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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we evaluated the sporontocidal activity of tafenoquine (WR-238605) and artelinic acid against naturally circulating isolates of Plasmodium vivax. Primaquine was used as a negative control5,6 and the dihydroacridine-dione WR-2505474–6 was used as a positive control. Criteria used to assess sporontocidal activity included 1) percentage of mosquitoes with oocysts, 2) mean number of oocysts per infected mosquito, 3) average oocyst diameter, and 4) percentage of mosquitoes with sporozoites in their salivary glands.4
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All research using animals was conducted in compliance with the Animal Welfare Act and other Federal statutes and regulations relating to animals and experiments involving animals and adhered to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. This study was reviewed by the Institutional Animal Care and Use Committee of the Armed Forces Research Institute of Medical Sciences and conducted under the auspices of Protocol 99–01 entitled Evaluation of Transmission-Blocking Antimalarials for the Control of Malaria.
Mosquitoes. Anopheles dirus A has been maintained at the Armed Forces Research Institute of Medical Sciences in Bangkok, Thailand for more than 25 years. Mosquitoes were reared at 26°C and a relative humidity of 70–80% and provided with 10% sucrose and water ad libitum. Groups of 100 mosquitoes were transported from Bangkok to the field sites in pint cartons placed inside a cooler. At the field sites, 6–8-day-old mosquitoes were provided with water only for 12 hours prior to feeding.
Study site and patients.
The study was conducted in Tak Province in western Thailand. Plasmodium vivax and P. falciparum are the predominant parasite species in the region, followed by P. malariae and P. ovale.10 Individuals who participated in the study were volunteers 
15 years old and older seeking treatment at the Mae Sod and Mae Kasa malaria clinics. Thick and thin blood smears were prepared for each individual and stained with 10% Giemsa by the malaria clinic staff. Gametocyte and trophozoite densities were determined for all P. vivax-positive patients by counting the number of parasites per 500 leukocytes using oil immersion microscopy, and converting raw counts to parasites/microliter by assuming a count of 7,000 leukocytes/µL. If gametocytes were present, the patient was asked to enroll in the study. After the patients were briefed on the project and completed consent forms, lithium-heparinized Vacutainer tubes were filled with 10 mL of blood drawn by venipuncture from each patient.
Infection of mosquitoes. Whole blood (1 mL) was added to a glass feeder (5 cm diameter) closed with a Baudruche membrane. To prevent exflagellation of microgametocytes, a constant temperature of 37°C was maintained using a water jacket circulation system. The mosquitoes were allowed to feed for 30 minutes, after which the glass membrane feeder was removed from the top of the carton and all unengorged mosquitoes were removed. Engorged mosquitoes were returned to Bangkok and maintained on a 5% sugar solution at 25–27°C and a relative humidity of 70–80%. After mosquito feeding was completed, volunteers were released from the study and received antimalarial treatment from the malaria clinic staff. The complete process (entry into the malaria clinic for initial diagnosis until receiving malaria treatment) took approximately 90 minutes.
Antimalarial drugs tested in this study. All compounds examined in this study were provided by the Department of Medicinal Chemistry, Division of Experimental Therapeutics, Walter Reed Army Institute of Research (Silver Spring, MD). The four compounds used were tafenoquine (WR-238605): 8-[(4-amino-1-methyl butyl) amino]-2,6-dimethoxy-4-methyl-5-(3-tri-fluoromethylphenoxy) quinoline succinate; artelinic acid: dihydroartemisinin 4-carboxybenzyl ether; primaquine: 6-methoxy-8-(4'-amino-1'-methylbutyl amino) quinoline; and WR-250547: (R)-7-chloro-3-(2',4'-dichlorophenyl)-1,2,3,4-tetrahydro-1-[(3-(dimethylamino)propyl)imino]-9-acridinol.
Drug preparation. All drugs were diluted in a 1:3 solution of 70% ethanol:phosphate-buffered saline (diluent).4 Infected mosquitoes received the drugs by feeding on mice that had received an intraperitoneal inoculation of a given drug 90 minutes earlier. Drugs were diluted so that each dose was delivered in 0.25–0.30 mL of diluent, with control animals receiving diluent only.
