Defining the biology component of the drug discovery strategy for malaria eradication

Review

Defining the biology component of the drug discovery strategy for malaria eradication Didier Leroy, Brice Campo, Xavier C. Ding, Jeremy N. Burrows, and Ste´phanie Cherbuin Medicines for Malaria Venture (MMV), PO Box 1826, 20 route de Pre´-Bois, 1215 Geneva 15, Switzerland

Malaria is still considered a deadly scourge in Africa, Asia, and South America despite improved vector control and curative treatments with new antimalarial combinations. The next challenge is to work towards disease eradication. To achieve this goal it is crucial to develop, validate, and integrate biological assays into test cascades that align with the key target product profiles. For anti-relapse, a parent molecule should kill hypnozoites or cause activation of Plasmodium vivax liver stages. For transmission blocking, dual equal-activity antimalarials killing both the asexual and the sexual parasite stages in human blood are favored. Finally, by assessing cross resistance and generating drug resistance in the laboratory, it is expected that new medicines with acceptable resistance profiles will be forthcoming. Disease context and need for novel antimalarials There are presently more than 200 million current cases of malaria worldwide. Most of the victims are children under the age of 5 years and expectant mothers; in 2012 the number of reported deaths was 627 000 – still an unacceptable number but one that reflects major progress in the control of malaria over the past decade [1]. Achieving this goal was made possible by improved usage of high-quality combination medicines, insecticide-treated nets (ITNs), and indoor residual spraying. It is expected that over the next decade, with sustained support from governments and funding bodies, the number of malaria cases will continue to decrease, with several countries embarking on ambitious plans for national elimination. However, despite strong control measures and a vaccine planned for launch in 2015 [2], there are currently insufficient tools to protect and cure patients and eradicate the disease. Thus new quality medicines focused on addressing the eradication agenda are required. Since the discovery in 1891 of methylene blue, the first synthetic antimalarial, multiple chemical classes of small molecules have been shown to efficiently clear blood-stage parasitemia in humans. The Achilles’ heel of this arsenal of treatments is that these molecules target the susceptible erythrocytic stage of the Plasmodium life cycle (Figure 1). Corresponding author: Leroy, D. ([email protected]). Keywords: malaria; anti-relapse drugs; transmission blocking; drug resistance. 1471-4922/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2014.07.004

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This stage, unfortunately, happens to be the point at which the parasites are most abundant and metabolically active; the combination of parasite density with high random mutation rates inevitably results in the emergence of drug-resistant phenotypes [3]. A further challenge is that these drugs often do not affect other more inert parasite stages that do not lead to symptoms. These unaffected forms remain effective for transmission from human to mosquito, and from mosquito to human. In addition, in the case of P. vivax, dormant parasites in the liver can relapse into a new symptomatic infection weeks or months after a mosquito bite. During the past decade new potential antimalarial targets have been proposed based on an increasing number of plasmodial genomes that have been successfully sequenced [4–7]. In parallel, new screening and imaging technologies have been applied to screen millions of compounds directly against the parasite in culture. These phenotypic assays have generated thousands of new active compounds against Plasmodium asexual blood stages, and some of these actives are currently in clinical development, their potential molecular targets having been identified through pull-down studies or reverse genetics [8–10]. It is interesting to note that such targets identified from mutants bear little overlap with those proposed from traditional target-validation approaches. However, despite this progress, and given the increasing effectiveness and safety of current antimalarials, simply curing malaria rapidly and efficiently with new candidate drugs is no longer sufficient. There is a crucial need for therapeutics that go beyond treating acute infections and have the potential to eradicate the disease [11]. Accordingly, four major goals have been identified: (i) efficient elimination of all human parasites that populate the liver as dormant forms (hypnozoites; see Glossary), notably those of P. vivax and Plasmodium ovale; (ii) blocking disease transmission by targeting parasite sexual stages in human blood; (iii) identifying and developing new chemical entities that overcome all known cross-resistance and miminize the risk of resistance emerging; and (iv) delivering molecules that protect vulnerable populations. To achieve these goals an in-depth understanding of the biology of each parasite stage is needed. In addition, biological assays, which constitute essential elements of the drug discovery test cascades necessary to build a strong and efficient process, need to be developed and implemented. Each in vitro assay is used as a

Review Glossary Gametocytes: non-dividing sexual forms of Plasmodium parasites that differentiate from asexual replicative forms in the blood of the host. Immature gametocyte stages I, II, and III are the precursors of the mature stages IV and V; the latter stage is transmitted to the mosquito midgut during a blood meal and then generates male and female parasite gametes. Glucose 6-phosphate dehydrogenase (G6PD deficiency): patients carrying mutations in the gene encoding for G6PD suffer from severe hemolysis when treated with the 8-aminoquinoline class of antimalarials and in particular with primaquine. The current hypothesis is that the primaquine metabolite responsible for the anti-hypnozoite activity would trigger massive lysis of the host erythrocytes in addition to the one due to the burst of parasites after the completion of every proliferative cycle (48 h for P. falciparum) in patient blood. High-throughput screening (HTS): key phase of the drug discovery process through which each single small molecule of large libraries (hundreds of thousands to millions of compounds) is tested at a given concentration (1– 10 mM) for its capacity to inhibit purified enzymes (target-based screening) or to kill cells/parasites/bacteria (phenotypic screening). These high-throughput assays require the simultaneous analysis of many compounds, and therefore microtiter plates with up to 1536 wells have been developed and are routinely filled with reagents by automated devices to test compounds. Hypnozoites: non-dividing liver forms of Plasmodium vivax and possibly Plasmodium ovale that stay quiescent (‘dormant’, from Greek hypnos) following the infection of host hepatocytes by sporozoites. These parasite forms stay small (2 mm in diameter) and have a rounded shape, in contrast to the developing liver forms that produce schizonts (>10 mm in diameter). Hypnozoites can exit dormancy anywhere between a few weeks and several months after the patient is cured of symptomatic malaria (proliferative stage in the blood). The reasons for its quiescence and activation are not known, and its biology is barely understood. Minimal inoculum for resistance (MIR): in the laboratory, incubation of antimalarials or drug candidates with various amounts of parasites (inocula) can lead to recrudescence after several days. This adaptation to growth in the presence of the drug depends on the capacity of the parasite to accumulate mutations leading to a modification of its drug targets or drug efflux pumps. The occurrence of mutated parasites correlates with the number of genomes (parasites) in the inoculum. The lower the inoculum allowing the appearance of a resistant mutant, the more the drug is assessed as inducing resistance. The MIR is the minimum necessary to produce resistance and is used to attribute a risk level to the molecule that is tested (a MIR of 105 parasites is a high risk, 107 a medium risk, and 109 a low risk). Medicines for Malaria Venture (MMV): product development partnership organization created in 1999 with the mission to discover, develop, and deliver safe, efficient, and inexpensive new antimalarials in endemic countries such as those in Africa, Asia, and South America. Currently, 70 staff members work in tight collaboration with more than 300 academic and industrial partners around the world to achieve such a mission. For several years now, and in conjunction with many members of the antimalarial community, the objective is to fulfill the eradication agenda. Oocyst: the ultimate proliferative Plasmodium form that develops in the midgut of the mosquito (Anopheles) and that contains thousands of sporozoites, the host liver infective forms. Oookinete: precursor parasite form of the oocyst that develops after the zygote is formed and that invades the mosquito midgut. The most striking features of this parasite form are that it develops an apical complex and is highly motile. The ookinete and the zygote are the only two Plasmodium forms which are diploid. Plasmodium berghei: one of the rodent parasites that is currently used to test new compounds in preclinical mouse models. Plasmodium cynomolgi: a parasite that infects monkeys (e.g., Rhesus monkeys) and that produces dormant forms in the liver of its host similarly to those produced in humans by Plasmodium vivax, its closest neighbor in the phylogenetic tree of Plasmodium spp. Plasmodium falciparum: one of the five human unicellular eukaryotic parasites (with P. vivax, P. ovale, Plasmodium malariae, and Plasmodium knowlesi). Primaquine: antimalarial of the 8-aminoquinoline chemical class that was discovered by the US army nearly 60 years ago. The drug is administered daily for 14 days to clear the liver of patients from P. vivax hypnozoites and provide a radical cure of patients by avoiding relapses. The current hypothesis is that a transient metabolite of primaquine formed in the human liver kills hypnozoites and developing liver forms of P. vivax extremely rapidly. Ring-stage assay (RSA): an assay developed by the group of D. Me´nard in Cambodia to synchronize P. falciparum cultures and study specifically the action of endoperoxides on both artemisinin-sensitive and -resistant clinical isolates. So far this assay is the only one that has led to the characterization of the artemisinin resistance phenotype in vitro. Single-exposure chemoprotection (SEC): a target product profile that has been defined to guide the discovery and development of the next-generation antimalarials that will protect vulnerable populations from infection by Plasmodium spp.

