Mechanism-Based Model of Parasite Growth and Dihydroartemisinin Pharmacodynamics in Murine Malaria

Murine models are used to study erythrocytic stages of malaria infection, because parasite morphology and development are comparable to those in human malaria infections. Mechanism-based pharmacokinetic-pharmacodynamic (PK-PD) models for antimalarials are scarce, despite their potential to optimize antimalarial combination therapy. The aim of this study was to develop a mechanism-based growth model (MBGM) for Plasmodium berghei and then characterize the parasiticidal effect of dihydroartemisinin (DHA) in murine malaria (MBGM-PK-PD). Stage-specific (ring, early trophozoite, late trophozoite, and schizont) parasite density data from Swiss mice inoculated with Plasmodium berghei were used for model development in S-ADAPT. A single dose of intraperitoneal DHA (10 to 100 mg/kg) or vehicle was administered 56 h postinoculation. The MBGM explicitly reflected all four erythrocytic stages of the 24-hour P. berghei life cycle. Merozoite invasion of erythrocytes was described by a first-order process that declined with increasing parasitemia. An efflux pathway with subsequent return was additionally required to describe the schizont data, thus representing parasite sequestration or trapping in the microvasculature, with a return to circulation. A 1-compartment model with zero-order absorption described the PK of DHA, with an estimated clearance and distribution volume of 1.95 liters h⁻¹ and 0.851 liter, respectively. Parasite killing was described by a turnover model, with DHA inhibiting the production of physiological intermediates (IC₅₀, 1.46 ng/ml). Overall, the MBGM-PK-PD described the rise in parasitemia, the nadir following DHA dosing, and subsequent parasite resurgence. This novel model is a promising tool for studying malaria infections, identifying the stage specificity of antimalarials, and providing insight into antimalarial treatment strategies.     

Treatment of Murine Cerebral Malaria by Artemisone in Combination with Conventional Antimalarial Drugs: Antiplasmodial Effects and Immune Responses

The decreasing effectiveness of antimalarial therapy due to drug resistance necessitates constant efforts to develop new drugs. Artemisinin derivatives are the most recent drugs that have been introduced and are considered the first line of treatment, but there are already indications of Plasmodium falciparum resistance to artemisinins. Consequently, drug combinations are recommended for prevention of the induction of resistance. The research here demonstrates the effects of novel combinations of the new artemisinin derivative, artemisone, a recently described 10-alkylamino artemisinin derivative with improved antimalarial activity and reduced neurotoxicity. We here investigate its ability to kill P. falciparum in a high-throughput in vitro assay and to protect mice against lethal cerebral malaria caused by Plasmodium berghei ANKA when used alone or in combination with established antimalarial drugs. Artemisone effects against P. falciparum in vitro were synergistic with halofantrine and mefloquine, and additive with 25 other drugs, including chloroquine and doxycycline. The concentrations of artemisone combinations that were toxic against THP-1 cells in vitro were much higher than their effective antimalarial concentration. Artemisone, mefloquine, chloroquine, or piperaquine given individually mostly protected mice against cerebral malaria caused by P. berghei ANKA but did not prevent parasite recrudescence. Combinations of artemisone with any of the other three drugs did completely cure most mice of malaria. The combination of artemisone and chloroquine decreased the ratio of proinflammatory (gamma interferon, tumor necrosis factor) to anti-inflammatory (interleukin 10 [IL-10], IL-4) cytokines in the plasma of P. berghei-infected mice. Thus, artemisone in combinations with other antimalarial drugs might have a dual action, both killing parasites and limiting the potentially deleterious host inflammatory response.     

Anthracene-Polyamine Conjugates Inhibit In Vitro Proliferation of Intraerythrocytic Plasmodium falciparum Parasites

Anthracene-polyamine conjugates inhibit the in vitro proliferation of the intraerythrocytic human malaria parasite Plasmodium falciparum, with 50% inhibitory concentrations (IC₅₀s) in the nM to μM range. The compounds are taken up into the intraerythrocytic parasite, where they arrest the parasite cell cycle. Both the anthracene and polyamine components of the conjugates play a role in their antiplasmodial effect.     

