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1 Introduction

The 8-aminoquinolines have a long history, being the first chemotype of synthetic antimalarials when pamaquine was used from the late 1920s [1]. In 1946, the screening of a large number of 8-aminoquinolines identified primaquine as a relatively safe and efficacious compound [2]. Today, additional 8-aminoquinolines have been synthesised as the search for safer and more efficacious compounds continues. From these efforts, tafenoquine is now in advanced clinical development and may become a new addition to the arsenal of antimalarial drugs.

The 8-aminoquinolines are effective against the exo-erythrocytic liver stages of the malaria parasite. This is central to preventing relapsing malaria as well as causal prophylaxis for malaria infections. Causal prophylaxis refers to the killing of parasites while they are in the liver, and thus prevents infection of erythrocytes and any signs of clinical disease. The efficacy of 8-aminoquinolines against liver-stage infection is especially valuable in the clearance of P. vivax and P. ovale, in which latent liver-stage forms known as hypnozoites can persist in the liver for months to years. Relapse infection occurs when the hypnozoites exit dormancy and differentiate into merozoites, which rupture from the hepatocyte to cause a blood-stage infection. In addition to being the only class of drugs with activity against hypnozoites, 8-aminoquinolines are active against gametocytes and thus interfere with malaria transmission.

2 Primaquine

2.1 Chemistry

The chemical name of primaquine is 6-methoxy-8-(4-amino-1-methylbutyl) aminoquinoline and its chemical formula is C15H12N3O, with a molecular weight of 259 (Fig. 1). Primaquine is a racemic mixture composed of d- and l-enantiomers, due to the presence of an asymmetric chiral center. It is water soluble and solutions are stable when protected from light. Primaquine tablets are given in the form of the diphosphate salt containing either 13.2 mg (= ~7.5 mg base) or 26.3 mg (= ~15 mg base).

Fig. 1
figure 1

Structure of Primaquine

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2.2 Clinical Use

There are three established indications for the use of primaquine: causal prophylaxis for all species of malaria, presumptive antirelapse therapy (terminal prophylaxis or postexposure prophylaxis) for P. vivax and P. ovale and radical cure of P. vivax and P. ovale infections. Since the use of primaquine depends on the species of parasite, an understanding of malaria endemicity is necessary for adequate prophylaxis use and highlights the importance of diagnostics for appropriate treatment. Primaquine primarily targets the exo-erythrocytic stage of the parasite’s life cycle. It is much less effective at killing the asexual blood-stage parasites of P. falciparum, as evidenced by in vivo and in vitro studies [3–5]. Asexual blood stages of P. vivax, however, are more sensitive to primaquine and administration of a daily dose of 45 mg for 14 days to human subjects demonstrated 80% efficacy in clearing parasitaemia with the primaquine-tolerant Chesson strain of P. vivax [6]. In less tolerant strains of P. vivax, such as those found in Thailand, a daily dose of 30 mg (0.5 mg/kg) of primaquine was 100% effective at clearing blood-stage parasitaemia [7, 8].

2.3 Chemoprophylaxis

Causal prophylaxis of primaquine is dependent on the dose and the timing of administration. Although primaquine is not currently labeled for use as a causal prophylactic agent, in experimental challenge studies, a single 30-mg primaquine dose given 1 day following infection with P. falciparum sporozoites prevented the development of blood-stage parasitaemia. However, lower doses or doses given before or on the day of challenge did not provide adequate protection against P. falciparum [3, 9]. Daily primaquine dosing before infection and throughout the exo-erythrocytic development prevented malaria [9, 10]. These studies demonstrate that there is a small therapeutic window for primaquine.

Discrepancy exists regarding the minimal effective dose of primaquine for causal prophylaxis against Plasmodium spp. [11, 12]. Several studies support that the most effective dose for primaquine prophylaxis is 30 mg daily. A daily dose of 30 mg primaquine was administered for one year to non-immune Indonesian adults and was found to be 90% and 94% efficacious against P. vivax and P. falciparum, respectively [13, 14]. Efficacy dropped when an alternate-day dosing scheme of 30 mg primaquine was administered [15]. Similar results were observed in a prophylaxis study of Colombian soldiers, with protective efficacies of 94% (P. falciparum) and 85% (P. vivax) following 30-mg primaquine daily for 16 weeks [16].