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Single-exposure radical cure and prophylaxis (SERCaP): a target product profile designed to guide the discovery and development of new combination therapies that will be administered to patients as a single dose, curing them from all malaria infections as well as providing protection against posttreatment reinfection. Standard membrane feeding assay (SMFA): a method of feeding mosquitoes through an artificial membrane (parafilm) in a device filled with human blood in the absence or presence of compounds. This assay is used to assess the transmission-blocking potential of compounds in the drug discovery and development pipeline. Sporozoites: Plasmodium forms that are released in the hemolymph and the salivary glands of the mosquito following the rupture of the oocyst membrane, and that infect the hepatocytes of the host, before evolving into replicative schizonts that ultimately liberate the erythrocyte infective forms, the merozoites. Tafenoquine: a new generation 8-aminoquinoline that will replace primaquine with a better efficacy and safety profile. Currently in Phase II clinical trial the drug is expected to be a single-dose treatment that will eliminate P. vivax hypnozoites from the liver of the host. Target candidate profile (TCP): pharmacodynamics, pharmacokinetics, and safety parameters that a drug candidate should exhibit to fulfill the radical cure or prophylaxis criteria. Target product profile (TPP): all the desired attributes of the final drug or drug combination, including pharmacodynamics, pharmacokinetic, safety and nonbiological properties, including cost, stability, dosage, etc.

filter to select molecules with the best activity profile and as a predictive tool for the next assay in the cascade, which is typically one with higher biological content, lower throughput, and higher cost. It is important to stress the need for cross-validation between assays; in particular, that the effect to be measured in patients is represented and validated in the context of preclinical in vitro and in vivo models. Antimalarial target product profiles (TPP) have recently been reevaluated and defined [12]. Future antimalarial combination treatments will need to cure the disease efficiently, by rapid clearance of parasitemia in patients, thereby reducing the risk of resistance and preventing recrudescence. In addition, these new medicines will be expected to block transmission and eliminate all liver forms of the parasite including dormant hypnozoites. This profile corresponds to the main treatment TPP at the Medicines for Malaria Venture (MMV) in which the ideal criterion constitutes a medicine administered as a single dose, the SERCaP (single-exposure radical cure and prophylaxis). In addition, a second TPP, called SEC (singleexposure chemoprotection), was defined to provide prophylaxis that protects populations at risk of infection. These antimalarials will need to provide long-duration prophylaxis through either the killing of asexual liver stages or by having an extended half-life. The SERCaP profile would ideally be met by a single molecule, but this goal is too ambitious and in any case, as recommended by the WHO, the medicine is likely to be a combination of several chemical entities fitting different target candidate profiles (TCPs) to reduce the risk of resistance. TCP1 corresponds to fast-acting molecules that could replace the artemisinins if resistance renders the class ineffective. TCP2 represents new molecules that could be either fast- or slow-acting, with an acceptable compound concentration above the minimum parasiticidal concentration (MPC) for 8 days, and which act as a partner of the TCP1 molecule in combination medications. TCP3 molecules target non-dividing parasites such as hypnozoites for TCP3a and mature gametocytes for TCP3b. The latter two TCPs focus on selecting molecules that kill the relapsing dormant forms in host liver and that block 479

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Figure 1. Life cycle of Plasmodium falciparum and Plasmodium vivax (modified with permission from [12]). The malaria parasite is transmitted to humans (green arrow) by a female Anopheles mosquito when taking a blood meal. The cells of the liver rapidly take up the parasites, where they become schizonts (A), multiply, and go on to invade blood cells. In the case of P. vivax and P. ovale, a dormant form of the parasite, known as hypnozoites (TCP3a), can reside in the liver and reactivate months or years later to induce new episodes of malaria. Current medicines mostly kill malaria parasites at the blood stage because this is when the parasite is most abundant (up to 1012 parasites per individual) (B), and is the stage that leads to clinical symptoms of malaria. Some of the blood-stage parasites develop into male and female gametocytes [broken line in (C)], which are taken up into the mosquito gut during a blood meal. In the gut, these gametocytes become gametes that mate and form a zygote. This is the sexual stage of the lifecycle. To eradicate malaria, medicines that can stop the parasite at this stage are needed (C–F), thereby halting transmission. This stage is also the most efficient to target because this is where the parasite density is as low as 10 oocysts per mosquito midgut (F). Malaria eradication will also depend on our ability to eliminate the relapsing parasite reservoir by killing hypnozoites efficiently. Abbreviations: TCP, target candidate profile. Adapted, with permission, from [12].

transmission, respectively. Finally, TCP4 describes molecules that protect populations against plasmodial infection. Such a chemoprotectant will focus on blocking the infection of the host liver by sporozoites, or on halting the development of liver proliferative and dormant forms of the parasite (schizonts and hypnozoites respectively) or the newly neo-asexual stages released in the blood. TCP4 molecules aim at replacing both the atovaquone–proguanil combination and mefloquine, the current gold standards for chemoprevention, which are non-optimal from a dosing frequency, cost, and/or safety perspective. Human malaria liver stages and P. vivax radical cure The biology of the liver-stage forms of Plasmodium is complex. Following a bite from an infected Anopheles mosquito, sporozoites injected into the host migrate to the liver where they first glide on epithelial cells, bind efficiently to sinusoidal cells, cross Kupffer cells, and then migrate through the hepatocytes, wounding several of them to finally invade a viable cell [13,14]. Inside the hepatocyte Plasmodium parasites multiply, generating schizonts that grow in size as Plasmodium replicates to generate thousands of new parasites. The culminating point of this phase is the release into the bloodstream of parasite-filled vesicles, termed merosomes, which 480

eventually burst, releasing erythrocyte-infective parasites termed merozoites [13,14]. This initiates the asexual erythrocytic cycle responsible for the symptomatic phase of the disease (Figure 1). Although the development of liver schizonts is common to all Plasmodium spp., liver infection by P. vivax can also produce a dormant liver form, the hypnozoite, that can reactivate weeks to months or even years after clearance of the original blood-stage parasitemia. This reactivation will lead to a new episode of malaria in the absence of a mosquito bite. Compounds aiming to block such relapses through their action on the hypnozoites are defined by the TCP3a profile. In the absence of experimental proof, the possibility that P. ovale hypnozoites exist relies mainly on biologic similarities with P. vivax. Moreover the few relapses reported so far are not convincing [15]. Effective treatments and control measures are anticipated to decrease malaria incidence and consequently reduce immunoprotection among the population at risk. The need to protect this emerging low-immunity population from infection, given the morbidity and mortality that will ensue, will be crucial. The short hepatic phase of the parasite life cycle invariably precedes the asexual logarithmic multiplication of the parasite in red blood cells that is responsible for the symptoms and which represents an

