat-inactivated AB human serum (Interstate Blood Bank,
Memphis, TN).
Asexual parasite SYBR Green I drug inhibition assay
Parasites were synchronized by sterile pre-warmed 5% D-sorbitol (wt/vol) (J.T. Baker,
Center Valley, PA) treatment for 9 minutes to enrich for ring stage parasites four days following
29 thawing from liquid nitrogen [28]. Prior to drug assay set-up, percentage parasitemia of cell culture was determined by microscopy using 10% Giemsa in distilled water at pH 6.8. In preparation for drug assay set up, a total of 1000 cells was counted and parasites diluted to 0.5% parasitemia and 2% hematocrit with addition of 50% freshly washed red blood cells in incomplete medium (MCM minus Albumax II or serum). Cells were pelleted prior to dilution from their complete medium by centrifugation with a Heraus Megafuge 16R centrifuge (Thermo Scientific,
Waltham, MA) at 900xg for 5 minutes. The prepared parasite sample was loaded into pre-loaded black 96-well plates with 2X 100ul antimalarial drug working solutions diluted 2-fold to a final volume of 200ul to generate a final hematocrit of 1%, 0.5% parasitemia and 1X drug concentration per well. Negative control wells without drug with either MCM, DMSO or 10%
1M HCl/90% methanol dissolved in MCM corresponding to total amounts in working drug solutions were set up in parallel for drugs dissolved in solvents other than water. Where possible,
DMSO concentrations were kept below 0.4%. Plates were incubated for 72 hours in at 37°C in a
5% humidified incubator followed by transfer to -20°C for at least 16 hours to facilitate cell lysis.
Lysis buffer (100ul) consisting of 20mM Tris ( pH 7.4), 5mM EDTA, 0.008% wt/vol saponin and 0.08%; vol/vol Triton X-100 with 0.2ul of SYBR Green I was added to each 96-well plate sample and mixed gently [29]. Plates were the then incubated at room temperature for at least 1 hour and SYBR Green I fluorescence corresponding to parasite density was determined using a
FluoStar Optima plate reader (BMG Labtech, Cary, NC). Median inhibitory concentrations
(IC50s) for each drug alone or in combination with TFQ were determined by three-parametric non-linear regression analysis of the resultant drug response growth curves using GraphPad Prism
5 software (La Jolla, CA).
30 Sexual parasite flow cytometry-based drug inhibition assay
Stages II-V, corresponding to days 4-12 post gametocyte induction) and stage IV gametocytes, corresponding to days 8 & 9 of the 3D7αtubIIGFP strain at 0.04% gametocytemia and
1% hematocrit were incubated in 2-fold serially diluted pre-loaded drug plates for 48 hours to determine stage-specific TFQ inhibitory effects and TFQ-ACT partner drug (drug X) interactions.
Starting drug concentrations for gametocytes ranged from 2mM to 10mM depending on the drug.
Following incubation, parasite samples per well were diluted in pre-warmed 1X Hank’s balanced salt solution (pH 7.4) containing 10mM D-Glucose, 10mM HEPES, 270mM KCl, 270mM NaCl,
1.5mM Na2HPO4 (Cold Spring Harbor Protocol 2008; doi: 10.1101/pdb.rec11561) to minimize auto-fluorescence of MCM to 0.4% hematocrit. Resulting green fluorescence from live parasites that survived antimalarial drug exposure were detected by flow cytometry; 25,000 events per sample were collected on a Guava EasyCyte HT flow cytometer (EMD Millipore Corp., Billerica,
MA). Fluorescence intensity (FI) calculated by FI= normalized events x mean green fluorescence using Flow Jo version 10 software plotted against drug concentration generated drug dose-effect curves from which IC50s for individual and TFQ-drug X combinations were calculated using
Graph Pad Prism 5 software .
Drug-drug combination assays
For asexual parasites, TFQ was combined with ACT partner drugs (drug X: LMF, PPQ,
MFQ, AMQ, NQ, PND) and CQ at fixed ratios reminiscent of both peak plasma concentrations in humans (Cmax) and in vitro IC50s (Tables 2 and 3). IC50s of individual drugs, and drugs in combination with TFQ against 5 laboratory adapted stains namely 3D7, 7G8, DD2, IPC5202 and
HB3 were determined. For sexual stage gametocytes, TFQ was combined in fixed ratios of 3:1,
31 1:1 and 1:3 in hundreds of µM concentrations as described elsewhere [16]. Three biological replicates, each in duplicate at a consistent 0.04% gametocytemia and 0.5% asexual parasitemia to minimize inoculum effects were performed [30]. Apparent IC50s of combinations were used to determine fractional inhibitory concentration indices (FICindex) using the formula (FICTFQ=
Apparent IC50 of drug X in combination with TFQ/IC50 of TFQ alone) + (FICX= Apparent IC50 of drug X in combination with TFQ/IC50 of drug X alone) where drug X represents the ACT-partner drugs under investigation or CQ. An average of FIC indices per fixed ratio represents an additive drug-drug interaction if = 1, a synergistic interaction if <1 and an antagonistic interaction if >1 corresponding to a straight diagonal line, concave curve (left of the diagonal) or convex curve
(right of the diagonal) isobologram of FICTFQ against FICx respectively [13].
Statistical analysis
One-way analysis of variance (ANOVA) and Tukey HSD tests were used to determine the differences between IC50s in different gametocyte stages as well as in IC50s for the different strains to the eight drugs under investigation using SAS University Edition Software (Cary, NC).
A Shapiro-Wilk test was used for testing the extent of data normality and a Levene’s test for homogeneity of weight variance used to determine equality of variances. A p-value of <0.05 was considered significant. A Tukey HSD test was used for multiple comparisons. Least square mean
FICindex was computed along with 95% confidence intervals to determine the extent of drug interaction deviation from additivity. A one sample t-test was performed to determine whether
FICs were equal to or unequal to 1 in either direction to determine synergism (significantly less than one) and antagonism (significantly greater than one).
32 Table 2-3 Pharmacokinetics of drugs used in this study. ACT-partner drugs, as well as CQ and
TFQ and their corresponding elimination half-lives (T1/2), peak plasma concentrations following a single dose (Cmax) and time to peak (Tmax). Amodiaquine’s active metabolite, monodesethyl amodiaquine (mdA) in vivo has a longer elimination half-life compared to its parent drug, AMQ. Data sources include [1] and the individual cited papers in the table. Piperaquine pharmacokinetics vary between starved and fed state, the value cited here was from a fed state.
Artemisinin and derivatives have Tmax values <1.8hr therefore are not used in this experiment
[17, 18]. Cmax micromolar (µM) values are computed from ng/ml or µg/l data reported in cited sources for easier translation to in vitro drug inhibitory concentrations. *TFQ and CQ are used in this study but are not ACT partner drugs.
