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Due to drug resistance, commonly used anti-Babesia drugs have limited efficacy against babesiosis and inflict severe side effects. Tafenoquine (TAF) was approved by the U.S. Food and Drug Administration in 2018 for the radical cure of Plasmodium vivax infection and for malaria prophylaxis. Here, we evaluated the efficacy of TAF for the treatment of Babesia infection and elucidated the suspected mechanisms of TAF activity against Babesia parasites. Parasitemia and survival rates of Babesia rodhaini-infected BALB/c and SCID mice were used to explore the role of the immune response in Babesia infection after TAF treatment. Parasitemia, survival rates, body weight, vital signs, complete blood count, and blood biochemistry of B. gibsoni-infected splenectomized dogs were determined to evaluate the anti-Babesia activity and side effects of TAF. Then, to understand the mechanism of TAF activity, hydrogen peroxide was used as an oxidizer for short-term B. rodhaini incubation in vitro, and the expression levels of antioxidant enzymes were confirmed using B. microti-infected mice by reverse transcription-quantitative PCR (qRT-PCR). Acute B. rodhaini and B. gibsoni infections were rapidly eliminated with TAF administration. Repeated administration of TAF or a combination therapy with other antibabesial agents is still needed to avoid a potentially fatal recurrence for immunocompromised hosts. Caution about hyperkalemia should be taken during TAF treatment for Babesia infection. TAF possesses a babesicidal effect that may be related to drug-induced oxidative stress. Considering the lower frequency of glucose-6-phosphate dehydrogenase deficiency in animals compared to that in humans, TAF use on Babesia-infected farm animals and pets is eagerly anticipated.

Tafenoquine is in development as a single-dose treatment for relapse prevention in individuals with Plasmodium vivax malaria. Tafenoquine must be coadministered with a blood schizonticide, either chloroquine or artemisinin-based combination therapy (ACT). This open-label, randomized, parallel-group study evaluated potential drug interactions between tafenoquine and two ACTs: dihydroartemisinin-piperaquine and artemether-lumefantrine. Healthy volunteers of either sex aged 18 to 65 years without glucose-6-phosphate dehydrogenase deficiency were randomized into five cohorts ( n = 24 per cohort) to receive tafenoquine on day 1 (300 mg) plus once-daily dihydroartemisinin-piperaquine on days 1, 2, and 3 (120 mg/960 mg for 36 to C max ) comparisons of tafenoquine plus ACT versus tafenoquine or ACT. All subjects receiving dihydroartemisinin-piperaquine experienced QTc prolongation (a known risk with this drug), but tafenoquine coadministration had no clinically relevant additional effect. Tafenoquine coadministration had no clinically relevant effects on dihydroartemisinin, piperaquine, artemether, or lumefantrine pharmacokinetics. Dihydroartemisinin-piperaquine coadministration increased the tafenoquine C max by 38% (90% confidence interval, 25 to 52%), the AUC from time zero to infinity (AUC 0–∞ ) by 12% (1 to 26%), and the half-life ( t 1/2 ) by 29% (19 to 40%), with no effect on the AUC from time zero to the time of the last nonzero concentration (AUC 0–last ). Artemether-lumefantrine coadministration had no effect on tafenoquine pharmacokinetics. Tafenoquine can be coadministered with dihydroartemisinin-piperaquine or artemether-lumefantrine without dose adjustment for any of these compounds. (This study has been registered at ClinicalTrials.gov under registration no. NCT02184637.)

Tafenoquine is a novel 8-aminoquinoline antimalarial drug recently approved by the U.S. Food and Drug Administration (FDA) for the radical cure of acute Plasmodium vivax malaria, which is the first new treatment in almost 60 years., Tafenoquine is a novel 8-aminoquinoline antimalarial drug recently approved by the U.S. Food and Drug Administration (FDA) for the radical cure of acute Plasmodium vivax malaria, which is the first new treatment in almost 60 years. A population pharmacokinetic (POP PK) analysis was conducted with tafenoquine exposure data obtained following oral administration from 6 clinical studies in phase 1 through phase 3 with a nonlinear mixed effects modeling approach. The impacts of patient demographics, baseline characteristics, and extrinsic factors, such as formulation, were evaluated. Model performance was assessed using techniques such as bootstrapping, visual predictive checks, and external data validation from a phase 3 study not used in model fitting and parameter estimation. Based on the analysis, the systemic pharmacokinetics of tafenoquine were adequately described using a two-compartment model. The final POP PK model included body weight (allometric scaling) on apparent oral and intercompartmental clearance (CL/F and Q/F, respectively), apparent volume of distribution for central and peripheral compartments (V2/F and V3/F, respectively), formulation on systemic bioavailability (F1) and absorption rate constant (Ka), and health status on apparent volume of distribution. The key tafenoquine population parameter estimates were 2.96 liters/h for CL/F and 915 liters for V2/F in P. vivax-infected subjects. Additionally, the analyses demonstrated no clinically relevant difference in relative bioavailability across the capsule and tablet formulations administered in these clinical studies. In conclusion, a POP PK model for tafenoquine was developed. Clinical trial simulations based on this model supported bridging the exposures across two different formulations. This POP PK model can be applied to aid and perform clinical trial simulations in other scenarios and populations, such as pediatric populations.

