Atovaquone is the major active component of the new antimalarial drug Malarone. Considerable evidence suggests that malaria parasites become resistant to atovaquone quickly if atovaquone is used as a sole agent. The mechanism by which the parasite develops resistance to atovaquone is not yet fully understood. Atovaquone has been shown to inhibit the cytochrome bc 1 (CYT bc 1 ) complex of the electron transport chain of malaria parasites. Here we report point mutations in Plasmodium falciparum CYT b that are associated with atovaquone resistance. Single or double amino acid mutations were detected from parasites that originated from a cloned line and survived various concentrations of atovaquone in vitro. A single amino acid mutation was detected in parasites isolated from a recrudescent patient following atovaquone treatment. These mutations are associated with a 25-to 9,354-fold range reduction in parasite susceptibility to atovaquone. Molecular modeling showed that amino acid mutations associated with atovaquone resistance are clustered around a putative atovaquone-binding site. Mutations in these positions are consistent with a reduced binding affinity of atovaquone for malaria parasite CYT b.The widespread resistance of malaria parasites to standard antimalarial drugs is a serious global health problem. The urgent need for new antimalarial drugs has led to the development of atovaquone (566C80) which, combined with proguanil, has been licensed as Malarone. There is some concern that parasites may develop resistance to Malarone. In one study, 33% of patients treated with atovaquone alone experienced a recrudescence of parasitemia after treatment. These parasites tolerated up to 1,000-fold higher concentrations of atovaquone than did the pretreated parasites (16). Atovaquone-resistant parasites have been readily selected in vitro. Up to 1 in 10 5 parasites became resistant to the drug after having been cultured in the presence of 10 Ϫ8 M atovaquone for 5 weeks (21, 23).Atovaquone has potent blood schizonticidal activity and is also effective against the preerythrocytic (2, 4, 5) and sexual stages (8, 9) of the malaria parasite. It acts by inhibiting mitochondrial electron transport (10) and collapsing mitochondrial membrane potential (25). From these observations and on the basis of its structural similarity to ubiquinol, it has been postulated that atovaquone binds to parasite cytochrome b (CYT b) (31). The inhibitors stigmatellin and 5-n-undecyl-4,7-dioxobenzoxythiazol (UHDBT), which are structurally similar to atovaquone, have been shown to bind at the ubihydroquinone (Q o ) site of CYT b and inhibit electron transport. Single point mutations within the Q o site confer resistance to these inhibitors in a variety of microorganisms (7). Two mutations in close proximity to the Q o site in Pneumocystis carinii are associated with atovaquone prophylaxis failure (33). Atovaquone-resistant Plasmodium yoelii lines have been derived from infected mice treated with suboptimal doses of atovaquone. All resistant lines ...

Artemisinin—the next generation: Efficacies of artemisone against the malaria parasite are substantially greater than those of the current artemisinin “gold standard”, artesunate. Also, in contrast to most current artemisinins it displays low lipophilicity and negligible neuro‐ and cytotoxicity in in vitro and in vivo assays. Thus, the drug offers promise for use in artemisinin‐based combination therapy.

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

In preclinical studies, artemisone (BAY 44-9585), a new artemisinin derivative, was shown to possess enhanced efficacy over artesunate, and it does not possess the neurotoxicity characteristic of the current artemisinins. In a phase I program with double-blind, randomized, placebo-controlled, single and multiple ascending oral-dose studies, we evaluated the safety, tolerability, pharmacokinetics, and ex vivo pharmacodynamic antimalarial activity of artemisone. Single doses (10, 20, 30, 40, and 80 mg) and multiple doses (40 and 80 mg daily for 3 days) of artemisone were administered orally to healthy subjects. Plasma concentrations of artemisone and its metabolites were measured by liquid chromatography/tandem mass spectrometry (LC/MS-MS). Artemisone was well tolerated, with no serious adverse events and no clinically relevant changes in laboratory and vital parameters. The pharmacokinetics of artemisone over the 10-to 80-mg range demonstrated dose linearity. After the single 80-mg dose, artemisone had a geometric mean maximum concentration of 140.2 ng/ml (range, 86.6 to 391.0), a short elimination half-life (t 1/2 ) of 2.79 h (range, 1.56 to 4.88), a high oral clearance of 284.1 liters/h (range, 106.7 to 546.7), and a large volume of distribution of 14.50 liters/kg (range, 3.21 to 51.58). Due to artemisone's short t 1/2 , its pharmacokinetics were comparable after single and multiple dosing. Plasma samples taken after multiple dosing showed marked ex vivo pharmacodynamic antimalarial activities against two multidrug-resistant Plasmodium falciparum lines. Artemisone equivalent concentrations measured by bioassay revealed higher activity than artemisone measured by LC/MS-MS, confirming the presence of active metabolites. Comparable to those of other artemisinin's, artemisone's t 1/2 is well suited for artemisinin-based combination therapy for the treatment of P. falciparum malaria.Artemisinin and its derivatives, artesunate, artemether, and dihydroartemisinin, are the most potent and rapidly acting antimalarial drugs available today (1, 33). They have very high parasite kill rates with a broad stage specificity of antimalarial action and produce a faster clinical and parasitological response than any other class of antimalarial drug (24,32,33). However, a therapeutic drawback of the artemisinins is the high recrudescence rates associated with monotherapy (6). For artemisinins to be effective when given alone, they must be administered for 7 days, but adherence to 7-day regimens may be poor in patients (34).The latest therapeutic approach to combat both malaria infections and the development of drug resistance has been the use of artemisinin-based combination therapy (ACT) administered over 3 days (1,23,24,34). However, 3-day regimens with the rapidly eliminated artemisinins are usually not curative unless they are given in combination with a slowly eliminated drug, such as mefloquine (34). Although the artemisinins have become the drug of choice for the treatment of multidrug-resistant Plasmodium falciparum malari...

A total of 34 analogues of the biguanide PS-15 (5s), a prodrug of the diaminotriazine WR-99210 (8s), have been prepared. Several of them, such as 5b (PS-33) and 5m (PS-26), maintain or exceed the in vivo activity of PS-15 while not requiring the use of highly regulated starting materials. The putative diaminotriazine metabolites of these new analogues (compounds 8) have also been prepared and shown to maintain the activity against resistant P. falciparum strains. The structure-activity relationships of biguanides 5 and putative metabolites 8 are discussed.