Partner Drug Elimination T1/2 Cmax (µM) Tmax(h) References mdA/ Amodiaquine (AMQ) 3hr/9-18 days 0.06 2.0 [31]
Naphthoquine (NQ) 12 days 0.02-0.04 3.5 [32, 33]
Lumefantrine (LMF) 4-5 days 13-18 6.0 [34, 35]
Mefloquine (MFQ) 14-21 days 4-7 45-52 [36, 37]
Pyronaridine (PND) 10 days 0.08-0.54 0.5-3.2 [38]
Piperaquine (PPQ) 35 days 0.2 4 [39]
Tafenoquine (TFQ)* (300mg) 14days 0.32-0.7 15 [4, 19]
Chloroquine (CQ)* (600mg) 9 days 0.7-2 6.6 [40–42]
33 Table 2-4 Ratios used for asexual TFQ- ACT partner drug interactions. ACT-partner drugs were combined, in 2-fold decreasing sequence ranging from 4 - 0.05 µM, to an increasing sequence of
TFQ ranging from 0.5 – 4 µM in fixed micromolar concentrations taking into account the Cmax values (table 2-3) but maintaining concentrations encompassing the in vitro IC50 to asexual parasites. 5-fold less of LMF Cmax was used so as not to leave out data points on the exponential and plateau part of the dose-effect curve. Ratio of TFQ (µM): 0.5 : 1: 2: 4: ACT partner ACT-Partner (µM) Amodiaquine (AMQ) 0.4 0.2 0.1 0.05 Naphthoquine (NQ) 0.4 0.2 0.1 0.05 Lumefantrine (LMF) 3.5 1.75 0.88 0.44 Mefloquine (MFQ) 4 2 1 0.5 Pyronaridine (PND) 0.64 0.32 0.16 0.08 Piperaquine (PPQ) 0.8 0.4 0.2 0.1 Chloroquine (CQ) 2.4 1.2 0.6 0.3
2.3 Results
ACT-partner drugs differentially inhibit asexual and sexual parasites
Prior to drug interaction studies, IC50s of each of the drugs under investigation were tested against 5 laboratory adapted asexual parasite strains that are either chloroquine sensitive
(CQS: HB3, 3D7), chloroquine resistant (CQR: 7G8, DD2 ) or artemisinin resistant (ART-R:
IPC5202) to determine whether genetic background significantly affects drug susceptibility. Drug susceptibilities were similar across all five strains for most drugs except for chloroquine as expected of the previously known resistant strains, as well as for IPC5202, the ART-R strain from
Cambodia. The 7G8 and IPC5202 strains also exhibited significantly lower and higher IC50
34 values to MFQ (ANOVA p<0.0001) (Table 2-5) respectively. TFQ drug susceptibilities across strains were not significant.
Drug susceptibilities for sexual stage transgenic parasite strain 3D7αtubIIGFP gametocytes were determined using flow cytometry methods to each of the eight antimalarial drugs under investigation. Additionally, TFQ inhibition of stages II-V gametocytes was tested to determine if gametocyte developmental stage affects TFQ efficacy. There was no significant difference
(ANOVA p>0.05) between the IC50s of TFQ in each of these stages (Table 2-6). As expected, median inhibitory drug concentrations were in the micromolar range for gametocytes, compared to asexual parasites that were in the nanomolar range for all drugs except TFQ (Table 2-5). LMF and PPQ had very low gametocyte inhibition compared to TFQ and AMQ. The rest of the drugs had moderate gametocytocidal activity in the micromolar (µM) range.
35 Table 2-5 Drug susceptibilities of asexual parasites (Mean ±SEM). Lab adapted strains were tested against ACT- partner drugs, as well as chloroquine (CQ), and tafenoquine (TFQ) using the SYBR Green I method. Parts of the world where the strains originate are shown in parentheses. CQS, CQR and ART-R stand for chloroquine sensitive, chloroquine resistant and the artemisinin metabolite, dihydroartemisinin resistant respectively. * indicates significantly different drug susceptibilities to a drug (ANOVA p<0.001). Drug HB3 (Honduras 3D7 (Africa 7G8 (Brazil DD2 (Indo- IPC5202 IC50s(nM)/ CQS) CQS) CQR) China CQR) (Cambodia STRAIN ART-R) AMQ 9.9±1.8 4.8±0.7 19.0±3.8 9.8±1.0 10.3±0.4
NQ 12.1±2.0 4.1±0.9 3.9±0.1 10.9±1.3 5.9±2.3
LMF 8.9±1.9 11.6±0.7 6.9±1.0 11.9±1.2 38.7±5.0
MFQ 16.5±1.6 13.0±0.4 1.6±0.6* 11.6±1.3 28.8±2.8*
PND 3.3±0.5 3.8±0.8 4.8±0.9 4.9±0.6 2.7±0.1
PPQ 17.8±1.8 23.6±0.6 31.6±2.6 15.3±1.5 19.1±0.7
CQ 18.5±1.5 24.5±1.4 318.0±14.4* 308.7±24.5* 416.3±27.7*
TFQ 1875.2±418.9 2132.7±369.3 2380.5±472.3 1129.8±201.0 2072.3±88.2
Table 2-6 Median TFQ inhibitory concentrations by gametocyte stage. Gametocyte developmental days post-induction corresponding to stage identification are as follows; day 4 (stage II), 6 (stage III), 8 or 9 (stage IV) and 11 or 12(stage V) as specified [43, 44]. Gametocyte Stage II III IV V
TFQ IC50 (µM ± SEM) 8.8±2.4 8.1±0.5 9.8±1.4 11.1±1.7
36 Table 2-7 Drug susceptibilities of 3D7αtubIIGFP gametocytes. Median inhibitory drug concentrations of stage IV 3D7αtubIIGFP gametocytes were obtained by flow cytometry followed by dose-effect curve IC50 determination.
αtubIIGFP Drug IC50s (µM) ±SEM of 3D7 gametocytes AMQ 5.9±1.26 NQ 142.5±15.75 LMF 723.6±79.2 MFQ 16.5±11.65 PND 25.1±7.9 PPQ 197.7±12.6 CQ 27.6±0.3 TFQ 9.8±1.4
TFQ synergizes ACT partner drugs in asexual parasites
In order to determine the drug-drug combinations between TFQ and ACT- partner drugs in asexual parasites, fractional inhibitory concentrations (FICs) were used to construct isobolograms and to elucidate whether the combinations are antagonistic (Mean sum of FIC >1), synergistic (Mean sum of FIC <1) or additive (Mean sum of FICs =1) suggesting decreased, enhanced or no effect drug interactions respectively. TFQ synergized the inhibition of ACT- partners in most strains, except in the 3D7 LMF- TFQ and DD2 NQ-TFQ at ratios of high drug X
(LMF or NQ): low TFQ where it showed an antagonistic relationship but a synergistic relationship to all other fixed ratios (Table 2-8, Appendix B).