2.4 Presumptive Antirelapse Therapy and Radical Cure

The prevention of P. vivax and P. ovale relapse infection is unique to primaquine. Primaquine is FDA approved and licensed for presumptive antirelapse therapy and radical cure of P. vivax and P. ovale. The licensure approval was based on data gathered from US soldiers returning from the Korean War in the 1950s. To prevent P. vivax relapse from returning soldiers, a daily dose of 15 mg primaquine for 14 days was used. This dose was used for two reasons; the Korean P. vivax strain infecting US military personnel was efficiently cleared with this dosing regimen and haemolytic anaemia in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency, a condition more prevalent in African Americans, was less likely to cause a life-threatening condition with this dose [17]. This approved dosing scheme persisted in the face of clinical data suggesting that P. vivax strains from Southeast Asia and Oceania were tolerant of a 15-mg dose and therefore higher doses were required [18]. Subsequently, reports from nearly all P. vivax endemic areas indicate that the standard daily 15 mg primaquine dose for 14 days has failed to prevent relapses [11, 12].

It has been suggested that the total dose of primaquine may be more important than the particular regimen used as presumptive antirelapse or radical cure therapy [19–21]. For example, a total dose of 420 mg was equally effective if given at 30 mg/day for 14 days or 60 mg/day for 7 days [22]. This observation was supported by a recent evaluation of numerous P. vivax antirelapse therapy trials in which total primaquine dose was assessed [23]. Collectively, it was demonstrated that treatment success positively correlated with increasing primaquine dose as a function of body weight. This observation supports recent recommendations of a primaquine therapeutic dose of 0.5 mg/kg daily for 14 days [12]. It was further recognised that additional factors such as geographical-specific relapse rates and tolerance to primaquine should be considered when advocating the most efficacious antirelapse therapy. It should be noted that although higher doses of primaquine have been advocated, US FDA approval is only for the 15 mg/day for 14 days regimen [11].

Poor compliance with the 14-day standard primaquine therapy and treatment studies suggesting total dose is more important than dosing schedule have led to the investigation of regimens using higher doses of primaquine over a shorter time period for radical cure of P. vivax. Studies conducted in Vietnam found that 96% of the subjects treated with 200 mg artesunate twice daily for 2 days followed by 22.5 mg primaquine twice daily for 7 days cleared blood and liver stage infection based on a 28-day follow-up period [24]. A study conducted in Thailand investigated the administration of either a 30-mg primaquine daily dose for 7 days versus a 60-mg primaquine daily dose for 7 days for the treatment of P. vivax. Both dosing regimens were well tolerated and 28-day relapse rates were 11% and 4%, respectively [25]. Similarly, a study testing the antirelapse efficacy of artesunate followed by various doses of primaquine found that 30 mg twice daily for 7 days was as effective as the standard 14 day regimen of 15 mg daily [26]. The short half-life of artesunate suggests that the higher dose of primaquine was the predominant factor for the relative efficacy. Collectively these results suggest that a 1-week course of primaquine could be effective for the treatment of P. vivax.

2.5 Drug Combinations

Several reasons exist to investigate partnering drugs with primaquine: to increase efficacy, shorten the 14-day regime to a dosing schedule that will improve compliance and circumvent the rise of chloroquine-resistant P. vivax. For antirelapse or radical cure therapy, primaquine is usually partnered with another antimalarial drug such as chloroquine (25 mg/kg over 3 days). Since primaquine lacks substantial activity against the asexual erythrocytic stages of P. falciparum and acts slowly against blood stages of P. vivax, a blood schizontocide drug should be administered with primaquine [11]. Early studies suggest that the addition of chloroquine or quinine potentiated the activity of primaquine [27]. A recent report demonstrates that synergy between primaquine and chloroquine may be attributed to the ability of primaquine to increase the accumulation of chloroquine within the parasite [28].

Evaluation of nine different trials that compared a 14-day primaquine plus chloroquine with a 5-day primaquine plus chloroquine regimen concluded that the 14-day primaquine regimen was superior to chloroquine alone or 5-day primaquine plus chloroquine [29]. This evaluation, however, did not take into account the dose of either chloroquine or primaquine. As an alternative to chloroquine plus primaquine for the treatment of vivax malaria, artesunate plus primaquine combinations have been shown to produce markedly shorter parasite and fever clearance times [24, 30]. Although artesunate has no radical curative activity, the rapid action of artemisinins on the blood stages of P. vivax is highly beneficial to the patient, in that infection and malaria symptoms are aborted at a much faster rate than with chloroquine. Furthermore, with increasing reports of chloroquine-resistant P. vivax malaria in Oceania, Southeast Asia, the Indian subcontinent and the Americas [14], artesunate may be considered a potential replacement for chloroquine for aborting an acute attack of vivax malaria.