Thiostrepton and derivatives exhibit antimalarial and gametocytocidal activity by dually targeting parasite proteasome and apicoplast

Ribosome-targeting antibiotics exert their antimalarial activity on the apicoplast of the malaria parasite, an organelle of prokaryote origin having essential metabolic functions. These antibiotics typically cause a delayed-death phenotype, which manifests in parasite killing during the second replication cycle following administration. As an exception, treatment with the antibiotic thiostrepton results in an immediate killing. We recently demonstrated that thiostrepton and its derivatives interfere with the eukaryotic proteasome, a multimeric protease complex that is important for the degradation of ubiquitinated proteins. Here, we report that the thiostrepton-based compounds are active against chloroquine-sensitive and -resistant Plasmodium falciparum, where they rapidly eliminate parasites before DNA replication. The minor parasite fraction that escapes the fast killing of the first replication cycle is arrested in the schizont stage of the following cycle, displaying a delayed-death phenotype. Thiostrepton further exhibits gametocytocidal activity by eliminating gametocytes, the sexual precursor cells that are crucial for parasite transmission to the mosquito. Compound treatment results in an accumulation of ubiquitinated proteins in the blood stages, indicating an effect on the parasite proteasome. In accordance with these findings, expression profiling revealed that the proteasome is present in the nucleus and cytoplasm of trophozoites, schizonts, and gametocytes. In conclusion, thiostrepton derivatives represent promising candidates for malaria therapy by dually acting on two independent targets, the parasite proteasome and the apicoplast, with the capacity to eliminate both intraerythrocytic asexual and transmission stages of the parasite.     

Mechanisms of artemisinin resistance in the rodent malaria pathogen Plasmodium yoelii

Artemisinin and its derivatives are important new antimalarials which are now used widely in Southeast Asia. Clinically relevant artemisinin resistance has not yet been reported but is likely to occur. In order to understand how the malaria parasite might become resistant to this drug, we studied artemisinin resistance in the murine malaria parasite Plasmodium yoelii. The artemisinin-resistant strain (ART), which is approximately fourfold less sensitive to artemisinin than the sensitive NS strain, accumulated 43% less radiolabeled drug in vitro (P < 0.01). Within the parasite, the drug appeared to react with the same parasite proteins in both strains. The translationally controlled tumor protein, one of the artemisinin target proteins, did not differ between the strains. No DNA sequence difference was found, but the resistant strain was found to express 2.5-fold-more protein than the sensitive strain (P < 0.01). Thus, the phenotype of artemisinin resistance in P. yoelii appears to be multifactorial.     

Plasmodium falciparum-based bioassay for measurement of artemisinin derivatives in plasma or serum

Artemisinin and its derivatives, artesunate and artemether, are rapidly acting antimalarials that are used for the treatment of severe and uncomplicated multidrug-resistant falciparum malaria. To optimize treatment regimens that use this new class of antimalarials, there is a need for readily available and reproducible assays to monitor drug levels closely in patients. A sensitive and reproducible bioassay for the measurement of the concentrations of artemisinin derivatives in plasma and serum is described. By modifying the in vitro drug susceptibility test, it was found that antimalarial activity in plasma or serum containing an unknown concentration of drug could be equated to the known concentrations of dihydroartemisinin (DHA) required to inhibit parasite growth. Dose-response curves for a Plasmodium falciparum clone (clone W2) and DHA were used as a standard for each assay. Assays with plasma or serum spiked with DHA proved to be reproducible (coefficient of variation, 相似文献   

Antimalarial quinolines and artemisinin inhibit endocytosis in Plasmodium falciparum

Endocytosis is a fundamental process of eukaryotic cells and fulfills numerous functions, most notably, that of macromolecular nutrient uptake. Malaria parasites invade red blood cells and during their intracellular development endocytose large amounts of host cytoplasm for digestion in a specialized lysosomal compartment, the food vacuole. In the present study we have examined the effects of artemisinin and the quinoline drugs chloroquine and mefloquine on endocytosis in Plasmodium falciparum. By using novel assays we found that mefloquine and artemisinin inhibit endocytosis of macromolecular tracers by up to 85%, while the latter drug also leads to an accumulation of undigested hemoglobin in the parasite. During 5-h incubations, chloroquine inhibited hemoglobin digestion but had no other significant effect on the endocytic pathway of the parasite, as assessed by electron microscopy, the immunofluorescence localization of hemoglobin, and the distribution of fluorescent and biotinylated dextran tracers. By contrast, when chloroquine was added to late ring stage parasites, followed by a 12-h incubation, macromolecule endocytosis was inhibited by more than 40%. Moreover, there is an accumulation of transport vesicles in the parasite cytosol, possibly due to a disruption in vacuole-vesicle fusion. This fusion block is not observed with mefloquine, artemisinin, quinine, or primaquine but is mimicked by the vacuole alkalinizing agents ammonium chloride and monensin. These results are discussed in the light of present theories regarding the mechanisms of action of the antimalarials and highlight the potential use of drugs in manipulating and studying the endocytic pathway of malaria parasites.     