37 ACT-partner + TFQ combinations differentially affects sexual stage parasites
In order to determine any enhancement or reduction in drug potency of ACT- partner drugs in combination with TFQ, TFQ and ACT- partner drugs were combined in fixed ratios of
1:3, 1:1 and 3:1. FICs and resultant isobolograms were used to determine the interaction effects.
Only piperaquine (PPQ) and pyronaridine (PND) appeared to interact with TFQ synergistically
(Table 2-8, Appendix B).
Table 2-8 Summary of TFQ-ACT-partner drug interactions. Asexual and sexual parasites are shown. Chloroquine or Artemisinin sensitive and resistant parasites, as well as 3D7αtubIIGFP gametocyte drug-drug interactions were investigated by combining TFQ and each drug in the ratios shown in Table 2-4 for asexual parasites and 1: 1, 1:3 and 3:1 in gametocytes. The sum of fractional inhibitory concentrations of each of the 4 ratios per drug were used to construct isobolograms (Appendix B) and the mean Sum of FICs used to interpret synergistic (Syng <1), antagonistic (Antg >1) and additive (Addt =1) interactions. Interactions that deviate from synergism in asexual and antagonism in sexual parasites are indicated in bold. * indicates that at least one ratio showed a different interaction from the overall result. DRUG + TFQ HB3 3D7 7G8 DD2 IPC5202 3D7αtubIIGFP Amodiaquine (AMQ) Syng Syng Syng Syng Syng Antg
Naphthoquine (NQ) Syng Syng Syng Syng* Syng Antg
Lumefantrine (LMF) Syng Syng* Syng Syng Syng Antg
Mefloquine (MFQ) Syng Syng Syng Syng Syng Antg
Pyronaridine (PND) Syng Syng Syng Syng Syng Syng
Piperaquine (PPQ) Syng Syng Syng Syng Syng Syng
Chloroquine (CQ) Syng Syng Syng Syng Syng Antg
38 2.4 Discussion
Resistance of P. vivax to CQ is spreading and a shift from CQ to ACT followed by PMQ to kill liver stage parasites in areas of emerging P. vivax CQ resistance is recommended [45].
Currently, PMQ is the only drug licensed for the radical cure of P. vivax. Its derivative, TFQ, is in late stage clinical development for a similar role, given as a single 300mg dose after a 3-day CQ regimen owing to its inhibitory properties against dormant liver stage parasites [5]. Since ACTs are already the first- line treatment for P. falciparum, this study aimed to investigate TFQ-ACT interactions in areas co-endemic for P. falciparum and P. vivax since mixed infections as well as mixed diagnoses are common [46, 47]. If deployed for P. vivax, TFQ will inevitably come into contact with either CQ or ACT, therefore assessing whether its presence is beneficial for drug potency is necessary for both P. falciparum and P. vivax parasites. As this was an in vitro study,
P. vivax was not used because of the difficulty of maintaining it in continuous culture and obtaining enough of it to use in drug susceptibility assays [11].
Using CQ and ART resistant or sensitive asexual and sexual P. falciparum parasites, it was determined that TFQ had overall mostly synergistic interactions with asexual parasites when combined with ACT-partner drugs (Table 2-8). This is desirable in the field because an augmentation of ACT drug inhibition in the presence of TFQ will likely result in faster cure rates and less asexual parasites surviving to later differentiate into infectious gametocytes. This synergism could be due to TFQ and the ACT-partner drugs having different mechanisms of action thereby evading competitive inhibition of the same drug binding site. TFQ is reported to exhibit low levels of inhibition of hematin polymerization which is likely to supplement the action of the quinolines in this study by further inhibiting hemoglobin digestion in the asexual parasites, but its action in the gametocyte stages remains speculative [48, 49]. In Leishmania donovani and Trypanosoma brucei, TFQ has been reported to interfere with mitochondrial
39 function leading to apoptosis [8, 9] However, in accordance with other findings, TFQ had low drug inhibition in asexual parasites, compared to the ACT-partner drugs in this study suggesting that its effects relating to hemoglobin digestion are negligible (Table 2-5) [13, 14, 16]. The TFQ-
LMF interaction of the CQ and ART sensitive 3D7 parasite was antagonistic at high LMF concentrations, but not at the high TFQ: low LMF concentrations. It is unclear why because LMF was never used in Africa, and this particular strain has single copy number and a wild-type allele for Pfmdr1that is associated with LMF tolerance. This observation might just be a factor of the highest drug concentrations used skewing the results towards antagonism. A similar effect was observed when a high LMF and low primaquine ratio were combined [16]. The CQ-R strain,
DD2 was also antagonistic at high NQ: low NQ ratios and synergistic in all others, suggesting either a drug concentration effect as seen in 3D7 LMF-TFQ or a possible role of its high Pfmdr1 copy number and Pfcrt mutation in reduced susceptibility to the closely related NQ to CQ, but no studies have reported this link to date since NQ, although used in China in the 1970s has received very little attention to date [50].
Few studies have investigated TFQ interactions in vitro, reporting synergistic interactions with CQ and ART respectively [14, 15]. Another study reported, instead, an antagonistic relationship of TFQ to CQ, however unlike the former CQ-TFQ study, ratios closer to in vitro
IC50s, rather than Cmax were used, resulting in pharmacologically relevant concentrations for CQ but not TFQ, because in order to fulfil a 1:3 ratio with a TFQ IC50 of approximately 2µM, a starting concentration of >20µM would have to be used, compared to the 0.5-4µM concentration range used here [13]. Taken together, TFQ appeared to have a positive inhibitory effect on asexual P. falciparum when combined to ACT-partner drugs, regardless of genetic background and CQ/ART susceptibility at fixed ratios taking the Cmax into consideration. It should be noted, however, that only the long- lasting ACT-partner drugs were used here because the artemisinin
40 components will have long been eliminated before the peak TFQ concentration is reached as shown in Table 2-3.
Compared to asexual parasites, gametocyte IC50 values were in the micromolar range, consistent with other studies [16, 51, 52]. Since many antimalarial drugs are active against gametocytes up to stage III, presumably coinciding with a halt in hemoglobin digestion, the drug susceptibilities of gametocytes by developmental stage was investigated [21]. Drug inhibition assays were performed on day 4 (late stage II), day 6 (stage III), day 8 or 9 (stage IV) and day 11 or 12 (stage V). Results showed no significant change in gametocyte susceptibility to TFQ over time (Table 2-6).
