A Drug Repurposing Approach Reveals Targetable Epigenetic Pathways in Plasmodium vivax Hypnozoites.

Radical cure of Plasmodium vivax malaria must include elimination of quiescent 'hypnozoite' forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets for hypnozoites, we screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library and a collection of epigenetic inhibitors against P. vivax liver stages. From both libraries, we identified inhibitors targeting epigenetics pathways as selectively active against P. vivax and P. cynomolgi hypnozoites. These include DNA methyltransferase (DNMT) inhibitors as well as several inhibitors targeting histone post-translational modifications. Immunofluorescence staining of Plasmodium liver forms showed strong nuclear 5-methylcystosine signal, indicating liver stage parasite DNA is methylated. Using bisulfite sequencing, we mapped genomic DNA methylation in sporozoites, revealing DNA methylation signals in most coding genes. We also demonstrated that methylation level in proximal promoter regions as well as in the first exon of the genes may affect, at least partially, gene expression in P. vivax. The importance of selective inhibitors targeting epigenetic features on hypnozoites was validated using MMV019721, an acetyl-CoA synthetase inhibitor that affects histone acetylation and was previously reported as active against P. falciparum blood stages. In summary, our data indicate that several epigenetic mechanisms are likely modulating hypnozoite formation or persistence and provide an avenue for the discovery and development of improved radical cure antimalarials.

The presence of leukocytes in ex vivo assays significantly increases the 50-percent inhibitory concentrations of artesunate and chloroquine against Plasmodium vivax and Plasmodium falciparum.

Plasmodium species ex vivo sensitivity assay protocols differ in the requirement for leukocyte removal before culturing. This study shows that the presence of leukocytes significantly increases the 50% inhibitory concentration (IC50) of P. vivax and P. falciparum to artesunate and chloroquine relative to results with the paired leukocyte-free treatment. Although leukocyte removal is not an essential requirement for the conduct of ex vivo assays, its use has important implications for the interpretation of temporal and spatial antimalarial sensitivity data.

The effects of alpha1-acid glycoprotein on the reversal of chloroquine resistance in Plasmodium falciparum.

An in-vitro model based on the semi-automated microdilution technique has been developed for selecting compounds that might be used clinically for the reversal of chloroquine resistance. This was used initially to test the susceptibility of Plasmodium falciparum clone W2 to chloroquine (CQ). The model was then employed to investigate the effects of each of four resistance-reversing agents (verapamil, desipramine, chlorpheniramine and promethazine, at 1 microM) on this parasite's susceptibility to CQ, with and without alpha(1)-acid glycoprotein (AGP), at a patho-physiological concentration (1.25 g/litre), in the culture medium. In the absence of AGP, each of the resistance-reversing agents reduced the median inhibitory concentrations of CQ by 82%-97%, from a baseline value of about 94 ng/ml. In the presence of AGP, however, most of the resistance-reversing agents had much less effect. There appears to be competitive interaction between CQ, the resistance-reversing agents and AGP. The binding kinetics between CQ, resistance-reversing agents, AGP and other plasma proteins will clearly need to elucidated if clinically effective resistance-reversing agents are to be selected in vitro.

Linkage disequilibrium between two distinct loci in chromosomes 5 and 7 of Plasmodium falciparum and in vivo chloroquine resistance in Southwest Nigeria.