Antibodies that Inhibit Malaria Merozoite Surface Protein–1 Processing and Erythrocyte Invasion Are Blocked by Naturally Acquired Human Antibodies

Merozoite surface protein–1 (MSP-1) of the human malaria parasite Plasmodium falciparum undergoes at least two endoproteolytic cleavage events during merozoite maturation and release, and erythrocyte invasion. We have previously demonstrated that mAbs which inhibit erythrocyte invasion and are specific for epitopes within a membrane-proximal, COOH-terminal domain of MSP-1 (MSP-1₁₉) prevent the critical secondary processing step which occurs on the surface of the extracellular merozoite at around the time of erythrocyte invasion. Certain other anti–MSP-1₁₉ mAbs, which themselves inhibit neither erythrocyte invasion nor MSP-1 secondary processing, block the processing-inhibitory activity of the first group of antibodies and are termed blocking antibodies. We have now directly quantitated antibody-mediated inhibition of MSP-1 secondary processing and invasion, and the effects on this of blocking antibodies. We show that blocking antibodies function by competing with the binding of processing-inhibitory antibodies to their epitopes on the merozoite. Polyclonal rabbit antibodies specific for certain MSP-1 sequences outside of MSP-1₁₉ also act as blocking antibodies. Most significantly, affinity-purified, naturally acquired human antibodies specific for epitopes within the NH₂-terminal 83-kD domain of MSP-1 very effectively block the processing-inhibitory activity of the anti-MSP-1₁₉ mAb 12.8. The presence of these blocking antibodies also completely abrogates the inhibitory effect of mAb 12.8 on erythrocyte invasion by the parasite in vitro. Blocking antibodies therefore (a) are part of the human response to malarial infection; (b) can be induced by MSP-1 structures unrelated to the MSP-1₁₉ target of processing-inhibitory antibodies; and (c) have the potential to abolish protection mediated by anti–MSP-1₁₉ antibodies. Our results suggest that an effective MSP-1₁₉–based falciparum malaria vaccine should aim to induce an antibody response that prevents MSP-1 processing on the merozoite surface.     

Current status of malaria chemotherapy and the role of pharmacology in antimalarial drug research and development

Antimalarial drugs have played a mainstream role in controlling the spread of malaria through the treatment of patients infected with the plasmodial parasites and controlling its transmissibility. The inadequate armory of drugs in widespread use for the treatment of malaria, development of strains resistant to currently used antimalarials, and the lack of affordable new drugs are the limiting factors in the fight against malaria. In addition, other problems with some existing agents include unfavorable pharmacokinetic properties and adverse effects/toxicity. These factors underscore the continuing need of research for new classes of antimalarial agents, and a re-examination of the existing antimalarial drugs that may be effective against resistant strains. In recent years, major advances have been made in the pharmacology of several antimalarial drugs both in pharmacokinetics and pharmacodynamics aspects. These include the design, development, and optimization of appropriate dosage regimens of antimalarials, basic knowledge in metabolic pathways of key antimalarials, as well as the elucidation of mechanisms of action and resistance of antimalarials. Pharmacologists have been working in close collaboration with scientists in other disciplines of science/biomedical sciences for more understanding on the biology of the parasite, host, in order to exploit rational design of drugs. Multiple general approaches to the identification of new antimalarials are being pursued at this time. All should be implemented in parallel with focus on the rational development of new agents directed against newly identified parasite targets. With major advances in our understanding of malaria parasite biology coupled with the completion of the malaria genome, has presented exciting opportunities for target-based antimalarial drug discovery.     

