Relevance of Higher-Order Epistasis in Drug Resistance.

We studied five chemically distinct but related 1,3,5-triazine antifolates with regard to their effects on growth of a set of mutants in dihydrofolate reductase. The mutants comprise a combinatorially complete data set of all 16 possible combinations of four amino acid replacements associated with resistance to pyrimethamine in the malaria parasite Plasmodium falciparum. Pyrimethamine was a mainstay medication for malaria for many years, and it is still in use in intermittent treatment during pregnancy or as a partner drug in artemisinin combination therapy. Our goal was to investigate the extent to which the alleles yield similar adaptive topographies and patterns of epistasis across chemically related drugs. We find that the adaptive topographies are indeed similar with the same or closely related alleles being fixed in computer simulations of stepwise evolution. For all but one of the drugs the topography features at least one suboptimal fitness peak. Our data are consistent with earlier results indicating that third order and higher epistatic interactions appear to contribute only modestly to the overall adaptive topography, and they are largely conserved. In regard to drug development, our data suggest that higher-order interactions are likely to be of little value as an advisory tool in the choice of lead compounds.

In Vivo Efficacy and Pharmacokinetics of the 2-Aminomethylphenol Antimalarial JPC-3210 in the Aotus Monkey-Human Malaria Model.

Nonimmune Aotus monkeys infected with Plasmodium falciparum and Plasmodium vivax were cured of their infections when treated with a single oral dose of 5 mg/kg and 10 mg/kg of the 2-aminomethylphenol, JPC-3210, respectively. Corresponding mean blood elimination half-lives of JPC-3210 were lengthy at 19.1 days and 20.5 days, respectively. This in vivo potency and lengthy half-life supports the further development of JPC-3210 as a promising, long-acting blood schizontocidal antimalarial for malaria treatment and prevention.

Multi-omic Characterization of the Mode of Action of a Potent New Antimalarial Compound, JPC-3210, Against Plasmodium falciparum.

The increasing incidence of antimalarial drug resistance to the first-line artemisinin combination therapies underpins an urgent need for new antimalarial drugs, ideally with a novel mode of action. The recently developed 2-aminomethylphenol, JPC-3210, (MMV 892646) is an erythrocytic schizonticide with potent in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum lines, low cytotoxicity, potent in vivo efficacy against murine malaria, and favorable preclinical pharmacokinetics including a lengthy plasma elimination half-life. To investigate the impact of JPC-3210 on biochemical pathways within P. falciparum-infected red blood cells, we have applied a "multi-omics" workflow based on high resolution orbitrap mass spectrometry combined with biochemical approaches. Metabolomics, peptidomics and hemoglobin fractionation analyses revealed a perturbation in hemoglobin metabolism following JPC-3210 exposure. The metabolomics data demonstrated a specific depletion of short hemoglobin-derived peptides, peptidomics analysis revealed a depletion of longer hemoglobin-derived peptides, and the hemoglobin fractionation assay demonstrated decreases in hemoglobin, heme and hemozoin levels. To further elucidate the mechanism responsible for inhibition of hemoglobin metabolism, we used in vitro ß-hematin polymerization assays and showed JPC-3210 to be an intermediate inhibitor of ß-hematin polymerization, about 10-fold less potent then the quinoline antimalarials, such as chloroquine and mefloquine. Further, quantitative proteomics analysis showed that JPC-3210 treatment results in a distinct proteomic signature compared with other known antimalarials. While JPC-3210 clustered closely with mefloquine in the metabolomics and proteomics analyses, a key differentiating signature for JPC-3210 was the significant enrichment of parasite proteins involved in regulation of translation. These studies revealed that the mode of action for JPC-3210 involves inhibition of the hemoglobin digestion pathway and elevation of regulators of protein translation. Importantly, JPC-3210 demonstrated rapid parasite killing kinetics compared with other quinolones, suggesting that JPC-3210 warrants further investigation as a potentially long acting partner drug for malaria treatment.

Validation of the triple timed up-and-go test in Lambert-Eaton myasthenia.

INTRODUCTION: There are no validated, practical, and quantitative measures of disease severity in Lambert-Eaton myasthenia (LEM). METHODS: Data from the Effectiveness of 3,4-Diaminopyridine in Lambert-Eaton Myasthenic Syndrome (DAPPER) trial were analyzed to assess triple timed up-and-go (3TUG) reproducibility and relationships between 3TUG times and other measures of LEM severity. RESULTS: The coverage probability technique showed ≥0.90 probability for an acceptable 3TUG difference of ≤0.2, indicating that it is reproducible in LEM patients. The correlation between 3TUG times and lower extremity function scores was significant in subjects who continued and in those who were withdrawn from 3,4-diaminopyridine free base. Worsening patient-reported Weakness Self-Assessment Scale and Investigator Assessment of Treatment Effect scores corresponded with prolongation of 3TUG times. DISCUSSION: The 3TUG is reproducible, demonstrates construct validity for assessment of lower extremity function in LEM patients, and correlates with changes in patient and physician assessments. These findings, along with prior reliability studies, indicate 3TUG is a valid measure of disease severity in LEM.