With the exception of PND and PPQ, all TFQ-ACT-partner interactions were antagonistic at the stage IV gametocyte stage, unlike at the asexual stage where synergism was the norm. For gametocytes, however, fixed- ratios for gametocytes used were highly divergent from Cmax values to account for the high (µM range) of in vitro gametocyte drug inhibition; pharmacologically relevant concentrations would not have resulted in dose-effect curves necessary to calculate IC50s , this could have affected the outcome, like in asexual studies using similar ratios as discussed earlier [16]. It is hard to deduce by genotype alone why gametocyte interactions are different from the asexual parasites in this study, because only one transgenic strain was used, that is otherwise susceptible to all drugs tested at the asexual stage. Bell, in a review of antimalarial drug interactions cites some probable causes for synergism namely that 1) both drug A and B being studied might bind the same protein such that a conformational change increases the binding of drug B 2) drug A might be binding to a transporter causing increased uptake of drug B 3) drugs A and B likely form a more toxic complex structure and 4) drug B activity might be enhanced by interacting with a more active metabolized form of drug A, although explanation 4) can be ruled out since this study was not carried out in the presence of liver microsomes that could metabolize any of these drugs [48]. All that can be deduced here is
41 that the 4-aminoquinolines and the aryl amino-alcohols have antagonistic interactions with TFQ in gametocytes. Interestingly, the definitions for synergy or antagonism, however, are arbitrary and it may very well be that had the threshold mean sum of fractional inhibitory combinations been set at 2 or 4 instead of 1, mostly synergistic or additive interactions with TFQ would have been observed [48]. It is worth noting that some of the drugs like LMF that in this in vitro assay displayed very high IC50s for gametocytes have been reported to have sporontocidal activity [53].
TFQ itself has some sporontocidal activity, at least in P. berghei and P. vivax therefore a compound effect of the synergism observed at the asexual stage following the rapid killing action of ART or its derivatives not tested here, as well as the increased comparative susceptibility of certain drugs in the early vs late gametocyte stages plus the sporontocidal effect at the mosquito transmission stages should severely suppress a P .falciparum infection, even though P. vivax is the primary target of TFQ development [54–56].
Limitations to this study were the omission of P. vivax in vitro culture, since it might act differently in the presence of ACT and TFQ. Only one transgenic parasite line was available for this study; another GFP expressing parasite strain under the direction of the αtubII [PlasmoDB:
PF3D7_042230] gene promoter with a background other than 3D7 might exhibit different drug- drug interactions. There is currently no female-gametocyte specific marker, so gametocyte drug susceptibilities were tested for both male and female gametocytes, and not individually. Studies have reported that there is differential tolerance to drugs between the sexually dimorphic sexual stages with females being more tolerant [57–59]. As discussed earlier, the fixed-ratios for gametocytes were higher than Cmax, possibly skewing the results. Lastly, AMQ was used here instead of its metabolite mono desethyl- amodiaquine that is more likely to interact with TFQ in vivo. It is suggested that a ratio of the parent drug: the metabolite be employed in vitro for more realistic results [60].
42 Significant contributions of this study however, include the investigation, for the first time of TFQ-ACT-partner drug-drug interactions on parasites with different genetic background at pharmacologically relevant drug concentrations and the use of gametocytes, in addition to asexual stage parasites in the interaction studies; only one other study has included these to investigate primaquine-ACT interactions [16]. In vivo studies will need to be carried out to validate these results as many host factors come into play. For example, some drugs like CQ and
MFQ are known to increase the number of gametocytes, which might be problematic assuming antagonism is reproduced in vivo [61, 62]. Other drugs like TFQ, MFQ, LMF and PPQ have a higher bioavailability when taken with food [4, 22, 39] . With regard to mixed infections, P. falciparum and P. vivax mixed infections have been reported to show differences in severity, onset of one or the other, the number of gametocytes etc compared to single infections [47, 63].
In addition, TFQ Cmax will also be variable depending on cytochrome P450 metabolism and gender, with women displaying higher plasma concentrations than males [64, 65].
Taken together, TFQ’s mostly synergistic effect on at least the asexual stage parasites is good news because although it is being targeted for P. vivax, it has some benefit to P. falciparum infection in the case of mixed infection. Hopefully if the presence of TFQ with ACT enhances the asexual inhibitory effect, less asexual parasites will commit to sexual stage gametocytes that are infectious to mosquitoes. Although being developed for P. vivax, it is important to note that earlier studies of TFQ against Plasmodium falciparum showed chemo-prophylactic activity [6,
66]. Care will need to be taken when choosing an ACT to take prior to TFQ dosage as the least desirable ones are those that show a consensus of antagonism in similar in vitro and in vivo studies. A study testing the dihydroartemisinin-piperaquine and artemether-lumefantrime ACT interactions with TFQ has been completed elsewhere (Unpublished Clinicaltrials.gov identifier:
NCT02184637). In the field, flow cytometry methods using transgenic parasites will not be possible but the sensitive real-time quantitative nucleic acid sequence-based amplification (QT-
43 NASBA) can perhaps be used to determine the duration of gametocyte clearance with the different combinations [67] . TFQ does however cause severe hemolysis in individuals with reduced G6PD activity so in vivo studies in humanized mice and humans with both normal and reduced G6PD activity studies will also need to be undertaken [68]. In summary, the future of malaria chemotherapy lies in the identification of combination therapies so as to maximize inhibitory effects to parasites and to slow down the evolution of resistance; drug-drug interactions such as this, with varied parasite strains and drug tolerance will therefore be imperative to test the different drug candidates prior to clinical trials.
2.5 Conclusions
Although TFQ is in late stage clinical trials for P. vivax, it appears to synergize ACTs against P. falciparum in vitro; this might be beneficial in mixed infections of world regions where
P. falciparum and P. vivax are co-endemic. Here we show that drug interactions between asexual and sexual stage parasites differ, prompting future drug interaction studies to include gametocytes instead of just the asexual stage as has been the norm. The flow cytometry based method used here, is one reproducible way to do this in vitro, although different methods may have to be applied in field isolates.
Cmax values vary from drug to drug, so fixed ratios for isobologram analysis should be chosen carefully to reflect in vivo pharmacokinetics, whilst maintaining in vitro inhibition efficacy. In vivo TFQ-ACT/CQ drug interaction work in humanized mice of varying G6PD activity as well as human clinical trials are a necessary follow-up to results reported here since host factors such as gender, immunity, drug metabolism as well as diet, among others, might influence the results obtained.
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50 Chapter 3
Conclusions and future directions
3.1 Summary of findings and relevance
This thesis investigates drug-drug interactions between tafenoquine (TFQ), an 8- aminoquinoline drug in late clinical drug development for P. vivax and six long-lasting ACT- partner drugs in Plasmodium falciparum asexual and sexual stage parasites. TFQ is being developed for the radical cure of P. vivax following a 3-day course of CQ, but an ongoing shift from CQ to ACT antimalarials prompted the assessment of ACT-TFQ interactions (Llanos-
Cuentas et al., 2013). Based on results obtained showing mostly a synergistic effect, at least in the asexual stages, it appears that TFQ dosage for P. vivax malaria following either a three-day drug regimen of CQ or ACT may have potential off-target benefits in the treatment of P. falciparum for individuals with a mixed infection. Care must be taken, if TFQ is deployed in the near future for P. vivax that the ACT of choice used does not have an antagonistic effect on either P. falciparum or P. vivax gametocyte carriage, as that might hinder efforts to control transmission to mosquitoes.