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Box 1. Current limitations in discovery of new anti-relapse antimalarials Hepatocytes. Freshly isolated or cryopreserved primary hepatocytes are in limited supply, do not proliferate in vitro, and give a very low rate of infection (<0.1% of infected hepatocytes). A human hepatocyte cell line (HC-04) allows complete Plasmodium falciparum liver-stage development but the infection rate is as low as 0.07% [63]. Culture of hepatocytes for 5–7 days to observe complete liver-stage development leads to loss of the cell monolayer. Sporozoites. Plasmodium vivax sporozoites can infect primary hepatocytes as well as hepatoma cell lines (HepG2 and HC-04) which allow full development of P. vivax liver stages [63,64], but infection rates remain low (0.1%), rendering the identification of hypnozoites difficult. No reliable culture system of the P. vivax asexual bloodstage is currently available. Therefore, P. vivax sporozoites are only produced from mosquitoes fed on blood from infected patients and should be used immediately because of their fragility. Access to cryopreservation is very limited, costly, and deleterious for sporozoite viability and/or infectivity. Hypnozoites. No sensitive and specific markers of hypnozoites exist at present. P. vivax subspecies produce variable levels of hypnozoites and relapse after very different time-periods. Anti-hypnozoite drugs. Primaquine is the only approved drug active against hypnozoites and which is effective exclusively in vivo. Primaquine suffers from poor compliance owing to the long treatment course (14 days), severe hemolytic toxicity in glucose 6phosphate dehydrogenase (G6PD)-deficient patients, contraindication in pregnant women, and association with significant gastrointestinal disturbance, which limit its use in the clinic. There is therefore an urgent need for new and safe drugs targeting P. vivax malaria [17,65]. A recent study using a microscale human liver platform (Figure I) for both P. falciparum and P. vivax demonstrated potential for medium-throughput antimalarial drug screening [24]. To adapt this assay to the HTS format it needs to be scaled up, to support the analysis of many compounds, and miniaturized to reduce cost. In addition, a homogenous, non-limiting supply of characterized sporozoites has to be available.

attractive target for prophylaxis drug discovery [13]. The profile of candidate molecules that will completely block the development of liver-stage (and thus the resulting blood-stage) parasites and leads to prophylaxis was recently formalized as TCP4. Moreover, interrupting this particular stage of the life cycle will contribute to blocking transmission of the parasite; not only is transmission blocked from an infected mosquito to an uninfected host, but the absence of gametocytes post-infection will prevent subsequent infection to a mosquito. Finally, the small number of hepatic forms (Figure 1) substantially reduces the risk of emergence of drug-resistant parasites [16]. The limitations in conducting liver-stage drug discovery (Box 1) relate predominantly to the biology of the Plasmodium liver stages and the inherent technical difficulties in studying them [17]. This is particularly true for the P. falciparum liver stages, which have proven much more difficult to study despite the description of complete liver-stage development in primary hepatocytes and the subsequent transition to a red blood cell infection in vitro more than 25 years ago [18]. At the research phase, no validated highthroughput screen (HTS) for P. vivax liver stages exists either, mainly due to the difficulty of accessing a sufficient amount of biomass. However, there has been encouraging progress (Box 1). Tafenoquine is the only anti-relapse drug presently in clinical development. Tafenoquine has recently completed a pivotal Phase II trial and demonstrated excellent antirelapse efficacy from a single 300 mg dose; a Phase III trial

The basic biology of hypnozoites is still unknown, and considerable research is required to inform drug discovery. The main difficulty at present is to produce sufficient hypnozoite biomass, a key step for fundamental research. Once these limitations are overcome P. vivax liver stages ‘omics’ approaches will reveal the transcripts and proteins that are produced to keep these dormant forms alive, enabling target-based drug discovery programs. Finally, an alternative to hypnozoiticidal compounds could be the search for an activator of hypnozoites that would trigger their evolution into liver schizonts. A major breakthrough was recently achieved when Plasmodium cynomolgi hypnozoites were shown to relapse in vitro and responded to activation by a small molecule [66]. Once asexual liver or blood stages are formed, classical antimalarials could then be used to kill the parasite. Again, the development of a highthroughput, low-cost, 1536-well P. vivax liver-stage in vitro assay will be key to such an endeavor.

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Figure I. Micropatterned cocultures of fibroblasts (left plate) and hepatocytes (middle plate).

has been initiated. This latter clinical trial is a randomized, double-blind treatment study to evaluate tafenoquine in adult patients infected by P. vivax. The efficacy, safety, and tolerability of tafenoquine to achieve radical cure when coadministered with chloroquine is analyzed in the context of the ‘DETECTIVE’ study (TAF112582) [19]. Tafenoquine is compared to primaquine for its safety, efficacy, and incidence on hemolysis in the ‘GATHER’ study (TAF116564). Medicines from the 8-aminoquinoline class are associated with hemolytic anemia in individuals with inherited glucose-6-phosphate dehydrogenase (G6PD) deficiency. Therefore, research is ongoing to develop a pointof-care diagnostic to identify individuals with G6PD deficiency to support well-tolerated and effective use of medicines for radical cure of patients infected with P. vivax. Thus, the development of new medicines which do display G6PD-dependent hemolysis is crucial. MMV test cascades and molecules that will be screened and profiled The current reality is that there are no P. vivax liver-stage in vitro assays that are robust enough, affordable, or with sufficient throughput to drive drug discovery efforts [20]. Therefore, the current strategy that has developed following work by the Biomedical Primate Research Center (BPRC) relies on a pragmatic approach using surrogate assays – with the obvious caveats that such assays often involve neither the human relevant pathogen nor host tissue (Figure 2A). First, compound libraries can be 481

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Figure 2. Current and ideal Medicines for Malaria Venture (MMV) testing cascades. (A) Identifying new anti-relapse molecules is extremely challenging without any culture system for Plasmodium vivax (right path boxes). Once P. vivax sporozoites will be available, in vitro assays and humanized liver mouse models will allow this. Until now, a surrogate high-throughput screening (HTS) assay is based on hepatocytes infected with Plasmodium berghei or Plasmodium yoelii parasites [22]. Molecules are then tested in vitro against Plasmodium cynomolgi [67] and in infected monkeys. (B) Chemoprotection: assays of liver-stage development (right path) suffer from poor hepatocyte infection and lack of P. vivax sporozoites. Similarly to TCP3a, P. berghei is used to invade hepatocytes and allow compound screening (left path). Then, potent compounds need to protect mice against infection. Ultimately, human sporozoite challenge will validate the chemoprotective potential of new drug candidates. (C) Transmission blocking: Plasmodium falciparum whole cell actives are screened against stage V gametocytes whose viability/fertility is assessed from a male/female gamete formation readout. This gametocyte functional assay selects molecules for oocyst inhibition (SMFA) after incubation with mature gametocytes [46]. Performed with the blood of patients [68] under treatment (ex vivo), SMFA will measure the capacity of molecules to block or decrease transmission. (D) Drug resistance: antimalarials and drug candidates tested on P. falciparum drug-resistant mutants allow detection of preexisting cross-resistance. Lack of cross-resistance against diverse known mutants may predict a new mechanism of action. Drug resistance can also be observed with new molecules tested at various inocula. Whole-genome sequencing of new resistant mutants identifies markers of resistance and suggests new drug targets.