Terpenes arrest parasite development and inhibit biosynthesis of isoprenoids in Plasmodium falciparum

Development of new drugs is one of the strategies for malaria control. The biosynthesis of several isoprenoids in Plasmodium falciparum was recently described. Interestingly, some intermediates and final products biosynthesized by this pathway in mammals differ from those biosynthesized in P. falciparum. These facts prompted us to evaluate various terpenes, molecules with a similar chemical structure to the intermediates of the isoprenoids pathway, as potential antimalarial drugs. Different terpenes and S-farnesylthiosalicylic acid were tested on cultures of the intraerythrocytic stages of P. falciparum, and the 50% inhibitory concentrations for each one were found: farnesol, 64 microM; nerolidol, 760 nM; limonene, 1.22 mM; linalool, 0.28 mM; and S-farnesylthiosalicylic acid, 14 microM. All the terpenes tested inhibited dolichol biosynthesis in the trophozoite and schizont stages when [1-(n)-(3)H]farnesyl pyrophosphate triammonium salt ([(3)H]FPP) was used as precursor. Farnesol, nerolidol, and linalool showed stronger inhibitory activity on the biosynthesis of the isoprenic side chain of the benzoquinone ring of ubiquinones in the schizont stage. Treatment of schizont stages with S-farnesylthiosalicylic acid led to a decrease in intensity of the band corresponding a p21(ras) protein. The inhibitory effect of terpenes and S-farnesylthiosalicylic acid on the biosynthesis of both dolichol and the isoprenic side chain of ubiquinones and the isoprenylation of proteins in the intraerythrocytic stages of P. falciparum appears to be specific, because overall protein biosynthesis was not affected. Combinations of some terpenes or S-farnesylthiosalicylic acid tested in this work with other antimalarial drugs, like fosmidomycin, could be a new strategy for the treatment of malaria.     

Artemisinin-Resistant Plasmodium falciparum Parasites Exhibit Altered Patterns of Development in Infected Erythrocytes

Artemisinin derivatives are used in combination with other antimalarial drugs for treatment of multidrug-resistant malaria worldwide. Clinical resistance to artemisinin recently emerged in southeast Asia, yet in vitro phenotypes for discerning mechanism(s) of resistance remain elusive. Here, we describe novel phenotypic resistance traits expressed by artemisinin-resistant Plasmodium falciparum. The resistant parasites exhibit altered patterns of development that result in reduced exposure to drug at the most susceptible stage of development in erythrocytes (trophozoites) and increased exposure in the most resistant stage (rings). In addition, a novel in vitro delayed clearance assay (DCA) that assesses drug effects on asexual stages was found to correlate with parasite clearance half-life in vivo as well as with mutations in the Kelch domain gene associated with resistance (Pf3D7_1343700). Importantly, all of the resistance phenotypes were stable in cloned parasites for more than 2 years without drug pressure. The results demonstrate artemisinin-resistant P. falciparum has evolved a novel mechanism of phenotypic resistance to artemisinin drugs linked to abnormal cell cycle regulation. These results offer insights into a novel mechanism of drug resistance in P. falciparum and new tools for monitoring the spread of artemisinin resistance.     

Broad-Spectrum Antimalarial Activity of Peptido Sulfonyl Fluorides,a New Class of Proteasome Inhibitors

Despite declining numbers of cases and deaths, malaria remains a major public health problem in many parts of the world. Today, case management relies heavily on a single class of antimalarial compounds: artemisinins. Hence, development of resistance against artemisinins may destroy current malaria control strategies. Beyond malaria control are elimination and eradication programs that will require drugs with good activity against acute infection but also with preventive and transmission-blocking properties. Consequently, new antimalarials are needed not only to ensure malaria control but also for elimination and eradication efforts. In this study, we introduce peptido sulfonyl fluorides (PSF) as a new class of compounds with antiplasmodial activity. We show that PSF target the plasmodial proteasome and act on all asexual stages of the intraerythrocytic cycle and on gametocytes. PSF showed activities at concentrations as low as 20 nM against multidrug-resistant and chloroquine-sensitive Plasmodium falciparum laboratory strains and clinical isolates from Gabon. Structural requirements for activity were identified, and cytotoxicity in human HeLa or HEK 293 cells was low. The lead PSF PW28 suppressed growth of Plasmodium berghei in vivo but showed signs of toxicity in mice. Considering their modular structure and broad spectrum of activity against different stages of the plasmodial life cycle, proteasome inhibitors based on PSF have a great potential for further development as preclinical candidate compounds with improved species-specific activity and less toxicity.     