Initial assessment of sporontocidal activity of antimalarials. Three days after feeding on gametocytemic malaria patients, mosquitoes were randomly separated into groups of 100 each and provided with water only. Twenty-four hours later (four days after the infectious feed), each group was allowed to feed for 30 minutes on three anesthetized mice that had received an intraperitoneal inoculation of a given drug (100 mg/kg of mouse body weight) 90 minutes earlier. Unengorged mosquitoes were removed from the cages, and mosquitoes were maintained as previously described until assessed for sporogonic development.
Determination of minimum effective doses. Minimum effective doses were determined for compounds that demonstrated sporontocidal activity at a dose of 100 mg/kg of mouse body weight. Procedures for establishing the minimum effective doses were identical to those previously described, except that serial dilutions of each drug were tested until no significant sporontocidal activity was detected at a given dose.
Determination of the stage of sporontocidal development in which drugs were active. This experiment was intended to determine the period of sporogonic development in which selected drugs could prevent transmission of P. vivax. Procedures followed those previously described; however, infected mosquitoes were refed on drug-treated mice (100 mg of drug/ kg of body weight) 4, 8, 11, or 16 days post-infection.
Assay for sporogonic development. Procedures used to determine oocyst and sporozoite production were modified from those described by Coleman and others.4–7 In each experiment, mosquitoes were sampled for oocysts on day 10 post-infection and for sporozoites on day 21 post-infection. Midguts were stained with mercurochrome and oocysts were counted by phase-contrast microscopy (200x and 400x). Oocyst development was quantified by measuring oocyst diameter using an ocular micrometer. Measurements were recorded for the five largest oocysts present in mosquitoes with at least five oocysts, and for all oocysts in mosquitoes with fewer than five oocysts. Salivary glands were removed on day 21 post-infection and examined for the presence or absence of sporozoites by phase-contrast microscopy (400x).
Statistical analysis. Three replicates were conducted for each trial and the results were pooled. Due to a limited number of P. vivax-infected mosquitoes, only two replicates were performed to determine minimum inhibitory concentrations. Chi-square analysis was used to determine if the percentage of drug-treated mosquitoes with oocysts or sporozoites was different from the percentage of control mosquitoes. Student’s t-test was used to determine if the number of oocysts per infected mosquito differed significantly, or if mean oocyst diameter differed significantly between treated and control groups.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, neither primaquine nor artelinic acid affected oocyst production. Although treatment with artelinic acid significantly reduced the size of oocysts, this drug did not significantly affect the percentage of mosquitoes with sporozoites (Table 1). Primaquine had no effect on sporozoite production. Since primaquine and artelinic acid had no significant affect on sporogonic development, their activity was not assessed in subsequent experiments.
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). As shown previously, administration of tafenoquine four days after the infectious feed inhibited sporozoite invasion of the salivary glands. In contrast, WR-250547 significantly inhibited sporozoite production when administered four or eight days post-infection (Table 2). Administration of this drug 11 days post-infection resulted in fewer mosquitoes developing salivary gland infections; however, this reduction was not significant. Administration of WR-250547 16 days post-infection had no effect on the percentage of mosquitoes with salivary gland sporozoites.
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). Doses of 25–100 mg/kg resulted in a significant reduction in the percentage of mosquitoes that developed salivary gland infections. In contrast, doses of 3.125–100 mg of WR-250547/ kg significantly affected the percentage of mosquitoes with oocysts; however, no reduction in the number of oocysts per mosquito was observed. Doses of 0.39–100 mg/kg significantly inhibited both oocyst development and sporozoite invasion of salivary glands (Table 4), but a lower dose (0.195 mg/kg) had no effect on any stage of sporogonic development.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gametocytocidal compounds interact directly with gametocytes, whereas sporontocidal compounds inhibit parasite development in infected mosquitoes.4,8 The gametocytocidal and sporontocidal modes of action can be differentiated by exposing the parasite to a particular drug during different stages of development. Determination of gametocytocidal activity is achieved by administering a particular drug with the infectious blood meal and evaluating subsequent mosquito infection rates, whereas sporontocidal activity is assessed by exposing previously infected mosquitoes to a drug. Methods of exposing previously infected mosquitoes to a drug include dilution of the drug in the sugar solution used to maintain adult mosquitoes11 or administration of the drug to an uninfected mouse that is then exposed to infected mosquitoes.4–7 The advantage of the latter method is that the drug may be metabolized prior to ingestion by the mosquito.