screened directly on liver stages, or following a first filtering procedure with a high-throughput P. falciparum bloodstage assay to decrease the number of compounds tested in the liver-stage assay. This initial liver stage assay uses human hepatocyte cell lines infected with the rodent parasites P. berghei or P. yoelii [21]. HTS techniques are coupled to high content imaging readouts to visualize the impact of compounds on development of liver schizonts in 384- or 1536-well plates [22]. Although liver schizonticidal activity 482

against rodent malaria is not directly relevant for relapse (and is more relevant for prophylaxis), it is a tool for prioritization of compounds for screening in low-throughput in vitro hypnozoite assays. To date all compounds blocking relapse have shown causal prophylactic activity. Therefore, to maximize the chance of finding small molecules that exhibit anti-hypnozoite activity, positives from the liverstage assay are tested in a surrogate of the P. vivax liverstage assay using Plasmodium cynomolgi. This parasite

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Box 2. Preclinical and clinical studies supporting new antimalarial discovery (i) Liver stages  Mouse model. On the in vivo side, a new preclinical model using humanized mice is currently in development to enable the characterization of potential anti-hypnozoite molecules [69].  Rhesus monkey model. This model allows relapse, the only physiological landmark of hypnozoites. In brief, following infection of rhesus monkeys with Plasmodium cynomolgi, the blood stage is cleared by chloroquine and followed by treatment with the anti-relapse candidate [70]. Blood-stage parasitemia due to relapsing hypnozoites is monitored in monkeys until 100 days post-treatment. Primaquine is used as rescue therapy. For prophylaxis, monkeys are treated with the drug candidate before infection.  Human challenge model. After preclinical safety and pharmacokinetic package is completed, the compounds are tested in a single rising-dose Phase I study. This offers the possibility to challenge human volunteers with Plasmodium vivax once the safe human dose has been predicted [71]. However, ethical approval remains a serious issue. This approach strictly depends on sporozoite availability and appropriate CYP2D6 testing to validate primaquine as appropriate rescue treatment. Safe anti-hypnozoite molecules entering Phase IIa can be tested in soldiers who have spent up to 12 months in P. vivax endemic areas (Papua) and then return to their malaria-free base (Java), receiving new anti-relapse agents and being followed-up over 12 months [72]. In this model, primaquine coadministered with a blood schizonticide has been shown to block relapses by 94% compared to 20% with blood schizonticide only.

Para sitology: infec ousness

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(ii) Transmission  Infectiousness. The standard membrane feeding assay (SMFA) is the crucial assay comparing the transmission-blocking activity of a compound preclinically versus in patients. In a clinical trial, patient blood can be taken and fed through a membrane to mosquitoes. The effects of the drug on sporogony are studied similarly to in vitro SMFA. Sustaining in patients a gametocytocidal concentration blocking oocyst formation in a SMFA is predicted to lead to meaningful transmission reduction.  Epidemiology. The full transmission is an epidemiological endpoint. The impact of an antimalarial on R0 (the basic reproduction number) reduces or fully blocks the transmission of Plasmodium from infected to non-infected vertebrates. Recently, a mouse to mouse preclinical model of Plasmodium berghei transmission has been developed [73]. A P. falciparum model will require humanized mice with a double engraftment of human erythrocytes and hepatocytes. and is a much longer term and more challenging goal. Interestingly, this P. berghei model incorporates a feeding assay element, leading to an interim assessment of parasites in the mosquito that links mosquito infectiousness and mouse to mouse transmission (Figure I). Similarly to primaquine, this model offers the possibility to test molecules whose activity depends on their metabolism in vivo. The statistical power of the study will permit comparison with data obtained in human populations such as the Phase IV study conducted by Novartis in Burkina Faso [40,41].

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Figure I. Mosquito infectiousness and epidemiological readouts in transmission studies.

infects primates, is the closest Plasmodium species to P. vivax based on phylogeny, and produces both large (schizont) and small (hypnozoite) exoerythrocytic forms in the liver of its host [23]. This assay uses P. cynomolgi sporozoite infections of primary rhesus hepatocytes and generates reproducibly small parasite forms. Recently hypnozoites were reported to reactivate in vitro. The P. cynomolgi assay represents the main assay through which all compounds identified as positive in the rodent liver-stage assays are tested to find series with potential hypnozoiticidal activity

similar to or better than primaquine (Figure 2A). The ultimate validation of anti-hypnozoite activity and a decision to proceed to preclinical development is made when the compound demonstrates anti-relapse activity in the relevant preclinical model: the P. cynomolgi infected rhesus monkey model (Box 2). The current screening cascade is not ideal because it uses many surrogate assays. As discussed previously, various organizations are working to deliver an industrialized P. vivax liver-stage in vitro assay [24] whose 483

Review throughput will be sufficiently high to permit direct screening of large libraries in an optimal manner (Figure 2A). The same screening cascade is also currently used to identify compounds with potential for chemoprophylaxis (Figure 2B). In the future, the ideal test cascade will consist in testing compounds against hepatocytes infected by P. falciparum and P. vivax sporozoites. Transmission-blocking strategy The eradication agenda proposed by BMGF and WHO in 2007 will only be possible if the cycle of transmission can be broken [25,26]. Transmission is a crucial step for the survival of the malaria parasite, and depends on the sequential infection of mosquitoes from infected human beings and then of non-infected human beings by the neoinfected mosquitoes. Infection of mosquitoes relies on gametocytes produced and matured in human blood. Gametocytes develop from committed trophozoites [27] that evolve to schizonts and ultimately liberate, in the case of falciparum malaria, immature sexual-stage parasites, the so-called stage I gametocytes, representing approximately 1% of the total parasitemia in the human blood (i.e., 1010 gametocytes at the peak of parasitemia). Successive differentiation leads to the emergence of further immature gametocytes termed stages II and III before converting into mature stages IV (sequestered) and V (observable on a blood smear), the latter being released into the blood and are responsible for mosquito infection when taken up in a blood meal (Figure 3). Once in the mosquito gut, and upon a temperature drop, an increase of the xanthurenic acid concentration, and pH change, mature gametocytes differentiate into male and female gametes that will mate to form a diploid zygote [28]. This zygote will subsequently transform into an ookinete that will invade the midgut before developing into the final reproductive stage – the oocyst. Oocysts produce thousands of sporozoites which, when liberated, reach the salivary glands of Anopheles and are released into human blood during their next feeding on a host. If no oocysts are produced, no sporozoites can form, and therefore the transmission of the disease is halted. The current MMV strategy aims at associating curative and transmission-blocking TCPs in a cost-effective and developable combination. Therefore, one option is to develop dual-activity agents targeting the asexual stage of the parasite and the gametocytes, which are accessible in human blood and are responsible for transmission to the mosquito; in particular stage V gametocytes. Ideally, molecules showing different modes of action/targets in asexual and sexual parasites will be selected to avoid potential resistance to propagate to sexual stages and to be transmitted rapidly. Moreover, combination therapy will surmount the increased transmissibility conferred to drug-resistant parasites [29]. Any additional activity found to inhibit development of sexual stages in Anopheles brings a strong benefit. Several biological assays targeting Plasmodium sexual stages have been developed so far [30–35]. In August 2012, experts, clinicians and WHO representatives met in Bangkok, Thailand, and recommended that a single low dose of primaquine (0.25 mg/kg) be administered to clear all gametocytes (Box 3) and, thus, deplete the 484