Polymorphism of a high molecular weight schizont antigen of the human malaria parasite Plasmodium falciparum

Intraspecies antigenic diversity in the blood stages of the human malaria parasite Plasmodium falciparum was investigated using a collection of murine monoclonal antibodies and clones of the parasite. The results were as follows: (a) The schizont and merozoite stages of the parasite express on their surface clonally restricted antigens detectable by strain-specific antibodies in indirect immunofluorescence tests. (b) These restricted antigens are phenotypically stable characteristics of clones grown in vitro. (c) The molecules carrying the specific antigens were isolated by immunoprecipitation and were found to be parasite proteins ranging in size from Mr 190,000 to 200,000 between clones. (d) Comparative immunoprecipitation and peptide mapping of these molecules showed that each parasite clone expresses a protein that is antigenically and structurally distinct from the equivalent products of several other clones. (e) The different clonal products are, however, immunologically interrelated, since they possess determinants in common with all tested isolates of the parasite. (f) These polymorphic molecules are closely related to a previously described schizont protein of P. falciparum that is posttranslationally cleaved into fragments located on the merozoite surface. These findings show the existence of a family of related polymorphic schizont antigens (PSA) of P. falciparum, whose expression is clonally restricted, and indicate that these proteins have regions of constant and variable antigenicity. We propose that a system of immunological classification of the parasite can be developed based on the polymorphism of these proteins.     

Cysteamine,the Molecule Used To Treat Cystinosis,Potentiates the Antimalarial Efficacy of Artemisinin

Malaria continues to be a major threat to global health. Artemisinin combination therapy (ACT) is the recommended treatment for clinical malaria; however, recent reports of parasite resistance to artemisinin in certain areas where malaria is endemic have stressed the need for developing more efficacious ACT. We report that cysteamine (Cys), the aminothiol used to treat nephropathic cystinosis in humans, strongly potentiates the efficacy of artemisinin against the Plasmodium parasite in vivo. Using a mouse model of infection with Plasmodium chabaudi AS, we observe that Cys dosing used to treat cystinosis in humans can strongly potentiate (by 3- to 4-fold) the antimalarial properties of the artemisinin derivatives artesunate and dihydroartemisinin. Addition of Cys to suboptimal doses of artemisinin delays the appearance of blood parasitemia, strongly reduces the extent of parasite replication, and significantly improves survival in a model of lethal P. chabaudi infection. Cys, the natural product of the enzyme pantetheinase, has a history of safe use for the clinical management of cystinosis. Our findings suggest that Cys could be included in novel ACTs to improve efficacy against Plasmodium parasite replication, including artemisinin-resistant isolates. Future work will include clinical evaluation of novel Cys-containing ACTs and elucidation of the mechanism underlying the potentiation effect of Cys.Malaria still represents a huge global health burden, with 500 to 600 million clinical cases resulting in 1 to 2 million deaths annually (www.who.int). The impact is particularly devastating in resource-poor countries of sub-Saharan Africa and Southeast Asia, where malaria is endemic and access to appropriate antimalarial drugs can be limited. Furthermore, as a result of widespread use and misuse of antimalarial drugs, the Plasmodium parasite has developed resistance to commonly used drugs, such as chloroquine, mefloquine, and sulfadoxine-pyrimethamine (23). Artemisinin is a sesquiterpene lactone endoperoxide (extracted from Artemisia annua) with potent antimalarial activity, and artemisinin combination therapy (ACT) is the strategy recommended by the World Health Organization for clinical care of malaria (www.who.int). However, recent reports of delayed parasite clearance times following standard ACT treatment in patients from the Pailin province of Cambodia have suggested emergence of resistance to artemisinin in the Plasmodium parasite (12). These alarming reports have highlighted the urgency for the development of novel and more effective chemotherapeutic strategies, including modification of current ACT composition.We have used a mouse model of blood-stage infection with P. chabaudi AS to identify novel genetic factors affecting host response to malaria. We mapped two major loci, Char4 (chromosome 3) and Char9 (chromosome 10), that control differential responses of innately susceptible A/J (high parasitemia, high mortality) and uniquely resistant AcB55 (low parasitemia, high rate of survival) mice to P. chabaudi infection. Char4-associated resistance was found to result from a loss-of-function mutation (I90N) in the erythrocyte enzyme pyruvate kinase (Pklr) (28). Subsequently, we observed that erythrocytes heterozygous or homozygous for defective variants in human PKLR are resistant to P. falciparum infection ex vivo (reduced parasite replication and increased phagocytosis), validating our initial observations with the mouse (5). On the other hand, we established that Char9-associated susceptibility is caused by a loss of activity in the pantetheinase enzyme encoded by the Vnn1/Vnn3 genes (27). Pantetheinase is an amidohydrolase that hydrolyzes pantetheine (product of coenzyme A degradation) to pantothenic acid (also called pantothenate or vitamin B5) and the small aminothiol cysteamine (Cys) (NH₂-CH₂-CH₂-SH₂) (13). Strikingly, exogenous administration of cysteamine to pantetheinase-deficient malaria-susceptible A/J mice reversed the phenotype by reducing blood parasitemia levels and increasing survival time (26, 27).Cysteamine, in the form of cysteamine bitartrate (Cystagon), is currently used for the clinical treatment of nephropathic cystinosis in humans (21). Cystinosis is a lysosomal storage disease caused by mutations in the lysosomal cystine transporter cystinosin, which result in lysosomal accumulation of cystine and concomitant cytopathic effects (3). Cysteamine depletes cells of cystine in vitro and in vivo and dramatically improves the prognosis for children with cystinosis. Using a mouse model of P. chabaudi infection, we have investigated the potential antimalarial activity of cysteamine at pharmacological doses that mimic those currently used in patients suffering from cystinosis. We have also tested the potential for cysteamine to enhance the antimalarial activity of known drugs, including artemisinin derivatives. Our results show that cysteamine can strongly potentiate and synergize with artemisinin derivatives to reduce blood parasitemia and rescue lethality in P. chabaudi-infected mice.     