2.6 Transmission Blocking

In a few malaria-endemic areas, the addition of a single dose of 45 mg primaquine to the treatment regimen had been advocated to reduce gametocyte burden and thus interfere with the transmission cycle of the malaria parasite. Early studies demonstrated that primaquine is a potent gametocytocidal and sporontocidal agent [31]. Several clinical studies demonstrated a primaquine-dependent reduction in gametocyte clearance times, when administered as a single 0.5-mg/kg dose to artesunate or quinine [32], a single 45-mg dose to chloroquine–sulfadoxine–pyrimethamine [33] and a single 0.75-mg/kg dose to artesunate–sulfadoxine–pyrimethamine [34] as compared with treatment groups receiving the various drug combinations without primaquine.

One study did not observe any significant advantage in adding a single 0.75-mg/kg primaquine dose to artesunate–sulfadoxine–pyrimethamine in reducing the gametocyte burden [35]. Discrepancies may be attributed to the methods and the accuracy of detecting submicroscopic levels of gametocytes, as artesunate possesses gametocytocidal activity. The absence of an additive effect of primaquine is consistent with the suggestion that the most effective way to prevent gametocytaemia is to clear asexual blood forms [36]. Since artesunate does not kill mature gametocytes [37], unlike primaquine, and treatment with particular antimalarials induces gametocytogenesis [38], additional transmission-blocking studies are required to address the benefit of adding primaquine to treatment regimens. Future studies are essential since ongoing efforts are aimed at eliminating malaria [39].

2.7 Mechanism of Action

The mechanism of action by which primaquine exerts its antimalarial activity is largely unknown but the mitochondria may be the biological target of primaquine. Specifically, primaquine accumulates within the mitochondria, resulting in swelling and structural changes within the inner membranes [40–44], thus destroying mitochondrial function [45–47]. Primaquine is quickly metabolised to several reactive intermediates that are responsible for toxicity to erythrocytes (discussed below) and also apparently for antimalarial activity [48, 49]. Several of the active metabolites are structurally similar to naphthoquinones [50]. The antimalarial activity of naphthoquinones, such as atovaquone, is due to inhibition of mitochondrial function [51, 52]. Atovaquone has been shown to collapse the mitochondrial electron membrane potential, resulting in disruption of pyrimidine biosynthesis [53, 54]. Since asexual blood stage parasites rely on glycolysis for their energy source rather than oxidative phosphorylation-generated ATP, a role in pyrimidine biosynthesis would support the essentiality of the mitochondria for asexual growth and explain the blood-stage antimalarial activity of atovaquone. Primaquine, however, is not an effective blood-stage antimalarial against P. falciparum. Interestingly, swelling of host cell mitochondria was not observed and hydroxynaphthoquinone and naphthoquinone are approximately 1,000-fold more potent against the plasmodial cytochrome bc 1 complex than the mammalian complex [51]. These selectivity differences are believed to be a result of structural differences within the plasmodial bc 1 complex that increases the affinity for selected antimalarials such as atovaquone and 8-aminoquinolines [55].

The metabolism of primaquine produces reactive intermediates that ultimately results in the accumulation of free radicals, hydrogen peroxides and superoxides which may be responsible for antimalarial activity [56]. Such weak activity of primaquine in vitro may be indicative of the fact that primaquine requires metabolism for antimalarial activity [57]. A similar mode of antimalarial action has been suggested for artemisinins, which are metabolized into free radicals [58]. These free radicals may disrupt oxidation–reduction systems, inactivate specific enzymes or attach to and disrupt biological membranes [59].

Although a generalised mechanism of action has been discussed for Plasmodium, it should be acknowledged that different mechanisms may exist depending on the species of Plasmodium. For example, primaquine appears to be effective against asexual blood stages of P. berghei [43, 60] and P. vivax [6–8]; however, it is a poor inhibitor of P. falciparum asexual stages [3]. Additionally, there are discrepancies and inconsistencies with the mechanisms of action of primaquine when compared with that of artemisinins (free radicals and oxidative stress) and atovaquone (collapse mitochondrial electron membrane potential) because the in vitro and in vivo efficiencies and stage specificity of primaquine are clearly different to artemisinins and atovaquone.