Characterization of the Preclinical Pharmacology of the New 2-Aminomethylphenol, JPC-3210, for Malaria Treatment and Prevention.

The new 2-aminomethylphenol, JPC-3210, has potent in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum lines, low cytotoxicity, and high in vivo efficacy against murine malaria. Here we report on the pharmacokinetics of JPC-3210 in mice and monkeys and the results of in vitro screening assays, including the inhibition of cytochrome P450 (CYP450) isozymes. In mice, JPC-3210 was rapidly absorbed and had an extensive tissue distribution, with a brain tissue-to-plasma concentration ratio of about 5.4. JPC-3210 had a lengthy plasma elimination half-life of about 4.5 days in mice and 11.8 days in monkeys. JPC-3210 exhibited linear single-oral-dose pharmacokinetics across the dose range of 5 to 40 mg/kg of body weight with high oral bioavailability (∼86%) in mice. Systemic blood exposure of JPC-3210 was 16.6% higher in P. berghei-infected mice than in healthy mice. In vitro studies with mice and human hepatocytes revealed little metabolism and the high metabolic stability of JPC-3210. The abundance of human metabolites from oxidation and glucuronidation was 2.0% and 2.5%, respectively. CYP450 studies in human liver microsomes showed JPC-3210 to be an inhibitor of CYP2D6 and, to a lesser extent, CYP3A4 isozymes, suggesting the possibility of a metabolic drug-drug interaction with drugs that are metabolized by these isozymes. In vitro studies showed that JPC-3210 is highly protein bound to human plasma (97%). These desirable pharmacological findings of a lengthy blood elimination half-life, high oral bioavailability, and low metabolism as well as high in vivo potency have led the Medicines for Malaria Venture to select JPC-3210 (MMV892646) for further advanced preclinical development.

3,4-diaminopyridine base effectively treats the weakness of Lambert-Eaton myasthenia.

INTRODUCTION: 3,4-diaminopyridine has been used to treat Lambert-Eaton myasthenia (LEM) for 30 years despite the lack of conclusive evidence of efficacy. METHODS: We conducted a randomized double-blind placebo-controlled withdrawal study in patients with LEM who had been on stable regimens of 3,4-diaminopyridine base (3,4-DAP) for ≥ 3 months. The primary efficacy endpoint was >30% deterioration in triple timed up-and-go (3TUG) times during tapered drug withdrawal. The secondary endpoint was self-assessment of LEM-related weakness (W-SAS). RESULTS: Thirty-two participants were randomized to continuous 3,4-DAP or placebo groups. None of the 14 participants who received continuous 3,4-DAP had > 30% deterioration in 3TUG time versus 72% of the 18 who tapered to placebo (P < 0.0001). W-SAS similarly demonstrated an advantage for continuous treatment over placebo (P < 0.0001). Requirement for rescue and adverse events were more common in the placebo group. DISCUSSION: This trial provides significant evidence of efficacy of 3,4-DAP in the maintenance of strength in LEM. Muscle Nerve 57: 561-568, 2018.

Reliability of the triple-timed up-and-go test.

INTRODUCTION: We report the reliability of a new measure, the triple-timed up-and-go (3TUG) test, for assessing clinical function in patients with Lambert-Eaton myasthenia (LEM). METHODS: Intrarater reproducibility and interrater agreement of the 3TUG test were assessed in 25 control participants, 24 patients with non-LEM neuromuscular disease, and 12 patients with LEM. The coverage probability (CP) method was the primary measure of reproducibility and agreement. The a priori acceptable range was < 20% difference in 3TUG test times and a CP ≥0.90 confirmed agreement. RESULTS: CP values > 0.90 for intrarater and interrater tests confirmed acceptable reproducibility and agreement for all groups. DISCUSSION: The 3TUG test is a quick, noninvasive, and reproducible measure that is easy to perform, measures clinically important weakness in LEM patients, and requires little training. Additional evaluation in a larger number of LEM patients is in progress to validate the 3TUG test as a clinical measure in LEM. Muscle Nerve 57: 136-139, 2017.

Population Pharmacokinetics/Pharmacodynamics of 3,4-Diaminopyridine Free Base in Patients With Lambert-Eaton Myasthenia.

Lambert-Eaton myasthenia (LEM) is a rare autoimmune disorder associated with debilitating muscle weakness. There are limited treatment options and 3,4-diaminopyridine (3,4-DAP) free base is an investigational orphan drug used to treat LEM-related weakness. We performed a population pharmacokinetic/pharmacodynamic (PK/PD) analysis using 3,4-DAP and metabolite concentrations collected from a phase II study in patients with LEM. The Triple Timed Up & Go (3TUG) assessment, which measures lower extremity weakness, was the primary outcome measure. A total of 1,270 PK samples (49 patients) and 1,091 3TUG data points (32 randomized patients) were included in the PK/PD analysis. A two-compartment and one-compartment model for parent and metabolite, respectively, described the PK data well. Body weight and serum creatinine partially explained the variability in clearance for the final PK model. A fractional inhibitory maximum effect (Emax ) model characterized the exposure-response relationship well. The PK/PD model was applied to identify a suggested dosing approach for 3,4-DAP free base.