Chloroquine (CQ) resistance in Plasmodium falciparum is associated with polymorphisms in loci on pfcrt and pfmdr1 genes. In this study, we determined the association and linkage disequilibrium between in vivo CQ resistance and P. falciparum polymorphisms in pfcrt gene at codon 76 and pfmdr1 gene at codon 86 in isolates obtained from 111 children with acute uncomplicated falciparum malaria in Nigeria. Patients were treated with standard dosage of CQ and followed up for 28 days. Filter paper samples were collected at enrollment and during follow-up for parasites genotypes and identification of pfcrt and pfmdr1 mutations. Association and linkage disequilibrium between mutant pfcrtT76 and pfmdr1Y86 alleles in pretreatment isolates of P. falciparum was determined. Fifty-five out of the 111 patients (49.5%) failed treatment. Single mutant pfcrtT76 or pfmdr1Y86 alleles were found in 55 out of 111 P. falciparum isolates screened at enrollment. Of these 55 isolates, the mutant pfcrtT76 and pfmdr1Y86 alleles were found in 84%. Both mutant pfcrtT76 (p=0.0196) and pfmdr1Y86 (p=0.000042) alleles were associated with in vivo CQ resistance. In addition, the mutant pfcrtT76 (p=0.047) and pfmdr1Y86 (p=0.006) alleles were significantly selected by CQ in patients who failed treatment. Association analysis between paired single alleles at pfcrt and pfmdr1 loci showed a significant association (p=0.0349 and chi(2)=4.45) between the pfcrt T76 allele on chromosome 7 and the pfmdr1Y86 allele on chromosome 5 and that these two mutant alleles were in linkage disequilibrium (p=0.000, D'=0.64, and r(2)=0.28). Considering the high level of CQ resistance and drug use in the study area, the observed linkage disequilibrium between the mutant pfcrtT76 and pfmdr1Y86 alleles is maintained epistatically through directional CQ selective pressure.

Association between mutations in Plasmodium falciparum chloroquine resistance transporter and P. falciparum multidrug resistance 1 genes and in vivo amodiaquine resistance in P. falciparum malaria-infected children in Nigeria.

This study investigated the association between Plasmodium falciparum chloroquine resistance transporter (pfcrt) T76 and P. falciparum multidrug resistance gene 1 (pfmdr1) Y86 alleles and in vivo amodiaquine (AQ) resistance, as well as the clearance of parasites harboring these two alleles in children treated with AQ in southwest Nigeria. One hundred one children with acute uncomplicated P. falciparum malaria infections were treated with the standard dosage of AQ and followed-up for 28 days. Blood samples were collected on filter paper samples at enrollment and during follow-up for identification of parasite genotypes and pfcrt and pfmdr1 mutations using polymerase chain reaction and restriction fragment length polymorphism approaches. Parasitologic assessment of response to treatment showed that 87% and 13% (RI) of patients were cured and failed treatment, respectively. Although infections in patients were polyclonal (as determined by merozoite surface protein 2 genotyping), the presence of both mutants pfcrtT76 and pfmdr1Y86 alleles in parasites is associated with in vivo AQ resistance (odds ratio = 7.58, 95% confidence interval = 1.58-36.25, P = 0.006) and is selected by the drug in children who failed AQ treatment. Treatment failure with the combination of mutant pfcrtT76 and pfmdr1Y86 alleles as well as the ability of patients to clear these resistant parasites is dependent on age, suggesting a critical role of host immunity in clearing AQ-resistant P. falciparum. The combination of mutant pfcrtT76 and pfmdr1Y86 alleles may be useful markers for monitoring the development and spread of AQ resistance, when combining this drug with other antimalarials for treatment of malaria in Africa.

Polymorphisms in Plasmodium falciparum dhfr and dhps genes and age related in vivo sulfadoxine-pyrimethamine resistance in malaria-infected patients from Nigeria.

Mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes have been used as means to predict treatment failure to sulfadoxine-pyrimethamine (SP) and for monitoring/surveillance of resistance to the drug in many areas where malaria is endemic. However, patients responses to treatment are significantly dependent on factors like host immunity profile of treated patients. In order to investigate the relationship between molecular markers of SP resistance, host immunity and clinical outcome, the association between pre-treatment dhfr and dhps genotypes, age and treatment outcomes was evaluated in 109 children treated with SP for acute uncomplicated malaria in Ibadan, Nigeria. Seventy-three percent of the children were cured with the drug, while 27% failed treatment after 28 days of follow-up. All children infected with parasites harboring less than two dhfr/dhps mutations were cured with SP. The dhfr triple (Asn-108/Ile-51/Arg-59) mutants or the dhps double mutants (Gly-437/Glu-540) were independently associated with SP treatment failure in children aged less than 5 years, but not in older children. The dhfr and dhps quintuple mutant (dhfr triple mutant+dhps double mutant) was the genotype most strongly associated with SP treatment failure (OR=24.72, 95%CI=8.24-74.15) in both younger and older children.

Molecular analysis of Plasmodium falciparum recrudescent malaria infections in children treated with chloroquine in Nigeria.

