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The technical genesis and practice of 8-aminoquinoline therapy of latent malaria offer singular scientific, clinical, and public health insights. The 8-aminoquinolines brought revolutionary scientific discoveries, dogmatic practices, benign neglect, and, finally, enduring promise against endemic malaria. The clinical use of plasmochin—the first rationally synthesized blood schizontocide and the first gametocytocide, tissue schizontocide, and hypnozoitocide of any kind—commenced in 1926. Plasmochin became known to sometimes provoke fatal hemolytic crises. World War II delivered a newer 8-aminoquinoline, primaquine, and the discovery of glucose-6-phosphate dehydrogenase (G6PD) deficiency as the basis of its hemolytic toxicity came in 1956. Primaquine nonetheless became the sole therapeutic option against latent malaria. After 40 years of fitful development, in 2018 the U.S. Food and Drug Administration registered the 8-aminoquinoline called tafenoquine for the prevention of all malarias and the treatment of those that relapse. Tafenoquine also cannot be used in G6PD-unknown or -deficient patients. The hemolytic toxicity of the 8-aminoquinolines impedes their great potential, but this problem has not been a research priority. This review explores the complex technical dimensions of the history of 8-aminoquinolines. The therapeutic principles thus examined may be leveraged in improved practice and in understanding the bright prospect of discovery of newer drugs that cannot harm G6PD-deficient patients.

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.

Cytochrome P450 (CYP) 2D metabolism is required for the liver-stage antimalarial efficacy of the 8-aminoquinoline molecule tafenoquine in mice. This could be problematic for Plasmodium vivax radical cure, as the human CYP 2D ortholog (2D6) is highly polymorphic. Diminished CYP 2D6 enzyme activity, as in the poor-metabolizer phenotype, could compromise radical curative efficacy in humans. Despite the importance of CYP 2D metabolism for tafenoquine liver-stage efficacy, the exact role that CYP 2D metabolism plays in the metabolism and pharmacokinetics of tafenoquine and other 8-aminoquinoline molecules has not been extensively studied. In this study, a series of tafenoquine pharmacokinetic experiments were conducted in mice with different CYP 2D metabolism statuses, including wild-type (WT) (reflecting extensive metabolizers for CYP 2D6 substrates) and CYP mouse 2D knockout (KO) (reflecting poor metabolizers for CYP 2D6 substrates) mice. Plasma and liver pharmacokinetic profiles from a single 20-mg/kg of body weight dose of tafenoquine differed between the strains; however, the differences were less striking than previous results obtained for primaquine in the same model. Additionally, the presence of a 5,6- ortho -quinone tafenoquine metabolite was examined in both mouse strains. The 5,6- ortho -quinone species of tafenoquine was observed, and concentrations of the metabolite were highest in the WT extensive-metabolizer phenotype. Altogether, this study indicates that CYP 2D metabolism in mice affects tafenoquine pharmacokinetics and could have implications for human tafenoquine pharmacokinetics in polymorphic CYP 2D6 human populations.

The protozoan parasite Trypanosoma brucei causes human African trypanosomiasis, or sleeping sickness, a neglected tropical disease that requires new, safer, and more effective treatments. Repurposing oral drugs could reduce both the time and cost involved in sleeping sickness drug discovery. Tafenoquine (TFQ) is an oral antimalarial drug belonging to the 8-aminoquinoline family which is currently in clinical phase III. We show here that TFQ efficiently kills different T. brucei spp. in the submicromolar concentration range. Our results suggest that TFQ accumulates into acidic compartments and induces a necrotic process involving cell membrane disintegration and loss of cytoplasmic content, leading to parasite death. Cell lysis is preceded by a wide and multitarget drug action, affecting the lysosome, mitochondria, and acidocalcisomes and inducing a depolarization of the mitochondrial membrane potential, elevation of intracellular Ca2+, and production of reactive oxygen species. This is the first report of an 8-aminoquinoline demonstrating significant in vitro activity against T. brucei., This study was supported by grants SAF2011-28215 (J.M.P.-V.) and SAF2012-34267 (F.G.) from the Spanish Ministerio de Economía y Competitividad, by grants BIO1786 (J.M.P.-V.) and CTS-7282 (F.G.) from the Junta de Andalucia, and by FEDER funds from the EU to J.M.P.-V. and F.G.

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

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.