2.8 Pharmacokinetics and Metabolism

An oral dose of primaquine is rapidly absorbed, with a mean bioavailability of 96% [61]. Primaquine exhibits linear and first-order kinetics over the dose range of 15–45 mg. The maximum drug concentration (C max) and the time to achieve the maximum concentration (t max) in plasma were 53 ng/mL and 2 h, respectively, following a single dose of 15 mg primaquine to healthy subjects [56]. Primaquine is extensively distributed into body tissues, with an apparent volume of distribution of 200–300 L and a systemic clearance varying between 30 and 40 L/h. The elimination half-life of primaquine is about 6 h [61, 62]. The pharmacokinetic properties of primaquine are comparable between G6PD-normal and G6PD-deficient healthy subjects [63]. Recently, sex-related differences were reported in the pharmacokinetics of primaquine, following multiple dosing of 30 mg primaquine for 14 days, with females having significantly slower clearance (0.31 L/h/kg versus 0.55 L/h/kg) and a lower apparent volume of distribution (3.42 L/kg versus 4.59 L/kg) when compared with males [64]. Further, studies are required to determine whether the increased exposure to primaquine in females leads to increased risk of toxicity compared with males, given the same maintenance dosage.

Excretion studies using 14C-labeled primaquine demonstrated that 64% of the radio label was found in the urine within 143 h after an oral dose [65]. Primaquine is rapidly and completely metabolised, as only 1–4% of the initial compound is found in the urine [61, 66]. Metabolism of primaquine results in the accumulation of numerous unstable intermediates [48]. The major plasma metabolite of primaquine is the inactive carboxyprimaquine but this is thought to be further metabolised as it is not found in urine [65]. Additional metabolites include 5-hydroxyprimaquine, 5,6-dihydroxy-8-aminoquinoline, 6-desmethylprimaquine, 5,6-dihydroxyprimaquine, and 6-methoxy-8-aminoquinoline. It is these later metabolites that are believed to generate oxygen-active species responsible for toxicity of parasite and host cells.

Several different approaches have been investigated to increase the bioavailability and the stability of primaquine. These include different mechanisms of drug administration such as transdermal delivery systems [67], galactose-coated polypropyleneimine nanoparticles as the primaquine vehicle [68] and primaquine encapsulation into liposomes and nanoparticles [69, 70]. These approaches increased stability or exposure time to drug; however, to date, these approaches have not advanced into clinical development to improve the quality of primaquine.

An additional approach used to increase stability and bioavailability is the conjugation of primaquine with amino acids [71] or with polymers of polyaspartamide [72]. The amino acid derivative demonstrated improved stability; however, these conjugates may be readily removed from primaquine via action of aminopeptidases [73]. Polyaspartamide conjugates significantly decreased parasitaemia levels and increased the survival times of mice infected with P. berghei compared with untreated or glucosamine-conjugated primaquine-treated mice. Radical cure, however, was not achieved, as all tested mice eventually died [72]. Nevertheless, these approaches support the proposal that modification or conjugation of primaquine and its analogs may be a viable alternative to increase the efficacy of primaquine. Detailed pharmacokinetic studies must be completed to assess the improved stability and bioavailability of these conjugates over the parent compound.

2.9 Safety and Tolerability

The toxicities associated with primaquine and other 8-aminoquinolines are well known [74]. Haemolytic anaemia is the most serious condition induced by primaquine in G6PD-deficient individuals [75, 76]. Erythrocytes, especially those infected with malaria parasites are prone to oxidative stress. Reduced glutathione (GSH) is important in the detoxification of free radicals. Once oxidised to glutathione disulfide (GSSG), GSH levels are reinstated by the activity of glutathione reductase and NAPDH. Because erythrocytes lack mitochondria, the pentose-phosphate pathway is the only source of NADPH. In G6PD-deficient individuals, NADPH levels are inadequate to restore GSH levels resulting in a compromised antioxidant system; thus, the erythrocytes do not have efficient protective mechanisms to handle oxidative stress. Primaquine is rapidly metabolised into hydroxylated intermediates that result in the generation of peroxides, superoxides and hydroxyated free radicals [49]. In G6PD-deficient individuals, erythrocytes are susceptible to these free radicals, which denature haemoglobin to form Heinz bodies that then react with erythrocyte membranes. This process causes premature lysis or subsequent clearing by the spleen [77, 78]. Although administration of primaquine to G6PD-deficient individuals can cause haemolytic anaemia, there are several factors such as polymorphic variation in G6PD alleles, total drug dose, and duration of the treatment that may modulate the severity of the haemolysis. Nevertheless, G6PD deficiency should be evaluated before the administration of primaquine [11]. Primaquine at the approved dosages for radical cure and presumptive antirelapse therapy is safe when administered to individuals with normal G6PD levels.