Parasite genotyping by a polymerase chain reaction was used to distinguish recrudescent from newly acquired Plasmodium falciparum infections in 50 of 160 Nigerian children taking part in a chloroquine efficacy study in Ibadan, Nigeria. A finger prick blood sample was taken from each child before and after treatment to identify recrudescent parasites. By investigating allelic variation in three polymorphic antigen loci, merozoite surface protein-1 (MSP-1), MSP-2, and glutamate-rich protein (GLURP), we determined parasite diversity in the population and in the infected host. DNA from pretreatment and post-treatment samples from 47 of the 50 patients who failed therapy was successfully amplified by the PCR. The MSP-1, MSP-2, and GLURP genotypes in all samples showed extensive diversity, indicating polyclonal infections. The average number of clones per infection in pre-treatment sample was 2.5 with MSP-1, 4.9 with MSP-2, and 2 with GLURP. The extent of multiplicity decreased significantly (P = 0.016) in posttreatment samples. Multiplicity of infection and initial parasite density were not age dependent. Comparison of the variant alleles in pretreatment and post-treatment samples of each patient indicates that 26 of the 47 children had genuinely recrudescent disease. Conversely, post-treatment samples from five children showed completely new genotypes, indicating either a previously sequestered population of parasites or a newly acquired infection. Overall, this study has shown the diversity and complexity of P. falciparum population in Ibadan, Nigeria. The study has also shown the dynamics of P. falciparum infections in this population before and after chloroquine treatment in an area of high malaria transmission.

Point mutations in the pfcrt and pfmdr-1 genes of Plasmodium falciparum and clinical response to chloroquine, among malaria patients from Nigeria.

Chloroquine (CQ) resistance in Plasmodium falciparum has been associated with specific point mutations in the pfcrt and pfmdr-1 genes. In the present study, 30 children aged 1-12 years, who were all suffering from acute, uncomplicated, P. falciparum malaria in Ibadan, Nigeria, were evaluated to assess the association between these mutations and clinical outcome following treatment with CQ. The parasites, in blood samples collected pre-treatment and, in those who failed treatment, on the day symptoms re-occurred post-treatment, were genotyped using the polymorphic MSP1, MSP2 and GLURP loci and PCR-RFLP. The results showed that, pre-treatment, all 30 patients had polyclonal infections, the mean numbers of P. falciparum clones detected per infection being 2.6 with MSP1, 4.2 with MSP2 and 2.8 with GLURP. The T76 allele of pfcrt and the Y86 allele of pfmdr-1 were found in 53% and 40%, respectively, of the pre-treatment samples from the 15 patients who failed CQ treatment, but the Y1246 mutation in pfmdr-1 was never detected. Although the parasites from the two patients with high-grade (RIII) resistance to CQ had both of these point mutations, the presence of the T76 allele of pfcrt or the Y86 allele of pfmdr-1 (considered individually) could not be used to predict treatment outcome. However, a high frequency of clonal multiplicity may confound attempts to associate the point mutations in pfcrt or pfmdr-1 with clinical response to CQ. It remains unclear whether the present results represent the characteristics of the predominant parasite populations in the study area. Further studies are needed before the strength of the association between the point mutations identified as markers of drug resistance and clinical outcome can be accurately evaluated, in this and other regions of intense transmission.

Efficacy comparison of intravenous artelinate and artesunate in Plasmodium berghei-infected Sprague-Dawley rats.

This paper reports the comparative antimalarial efficacy of intravenous artelinate and artesunate in rats. Prior to efficacy experiments, a Plasmodium berghei-Sprague-Dawley rat model of malaria was developed, in which the clearance effects of intravenous drugs could be readily compared. In efficacy experiments, groups of P. berghei-infected rats were given 3 daily intravenous treatments of artelinate or artesunate at molar equivalent dose rates (total of 0-191.2 micromoles/kg). Artelinate was superior to artesunate in terms of clearance (100% clearance dose of 95.6 micromoles/kg (40 mg/kg) versus 191.2 micromoles/ kg for AS (73.4 mg/kg)) and parasite clearance time (1.7 +/- 0.5 days for AL versus 2.7 +/- 0.5 days for AS at a dose rate of 191.2 micromoles/kg, P < 0.01). No frank clinical toxicity was observed, though both artesunate and artelinate induced dose-related vascular necrosis at the site of injection. The necrosis was less severe and reversible when the drugs were administered via femoral, rather than tail/foot veins. The data suggest that the P. berghei-7-week-old Sprague-Dawley rat model of malaria is reproducible and useful for assessing the efficacy of antimalarials and that artelinate is at least as potent, and safe, as artesunate, the leading clinical treatment for severe malaria.