With the exception of primaquine, tafenoquine, and atovaquone, there are very few antimalarials that target liver stage parasites. In this study, a transgenic Plasmodium berghei parasite (1052Cl1; Pb GFP-Luc con ) that expresses luciferase was used to assess the anti-liver stage parasite activity of ICI 56,780, a 7-(2-phenoxyethoxy)-4(1 H )-quinolone (PEQ), as well as two 3-phenyl-4(1 H )-quinolones (P4Q), P4Q-146 and P4Q-158, by using bioluminescent imaging (BLI). Results showed that all of the compounds were active against liver stage parasites; however, ICI 56,780 and P4Q-158 were the most active, with low nanomolar activity in vitro and causal prophylactic activity in vivo . This potent activity makes these compounds ideal candidates for advancement as novel antimalarials.

The aim of this study was to develop a simple, field-practical, and effective in vitro method for determining the sensitivity of fresh erythrocytic Plasmodium vivax isolates to a range of antimalarials. The method used is a modification of the standard World Health Organization (WHO) microtest for determination of P. falciparum drug sensitivity. The WHO method was modified by removing leukocytes and using a growth medium supplemented with AB + serum. We successfully carried out 34 in vitro drug assays on 39 P. vivax isolates collected from the Mae Sod malaria clinic, Tak Province, Thailand. The mean percentage of parasites maturing to schizonts (six or more merozoites) in control wells was 66.5% ± 5.9% (standard deviation). This level of growth in the control wells enabled rapid microscopic determination (5 min per isolate per drug) of the MICs of chloroquine, dihydroartemisinin, WR238605 (tafenoquine), and sulfadoxine. P. vivax was relatively sensitive to chloroquine (MIC = 160 ng/ml, 50% inhibitory concentration [IC 50 ] = 49.8 ng/ml) and dihydroartemisinin (MIC = 0.5 ng/ml, IC 50 = 0.47 ng/ml). The poor response of P. vivax to both tafenoquine (MIC = 14,000 ng/ml, IC 50 = 9,739 ng/ml) and sulfadoxine (MIC = 500,000 ng/ml, IC 50 = 249,000 ng/ml) was due to the slow action of these drugs and the innate resistance of P. vivax to sulfadoxine. The in vitro assay developed in our study should be useful both for assessing the antimalarial sensitivity of P. vivax populations and for screening new antimalarials in the absence of long-term P. vivax cultures.

Initial field malaria prophylaxis trials with azithromycin revealed insufficient efficacy against falciparum malaria to develop azithromycin as a single agent. The objective of this in vitro study was to determine the best drug combination(s) to evaluate for future malaria treatment and prophylaxis field trials. In vitro, azithromycin was tested in combination with chloroquine against 10 representative Plasmodium falciparum isolates. Azithromycin was also assessed in combination with eight additional antimalarial agents against two or three multidrug-resistant P. falciparum isolates. Parasite susceptibility testing was carried out with a modification of the semiautomated microdilution technique. The incubation period was extended from the usual 48 h to 68 h. Fifty percent inhibitory concentrations (IC 50 s) were calculated for each drug alone and for drugs in fixed combinations of their respective IC 50 s (1:1, 3:1, 1:3, 4:1, 1:4, and 5:1). These data were used to calculate fractional inhibitory concentrations and isobolograms. Chloroquine-azithromycin studies revealed a range of activity from additive to synergistic interactions for the eight chloroquine-resistant isolates tested, while an additive response was seen for the two chloroquine-sensitive isolates. Quinine, tafenoquine, and primaquine were additive to synergistic with azithromycin, while dihydroartemisinin was additive with a trend toward antagonism. The remaining interactions appeared to be additive. These results suggest that a chloroquine-azithromycin combination should be evaluated for malaria prophylaxis and that a quinine-azithromycin combination should be evaluated for malaria treatment in areas of drug resistance.