In vivo studies in model organisms or in humans in malaria-endemic areas will have to be undertaken to verify drug-drug interactions since host factors such as immunity or drug metabolism might come into play. A study to investigate the drug-drug interactions of TFQ treatment following treatment with two ACTs, dihydroartemisinin- piperaquine and artemether- lumefantrine has been completed, but is yet to be published; this should yield some in vivo data on beneficial/adverse effects of TFQ when combined with ACT, rather than the still widely used
CQ for P. vivax due to CQ resistance in P. vivax not being as widespread as it is in P. falciparum
(ClinicalTrials.gov identification: NCT02184637; Green et al., 2015: ASTMH abstract 1209). In
51 addition, GlaxoSmithKline, the pharmaceutical company developing TFQ, is carrying out a clinical trial to compare PMQ and TFQ toxicity in people with and without a G6PD deficiency since both drugs cause severe hemolysis in these people, and TFQ having a longer elimination half-life than PMQ, might cause additional complications (ClinicalTrials.gov identifier:
NCT01376167). A hemolytic effect equal to or lower than that of PMQ is desirable.
To my knowledge, this is the first study in vitro that tests the drug interactions between
TFQ and ACT-partner drugs using both sexual and asexual stage parasites at ratios reflective of the pharmacokinetics of each individual drug in the human body. Most studies use arbitrary ratios of 1:1, 1:3 and 3: 1 based on in vitro IC50s, but these may not necessarily reflect drug peak concentrations in vivo, so the conclusions cannot easily be translated to the clinic (Gorka, et al.,
2013; Akoachere et al., 2005).
Lastly, it is my hope that with the rise of drug-drug interaction studies in coming years for antimalarial combination drugs, of which there are many in clinical trials, drug interaction assays will become more standardized to better reflect in vivo drug exposure, since as shown here, the experimental conditions as well as drug concentrations used can give differing results, and that more stringent definitions will be developed to define synergy and antagonism, because these vary, with some papers citing that synergy is any drug combination of <1, and others using
0.5 as a cut- off point. Antagonism has been reported at thresholds ranging from an FIC index of above 1 to above 4; suggesting that anyone can use whichever guideline they would like to avoid their drug combinations of interest being classified as antagonistic (Fivelman et al., 2004; Bell,
2005; Wells et al., 2015).
52 3.2 Perspectives on malaria control
Recently published work on the 8-aminoquinoline PMQ, used for both gametocytocidal purposes in P. falciparum and for elimination of the liver stage in P. vivax, has shown that when taken concomitantly with ACT partner drugs, the interactions are predominantly additive or synergistic, except in the case of lumefantrine (LMF) at certain concentrations (Cabrera and Cui,
2015). This is great news considering that the PMQ used in vitro is less effective compared to its metabolite, carboxyprimaquine, in vivo, suggesting that the combinations are likely even more synergistic in vivo (Ganesan et al., 2009). This study comes in 9 years after ACTs were officially recommended by the WHO as the first line treatment for malaria, and PMQ dosage being recommended in addition to either CQ or ACT antimalarials in areas of emerging ART resistance to suppress transmission of infectious gametocytes from humans to mosquito vectors and to kill dormant liver parasites in P. vivax, ovale and malariae (Wells et al., 2015; WHO 2014).
Considering how long these drugs have been co-administered, it is astonishing that only a handful of studies have studied in vitro drug-drug interactions to currently used antimalarials, moreover most have been performed with only asexual and not sexual stage parasites because if the goal is to increase efficacy of gametocyte killing, then a suitable synergistic combination of drugs is desired (Gorka et al., 2013, Bray et al., 2005). Also, with the anticipated evolution of resistance to every monotherapy drug developed, fixed-dose combination drugs like ACTs are likely to remain the preferred drug regimens as they are presumed to lower the chances of resistance developing rapidly (White, 2004). Therefore, in vitro and in vivo drug-drug combination studies, in my opinion, will likely become even more routine because they will help guide the process of choosing which drugs to co-formulate.
Malaria deaths have certainly decreased in the last decade, owing to the effectiveness of
ACT, and increased funding for vector control through insecticide treated nets, site-specific
53 elimination through mass drug administration, and more research funding on basic research of the malaria parasite and the mosquito vector (WHO, 2014). In 2013 alone, about $2.7 billion was spent worldwide on malaria control efforts. There has also been significant progress towards the development of a protective vaccine for malaria, although currently the front-runner RTS,S vaccine has only limited protective efficacy of no greater than 55% and only for a few months
(Wells et al., 2015). Antimalarial chemotherapy, therefore still plays a large role in the control of the spread of malaria.
It is known that gametocytes are the sole link between mosquito and human malaria transmission, so a focus on developing drugs that target both asexual parasites that cause malaria symptoms and sexual stage parasites that facilitate mosquito transmission is key. A few candidates in the pipeline include a previously used chemotherapeutic dye, and two new classes of drugs, the spiroindolones and imidazolopiperazines etc to mention but a few (Leong et al.,
2014; van Pelt-Koops et al., 2012; Coulibaly et al., 2015). Currently, the 8-aminoquinoline, PMQ is the only transmission suppressing drug approved for human use on the market, although bulaquine/ elubaquine, an 8-aminoquinoline that is metabolized to PMQ is being used in India for transmission blocking, as well as for the prevention of P. vivax relapses of malaria but has yet to be approved by the Stringent Regulatory Authority of the European Medicine s Agency for broader usage (White, 2013; Krudsood et al., 2006).
Currently, the problem with PMQ and TFQ is that they cause severe hemolysis in people with a G6PD deficiency. The classic test for G6PD has been the fluorescent spot test but it has a few shortcomings, namely that it requires a UV light, cannot accurately measure G6PD activity in heterozygous females because it is an X chromosome recessive condition, is dependent on temperature and requires a series of cold chain experiments. (Beutler, 2008; Baird et al., 2015;
WHO, 2015). The recently developed CareSTART (AccessBio Inc) has the potential to improve
G6PD screening because it is cheap, fast and is not dependent on temperature, and has been used
54 successfully in the field with 96% sensitivity (von Fricken et al., 2014; Adu-Gyasi et al., 2015)
The use of humanized mice will also aid in the initial investigation of effects of experimental drugs in vivo, before experiments are translated to monkeys/ humans (Rochford et al., 2013).
Another complication with transmission blocking drugs is the differential effects in male and female gametocytes, as some studies have suggested that females are more tolerant to many antimalarial drugs and are likely to persist in circulation following treatment (White et al., 2014).