Methaemoglobinaemia is also a common toxicity associated with primaquine, which can, in some cases, require treatment with methylene blue. Methaemoglobin (MetHb) is an oxidised form of haemoglobin that cannot bind and transport oxygen to various tissues. Normal MetHb levels are less than 1% of total haemoglobin; however, in individuals deficient for G6PD or methaemoglobin reductase (an NADH-dependent enzyme that converts MetHb to haemoglobin) or under extreme oxidative stress, the levels of MetHb may increase to harmful levels, resulting in cyanosis. Primaquine increases the rate of MetHb formation [79] through oxidative stress via the free-radical metabolites of primaquine. MetHb levels as high as 11% have been reported in healthy Caucasians treated with primaquine [80]. In individuals without anemia, primaquine-induced methaemoglobinaemia, however, is a well-tolerated condition that is alleviated upon the discontinuation of primaquine dosing [11].

Gastrointestinal (GI) discomfort has been associated with primaquine in a dose-dependent manner [80–82]. Symptoms include cramping, nausea, diarrhoea and vomiting. Most of these symptoms are mild and are often avoided, if primaquine is taken with food [80].

2.10 Primaquine Resistance

Experimentally induced primaquine resistance has been developed in P. berghei and P. knowlesi [83, 84]. These controlled experiments were later supported with field reports that indicated the existence of primaquine-tolerant P. vivax [85]. Several reports suggest resistance to standard antirelapse primaquine therapy; however, factors such as noncompliance with the 14-day treatment [12] or inadequate weight-based dose could also explain the observed failures rather than inherited resistance [86]. P. vivax strains from Southeast Asia and the Southwest Pacific are more tolerant to primaquine than elsewhere [19]. These tolerant strains, however, can be effectively treated with increased doses of primaquine [11]. Although little evidence exists to support primaquine-resistant exo-erythrocytic stages including hypnozoites, several reports have described multiple relapses of P. vivax in military personnel after primaquine treatment [87, 88]. Further well-controlled studies where treatment compliance is known and primaquine is administered in a weight-based dose would help resolve the resistance issue.

3 Tafenoquine

3.1 Historical Development

Originally labeled as WR238605 or SB-252263 and now named tafenoquine, the drug is a new 8-aminoquinoline antimalarial being codeveloped by GlaxoSmithKline Pharmaceuticals and the US Army as a replacement for primaquine for radical cure of P. vivax malaria and as a potential prophylactic agent [89–91]. In an effort to develop less toxic, more active and longer acting 8-aminoquinolines, tafenoquine was first synthesised by the US Army at the Walter Reed Army Institute of Research in 1979. Although tafenoquine is a primaquine analog, it possesses different physicochemical properties, antimalarial potency and toxicological and pharmacokinetic properties compared with primaquine. In in vitro testing and in vivo preclinical animal models tafenoquine is more active than primaquine. To date, it has been evaluated in more than 2,000 human subjects in clinical studies.

On an equimolar basis, in vitro antimalarial susceptibility studies have shown tafenoquine to exhibit equivalent activity (IC50 of 0.7–1.5 μM) to primaquine against culture-adapted chloroquine-sensitive strains, but was considerably more active than primaquine against multidrug-resistant P. falciparum lines, with IC50 values ranging from 0.06 to 0.3 μM [92]. It is conceivable that tafenoquine’s enhanced blood schizontocidal potency compared with primaquine is because it exerts greater oxidative stress on multidrug-resistant parasitised erythrocytes [93]. In contrast to culture-adapted multidrug-resistant P. falciparum lines, tafenoquine was only marginally more active than primaquine against wild isolates of P. falciparum from central, west and east Africa (mean IC50 values of 4.43 μM versus 6.82 μM) [94, 95]. The enantiomers of tafenoquine have similar levels of in vitro antimalarial activity against the drug-sensitive D6 and multidrug-resistant W2, TM90-C2a and TM90-C2b strains of P. falciparum (D. K. Kyle personal communication).

In the rodent–P. berghei Peters 4-day suppressive test, tafenoquine was about 9 times more active as a blood schizontocide than primaquine against the drug-sensitive P. berghei N strain and 4–5 times as active as primaquine against highly resistant chloroquine, mefloquine or halofantrine strains of P. berghei [95]. In addition to developing new schizontocidal drugs, the capacity to interrupt malaria transmission is also of great importance. Tafenoquine possesses significant sporontocidal activity against P. berghei, with a minimum effective dose of 25 mg/kg that prevents mosquitoes from developing sporozoites [96]. Tafenoquine also has gametocytocidal activity, with a significant reduction in the number of gametocytes in the blood of P. berghei-infected mice treated with 25 mg/kg, resulting in a twofold extension of mice survival time [90].