The neglected tropical diseases leishmaniasis, Chagas' disease, and human African trypanosomiasis (HAT), caused by trypanosomatid parasites, have a limited number of drugs for treatment and control, all with limitations of toxicity, variable efficacy, long dosing regimens, and/or parenteral administration. Recent reviews have outlined the advances made in the chemotherapy of these diseases over the past decade for visceral leishmaniasis (VL) (1), cutaneous leishmaniasis (CL) (18), Chagas' disease (22), and human African trypanosomiasis (2). The search for new treatments for these diseases has adopted various strategies, including rational design of drugs (7, 15), screening libraries of synthetic and natural products (11), and therapeutic switching. The more rapid development of a new treatment by the latter approach has been recently demonstrated for Chagas' disease with ergosterol biosynthesis inhibitors (22) and for leishmaniasis with miltefosine and paromomycin (8, 20). The 8-aminoquinolines (Fig. ​(Fig.1)1) have a long history as antiprotozoal drugs, in particular as antimalarials. Since the 1950s, several have also been reported as being active against Leishmania and Trypanosoma parasites (13, 21). Interest in the activity of this class of compounds for these diseases has been kept in focus by the clinical trials of sitamaquine (WR6026) for VL (12, 23). Sitamaquine also has anti-Trypanosoma cruzi activity (6). Research on another 8-aminoquinoline, NPC1161, has identified an enantiomer with significant antileishmanial activity and a lower toxicity profile (17). Tafenoquine (TFQ) (WR238605), developed, like many agents of this class, by the Walter Reed Army Institute of Research (WRAIR), is now in clinical trials for the radical cure of Plasmodium vivax by GlaxoSmithKline (GSK) and the Medicines for Malaria Venture (MMV) (16). We present here the results of studies of the in vitro and in vivo activities of TFQ against Leishmania donovani and Trypanosoma cruzi. Studies on the mechanism of action of TFQ against Leishmania and activity against Trypanosoma brucei subsp. will be reported elsewhere. FIG. 1. Structures of tafenoquine, sitamaquine, and primaquine. Early tests of TFQ against the promastigotes of different Leishmania species demonstrated 50% inhibitory concentrations (IC50s) below 3 μM (data not shown). Of more clinical relevance, TFQ (GSK, United Kingdom) activity was evaluated, in vitro, against intracellular amastigotes of L. donovani MHOM/ET/67/HU3 (from East Africa), L. donovani MHOM/IN/82/DD8 (from India), and L. donovani BHU1 and BHU3 (antimony-resistant strains from India generously donated by Shyam Sundar). Infected murine peritoneal macrophages were exposed to the drug as previously described (24). The percent infection was calculated, and the IC50s were derived (Prism). Subsequently, TFQ was further evaluated in the BALB/c mouse-L. donovani model of infection (9). Eight-week-old, female mice (Charles River, United Kingdom) were infected with amastigotes harvested from a donor animal. After 7 days, the mice were treated with TFQ formulated in 10% Tween 80-ethanol (EtOH) 70:30 double-distilled water (ddH2O), at 5 mg/kg, by the oral route, for 5 consecutive days. On day 14, the mice were euthanized and liver impression smears were made at necropsy. The amastigote burden was calculated (Leishman-Donovan units [LDUs]) (4), the percent inhibition was derived, and 50% effective dose (ED50) values were calculated. TFQ hydrochloride (racemate batch R146390, positive enantiomer batch R206420, and negative enantiomer batch R206422) and sitamaquine tosylate (batch SLV3L004) were donated by GSK. Miltefosine was donated by Astra Zeneca, United Kingdom, and amphotericin B deoxycholate (Fungizone) was purchased from a commercial supplier. All in vivo experiments were carried out under license at the London School of Hygiene & Tropical Medicine (LSHTM) according to UK Home Office regulations. The efficacy of TFQ against T. cruzi (Tulahuen-LacZ strain) (5) was tested against amastigotes in vitro. Peritoneal macrophages were infected with T. cruzi harvested from feeder cell layers and exposed to TFQ. β-Galactosidase activity was measured by the addition of Nonidet P-40 (detergent) and chlorophenol red β-d-thiogalactopyranoside (CPRG; developer). Ninety-six-well assay plates were read at 570 λ, and IC50s were calculated. Benznidazole (Roche, Switzerland) was used as a positive control. Both the racemate and positive and negative enantiomers of TFQ were active against intracellular amastigotes of all of the L. donovani strains tested (see Table ​Table11 for IC50s) and compared favorably with the standard drugs tested alongside. In the BALB/c mouse model, TFQ was equally active against both antimony-sensitive and antimony-resistant strains (BHU1 and BHU3), with no difference seen between the racemate and enantiomers. At 5 mg/kg, TFQ achieved 99% inhibition against all L. donovani species, with the enantiomers performing similarly. In a subsequent dose-response experiment, the ED50 values ranged from 1.01 to 3.5 mg/kg (Table ​(Table2)2) . TABLE 1. Activity of TFQ enantiomers against L. donovani SbVa-sensitive strains by the amastigote-peritoneal exudate macrophage model TABLE 2. In vivo activities of TFQ, sitamaquine, and SbVa in L. donovani-BALB/c mouse models We have shown that TFQ, an 8-aminoquinoline in development for the treatment of malaria (21) has, like other drugs of the same class, potential as an oral antileishmanial agent. In both in vitro and rodent models of Leishmania infection, TFQ had similar potency to sitamaquine, the drug currently in clinical development for VL, and NPC111B, which is in preclinical development (21). The limitation of this class has been toxicity, which is of special concern for glucose-6-phosphate dehydrogenase (G6PD)-deficient patients. The extensive antimalarial safety data for TFQ, along with clinical data on sitamaquine for VL, could support the design of appropriate treatment regimes for VL with TFQ. TFQ might also be an oral partner of interest in combination therapies for the treatment of VL (10, 19). The activities of several series of 8-aminoquinolines against the causative pathogen of Chagas' disease have been published (13). Some have undergone preclinical development (23): for example, moxipraquine for Chagas' disease (3). Sitamaquine showed potential for prevention of T. cruzi transmission through blood transfusion, with activity against trypomastigotes at 4°C (6). We did not find TFQ to be as active in vitro against T. cruzi as other 8-aminoquinolines (Table ​(Table3),3), as others have previously reported (3, 13, 14). TABLE 3. In vitro activity of TFQ versus T. cruzi