Biosynthesis and processing of a Plasmodium falciparum schizont antigen recognized by immune serum and a monoclonal antibody

Stage-specific protein synthesis by the erythrocytic forms of the malaria parasite Plasmodium falciparum was investigated by pulse labeling synchronous parasite cultures with [35S]methionine at 6-h intervals during a complete 48-h developmental cycle. About 40 labeled parasite proteins could be immunoprecipitated with human immune serum, and most of these were associated with the schizont stage of development. In particular, one schizont protein was a 195,000-mol wt species against which a murine monoclonal antibody was produced. This monoclonal antibody, 89.1 reacted with the parasite membrane in schizonts and also with the surface of free merozoites in the indirect immunofluorescence test. In addition to the 195,000-mol wt protein, antibody 89.1 immunoprecipitated a series of lower-molecular weight polypeptides from extracts of labeled asynchronous P. falciparum parasite cultures. These were shown to be related to the 195,000-mol wt protein by peptide mapping. Pulse-chase labeling of synchronized cultures, and immunoprecipitation with antibody 89.1, showed that specific processing of the 195,000-mol wt polypeptide to the lower- molecular-weight products in concomitant with schizont maturation and merozoite release. It is suggested that this P. falciparum protein may be analogous to a similarly processed 230,000-mol wt protective antigen of the rodent malaria parasite, P. yoelii.     

Potent Plasmodium falciparum Gametocytocidal Activity of Diaminonaphthoquinones,Lead Antimalarial Chemotypes Identified in an Antimalarial Compound Screen

Forty percent of the world''s population is threatened by malaria, which is caused by Plasmodium parasites and results in an estimated 200 million clinical cases and 650,000 deaths each year. Drug resistance has been reported for all commonly used antimalarials and has prompted screens to identify new drug candidates. However, many of these new candidates have not been evaluated against the parasite stage responsible for transmission, gametocytes. If Plasmodium falciparum gametocytes are not eliminated, patients continue to spread malaria for weeks after asexual parasite clearance. Asymptomatic individuals can also harbor gametocyte burdens sufficient for transmission, and a safe, effective gametocytocidal agent could also be used in community-wide malaria control programs. Here, we identify 15 small molecules with nanomolar activity against late-stage gametocytes. Fourteen are diaminonaphthoquinones (DANQs), and one is a 2-imino-benzo[d]imidazole (IBI). One of the DANQs identified, SJ000030570, is a lead antimalarial candidate. In contrast, 94% of the 650 compounds tested are inactive against late-stage gametocytes. Consistent with the ineffectiveness of most approved antimalarials against gametocytes, of the 19 novel compounds with activity against known anti-asexual-stage targets, only 3 had any strong effect on gametocyte viability. These data demonstrate the distinct biology of the transmission stages and emphasize the importance of screening for gametocytocidal activity. The potent gametocytocidal activity of DANQ and IBI coupled with their efficacy against asexual parasites provides leads for the development of antimalarials with the potential to prevent both the symptoms and the spread of malaria.     