In the rhesus monkey–P. cynomolgi model, tafenoquine was effective as a causal prophylactic agent against pre-erythrocytic tissue stages of sporozoite-induced P. cynomolgi malaria [97]. The causal prophylactic ED50 (50% effective dose) of tafenoquine was 0.125 mg/kg/day or 0.27 μM/kg/day for 3 days, which was 14 times more effective than primaquine, with an ED50 of 1 mg/kg/day or 3.86 μM/kg/day for 3 days. Tafenoquine was also a highly effective agent against liver stages of P. cynomolgi, with an ED50 of 0.172 mg/kg/day or 0.371 μM/kg/day for 7 days and was 7 times more potent than primaquine, with an ED50 of 0.712 mg/kg/day or 2.75 μM/kg/day for 7 days [98].

Although developed primarily as an antirelapse agent, tafenoquine has also been found to possess significant blood schizontocidal activity against trophozoite-induced infections in simian–malaria models. Against P. cynomolgi B and P. fragile, which are recognised as biological counterparts of P. vivax and P. falciparum infections in humans, respectively [99], tafenoquine at a dose of 3.16 mg/kg/day for 7 days led to a cure for established trophozoite induced infections in monkeys with both these parasites [100]. In contrast, primaquine was only partially curative (25% for P. cynomolgi B and 67% for P. fragile) at a dose of 10 mg/kg/day for 7 days. Tafenoquine was also effective against blood-induced vivax malaria infections of the chloroquine-resistant AMRU1 strain in the Aotus monkey–P. vivax model. Parasite clearance of the AMRU1 strain occurred at a dose of 0.3 mg/kg tafenoquine daily for 3 days and cures were achieved at 3 mg/kg daily for 3 days [101].

In addition to tafenoquine’s greater in vitro and in vivo antimalarial activities compared with primaquine in preclinical studies, it is less toxic than primaquine. In acute oral toxicity studies, tafenoquine’s LD50 (50% lethal dose) of 0.78 and 0.64 mM/kg in rats and guinea pigs, respectively, was markedly less toxic than primaquine, with corresponding LD50 values of 0.46 and 0.12 mM/kg [98]. In subchronic and chronic studies of tafenoquine (WR 238605 IND #38503), the compound was also found to be less toxic than primaquine. For example, in dog toxicology studies, 3 and 9 mg/kg/day of primaquine orally for 28 days resulted in muscle necrosis, coma and death, whereas tafenoquine up to a maximum tested dose of 16 mg/kg/day for 28 days did not produce these adverse events [102].

3.2 Chemistry

The chemical name for the racemic tafenoquine is (±)-8-[(4-amino-1-methylbutyl)amino]-2,6-dimethoxy-4-methyl-5-(3-trifluoromethylphenoxy) quinoline succinate. The structural formula for tafenoquine is shown in Fig. 2. Its chemical formula is C24H28N3O3·C4H6O4, with molecular weights of 463 for the base and 581 for the succinate salt. Tafenoquine is an off-white to pink/orange/brown solid powder with a strong phenolic odor. It is poorly soluble in water and stable at room temperature, when stored in amber bottles for at least 10 years. The formulated product of tafenoquine is a hard gelatin capsule containing 250 mg tafenoquine succinate equivalent to 200 mg of the free base. Tafenoquine capsules should be stored below 30°C and protected from light.

Fig. 2
figure 2

Structure of Tafenoquine

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3.3 Mechanism of Action and Development of Resistance

As already indicated, the exact mechanism of action of 8-aminoquinolines is not well understood. It has been proposed that the blood-stage activity of 8-aminoquinolines may be derived from an oxidative stress mechanism since it is known that primaquine stimulates the hexose monophosphate shunt, increases hydrogen peroxide and MetHb production and decreases glutathione levels in the erythrocyte [93, 103, 104]. Similar to chloroquine, the blood-stage activity of tafenoquine may be through inhibition of haematin polymerisation. In contrast to the inactive primaquine (IC50 > 2,500 μM), tafenoquine (IC50 of 16 μM) inhibited haematin polymerisation more efficiently than did chloroquine (IC50 of 80 μM) [92]. Other suggested modes of action of tafenoquine include drug-induced mitochondrial dysfunction or inhibition of receptor recycling by endosomes [105, 106].

In vitro studies have also shown a positive correlation between tafenoquine and primaquine (r 2 = 0.61) against seven P. falciparum lines, with different levels of susceptibility to chloroquine and mefloquine [92]. In contrast, no correlation exists between tafenoquine and either chloroquine or mefloquine, suggesting a lack of cross-resistance between tafenoquine and chloroquine or mefloquine.