Several assays have been developed to investigate sex differences to antimalarial therapy, but these are conflicting, with some drugs showing gametocytocidal activity in one study, but not in others (Delves et al., 2013; Adjalley et al., 2011; Reader et al., 2015; Ruecker et al., 2014). One difficulty is that so far there is no reported female specific marker that could aid in drug screening efforts targeting female gametocytes by flow cytometry (Schwank et al., 2010).
Taken together, malaria control is continuing to make steady progress in the right direction, but for this trend to persist there is a need for continuous funding, as well as co- operation between academics and pharmaceutical companies (Wells et al., 2015). The fact that a lot of pharmaceutical companies developing antimalarials are sharing their data publicly, and are willing to perform this research despite a lack of a foreseeable profit since their target market is in mostly low-income countries, is commendable. Of course the downstream benefits will be felt by everybody when economies of malaria-inflicted countries perform better and require less aid from the developed world, and when expatriates, exchange students, tourists and soldiers can safely travel to malaria-endemic countries. Here at The Pennsylvania State University, there is noteworthy research concerning the use of viruses, bacteria, transgenic mosquitoes and fungi for mosquito control and suppression of mosquito stage parasite growth, all of which will be valuable additions to the multi-target effort to develop safe, resistance-proof and relatively cheap methods in the fight against malaria (Heinig et al., 2015; Suzuki et al., 2015; Murdock et al., 2014).
55 3.3 References
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Leslie W. Deady, and Leann Tilley. "Primaquine synergises the activity of chloroquine against chloroquine-resistant P. falciparum." Biochemical pharmacology 70, no. 8 (2005): 1158-1166.
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60 Appendix A
A1: Gametocyte induction
A1.1 Equipment and Materials
- Heraeus Megafuge 16R Centrifuge (Thermo Sci)
- Eclipse 50i Microscope 100x (Nikon)
- Humidified 5% CO2 incubator
- Blue vented cap culture flasks (Corning)
A1.2 Reagents
- Parasite thawing solutions
Purpose: Recovery of previously cryopreserved parasites in liquid nitrogen (-196°C).
12% NaCl in distilled water
1.8% NaCl in distilled water
0.9% NaCl/ 0.2% Glucose in distilled water
Critical steps: All three solutions should be filter sterilized by passing through a 0.2µm membrane and pre-warmed at 37°C before use.
- Heparin sodium salt from porcine intestinal mucosa (Sigma)
Purpose: To inhibit merozoite invasion hence eliminating asexual stage proliferation in gametocyte culture (Miao et al., 2013).
Critical steps: Dissolve 50mg/ml in distilled water and filter sterilize using 0.2µm membrane and syringe.
61 - Percoll (Sigma)
Purpose: For high density centrifugation to separate blood, asexual stage and sexual stage parasites.
Critical steps: Make up to 90% with incomplete medium (pH 7.4) and filter sterilize, store at 4°C.
Pre- warm to 37°C before gametocyte culture centrifugation.
- Malaria Complete Medium (MCM). RPMI 1640 + Albumax II (Lipid rich bovine serum).
Purpose: Provide necessary nutrients for read blood cell culture
Recipe below makes 4 liters of medium pH 7.4.
RPMI 1640 (Gibco) 41.6g
HEPES (Fisher Sci) 23.8g
Hypoxanthine (Calbiochem) 0.2g
NaHCO3 8.4g
Gentamycin antibiotic (Gibco) 300uL of 50mg/ml
MilliQ water (Millipore) Make up to 4L
Albumax II (Gibco) : Asexual culture MCM 20g
Critical steps: RPMI 1640 and Albumax II powder are stored at 4°C. Stir for at least 4 hours after adding hypoxanthine before adding the rest of the ingredients (list is written in chronological order of ingredient addition). Filter- sterilize using 0.2µm membrane (VWR). For gametocyte cultures, add 5% O+ heat inactivated human serum to 0.25% Albumax II (gametocyte MCM).
- Incomplete medium: Same as above, without Albumax II or 5% Human serum
Purpose: For washing whole blood to remove buffy coat and for temporary cell suspensions and washes.
- Whole human Blood: O+ from male donor (Biological Specialty Corp.)
Purpose: Asexual P. falciparum malaria parasites infect human red blood cells (RBCs) and digest host cell hemoglobin for nutrients.
62 Critical steps: Wash 3 times to remove buffy coat that contains leukocytes and complement system at 3500rpm for 5 minutes. Store in 50% incomplete medium at 4°C and use within 10 days, making sure to re-wash and re-suspend every 72 hrs.
- 5% Sorbitol (J.T Baker)
Purpose: Used for mixed stage culture synchronization to enrich ring stage parasites because red blood cells containing mature parasites will be preferentially lysed (Lambros and Vanderberg,
1979)
- 10% Giemsa Azure Eosin methylene Blue pH 6.8 (Fluka)
Purpose: For differentially staining infected and uninfected red blood cells
Critical steps: Dissolve in distilled water and use within 24 hrs. For better resolution, use phosphate buffered giemsa, pH 7.4
A1.3 Procedure (Modified from Fivelman et al., 2007; Lucantoni et al., 2013)
1. Thaw out parasite strains from liquid nitrogen using 12% NaCl, 1.8% and
0.9%NaCl/0.2% Glucose solutions in succession, incubating for 5 minutes and shaking
between addition steps. Keep culture medium in small T25 blue vented cap flasks in
humidified 37°C incubator.
Note: Gametocytogenesis in my hands is better with recently thawed out parasites (no
older than 2 week-old culture).
2. Maintain culture at 2.5% hematocrit in 50ml MCM (with Albumax II) in 37ºC incubator.
3. At about 5% ring parasitemia determined by 10% giemsa staining, perform sorbitol
synchronization (5% D-Sorbitol) for 9 minutes to enrich for ring stage parasites. Split the
culture into 3 flasks and make up to 30ml total MCM (with 0.25% Albumax II and 5%
heat inactivated human serum) at 2.5% hematocrit.
63 4. Grow parasites for an additional 48 hr cycle and perform a 2nd sorbitol synchronization
for 9 minutes. This is termed day -4. Make cell culture up to 50ml at 1.5% hematocrit.
5. Day -3: Count 2.5-3% trophozoite parasites and make up to 50ml with gametocyte MCM.
6. Day -2: Count ring parasitemia in order to determine how much conditioned medium
(CM) to leave and how much to take out and add fresh media. This nutritional stress step
is essential for stress- induced gametocytogenesis. If 10% parasitemia, aspirate 20ml of
CM, leaving 30ml add 20 ml fresh gametocyte MCM to the flask, if 12%, leave 25 ml
CM and add 25ml of fresh gametocyte MCM.
7. Day -1: At about 11am, check for parasite stress, and add 5-10ml of gametocyte MCM to
boost growth and prevent excessive cell death, but still maintain stressful conditions.
8. At about 3.30pm, still on day -1, transfer all stressed culture to a large T225 culture
vented flask with freshly washed blood to a total of 150ml gametocyte MCM (including
the 50ml CM that was used to stress the parasites at 3% hematocrit.