Tafenoquine (TFQ), an 8-aminoquinoline analogue of primaquine, which is currently under clinical trial (phase IIb/III) for the treatment and prevention of malaria, may represent an alternative treatment for leishmaniasis. In this work, we have studied the mechanism of action of TFQ against Leishmania parasites. TFQ impaired the overall bioenergetic metabolism of Leishmania promastigotes, causing a rapid drop in intracellular ATP levels without affecting plasma membrane permeability. TFQ induced mitochondrial dysfunction through the inhibition of cytochrome c reductase (respiratory complex III) with a decrease in the oxygen consumption rate and depolarization of mitochondrial membrane potential. This was accompanied by ROS production, elevation of intracellular Ca2+ levels and concomitant nuclear DNA fragmentation. We conclude that TFQ targets Leishmania mitochondria, leading to an apoptosis-like death process. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

The population pharmacokinetics of tafenoquine were studied in Australian soldiers taking tafenoquine for malarial prophylaxis. The subjects (476 males and 14 females) received a loading dose of 200 mg tafenoquine base daily for 3 days, followed by a weekly dose of 200 mg tafenoquine for 6 months. Blood samples were collected from each subject after the last loading dose and then at weeks 4, 8, and 16. Plasma tafenoquine concentrations were determined by liquid chromatography-tandem mass spectrometry. Population modeling was performed with NONMEM, using a one-compartment model. Typical values of the first-order absorption rate constant ( K a ), clearance (CL/ F ), and volume of distribution ( V / F ) were 0.243 h −1 , 0.056 liters/h/kg, and 23.7 liters/kg, respectively. The intersubject variability (coefficient of variation) in CL/ F and V / F was 18% and 22%, respectively. The interoccasion variability in CL/ F was 18%, and the mean elimination half-life was 12.7 days. A positive linear association between weight and both CL/ F and V / F was found, but this had insufficient impact to warrant dosage adjustments. Model robustness was assessed by a nonparametric bootstrap (200 samples). A degenerate visual predictive check indicated that the raw data mirrored the postdose concentration-time profiles simulated ( n = 1,000) from the final model. Individual pharmacokinetic estimates for tafenoquine did not predict the prophylactic outcome with the drug for four subjects who relapsed with Plasmodium vivax malaria, as they had similar pharmacokinetics to those who were free of malaria infection. No obvious pattern existed between the plasma tafenoquine concentration and the pharmacokinetic parameter values for subjects with and without drug-associated moderate or severe adverse events. This validated population pharmacokinetic model satisfactorily describes the disposition and variability of tafenoquine used for long-term malaria prophylaxis in a large cohort of soldiers on military deployment.