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infectious parasite reservoir and block transmission [36]. Currently, however, there have been no suitably powered clinical trials to confirm the efficacy of single, low-dose primaquine, although these are now in progress [37,38]. The blood of patients treated with a single exposure of primaquine will be fed to mosquitoes in a standard membrane feeding assay (SMFA) and the development of oocysts and sporozoites will be measured. Interestingly, the benefit of adding primaquine to ACTs has been modeled very recently [39]. Ultimately, for mass drug administration to target asymptomatic carriers, a formal transmission-blocking study comparing the numbers of cases in villages treated with primaquine to untreated, as was performed with Coartem by Novartis [40,41], would reveal the transmission blocking efficacy of the drug. In parallel to the evaluation of the efficiency of primaquine against the transmission of the disease, the safety risk in G6PD-deficient patients, young children, and pregnant women will need to be mitigated [42]. Test cascades and molecules that will be screened and profiled The biology of the parasite sexual and mosquito stages is complex and there are limitations regarding the throughput of screening approaches that target these stages compared with the asexual blood stage. In the present case, a pragmatic approach focused on profiling preclinical candidates and late leads is favored. This low number of molecules (<10 at steady-state) is perfectly suited to fit within a low-throughput/high biological content assay strategy which spans almost the entire development of the sexual stages from mature gametocytes to oocysts. The in vitro SMFA is currently the best proxy and most pertinent filter upstream to the ex vivo equivalent assay performed with the blood of patients treated with the candidate drug (Box 2). Active compounds selected based on their potency against asexual parasites, as well as on their physicochemical properties and tractability, can similarly be profiled for transmission-blocking potential. Historical and more recent data show that some (but not all) molecules that clear blood schizonts also eliminate stage I–III gametocytes. Consequently, it is also necessary to profile the early-stage gametocytocidal activity of a compound to avoid a drug combination in which neither compound kills early-stage gametocytes. Otherwise there is a risk of transmission from any surviving early stage gametocytes. At the drug candidate level, the SMFA will be required to evaluate whether the concentration–time profile associated with the predicted asexual blood-stage therapeutic dose can deliver the required transmission-blocking efficacy. If the potencies against the sexual and asexual stage are similar there is likely to be a significant contribution to blocking transmission to the mosquito. Where many molecules need to be tested, for example the set of 20 000 actives identified by phenotypic screening against asexual blood-stage parasites [43–45], a filter assay is necessary to narrow-down those with transmissionblocking potential. Such a role can be played by a functional gametocyte assay measuring the production of gametes

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Figure 3. Medicines for Malaria Venture (MMV) strategy to identify transmission-blocking compounds. A cascade of tests has been developed to assess the capacity of each lead molecule to kill or block the development of the key sexual stages of the parasite. Blood-stage active compounds are screened against gametocytes in a functional assay that detects the formation of male and female gametes. The ultimate assay allows quantification of the number of oocysts produced followed by incubation of compounds with mature gametocytes and the standard membrane feeding procedure.

following gametocyte maturation. This test cascade enables the funneling of asexual blood-stage hits towards, ideally, equipotent leads with dual activity and preclinical candidates with transmission-blocking potential. Molecules can impact mature gametocytes but exert their effects on a later sexual stage – for example, the

gametocytes may still be viable but are unable to complete the next sexual development steps in the Anopheles vector. Thus, it is important to analyze the formation of male and female gametes as a combined assay readout after the molecules have been applied to mature gametocytes (Figures 2C, 3) for at least 24 h. From previous work, 485

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Box 3. Marketed drugs as potential transmission-blocking weapons gametocytes, oocysts, and sporozoites are totally cleared after drug administration [76]; even if this is a result of insufficient dosing for asexual blood stages, it highlights the particular sensitivity of gametocytes to primaquine. An associated unknown risk is whether the demonstration of resistance strains to primaquine in mice suggests that there is the future possibility of emergence of clinical parasite phenotypes resistant to the drug.

Primaquine A drug known to cause methemoglobinemia and hemolysis in patients with G6PD deficiency. The extent of this depends potentially on the variant and the expression/activity of patient CYP2D6 given that a metabolite appears to be associated with both activity and toxicity of the drug [74]. Primaquine (Figure I) is weakly potent when tested in a standard Plasmodium falciparum in vitro growth-inhibition assay (EC50 > 1.1 mM) [46]. However, primaquine is extremely efficacious in vivo in the Plasmodium berghei mouse model (ED90 4.8 mg/kg) [75]. It is likely, therefore, that this effect on asexual blood stages is a result of an active metabolite(s). It is hypothesized that primaquine has the ability, through such active metabolite(s), to clear asexual blood-stage parasites in patients if the dosing is sufficient. The observed low clinical blood-stage efficacy of the drug, however, suggests otherwise; presumably the doses used for radical cure or transmission-blocking are insufficient to achieve efficacious concentrations of blood-stage active metabolites in humans. These few relevant clinical trials show no impact of a single exposure of primaquine on the asexual parasites in the patients, whereas mature

Primaquine

Ivermectin A safe and effective endectocide (Figure I) used for the treatment of onchocerciasis, and delivered freely as part of Merck’s commitment to Global Health, has recently emerged as a potential malaria transmission-blocking molecule through its activity on the mosquito. However, further investigations are needed before it is ready for consideration as a mass drug administration tool to block transmission. Consequently, the MMV strategy is to wait for confirmatory clinical data on safety and efficacy while proactively delivering new molecules with transmission-blocking potential through effects on gametocytes as risk mitigation.

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Figure I. Chemical structures of primaquine and ivermectin.

pyronaridine, thiostrepton, and cycloheximide are known to inhibit P. falciparum male gamete exflagellation and can therefore be considered as positive controls [46]. Positive controls to be used in the female gamete assay are less obvious because this assay has been developed only recently and background/historical information is very limited. Although gamete formation, and especially the male gamete exflagellation process, is an explosive transformation that takes no more than 20 min, it is possible that a compound might specifically interfere with this event and not act at the level of the mature gametocytes. To discriminate between these possibilities, a wash-out step can be introduced to eliminate the compounds before gamete induction. The next key assay in the transmission-blocking test cascade is the SMFA, an assay that focuses on mosquitoes ingesting a blood meal containing gametocytes previously incubated with the compound of interest for at least 24 h (Figures 2C and 3). The potency of the compound to inhibit the development of the sporogony (oocysts and sporozoites) in the mosquito is evaluated by quantifying the numbers of oocysts produced (intensity) and mosquitoes infected (prevalence). Previous results show that dihydroartemisinin and lumefantrine are antimalarials that fully inhibit oocyst formation at 10 mM and can be used as positive controls in SMFA experiments [46]. 486

Today, progress has been made such that full concentration responses of molecules can be analyzed with this assay, and consequently IC50 values can be determined for comparison with potencies against gametes and gametocytes. Extensive screening on mature gametocytes to search for molecules only active in transmission blocking is not part of the strategy for the time being. If additional gametocytocidal agents from the available hits are not identified, and provided that a low cost HTS assay becomes available in the 1536-well plate format, mass screening on mature gametocytes could be a viable option. The immature gametocytes will not be targeted using HTS with a large compound library but will be used in assays to profile frontrunners and candidates. Drug resistance The history of each antimalarial has been one of ultimate failure owing to the emergence of drug resistance. It is as true for historical antimalarials such as chloroquine and sulfadoxine-pyrimethamine as it is today for the newer artemisinins, where ring-stage resistance has emerged in several areas of Southeast Asia, thus threatening to add ACTs to this list [47–51]. Recently the situation worsened with evidence of clinically resistant parasites against