9. Replace gametocyte MCM every day with 100ml fresh MCM making sure not to aspirate
too much CM since gametocytes become lighter as they mature and could be accidentally
aspirated.
10. On day 1, add heparin to prevent asexual proliferation in the gametocyte induced culture.
Make sure culture stays at 37°C because gametocytes are very sensitive to temperature
change.
11. Culture as in 9 for an additional 12 days, adding heparin up to day 5 of gametocyte
culture.
Note: If gametocytes are needed earlier than day 12, percoll purify them using carefully
pre-layered 75%/35%/ percoll, centrifuged at 4000rpm for 15 minutes with a 0
deceleration in 50ml conical flasks with equal volumes of percoll layers (75% layer at the
bottom, 35% in the middle, 3.5x diluted parasitized red blood cell culture at the top) and
64 parasite culture (diluted 3.5x). Collect the interface layer and re-suspend in fresh RBCs
and fresh gametocyte MCM. 11mls per layer for each 50ml conical flask for a total of
33ml/tube works well. Keep the pipet tip just above the liquid over a slightly tilted tube
when applying percoll layers. Be sure to balance the centrifuge with tubes opposite the
sample with the same volume of incomplete media. The top most floating layer will be
dead cells because they are the lightest, followed by gametocytes at the interface layer,
and then RBCs will be at the bottom layer. Check the smear via microscope for each
layer to verify that you have the right parasites you are looking for. Wash out the percoll
in pre-warmed incomplete medium three times before resuspension in culture.
A.2 Flow cytometry method to determine gametocyte drug inhibition
A2.1 Materials and Equipment
- Eppendorf centrifuge 5415D
- Integra ViaFlo 96 multi-channel pipettor
- Guava EasyCyte HT flow cytometer
Purpose: Flow cytometry separates cells by size and complexity as a diluted liquid sample flows through a capillary. In this case the cells desired are fluorescent green, and are separated from the background red blood cells by a green laser.
- Purified transgenic parasites tagged with GFP or fluorescent dye
- Non- sterile clear round bottomed 96-well plate (Falcon)
Purpose: Following parasite drug- incubation of 48 hours, surviving parasites must be counted using a flow cytometer. The sample is prepared by diluting the parasite sample in Hanks Buffer
Saline to maintain osmotic potential of the parasite and to minimize auto- fluorescence of the
65 MCM. These plates must be round-bottomed, not flat; to prevent damage to the capillary as it reads samples.
- Sterile Black 96-well plates (Costar)
Purpose: Drug dilutions are prepared in these and parasite culture added to them for a 48 hour incubation. Unlike the round bottomed plates, these must be sterile.
Critical steps: Pre-warm plates at 37°C to prevent moisture formation and to maintain a stable temperature for the parasite culture when it is transferred to the plate.
A2.2 Reagents
- 2X HBS
Purpose: To maintain osmotic pressure of cells when re-suspended for flow cytometry.
Diluting the sample in MCM is not recommended as RPMI 1640 contains phenol red that is
likely auto- fluorescent.
To make 250ml, use the following amounts.
D-Glucose 0.5g
HEPES 2.5g
KCl 0.18
NaCl 4g
Na2HPO4 0.05g
Critical steps: Dissolve up to 250ml of distilled water and make up to pH 7.4 with NaOH.
Filter-sterilize with 0.2µm membrane and store at 4°C. Make up to 1X HBS with sterile water
and pre-warm at 37°C before use.
- 100% DMSO
Purpose: To dissolve non-water soluble antimalarial drugs in preparation for drug assays.
66 Critical steps: Filter DMSO over a nylon 0.2µm membrane prior to adding it to drug powders
to remove any particulates. You do not need to re-filter the drug stocks after preparing them
because virtually no contaminants can grow in DMSO, as it is toxic to cells.
A2.3 Procedure (Modified from Wang et al., 2014)
1. Perform a guava clean on the flow cytometer in preparation for sample reading 15
minutes before preparing a sample.
Note: Make sure to have defined your gates for your cell sample that will clearly show a
separation between your desired cell population, in this case, green fluorescent parasites,
and the background red blood cells. You will use the same gates for multiple cell samples
of the same experiment for consistency.
2. On either day 8 or 9 of gametocyte induction (from Procedure A1), take out a 110µl
aliquot of culture at 2.5% hematocrit and spin it down for 1.5 minutes using the
Eppendorf Microfuge at 3.6rpm in a 1.5ml microfuge tube to pellet out the red blood
cells from the gametocyte MCM.
Note: Prior to drug treatment of 3 replicate experiments, one must maintain the same
hematocrit and number of initial gametocytes to avoid fluctuations in data due to an
inoculum effect (whereby an increase/decrease in parasite-load or hematocrit results in
differential drug inhibition). This step is used to measure the existing number of
gametocytes in the initial culture in order to be eventually diluted to a chosen constant
starting gametocytemia of 0.04% in all drug exposure my experiments.
3. In a new 1.5ml tube, add 600ul of pre-warmed 1x HBS (pH 7.4) and re-suspend 2.4µl of
the pelleted infected red blood cells to a total hematocrit of 0.4%.
67 4. Run the sample on the Guava EasyCyte flow cytometer using Guavasoft 2.7 software at
defined parameters. For 3D7αtubIIGFP parasites, use a very low flow rate (0.12µl/s) to
collect 150,000 events at a Forward scatter of 1.61, Side scatter of 2.48, Green laser gain
control of 2.18, Yellow gain at 8 and a Red gain of 22.6.
Note: These parameters worked well in separating out the red blood cells (clustering on
the left edge of my red vs green fluorescence dot plot) and the desired green fluorescent
parasites (clustering to the right of the red blood cells).
Fig 4-1. Dot plots of control and fluorescent parasites. Flow cytometer dot plots from left
to right of red blood cells, wild type non-fluorescent 3D7 gametocyte parasites and
3D7αtubIIGFP parasites showing differential distribution within defined low and high GFP
gates. 150,000 events were collected per sample at a very low flow rate of 0.12µl/s.
5. Record the percent number of gametocytes and dilute up to 0.04% gametocytemia with
freshly washed RBCs at a hematocrit of 2% to prepare for drug screening in sterile black
plates.
Note: 14 ml of parasitized culture at 2200 rpm for 5 minutes for all my drug assays for
consistency was centrifuged each time, as the packed cell volume can vary at higher or
lower speeds resulting in addition of more or less cells than desired to my dilution to
0.04%.
68 6. Prepare the desired concentration of antimalarial drug stocks in either water/ DMSO
depending on solubility; vortex to mix thoroughly and syringe- filter them through a
0.2µm cellulose acetate membrane for water, and 0.2µm nylon membrane for DMSO.
Depending on the Molecular weight of the drugs, 10 mM- 100mM stocks are prepared
and store them at -80°C to minimize degradation.