Tafenoquine and artelinic acid are relatively new antimalarials that are currently undergoing testing in humans and non-human primates. Tafenoquine was originally developed as a replacement for primaquine because it is less toxic and more effective than primaquine.12,13 This drug is unique among antimalarials in that it affects all stages of parasites,13,14 including exo-erythrocytic (liver)15–17 and erythrocytic asexual forms,17–20 sexual (gametocytes) stages,14,20 and sporogonic development in the mosquito.5–7 Artelinic acid is a water-soluble, semi-synthetic derivative of artemisinin (or Qinghaosu), which is the parent compound of a completely new class of antimalarials.21 The artemisinin compounds are fast-acting blood shizontocides with potent gametocytocidal activity.21–24 Although they show excellent efficacy in both severe and uncomplicated malaria, dosage regimens still need to be optimized and pharmacokinetic profiles defined. In the treatment of uncomplicated malaria, the artemisinin drugs are used in combination with a long-acting antimalarial to protect both drugs against the emergence of resistance.24,25 Artelinic acid is currently undergoing pre-clinical testing in non-human primates.
Primaquine is commonly used for the eradication of hepatic stage P. vivax,11,26 while in some areas (e.g., Thailand) it may be used as a gametocytocidal agent against P. falciparum.26,27 In addition, primaquine is currently being evaluated as a potential prophylactic drug.28–30 Although primaquine does not affect the sporogonic development of P. berghei or P. falciparum,5,6 it is not known whether it has sporontocidal activity against P. vivax.
The dihydroacridine-dione WR-250547 has significant sporontocidal activity against P. berghei,5,6 chloroquine-sensitive and multidrug-resistant P. falciparum,5 and P. vivax.4 Although WR-250547 was screened for antimalarial activity by the Walter Reed Army Institute of Research Antimalarial Screening Program, this compound is not currently under development as an antimalarial agent. In this study, we used WR-250547 as a positive control agent because it has documented sporontocidal activity against which the efficacy of tafenoquine and artelinic acid could be compared.
Transmission-blocking antimalarials may provide an elegant yet simple method of controlling malaria. Laboratory studies have clearly demonstrated that selected antimalarials can prevent the mosquito transmission of P. berghei, P. vivax, and drug- sensitive and multidrug-resistant strains of P. falciparum.4–7 In studies conducted more than 40 years ago, mosquitoes were fed on P. vivax-infected patients and then maintained on sugar solutions spiked with an antimalarial agent.11 However, use of spiked solutions does not simulate the normal route of parasite exposure to the drug because no metabolism of the drug occurs. In the model system used in this study, we expose mice to the drugs for 90 minutes prior to feeding mosquitoes on the animals, thereby allowing for absorption and metabolism of the compound by the mice. This mode of drug delivery is presumably more realistic than feeding mosquitoes on sugar pads impregnated with the drugs, although drug metabolism by mice may differ significantly from metabolism of the same drug by humans.
In contrast to most studies that have assessed sporontocidal drug activity using either rodent malaria models or gametocyte-producing P. falciparum cultures, we used naturally circulating isolates of P. vivax to evaluate drug activity. Although the heterogeneous parasite populations used in this system may result in greater variability in results, the use of naturally circulating parasite populations provides a more realistic understanding of how the drugs perform in nature. By using multiple criteria (oocyst size and numbers and sporozoite invasion of salivary glands) to assess sporontocidal activity, we get a more comprehensive view of drug activity, thereby allowing for differentiation of active and inactive compounds.
We used primaquine and the dihydroacridine-dione WR-250547 as negative and positive controls, respectively. Although Young11 reported that primaquine exhibited sporontocidal activity against P. vivax when infected mosquitoes were fed on a primaquine-spiked sugar solution, previous studies in our laboratory found that primaquine had no sporontocidal activity against drug-sensitive or multidrug-resistant P. falciparum.5 Data on the sporontocidal activity of primaquine in a P. berghei mouse model is less clear because sporontocidal activity was detected in one study6 but not in another.5 In this study, we found no evidence that primaquine (100 mg/kg of mouse body weight) had sporontocidal activity against P. vivax in An. dirus mosquitoes (Table 1
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Although WR-250547 is currently not under development as an antimalarial, several previous studies reported that WR- 250547 had significant sporontocidal activity against P. berghei, drug-sensitive and drug-resistant P. falciparum, and P. vivax.4–6 The minimum inhibitory dose (MID) of WR-250547 against P. berghei was 0.1563 mg of base drug/kg of mouse body weight.6 In this study, the MID was 0.39 mg/kg against P. vivax (Table 4); however, the mode of administration was not identical to the P. berghei study. These data clearly demonstrate that WR-250547 has sporontocidal activity.