Antibodies against merozoite surface protein (MSP)-1(19) are a major component of the invasion-inhibitory response in individuals immune to malaria.

Antibodies that bind to antigens expressed on the merozoite form of the malaria parasite can inhibit parasite growth by preventing merozoite invasion of red blood cells. Inhibitory antibodies are found in the sera of malaria-immune individuals, however, the specificity of those that are important to this process is not known. In this paper, we have used allelic replacement to construct a Plasmodium falciparum parasite line that expresses the complete COOH-terminal fragment of merozoite surface protein (MSP)-1(19) from the divergent rodent malaria P. chabaudi. By comparing this transfected line with parental parasites that differ only in MSP-1(19), we show that antibodies specific for this domain are a major component of the inhibitory response in P. falciparum-immune humans and P. chabaudi-immune mice. In some individual human sera, MSP-1(19) antibodies dominated the inhibitory activity. The finding that antibodies to a small region of a single protein play a major role in this process has important implications for malaria immunity and is strongly supportive of further understanding and development of MSP-1(19)-based vaccines.     

In Vitro Susceptibility of Plasmodium vivax to Antimalarials in Colombia

The in vitro susceptibilities of 30 isolates of Plasmodium vivax to a number of antimalarials (chloroquine [CQ], mefloquine, amodiaquine, quinine, and artesunate [AS]) were evaluated. The isolates came from the region of Urabá in Colombia, in which malaria is endemic, and were evaluated by the schizont maturation test. The 50% inhibitory concentration (IC₅₀) was 0.6 nM (95% confidence interval [CI], 0.3 to 1.0 nM) for artesunate, 8.5 nM (95% CI, 5.6 to 13.0 nM) for amodiaquine, 23.3 nM (95% CI, 12.4 to 44.1 nM) for chloroquine, 55.6 nM (95% CI, 36.8 to 84.1 nM) for mefloquine, and 115.3 nM (95% CI, 57.7 to 230.5 nM) for quinine. The isolates were classified according to whether the initial parasites were mature or immature trophozoites (Tfz). It was found that the IC₅₀s for chloroquine and artesunate were significantly different in the two aforementioned groups (P < 0.001). The IC₅₀s of CQ and AS were higher in the isolates from mature Tfz (CQ, 39.3 nM versus 17 nM; AS, 1.4 nM versus 0.3 nM), and 10% of the isolates showed lower susceptibilities to one of the antimalarial drugs, 13.3% to two antimalarial drugs, and 3.3% to more than three antimalarial drugs. It should be highlighted that despite the extensive use of chloroquine in Colombia, P. vivax continues to be susceptible to antimalarials. This is the first report, to our knowledge, showing in vitro susceptibilities of P. vivax isolates to antimalarials in Colombia.     

In Vitro and In Vivo Antimalarial Efficacies of Optimized Tetracyclines

With increasing resistance to existing antimalarials, there is an urgent need to discover new drugs at affordable prices for countries in which malaria is endemic. One approach to the development of new antimalarial drugs is to improve upon existing antimalarial agents, such as the tetracyclines. Tetracyclines exhibit potent, albeit relatively slow, action against malaria parasites, and doxycycline is used for both treatment (with other agents) and prevention of malaria. We synthesized 18 novel 7-position modified tetracycline derivatives and screened them for activity against cultured malaria parasites. Compounds with potent in vitro activity and other favorable drug properties were further tested in a rodent malaria model. Ten compounds inhibited the development of cultured Plasmodium falciparum with a 50% inhibitory concentration (IC₅₀) after 96 h of incubation of <30 nM, demonstrating activity markedly superior to that of doxycycline (IC₅₀ at 96 h of 320 nM). Most compounds showed little mammalian cell cytotoxicity and no evidence of in vitro phototoxicity. In a murine Plasmodium berghei model, 13 compounds demonstrated improved activity relative to that of doxycycline. In summary, 7-position modified tetracyclines offer improved activity against malaria parasites compared to doxycycline. Optimized compounds may allow lower doses for treatment and chemoprophylaxis. If safety margins are adequate, dosing in children, the group at greatest risk for malaria in countries in which it is endemic, may be feasible.