Review piperaquine. The existence (or threat of emergence) and spread of mutant parasites resistant to antimalarials in the field is therefore one of the main drivers for the search of new chemotypes. Resistance is generally achieved either by excluding antimalarials from their subcellular site of action or by altering their biological targets in a way that limits efficacy. For instance, single-nucleotide polymorphisms (SNPs) in pfmdr1 and pfcrt, which both encode putative transporters localized on the parasitophorous food vacuole, as well as copy-number variations of pfmdr1, have been directly linked to resistance against antimalarials that act within this vacuole, such as chloroquine and other 4-aminoquinolines [52–55]. Typical examples of target mutations that abolish drug-mediated inhibition include the pfdhodh gene [56] and the folate pathway genes pfdhps and pfdfhr which encode enzymes targeted by sulfadoxine and pyrimethamine, respectively [53]. More recently, parasites were identified in Southeast Asia with a phenotype that results in a clinically significant increase of the parasite clearance time in response to artesunate treatment [54]. A specific loss of sensitivity of ring-stage parasites is believed to underlie the phenomenon, and this is likely ultimately to affect the clinical efficacy of ACTs and therefore represents a major public health concern [55]. For the first time, a marker associated with clinical resistance to artemisinin was identified as the kelch domain of the ‘K13-propeller’ protein [57]. Research guided by the clinical reality of implementing drug resistance assays in the laboratory would be the ideal situation. However, the difficulty here is to align availability of field isolates with the necessity to test newly synthesized molecules on a regular basis along iterative medicinal chemistry programs. A quantitative framework was recently designed and applied for assessing the risk of resistance selection by antimalarials in development [58]. This strategy aims to minimize the risk of cross-resistance with genetically defined resistance mechanisms known to occur in the wild, or generated in the laboratory, as well as to characterize the compound-specific propensity to select new resistance traits in P. falciparum (Figure 2D). The first goal is achieved by evaluating the activity of new compounds against K1, a standard multi-drug resistant (MDR) P. falciparum strain. Optimized compounds are further tested against a more diverse panel of resistant strains, including recently adapted laboratory cultures, and a set of P. falciparum and P. vivax ex vivo isolates, the latter being a ring-stage maturation assay. Furthermore, cross-resistance with artesunate is assessed in a ringstage survival assay using Cambodian parasites from patients experiencing decreased artesunate parasite clearance times. The frequency of de novo resistance selection is measured by exerting a controlled level of drug pressure on various initial inocula, ranging typically from 106 to 109 parasites using the Dd2 strain. The minimal inoculum for resistance (MIR) – the minimal number of parasites from which resistant clones can be reproducibly selected – is a relative measure of the genetic ability of P. falciparum to evolve a significant resistance mechanism against a given compound, and an MIR

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equal or inferior to 105 Dd2 would be considered as an increased risk of seeing resistance developing rapidly in patients [58]. Except if demonstrated as highly indicative of a high risk of resistance (very low inoculum and major loss of potency), these data are not anticipated to be used for decision-making in the drug discovery pipeline. Indeed, the next goal, over time, is to compare the resistance data acquired in the lab with those collected from clinical studies. Isolation of in vitro resistant clones allows further study of the impact of resistance on parasite fitness, gametocyte production, viability, or sensitivity, as well as cross-resistance with potential partner drugs. Ultimately the identification of the genetic determinants of resistance provides not only potential markers of resistance, that are useful for monitoring resistance during clinical development and beyond, but also information about the compound mode-of-action itself [59]. The latter is particularly important for the large set of compounds initially identified from whole cell phenotypic screens and for which the mode of action is rarely known [60]. The establishment of a repository of clones resistant to the various molecules in development will enable prioritization of novel modes of action [61]. The absence of in vitro assays able to recapitulate the artesunate delayed clearance phenotype observed with some parasites in the clinic has hampered the evaluation of the risk that other endoperoxides, including the drug candidate OZ439, might also be subject to this emerging phenomenon. The recent development of the ring-stage assay (RSA), in which resistance can be readily observed when tightly synchronized early ring-stage parasites are subjected to short exposures of artesunate [62], allows in principle the evaluation of other drug candidates. Overall, resistance studies should demonstrate the ability of new antimalarial compounds to overcome existing resistance mechanisms as well as having a reasonably low propensity to select for de novo resistance mechanisms in vitro. A comprehensive understanding of the resistance liabilities of new antimalarials will also help to guide their development and ultimately to optimize their clinical usage through sensible combination strategies. Concluding remarks and future directions Eradicating malaria places demands on medicines, and therefore drug discovery, beyond the need to cure the patient. Medicines are needed to stop the relapse of P. vivax and block transmission from patient to patient. As a result, we must look beyond the blood stage and seek molecules able to eliminate all forms of the parasite in patients, leading to the elusive radical cure. To identify these molecules, biological assays permitting the measurement of the effect of compounds on both hypnozoites and gametocytes are crucial. Although there are some exciting developments towards the development of a P. vivax liverstage assay using microscale human liver platforms, the reality is that currently there is no assay that is sufficiently robust and affordable. In its place, a pragmatic screening cascade has been established, first using a surrogate 487

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Box 4. Outstanding questions and future directions  Is SERCaP an achievable goal? As discussed [12], it is a major challenge to deliver a single molecule with all attributes necessary to fulfill the ideal TCP1 (SERCaP) criteria. Even as a combination medicine with two or three components it remains a significant but not impossible challenge. Already, within the global portfolio, there are compounds in clinical development which have the potential to achieve this goal if combined – although there is still a long way to go. Furthermore, new molecules in early-stage development selected for long duration and EE-stage activity will be able to tackle eradication TCPs in an intentional way that has not been possible previously.  What is the benefit/risk balance of SEC? Any single-exposure chemoprotection combination will provide drug concentrations necessary to protect vulnerable populations over time. As with all chemoprotectants, however, safety is absolutely crucial because the individuals receiving drug are not infected.  Will Plasmodium vivax culture systems and related drug-screening assays shortly be available to the antimalarial research community? This is a hugely challenging area with major logistical issues. It is unclear yet if a breakthrough in P. vivax continuous culture is imminent, but there has been huge progress there, and in the liverstage work both in vitro and in vivo. Thus it is expected that, in the near term, P. vivax sporozoites from mosquitoes feeding on patient blood will be available for limited screening in robust liver assays in endemic regions. Success in cryopreservation would be another trigger to expand dramatically the accessibility of sporozoites to the community, and is thus highly desirable. Finally, in the longer term, the paradigm shift will occur once P. vivax blood stages can be continuously cultured.

rodent malaria liver-stage assay followed by an in vitro P. cynomolgi assay and in vivo P. cynomolgi model. To date, these are the only assays to determine whether a small molecule is active against the dormant small liver forms. To identify molecules able to block transmission, a functional test cascade is in operation. Given the complexity of the parasite sexual and mosquito stages, the throughput of these assays is limited; nonetheless, preclinical candidates and late leads have been successfully profiled. Nevertheless, efforts to identify molecules to block transmission and cure patients from uncomplicated and relapsing malaria will be in vain if the parasite is easily able to develop resistance. Today, there is no simple rationale that can help to categorize chemotypes according to their capacity to lead to or select for drug resistance phenotypes in Plasmodium. Over the past 5 years the spread of artemisinin resistance has been anticipated, and medicinal chemistry has focused on the identification of novel acting chemotypes. Using drug-resistant strains that are either isolated from patients or generated following exposure of the parasite to development molecules, and assessing whether the targets/pathways hit by new compounds are likely to induce drug resistance, enables prioritization of the compounds. Outstanding questions are listed in Box 4. Owing to concerted research efforts in recent years, we have come a long way towards developing the assays and tools needed to identify efficiently the next generation of medicines that will enable the elimination and eradication of malaria. Importantly, efforts are also underway to fill the gaps. The foundations of the pathway towards discovering medicines for malaria elimination and eradication are being laid; we now need to strengthen those foundations and follow the path to the end. 488