Neurotoxicity and efficacy of arteether related to its exposure times and exposure levels in rodents.

The neurotoxicity of beta-arteether (AE) is related to drug accumulation in blood due to slow and prolonged absorption from the intramuscular injection sites. In this efficacy and toxicity study of AE, the traditional sesame oil vehicle was replaced with cremophore to decrease the accumulation and toxicity of AE. Dihydroartemisinin (DQHS), a more toxic and active metabolite of AE, was also analyzed. When administered at a daily dosage of 25 mg/kg for seven days, blood accumulation of AE with sesame oil (AESO) was used had a 7.5-fold higher area under the curve (AUC) (on last versus first day dosing), while AE with cremophore (AECM) had only a 1.8-fold higher AUC. Although the accumulation of AECM was greatly reduced, its total exposure level (46.29 microg x h/ml) was 2.7-fold higher than with AESO (16.92 microg x h/ml) due to a higher bioavailability of AECM (74.5%) compared with AESO (20.3%). Total exposure time (calculated at over the minimal detected neurotoxicity level of 41.32 ng/ml) of AECM was 103 hours during the whole treatment period (192 hours), which was more than one-third (37%) less than with AESO (162 hours). Similar pharmacokinetic results were also shown with the active metabolite, DQHS. Anorexia and gastrointestinal toxicity with AESO were significantly more severe than with AECM (P < 0.001). Histopathologic examination of the brain demonstrated neurotoxic changes; the AESO rat group was significantly more severe than the AECM rat group. The brain injury scores with AECM were mild to moderate (2.3-3.0), and with AESO they were moderate to severe (3.0-4.7) on day 7 and day 10, respectively. In addition, the results of a 50% cure dose (CD50) against Plasmodium berghei in mice were 34.1 mg/kg for AESO and 14.2 mg/kg for AECM, indicating a significant higher efficacy was found in the AECM animals. Toxicity and efficacy of DQHS were also dependent on its exposure time and level, which was the same as its parent drug (AE). In conclusion, following the seven-day treatment in rats, AE and DQHS exposure time and level varied based on the vehicle used. The extension of drug exposure time and the low peak level of AE and DQHS were more associated with severe neurotoxicity and lower antimalarial efficacy, whereas the high level and short exposure time of AE and DQHS resulted in higher efficacy and milder toxicity.

Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with pfcrt polymorphism in Papua New Guinea and South America.

The mechanistic basis for chloroquine resistance (CQR) in Plasmodium falciparum recently has been linked to the polymorphic gene pfcrt. Alleles associated with CQR in natural parasite isolates harbor threonine (T), as opposed to lysine (K) at amino acid 76. P. falciparum CQR strains of African and Southeast Asian origin carry pfcrt alleles encoding an amino acid haplotype of CVIET (residues 72-76), whereas most South American CQR strains studied carry an allele encoding an SVMNT haplotype; chloroquine-sensitive strains from malarious regions around the world carry a CVMNK haplotype. Upon investigating the origin of pfcrt alleles in Papua New Guinean (PNG) P. falciparum we found either the chloroquine-sensitive-associated CVMNK or CQR-associated SVMNT haplotypes previously seen in Brazilian isolates. Remarkably we did not find the CVIET haplotype observed in CQR strains from Southeast Asian regions more proximal to PNG. Further we found a previously undescribed CQR phenotype to be associated with the SVMNT haplotype from PNG and South America. This CQR phenotype is significantly less responsive to verapamil chemosensitization compared with the effect associated with the CVIET haplotype. Consistent with this, we observed that verapamil treatment of P. falciparum isolates carrying pfcrt SVMNT is associated with an attenuated increase in digestive vacuole pH relative to CVIET pfcrt-carrying isolates. These data suggest a key role for pH-dependent changes in hematin receptor concentration in the P. falciparum CQR mechanism. Our findings also suggest that P. falciparum CQR has arisen through multiple evolutionary pathways associated with pfcrt K76T.