Antimalarial drugs, when used as monotherapies, are rapidly losing their effectiveness. One promising new drug is the antimalarial 8-aminoquinoline tafenoquine (SB-252263 [formerly WR-238605]), a new synthetic primaquine analogue codeveloped by the U.S. Army and GlaxoSmithKline, which has been shown effective not only against the liver stages, gametocytes, and sporozoites of Plasmodium falciparum (4), but also against the blood stages of the parasite (13). Tafenoquine demonstrated significant protection against P. falciparum infection in Gabon, Ghana, and Kenya (6, 7, 12). Tafenoquine has been reported to be well tolerated, with only mild gastrointestinal effects (8). Isolates were collected in 1999 from malaria patients from Libreville (Gabon, Central Africa), Dielmo and Ndiop (Senegal, West Africa), and Djibouti (East Africa). The isotopic, microdrug susceptibility test used was described previously (10). The 50% inhibitory concentration (IC50) values for tafenoquine were in the range 0.9 to 9.7 μM in Djibouti, 0.6 to 33.1 μM in Gabon, and 0.5 to 20.7 μM in Senegal. The geometric mean IC50 was 2.68 μM in Djibouti, versus 4.62 μM in Gabon and 5.06 μM in Senegal (Table ​(Table1).1). Tafenoquine was found to possess marked blood schizonticidal activity in P. falciparum in areas with high percentages of multidrug-resistant parasite populations. There was no difference in the tafenoquine mean IC50 values between Dielmo-Ndiop and Libreville, even though the levels of reduced susceptibility for chloroquine, mefloquine, cycloguanil, and pyrimethamine were different. Conversely, tafenoquine was significantly more active in Djibouti than in Gabon or Senegal (P = 0.016). The results of these in vitro tests were comparable with those reported by other authors in culture-adaptated P. falciparum clones and strains (2, 11). TABLE 1. In vitro susceptibilities and prevalence of resistance or reduced susceptibility of wild isolates of Plasmodium falciparum from Djibouti, Gabon, and Senegal to the drugs shown Published in vitro data for the blood schizonticidal activity of primaquine in P. falciparum show a range of IC50 values between 0.3 μM and 14 μM (1, 2, 13). In this study, there was no difference in the tafenoquine mean IC50 values between the three areas (P = 0.111). Tafenoquine is more active in vitro than primaquine, wherever the area. Tafenoquine exerts a blood schizonticidal activity 4 to 100 times higher than that of primaquine in the Plasmodium berghei and Plasmodium yoelii mouse model (9). Tafenoquine had a half-life that is more than 50 times longer than that of primaquine (3, 5). The difference in kinetics results in more prolonged, high concentrations of tafenoquine in the blood. These properties permit weekly dosing for prophylaxis and short-term or single-dose therapy for radical cure. Only 3.5% of the variation of response to tafenoquine is explained by response variation to primaquine. The coefficients of determination, r2, ranging from 0.001 to 0.113, are too weak to consider that cross-resistance may exist between tafenoquine and standard antimalarial drugs. Since correlation analysis provides an insight into the mode of action and cross-susceptibilities between different drugs, these data may be seen as an indication of the relative independence of tafenoquine from the susceptibility of P. falciparum to standard antimalarial drugs. In conclusion, these data permit definition of the baseline of in vitro susceptibility to tafenoquine before its use and will allow the monitoring of its resistance or its reduced susceptibility when tafenoquine will be commonly used. Given its greater schizonticidal activity, tafenoquine is a promising candidate as a short treatment for P. falciparum and Plasmodium vivax malaria. However, the potential side effects of tafenoquine, such as the production of methemoglobin and the risk of hemolysis in glucose-6-phosphate dehydrogenase-deficient patients, have to be taken into consideration (14).

The prophylactic efficacy of WR 238605, a primaquine analog, was studied with a human Plasmodium falciparum challenge model. A single oral dose of 600 mg, administered 1 day prior to challenge, successfully protected three of four subjects. The fourth subject developed mild, oligosymptomatic malaria on day 31, with drug concentrations one-half of those in the protected individuals. WR 238605 appears to be a promising prophylactic drug for P. falciparum malaria.