3.4 Pharmacokinetics and Metabolism

The pharmacokinetics of tafenoquine has been investigated following both single and multiple oral administration of the drug in healthy subjects. Single-dose studies ranging from 4 to 600 mg tafenoquine have been carried out in 48 healthy males (Caucasian [n = 20], African American [n = 12] and Hispanic [n = 16]) in the fasting state [98]. The absorption half-life of tafenoquine was 1.7 h, suggesting rapid absorption of the compound. However, the t max of 13.8 h implied prolonged absorption of tafenoquine from the gut. Plasma tafenoquine concentrations declined in a mono-exponential manner and the drug was slowly cleared, with an elimination half-life of 14 days. The C max and area under the drug concentration curve of tafenoquine were linear over the doses studied. The tafenoquine concentration–time data were best described by a one-compartment model, with first-order absorption and elimination. Tafenoquine had a low oral clearance (CL/F, 5.7 L/h) and a large apparent volume of distribution (V/F, 2,558 L), suggesting extensive tissue binding. Whole blood concentrations of tafenoquine were 1.8-fold higher than corresponding plasma concentrations, reflecting an accumulation of the drug in erythrocytes, which may contribute to the greater potency of tafenoquine compared with primaquine, which does not concentrate in erythrocytes [61].

The population pharmacokinetics of tafenoquine has also been determined in healthy Thai and Australian soldiers after receiving tafenoquine for malaria prophylaxis. A one-compartment model with first-order absorption and elimination was found to best describe the population pharmacokinetics of tafenoquine. In the Thai study, 104 soldiers received a loading dose of 400 mg tafenoquine daily for 3 days followed by 400 mg tafenoquine monthly for 5 consecutive months [107]. Blood samples were randomly collected from each soldier on several occasions each month. The population estimates of the first-order absorption rate constant (K a), CL/F and V/F were 0.69/h, 3.20 L/h and 1,820 L, respectively. The absorption and elimination half-lives were 1.0 h and 16.4 days, respectively. The covariants, age and weight influenced the volume of distribution. The one subject who contracted malaria had a higher plasma clearance, but this was not considered to have sufficient impact to warrant a change in dosing.

In the Australian study, 490 soldiers received a loading dose of 200 mg tafenoquine daily for 3 days followed by a weekly dose of 200 mg tafenoquine for 6 months [108]. Blood samples were collected from each soldier after the last loading dose and then at weeks 4, 8 and 16. Typical values of K a, CL/F and V/F were 0.24/h, 4.37 L/h and 1,901 L, respectively. The V/F was similar to that reported in the Thai soldiers, but the systemic CL/F was greater (4.37 L/h versus 3.20 L/h). The derived elimination half-life of tafenoquine in the Australian soldiers of 12.7 days was slightly shorter than the 14 and 16 days reported previously in healthy Caucasians, African-Americans and Hispanic subjects [98] and in Thai soldiers [107], respectively, which may partly reflect the fact that the last samples were drawn at only up to 1 week post dose and therefore, the presumed “terminal” phase may have included some components of the distribution phase. The mean values for CL/F and V/F obtained in the fed Australian soldiers were 30–35% lower than values derived in the fasted healthy subjects participating in the single dose escalating study of tafenoquine. A possible explanation for the disparity is that a high-fat meal can increase the oral bioavailability (F) of tafenoquine by up to 40% (A. K. Miller personal communication), which when comparing the two studies would bring the respective CL/F and V/F values into closer agreement after correcting for F.

Limited investigations have been carried out on the metabolism of tafenoquine. In vitro rat liver microsomal studies have identified tafenoquine to be metabolised to aminophenolic compounds that undergo air oxidation to a mixture of quinones and quinoneimines [109]. Similar to primaquine, the metabolism of tafenoquine is difficult to study, because its structure contains several metabolically labile constituent groups, and its intermediates are unstable and possess amphoteric properties [74]. So far, no metabolites of tafenoquine have been identified in either human plasma or urine.

3.5 Safety and Tolerability

In single dose escalating pharmacokinetic studies in healthy subjects, only a few GI side effects such as heartburn, flatulence, vomiting and diarrhoea were seen in those subjects who received the higher doses of 300–600 mg tafenoquine [98]. These side effects were few and were not unexpected, based on past experiences with primaquine. Methaemoglobinaemia, haemolytic anaemia, thrombocytopenia, or changes in white blood cell counts or electrocardiograms were not observed in the subjects. Because tafenoquine is related to primaquine, it can cause methaemoglobinaemia and haemolytic anaemia in individuals with deficiency of G6PD. Thus, all individuals who receive an 8-aminoquinoline should undergo laboratory testing for confirmation of a normal G6PD status [110]. This is potentially tafenoquine’s major drawback for use worldwide as G6PD is one of the most common human genetic polymorphisms. Although malaria patients with anemia may be at greater risk, methaemoglobinaemia generally is not a serious concern when <20% of haemoglobin is in the MetHb form and only rarely will testing for methaemoglobinaemia be indicated on clinical grounds, such as the presence of bluish mucous membranes [111].