 What is the current understanding of the mechanism of artemisinin resistance, and how can this risk be mitigated with new drugs? Resistance to artemisinin-like molecules reported in South-East Asia [49] is not a ‘classical’ resistance phenotype. Parasite clearance times are doubled in patients whereas the in vitro assessed potency of these parasites, in a standard growth-inhibition assay, remains similar to those of sensitive parasites. Recently, Me´nard and colleagues have developed a ring-stage survival assay (RSA) and showed that, in their system, artemisinin derivatives such as dihydroartemisinin (DHA) applied over 6 h were unable to block the growth of the ‘resistant’ parasite, but showed high potency in the same assay with a wild type strain. It is clear that new compounds that target ring-stage parasites will need to be assessed in this RSA, comparing drug activity against strains with clinical resistance to artemisinins and wild type strains. This will provide data on the likely cross-resistance profile with DHA and help discovery projects to select those compounds predicted to show equal efficacy across all parasite populations.  Will the drug discovery approach to target malaria elimination alter over time? Drug discovery for malaria is an extremely complex discipline, and the approach to discover new drugs will alter as MMV and the community receive new insights about the parasite and the strengths and deficiencies of advanced molecules in the pipeline. Any discovery strategy needs to be dynamic and sufficiently flexible to respond properly to the threat of the parasite and, if focused on elimination, must incorporate a major component aimed at breaking the lifecycle and transmission, thus moving beyond only the asexual blood stages.

Acknowledgments We are thankful to our MMV colleagues for help with the manuscript. We thank all the MMV partners (see MMV website) for their support in the assembly of the largest pipeline of antimalarials ever, and the anonymous reviewers for constructive comments. We thank Pierre Chassany (Comstone) for professional support with the figures.

References 1 World Health Organization (2012) World Malaria Report 2012, World Health Organization 2 Roll Back Malaria: Malaria Case Management Working Group (2005) Roll Back Malaria Consultative Meeting on the Role of Medicine Sellers in the Management of Malaria: What’s Worked and Where Do We Go From Here? Roll Back Malaria 3 Fidock, D. (2013) Eliminating Malaria. Science 340, 1531–1533 4 Pain, a et al. (2008) The genome of the simian and human malaria parasite Plasmodium knowlesi. Nature 455, 799–803 5 Gardner, M.J. et al. (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419, 498–511 6 Carlton, J.M. et al. (2008) Comparative genomics of the neglected human malaria parasite Plasmodium vivax. Nature 455, 757–763 7 Tachibana, S.I. et al. (2012) Plasmodium cynomolgi genome sequences provide insight into Plasmodium vivax and the monkey malaria clade. Nat. Genet. 44, 1051–1055 8 Moehrle, J.J. et al. (2012) First-in-man safety and pharmacokinetics of synthetic ozonide OZ439 demonstrates an improved exposure profile relative to other peroxide antimalarials. Br. J. Clin. Pharmacol. 75, 524–537 9 Charman, S.A. et al. (2011) Synthetic ozonide drug candidate OZ439 offers new hope for a single-dose cure of uncomplicated malaria. Proc. Natl. Acad. Sci. U.S.A. 108, 4400–4405 10 Van Pelt-Koops, J.C. et al. (2012) The spiroindolone drug candidate NITD609 potently inhibits gametocytogenesis and blocks Plasmodium falciparum transmission to anopheles mosquito vector. Antimicrob. Agents Chemother. 56, 3544–3548 11 White, N.J. et al. (2014) Malaria. Lancet 383, 723–735 12 Burrows, J.N. et al. (2013) Designing the next generation of medicines for malaria control and eradication. Malar. J. 12, 187 13 Prudeˆncio, M. et al. (2006) The silent path to thousands of merozoites: the Plasmodium liver stage. Nat. Rev. Microbiol. 4, 849–856

Review 14 Frevert, U. et al. (2005) Intravital observation of Plasmodium berghei sporozoite infection of the liver. PLoS Biol. 3, 1034–1046 15 Richter, J. et al. (2010) What is the evidence for the existence of Plasmodium ovale hypnozoites? Parasitol. Res. 107, 1285–1290 16 Lindner, S.E. et al. (2012) Malaria parasite pre-erythrocytic infection: preparation meets opportunity. Cell. Microbiol. 14, 316–324 17 Wells, T.N.C. et al. (2010) Targeting the hypnozoite reservoir of Plasmodium vivax: the hidden obstacle to malaria elimination. Trends Parasitol. 26, 145–151 18 Mazier, D. et al. (1985) Complete development of hepatic stages of Plasmodium falciparum in vitro. Science 227, 440–442 19 Llanos-Cuentas, A. et al. (2013) Tafenoquine plus chloroquine for the treatment and relapse prevention of Plasmodium vivax malaria (DETECTIVE): a multicentre, double-blind, randomised, phase 2b dose-selection study. Lancet 6736, 1–10 20 Mazier, D. et al. (2009) A pre-emptive strike against malaria’s stealthy hepatic forms. Nature 8, 854–864 21 Prudeˆncio, M. et al. (2011) A toolbox to study liver stage malaria. Trends Parasitol. 27, 565–574 22 Meister, S. et al. (2011) Imaging of Plasmodium liver stages to drive next-generation antimalarial drug discovery. Science 334, 1372–1377 23 Dembele, L. et al. (2011) Towards an in vitro model of Plasmodium hypnozoites suitable for drug discovery. PLoS ONE 6, e18162 24 March, S. et al. (2013) A microscale human liver platform that supports the hepatic stages of Plasmodium falciparum and vivax. Cell Host Microbe 14, 104–115 25 Roberts, L. and Enserink, M. (2007) Did they really say. . .eradication? Science 318, 1544–1545 26 Sinden, R.E. et al. (2012) The biology of sexual development of Plasmodium: the design and implementation of transmissionblocking strategies. Malar. J. 11, 70 27 Talman, A.M. et al. (2004) Gametocytogenesis: the puberty of Plasmodium falciparum. Malar. J. 3, 24 28 Billker, O. et al. (2000) Determination of mosquito bloodmeal pH in situ by ion-selective microelectrode measurement: implications for the regulation of malarial gametogenesis. Parasitology 120, 547–551 29 Hallett, R.L. et al. (2004) Combination therapy counteracts the enhanced transmission of drug-resistant malaria parasites to mosquitoes. Antimicrob. Agents Chemother. 48, 3940–3943 30 Lelie`vre, J. et al. (2012) Activity of clinically relevant antimalarial drugs on Plasmodium falciparum mature gametocytes in an ATP bioluminescence ‘transmission blocking’ assay. PLoS ONE 7, e35019 31 Peatey, C.L. et al. (2012) Anti-malarial drugs: how effective are they against Plasmodium falciparum gametocytes? Malar. J. 11, 34 32 Buchholz, K. et al. (2011) A high-throughput screen targeting malaria transmission stages opens new avenues for drug development. J. Infect. Dis. 203, 1445–1453 33 Tanaka, T.Q. et al. (2013) A quantitative high throughput assay for identifying gametocytocidal compounds. Mol. Biochem. Parasitol. 188, 20–25 34 Lucantoni, L. and Avery, V. (2012) Whole-cell in vitro screening for gametocytocidal compounds. Future Med. Chem. 4, 2337–2360 35 Delves, M.J. et al. (2012) A high-throughput assay for the identification of malarial transmission-blocking drugs and vaccines. Int. J. Parasitol. 42, 999–1006 36 World Health Organization (2012) WHO Evidence Review Group: The Safety and Effectiveness of Single Dose Primaquine as a P. falciparum gametocytocide, World Health Organization 37 Graves, P. et al. (2012) Primaquine for reducing Plasmodium falciparum transmission. Cochrane Database Syst. Rev. 9, http:// dx.doi.org/10.1002/14651858.CD008152.pub2 CD008152 38 White, N.J. et al. (2012) Rationale for recommending a lower dose of primaquine as a Plasmodium falciparum gametocytocide in populations where G6PD deficiency is common. Malar. J. 11, 418 39 Johnston, G.L. et al. (2014) Modeling within-host effects of drugs on Plasmodium falciparum transmission and prospects for malaria elimination. PLoS Comput. Biol. 10, e1003434 40 Kern, S.E. et al. (2011) Community screening and treatment of asymptomatic carriers of Plasmodium falciparum with artemether– lumefantrine to reduce malaria disease burden: a modelling and simulation analysis. Malar. J. 10, 210