Note: DMSO will dissolve a cellulose acetate filter, so a recommended filter like nylon
should be used. Read the MSDS of the drugs before use because some are light sensitive
e.g chloroquine or dihydroartemisinin and must be covered with aluminum foil or
prepared in an amber light proof 1.5ml microfuge tube instead.
7. Make 4x working drug concentrations of the highest inhibitory concentration of choice of
your drugs under investigation. This is because the desired final liquid volume per well of
a 96-well plate is 200µl. We start off by adding 100µl of gametocyte MCM per well.
8. 100 µl of the 4x drug is then added to all wells in column A to make it up to 2x of the
desired concentration. 2- fold serial dilutions are then carried out using a ViaFlo
multichannel pipetting machine by aspirating 100µl from each well on the left and
dispensing it in an adjacent well on the right, mixing thoroughly, and taking out 100µl of
the resulting drug medium and dispensing it in the following adjacent well on the right
until the second to last well. The final well in column H is a negative control well with no
drug, just the drug solvent + MCM. Total DMSO percentage should be maintained below
0.4% for drugs that are dissolved in this solvent.
9. Gently mix the previously prepared 0.04% gametocyte culture in 2% red blood cells and
add 100µl to each well in the 96-well plate for a total hematocrit of 1%.
10. Store the completed drug dosed parasite plates at 37°C in a humidified CO2 incubator for
48 hours without agitation. Note the time because 48 hours later, the plates will be read
by flow cytometry.
69 11. After 47 hours, start preparing the sample for flow cytometry to detect surviving
parasites. 1X HBS and clear round bottomed 96-well plates for flow cytometry analysis
should be warmed to 37°C at this point. The flow cytometer should be cleaned at least
fifteen minutes prior to sample analysis.
12. At the 48- hour time point, obtain a pre-warmed clear round bottom 96-well plate from
the 37°C incubator. Add 120µl of pre-warmed 1X HBS to each well in the 96-well plate.
Mix gently and add 80µl of each sample per well for a total of 200ul cell sample for a
final hematocrit of 0.4%.
Note: Check the upper limit of cell concentration of your flow cytometer. This particular
flow cytometer clogs up if the cell concentration goes above 3500 cells/µl, so a
hematocrit of ~0.25 and 0.45% is ideal for sample analysis.
13. Read the sample using the flow cytometer making sure to collect at least 25,000 cells at a
high flow rate.
Note: One 96-well plate takes exactly 1 hour to read if 25,000 cells are collected.
Medium or low flow rates may be chosen for increased precision if one has fewer
samples, however, waiting for >2 hours/plate may not be ideal if one has more than 3
plates to sample. Make sure to save the sample files as .FCS for further analysis using
Flow Jo software.
14. Flow Jo V10 software is used to analyze the dot plots obtained by plotting the red
fluorescence versus green fluorescence and defining a gate for the desired GFP cell
population.
Note: In order to determine inhibitory drug concentrations, data are normalized by the
formula Normalized gating count= (Number of events in defined gate/Total events)*
25,000. The fluorescence intensity (FI) is then obtained by FI= Normalized gating count
* Mean green fluorescence. FI on the y axis is then plotted against the drug concentration
70 to obtain sigmoid curves that are analyzed by non-linear regression (GraphPad Prism) in order to obtain the median inhibitory drug concentrations per drug on the fluorescent gametocytes tested.
71 A2.4 References
Fivelman, Quinton L., Louisa McRobert, Sarah Sharp, Cathy J. Taylor, Maha Saeed,
Claire A. Swales, Colin J. Sutherland, and David A. Baker. "Improved synchronous production of
Plasmodium falciparum gametocytes in vitro."Molecular and biochemical parasitology 154, no. 1
(2007): 119-123.
Lambros, Chris, and Jerome P. Vanderberg. "Synchronization of Plasmodium falciparum erythrocytic stages in culture." The Journal of parasitology (1979): 418-420.
Lucantoni, Leonardo, Sandra Duffy, Sophie H. Adjalley, David A. Fidock, and Vicky M.
Avery. "Identification of MMV malaria box inhibitors of Plasmodium falciparum early-stage gametocytes using a luciferase-based high-throughput assay." Antimicrobial agents and chemotherapy 57, no. 12 (2013): 6050-6062.
Miao, Jun, Zenglei Wang, Min Liu, Daniel Parker, Xiaolian Li, Xiaoguang Chen, and
Liwang Cui. "Plasmodium falciparum: Generation of pure gametocyte culture by heparin treatment." Experimental parasitology 135, no. 3 (2013): 541-545.
Smilkstein, Martin, Nongluk Sriwilaijaroen, Jane Xu Kelly, Prapon Wilairat, and Michael
Riscoe. "Simple and inexpensive fluorescence-based technique for high-throughput antimalarial drug screening." Antimicrobial agents and chemotherapy 48, no. 5 (2004): 1803-1806.
Wang, Zenglei, Min Liu, Xiaoying Liang, Salil Siriwat, Xiaolian Li, Xiaoguang Chen,
Daniel M. Parker, Jun Miao, and Liwang Cui. "A flow cytometry-based quantitative drug sensitivity assay for all Plasmodium falciparum gametocyte stages." PloS one 9, no. 4 (2014): e93825.
72 Appendix B: Drug-Drug interaction isobolograms for each lab strain
1.2 MFQ 1 HB3 PPQ 0.8 AMQ 0.6 LMF
FIC TFQ FIC 0.4 NQ PND 0.2 CQ 0 0 0.5 1 1.5 FIC X
1.2 DD2 MFQ 1 PPQ
0.8 AMQ 0.6 LMF
FIC TFQ FIC 0.4 NQ PND 0.2 CQ 0 0 0.5 1 1.5 FIC X
1.2 IPC5202 1 MFQ PPQ
0.8 AMQ 0.6 LMF FIC TFQ FIC 0.4 NQ PND 0.2 CQ 0 0 0.5 1 1.5 FIC X
73
1.2 7G8 MFQ 1 PPQ AMQ 0.8 LMF 0.6
NQ FIC TFQ FIC 0.4 PND 0.2 CQ 0 0 0.5 1 1.5 FIC X
1.2 3D7 1 MFQ PPQ
0.8 AMQ 0.6 LMF FIC TFQ FIC 0.4 NQ PND 0.2 CQ 0 0 0.5 1 1.5 2 2.5 FIC X
3 3D7αtubIIGFP 2.5 MFQ
2 PPQ
1.5 AMQ FIC TFQ FIC 1 LMF NQ 0.5 PND 0 0 1 2 3 4 5 CQ FIC X
Fig 5-1 Isobolograms of asexual and sexual parasite strains. Mean fractional inhibitory concentrations per fixed ratio of tafenoquine are plotted against those for the ACT-partner drug
(FICTFQ vs FICX).