In individuals with severe G6PD deficiency, such as the Mediterranean variety, tafenoquine or primaquine should not be used. Even individuals with the low-grade deficiency (A-) variant of G6PD, which is most commonly found in Africa, can be at risk of developing haemolysis when exposed to tafenoquine. In a Kenyan field study, two women who were inadvertently given tafenoquine (400 mg daily for 3 days) experienced a haemolytic reaction when their G6PD deficiency status was incorrectly recorded during screening [112]. One woman, who was later found to be heterozygous for the (A-) G6PD variant, developed intravascular haemolysis and required a 2-unit blood transfusion. Haemolysis did not continue after the acute event, no renal compromise was seen in spite of blackwater urine, and she restored and maintained normal haematologic parameters for 6 months after the event. The other woman, who was later found to be homozygous for the (A-) G6PD variant, remained asymptomatic despite an acute 30 g/L decrease in haemoglobin, which was noticed only because of routine blood tests. She restored her haemoglobin level without intervention.

3.6 Clinical Use

3.6.1 Chemoprophylaxis against P. falciparum and P. vivax Malaria

The development and spread of multiple drug-resistant P. falciparum malaria in many parts of the world highlights the need to develop new, safe, well-tolerated and effective chemoprophylactic agents for travellers and in special risk groups such as military personnel. A long-acting drug that acts on all stages of the malaria parasite could be a significant addition to the limited armamentarium for protecting individuals against malaria infections. Tafenoquine is a long-acting antimalarial and, based on preclinical studies acts on all stages of the parasite, including the pre-erythrocytic stages providing causal prophylactic activity. Table 1 summarises the Phase II and III studies on the safety, tolerability and protective efficacy of tafenoquine in its clinical development.

Table 1 Studies on the safety, tolerability and protective efficacy of tafenoquine
Full size table

3.6.2 Presumptive Antirelapse Therapy and Radical Cure

Tafenoquine was also developed as a potential replacement of primaquine for presumptive antirelapse therapy and radical cure. Table 2 summarises the clinical development of tafenoquine for antirelapse therapy.

Table 2 Studies on the safety, tolerability and efficacy of tafenoquine for anti-relapse therapy
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3.7 Future Potential

Tafenoquine is a unique antimalarial drug that is active against all stages of Plasmodium spp. Although clinical studies of tafenoquine have shown the long-acting 8-aminoquinoline to have comparable efficacy to primaquine for radical cure and presumptive antirelapse therapy, the markedly shorter regime of tafenoquine compared with primaquine (3 days versus 14 days) is more convenient and with improved compliance one could expect the number of relapses of P. vivax malaria to decrease markedly. For the treatment of uncomplicated P. falciparum, artemisinin-based combination therapies (ACTs) are now recommended for first-line treatment worldwide. Because of tafenoquine’s long elimination half-life of 14 days, it could be considered as a partner drug with an artemisinin derivative such as artesunate. Today, however, we have very efficacious and well-tolerated ACTs for the treatment of falciparum malaria [125]. Thus, it may be more prudent to limit the use of tafenoquine to treating P. vivax and P. ovale infections, and for selected applications, including prophylaxis (short and long-term) for special risk groups such as military personnel.

Furthermore, since tafenoquine possesses both gametocytocidal and sporontocidal activity it is a promising candidate agent for transmission-blocking public health applications. Because of its long half-life, tafenoquine has enormous potential for malaria control and possibly the elimination of the disease. To test this latter concept will be difficult. Perhaps tafenoquine could be evaluated for transmission blocking in an area of low endemicity, with controlled geographical access such as an island. For malaria elimination, tafenoquine could be used in mass drug administration to eliminate residual parasites in an entire population [112] and, thus, would be an excellent drug for the eradication of malaria under the new initiative by the Bill and Melinda Gates Foundation [126].

Before these possible public health applications of tafenoquine can be implemented, a regimen that can safely be given to G6PD-deficient individuals needs to be developed. Alternatively, a field friendly, rapid and inexpensive G6PD test needs to be produced so that the G6PD status of the individual can be ascertained prior to tafenoquine administration. A clinical dose-escalating study in G6PD-deficient subjects is planned to better quantify and characterise the risk of tafenoquine use in this important risk group [117].