New class of small nonpeptidyl compounds blocks Plasmodium falciparum development in vitro by inhibiting plasmepsins.

Malarial parasites rely on aspartic proteases called plasmepsins to digest hemoglobin during the intraerythrocytic stage. Plasmepsins from Plasmodium falciparum and Plasmodium vivax have been cloned and expressed for a variety of structural and enzymatic studies. Recombinant plasmepsins possess kinetic similarity to the native enzymes, indicating their suitability for target-based antimalarial drug development. We developed an automated assay of P. falciparum plasmepsin II and P. vivax plasmepsin to quickly screen compounds in the Walter Reed chemical database. A low-molecular-mass (346 Da) diphenylurea derivative (WR268961) was found to inhibit plasmepsins with a K(i) of 1 to 6 microM. This compound appears to be selective for plasmepsin, since it is a poor inhibitor of the human aspartic protease cathepsin D (K(i) greater than 280 microM). WR268961 inhibited the growth of P. falciparum strains W2 and D6, with 50% inhibitory concentrations ranging from 0.03 to 0.16 microg/ml, but was much less toxic to mammalian cells. The Walter Reed chemical database contains over 1,500 compounds with a diphenylurea core structure, 9 of which inhibit the plasmepsins, with K(i) values ranging from 0.05 to 0.68 microM. These nine compounds show specificity for the plasmepsins over human cathepsin D, but they are poor inhibitors of P. falciparum growth in vitro. Computational docking experiments indicate how diphenylurea compounds bind to the plasmepsin active site and inhibit the enzyme.

Structural analysis of chloroquine resistance reversal by imipramine analogs.

For imipramine, desipramine, and eight analogs of these well-known drugs, an N-5-aminoalkyl substitution was a minimum but insufficient structural feature associated with chloroquine resistance reversal. Although a second distal aliphatic nitrogen atom was unnecessary for resistance reversal, the direction of the dipole moment vector was critical.

Effects of hydroxypyridinone iron chelators in combination with antimalarial drugs on the in vitro growth of Plasmodium falciparum.

Using standard in vitro drug susceptibility methods, we assessed the antimalarial activity of 3 orally administered iron chelators (hydroxypyridinones) alone and in combination with conventional antimalarials drugs (quinine, mefloquine, artesunate, tetracycline, atovaquone) against a chloroquine-resistant Plasmodium falciparum isolate. When tested alone, all iron chelators and antimalarial compounds inhibited the growth of the parasites. IC50 values for iron chelators were 60-70 microM, whereas the IC50 values for antimalarial drugs were in nM ranges, with artesunate being the most potent. The derived isobolograms for the interaction of hydroxypyridinones and antimalarial drugs showed addition or mild antagonism, similar to desferroxamine (Sum of Fractional Inhibitory Concentration, sigma FIC < 0.5 or > 4.0). Despite the absence of synergy with conventional drugs, intrinsic antimalarial activity of hydroxypyridinones supports the continued assessment of these iron chelators as treatment adjuncts.

Serial analysis of gene expression (SAGE) in Plasmodium falciparum: application of the technique to A-T rich genomes.

The advent of high-throughput methods for the analysis of global gene expression, together with the Malaria Genome Project open up new opportunities for furthering our understanding of the fundamental biology and virulence of the malaria parasite. Serial analysis of gene expression (SAGE) is particularly well suited for malarial systems, as the genomes of Plasmodium species remain to be fully annotated. By simultaneously and quantitatively analyzing mRNA transcript profiles from a given cell population, SAGE allows for the discovery of new genes. In this study, one reports the successful application of SAGE in Plasmodium falciparum, 3D7 strain parasites, from which a preliminary library of 6880 tags corresponding to 4146 different genes was generated. It was demonstrated that P. falciparum is amenable to this technique, despite the remarkably high A-T content of its genome. SAGE tags as short as 10 nucleotides were sufficient to uniquely identify parasite transcripts from both nuclear and mitochondrial genomes. Moreover, the skewed A-T content of parasite sequence did not preclude the use of enzymes that are crucial for generating representative SAGE libraries. Finally, a few modifications to DNA extraction and cloning steps of the SAGE protocol proved useful for circumventing specific problems presented by A-T rich genomes.

Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I.

Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis. It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi. Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid, plant-like single chain enzyme or more like the animal complex polypeptide chain. We identified a plant-like Fab I in Plasmodium falciparum and modelled the structure on the Brassica napus and Escherichia coli structures, alone and complexed to triclosan (5-chloro-2-[2,4 dichlorophenoxy] phenol]), which confirmed all the requisite features of an ENR and its interactions with triclosan. Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites P. falciparum and Toxoplasma gondii at low (i.e. IC50 congruent with150-2000 and 62 ng/ml, respectively) concentrations. Discovery and characterisation of an apicomplexan Fab I and discovery of triclosan as lead compound provide means to rationally design novel inhibitory compounds.

Comparative pharmacokinetics and effect kinetics of orally administered artesunate in healthy volunteers and patients with uncomplicated falciparum malaria.

The single-dose pharmacokinetics of 100 mg of orally administered artesunate (AS) were studied in 6 patient volunteers with uncomplicated falciparum malaria and in 6 healthy volunteers. Plasma concentrations of both the parent drug, AS, and its major metabolite, dihydroartemisinin (DHA), were measured simultaneously by high-performance liquid chromatography (HPLC) with electrochemical detection (ECD). The antimalarial activity of each plasma sample measured by an in vitro bioassay (BA) was used to derive activity concentrations. Artesunate was absorbed rapidly and then almost completely hydrolyzed to DHA in patients, whereas hydrolysis was incomplete in healthy volunteers. The mean +/- standard deviation (SD) maximum concentration (Cmax) of AS was 296+/-110 nmol/L, the time to peak blood level (tmax was 0.71+/-0.66 hr, the half-life (t1/2,z) was 0.41+/-0.34 hr, and the bioavailability over 12 hr (area under the curve [AUC](0-12)) was 253+/-185 nmol hr/L. Measured by HPLC, the Cmax and AUC(0-12) values of DHA in patients with malaria were significantly greater than in volunteers (1,948+/-772 and 1,192+/-315 nmol/L; 4,024+/-1,585 and 1,763+/-607 nmol hr/L, respectively; P < or = 0.05). These differences were even greater when measured by BA. The Cmax for patients with malaria was 2,894+/-2,497 and 795+/-455 nmol/L for volunteers, and AUC(0-12) was 5,970+/-3,625 and 1,307+/-391 nmol hr/L, respectively (P < or = 0.05). In contrast, DHA parameter estimates for t1/2,z and tmax were similar between patients and healthy volunteers, with values of 0.80+/-0.30 versus 0.87+/-0.06 hr and 1.50+/-0.55 versus 1.13+/-0.52 hr, respectively (P > 0.5). Both drug metabolism and tissue protein binding could contribute to the differences between the antimalarial activity of artemisinin drugs in healthy volunteers and malaria infected patients.

Syntheses and antimalarial activities of 10-substituted deoxoartemisinins.

Two series of 10-substituted deoxoartemisinin derivatives have been synthesized. The first employed the reaction of dihydroartemisinin acetate with several silyl enol ethers in the presence of titanium tetrachloride. The second utilized the reaction of 10-(2-oxoethyl)deoxoartemisinin with several Grignard reagents. The in vitro antimalarial activities of both series were determined against two drug-resistant clones of P. falciparum. The activities of 13 beta and 15 beta were 5-7 times greater than that of artemisinin.

Mefloquine pharmacokinetic-pharmacodynamic models: implications for dosing and resistance.

Antimalarial resistance develops and spreads when spontaneously occurring mutant malaria parasites are selected by concentrations of antimalarial drug which are sufficient to eradicate the more sensitive parasites but not those with the resistance mutation(s). Mefloquine, a slowly eliminated quinoline-methanol compound, is the most widely used drug for the treatment of multidrug-resistant falciparum malaria. It has been used at doses ranging between 15 and 25 mg of base/kg of body weight. Resistance to mefloquine has developed rapidly on the borders of Thailand, where the drug has been deployed since 1984. Mathematical modeling with population pharmacokinetic and in vivo and in vitro pharmacodynamic data from this region confirms that, early in the evolution of resistance, conventional assessments of the therapeutic response