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41 Tiono, A.B. et al. (2013) A controlled, parallel, cluster-randomized trial of community-wide screening and treatment of asymptomatic carriers of Plasmodium falciparum in Burkina Faso. Malar. J. 12, 79 42 Howes, R.E. et al. (2013) G6PD deficiency: global distribution, genetic variants and primaquine therapy. Adv. Parasitol. 81, 133–201 43 Plouffe, D. et al. (2008) In silico activity profiling reveals the mechanism of action of antimalarials discovered in a highthroughput screen. Proc. Natl. Acad. Sci. U.S.A. 105, 9059–9064 44 Gamo, F.J. et al. (2010) Thousands of chemical starting points for antimalarial lead identification. Nature 465, 305–310 45 Guiguemde, W.A. et al. (2010) Chemical genetics of Plasmodium falciparum. Nature 465, 311–315 46 Delves, M. et al. (2012) The activities of current antimalarial drugs on the life cycle stages of Plasmodium: a comparative study with human and rodent parasites. PLoS Med. 9, e1001169 47 Wongsrichanalai, C. et al. (2002) Epidemiology of drug-resistant malaria. Lancet Infect. Dis. 2, 209–218 48 Noedl, H. et al. (2008) Evidence of artemisinin-resistant malaria in western Cambodia. N. Engl. J. Med. 359, 2619–2620 49 Dondorp, A. et al. (2009) Artemisinin resistance in Plasmodium falciparum malaria. N. Engl. J. Med. 361, 455–467 50 Phyo, A.P. et al. (2012) Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study. Lancet 379, 1960–1966 51 Kyaw, M.P. et al. (2013) Reduced susceptibility of Plasmodium falciparum to artesunate in southern Myanmar. PLoS ONE 8, e57689 52 Sanchez, C.P. et al. (2010) Transporters as mediators of drug resistance in Plasmodium falciparum. Int. J. Parasitol. 40, 1109–1118 53 Nzila, A. (2006) The past, present and future of antifolates in the treatment of Plasmodium falciparum infection. J. Antimicrob. Chemother. 57, 1043–1054 54 Fairhurst, R.M. et al. (2012) Artemisinin-resistant malaria: research challenges, opportunities, and public health implications. Am. J. Trop. Med. Hyg. 87, 231–241 55 Dondorp, A.M. and Ringwald, P. (2013) Artemisinin resistance is a clear and present danger. Trends Parasitol. 29, 359–360 56 Ross, L.S. et al. (2014) In vitro resistance selections for Plasmodium falciparum dihydroorotate dehydrogenase inhibitors give mutants with multiple point mutations in the drug-binding site and altered growth. J. Biol. Chem. 289, 17980–17995 57 Ariey, F. et al. (2014) A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505, 50–55 58 Ding, X.C. et al. (2012) A framework for assessing the risk of resistance for antimalarials in development. Malar. J. 11, P23 59 Flannery, E.L. et al. (2013) Using genetic methods to define the targets of compounds with antimalarial activity. J. Med. Chem. 56, 7761–7771 60 Guiguemde, W.A. et al. (2012) Global phenotypic screening for antimalarials. Chem. Biol. 19, 116–129 61 Kuhen, K.L. et al. (2014) KAF156 is an antimalarial clinical candidate with potential for use in prophylaxis, treatment and prevention of disease transmission. Antimicrob. Agents Chemother. 02727–2813 62 Witkowski, B. et al. (2013) Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in Western Cambodia. Antimicrob. Agents Chemother. 57, 914–923 63 Sattabongkot, J. et al. (2006) Establishment of a human hepatocyte line that supports in vitro development of the exo-erythrocytic stages of the malaria parasites Plasmodium falciparum and P. vivax. Am. J. Trop. Med. Hyg. 74, 708–715 64 Hollingdale, M.R. et al. (1986) In vitro culture of exoerythrocytic parasites of the North Korean strain of Plasmodium vivax in hepatoma cells. Am. J. Trop. Med. Hyg. 35, 275–276 65 Price, R.N. et al. (2011) Plasmodium vivax treatments: what are we looking for? Curr. Opin. Infect. Dis. 24, 578–585 66 Dembe´le´, L. et al. (2014) Persistence and activation of malaria hypnozoites in long-term primary hepatocyte cultures. Nat. Med. 20, 307–312 67 Zeeman, A.M. et al. (2014) KAI407, a potent non-8-aminoquinoline compound that kills Plasmodium cynomolgi early dormant liver stage parasites in vitro. Antimicrob. Agents Chemother. 58, 1586–1595 68 Bousema, J.T. et al. (2007) A longitudinal study of immune responses to Plasmodium falciparum sexual stage antigens in Tanzanian adults. Parasite Immunol. 29, 309–317 489

Review 69 Vaughan, A.M. et al. (2012) Complete Plasmodium falciparum liver stage development in liver-chimeric mice. J. Clin. Invest. 122, 1–11 70 Deye, G. and a et al. (2012) Use of a rhesus Plasmodium cynomolgi model to screen for anti-hypnozoite activity of pharmaceutical substances. Am. J. Trop. Med. Hyg. 86, 931–935 71 Mccarthy, J.S. et al. (2013) Experimentally induced blood-stage Plasmodium vivax infection in healthy volunteers. J. Infect. Dis. 208, 1688–1694 72 Sutanto, I. et al. (2013) Randomized, open-label trial of primaquine against vivax malaria relapse in Indonesia. Antimicrob. Agents Chemother. 57, 1128–1135

490

Trends in Parasitology October 2014, Vol. 30, No. 10

73 Blagborough, a.M. et al. (2013) Transmission-blocking interventions eliminate malaria from laboratory populations. Nat. Commun. 4, 1812 74 Pybus, B.S. et al. (2013) The metabolism of primaquine to its active metabolite is dependent on CYP2D6. Malar. J. 12, 212 75 Peters, W. (1988) Chemotherapy of Rodent Malaria (Annual Report), US Army Medical Research & Development Command 76 Rieckmann, K.H. et al. (1968) Gametocytocidal and sporontocidal effects of primaquine and of sulfadiazine with pyrimethamine in a chloroquine-resistant strain of Plasmodium falciparum. Bull. World Health Organ. 38, 625–632