A recent study suggested that artelinic acid was an ideal antimalarial candidate because it had higher plasma concentrations, higher red blood cell binding capacity, longer half-life, and lower toxicity than equivalent doses of other artemisinin derivatives.31 This drug was developed by the Walter Reed Army Institute of Research for treatment of uncomplicated multidrug-resistant P. falciparum malaria; however, it is also a potent gametocytocide.21 We injected mice with artelinic acid 90 minutes prior to feeding mosquitoes on the animals. Since the elimination half-life of artelinic acid in rats is 1.35 hours,31 we believe that our model should have detected any sporontocidal activity. Although we did observe a reduction in the size of oocysts treated with 100 mg of artelinic acid/kg of mouse body weight, this reduction was not significant and no other phases of sporogony were affected (Table 1). Although Tripathi and others21 demonstrated that artelinic acid was a potent gametocytocide using a P. cynomolgi/ rhesus monkey model, they detected no sporontocidal activity.
In contrast to primaquine, tafenoquine (a primaquine analog) exhibited significant sporontocidal activity against naturally circulating strains of P. vivax (Tables 1–3). This data supports our previous studies with P. berghei and P. falciparum and extends it to a new species. Tafenoquine has an elimination half-life of approximately 14 days, suggesting an increased probability that sporogonic-stage parasites might be exposed to the drug (when compared with artelinic acid). Although tafenoquine was less effective than WR-250547 on a mg/kg basis (MID = 25 mg of tafenoquine/kg versus 0.39 mg of WR-250547/kg), when molarity of the compound was considered, tafenoquine was actually active at lower doses than WR-250547 (MID = 0.004 moles of tafenoquine/L versus 0.08 moles of WR-250547/L).
Neither tafenoquine or WR-250547 completely eliminated oocysts; however, oocyst development was significantly reduced even when oocyst numbers were not affected (Tables 1, 3, and 4). Some of these oocysts eventually produced sporozoites, which succeeded in invading the salivary gland. Although the ideal drug would completely prevent sporozoite invasion of salivary glands, a delay in invasion could have a significant impact on transmission rates. The extrinsic incubation (EI) period is one of the key parameters that drives transmission of any vector-borne disease.32 Although we did not evaluate the impact of the drugs on the EI period, the incomplete reduction in the percentage of mosquitoes with sporozoites in the salivary glands may have reflected a delay in the EI period. Since even minimal increases in the EI period can have significant impacts on disease transmission, both tafenoquine and WR-250547 may be even more effective than our data would suggest.
The model system presented here and in our previous study4 provides advantages over other models used to assess sporontocidal activity; however, this model is clearly not perfect since drug metabolism by mice may be different from that of humans. The results obtained in this study need to be confirmed with further testing in humans. Since tafenoquine is currently undergoing clinical testing in humans, it should be possible to more fully elucidate its role as a sporontocidal agent.
Received October 31, 2002. Accepted for publication January 20, 2003.
Acknowledgments: We are grateful to Drs. Gabby Zollner, James Jones, and R. Scott Miller for reviewing the manuscript and to the staff of the Mae Sod and Mae Kasa malaria clinics for their support of this study. The test compounds originated from the Chemical Synthesis Program of the Department of Medicinal Chemistry, Division of Experimental Therapeutics, Walter Reed Army Institute of Research (Washington, DC).
Financial support: Funding for this project was provided by the Military Infectious Diseases Research Program of the U.S. Army Medical Research and Materiel Command, Fort Detrick (Frederick, MD).
Disclaimer: The opinions of assertions contained in this manuscript are the private ones of the authors and are not to be construed as the official or reflecting views of the Department of Defense or the Armed Forces Research Institute of Medical Sciences.
Authors’ addresses: Narong Ponsa and Pattamaporn Kittayapong, Center of Vectors and Vector-Borne Diseases and Department of Biology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. Jetsumon Sattabongkot, Nantana Eikarat, and Russell E. Coleman, United States Army Medical Component, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok 10400, Thailand.
Reprint requests: Department of Entomology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok 10400, Thailand.
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