Lead Selection of a New Aminomethylphenol, JPC-3210, for Malaria Treatment and Prevention.

Structure-activity relationship studies of trifluoromethyl-substituted pyridine and pyrimidine analogues of 2-aminomethylphenols (JPC-2997, JPC-3186, and JPC-3210) were conducted for preclinical development for malaria treatment and/or prevention. Of these compounds, JPC-3210 [4-(tert-butyl)-2-((tert-butylamino)methyl)-6-(5-fluoro-6-(trifluoromethyl)pyridin-3-yl)phenol] was selected as the lead compound due to superior in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum lines, lower in vitro cytotoxicity in mammalian cell lines, longer plasma elimination half-life, and greater in vivo efficacy against murine malaria.

Evolution from double to triple-antimalarial drug combinations.

Drug combinations are used to treat multiple-drug resistant malaria parasites and to attempt to further delay the evolution of drug resistance. Most current antimalarial combinations are binary but it is likely that new triple drug combinations will be required in the future. A review of previous triple combinations of antimalarial drugs was done to focus attention on past problems and possible future combinations. The advantages of such triple drug combinations include greater efficacy against multiple-drug resistant strains, synergistic action between the different medications and simplification of the regimen so that it could be administered under direct observation and possibly as single-dose therapy. The disadvantages of poly-pharmacy include increased cost of medication, difficulty preparing robust regulatory packages and problems constructing combined formulations due to drug-drug interactions. Given the arrival of artemisinin tolerance/resistance in Southeast Asia, it is likely that new drugs introduced for malaria treatment will be in triple drug combinations.

JPC-2997, a new aminomethylphenol with high in vitro and in vivo antimalarial activities against blood stages of Plasmodium.

4-(tert-Butyl)-2-((tert-butylamino)methyl)-6-(6-(trifluoromethyl)pyridin-3-yl)-phenol (JPC-2997) is a new aminomethylphenol compound that is highly active in vitro against the chloroquine-sensitive D6, the chloroquine-resistant W2, and the multidrug-resistant TM90-C2B Plasmodium falciparum lines, with 50% inhibitory concentrations (IC50s) ranging from 7 nM to 34 nM. JPC-2997 is >2,500 times less cytotoxic (IC50s > 35 µM) to human (HepG2 and HEK293) and rodent (BHK) cell lines than the D6 parasite line. In comparison to the chemically related WR-194,965, a drug that had advanced to clinical studies, JPC-2997 was 2-fold more active in vitro against P. falciparum lines and 3-fold less cytotoxic. The compound possesses potent in vivo suppression activity against Plasmodium berghei, with a 50% effective dose (ED50) of 0.5 mg/kg of body weight/day following oral dosing in the Peters 4-day test. The radical curative dose of JPC-2997 was remarkably low, at a total dose of 24 mg/kg, using the modified Thompson test. JPC-2997 was effective in curing three Aotus monkeys infected with a chloroquine- and pyrimethamine-resistant strain of Plasmodium vivax at a dose of 20 mg/kg daily for 3 days. At the doses administered, JPC-2997 appeared to be well tolerated in mice and monkeys. Preliminary studies of JPC-2997 in mice show linear pharmacokinetics over the range 2.5 to 40 mg/kg, a low clearance of 0.22 liters/h/kg, a volume of distribution of 15.6 liters/kg, and an elimination half-life of 49.8 h. The high in vivo potency data and lengthy elimination half-life of JPC-2997 suggest that it is worthy of further preclinical assessment as a partner drug.

Discrimination of potent inhibitors of Toxoplasma gondii enoyl-acyl carrier protein reductase by a thermal shift assay.

Many microbial pathogens rely on a type II fatty acid synthesis (FASII) pathway that is distinct from the type I pathway found in humans. Enoyl-acyl carrier protein reductase (ENR) is an essential FASII pathway enzyme and the target of a number of antimicrobial drug discovery efforts. The biocide triclosan is established as a potent inhibitor of ENR and has been the starting point for medicinal chemistry studies. We evaluated a series of triclosan analogues for their ability to inhibit the growth of Toxoplasma gondii, a pervasive human pathogen, and its ENR enzyme (TgENR). Several compounds that inhibited TgENR at low nanomolar concentrations were identified but could not be further differentiated because of the limited dynamic range of the TgENR activity assay. Thus, we adapted a thermal shift assay (TSA) to directly measure the dissociation constant (Kd) of the most potent inhibitors identified in this study as well as inhibitors from previous studies. Furthermore, the TSA allowed us to determine the mode of action of these compounds in the presence of the reduced nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide (NAD⁺) cofactor. We found that all of the inhibitors bind to a TgENR-NAD⁺ complex but that they differed in their dependence on NAD⁺ concentration. Ultimately, we were able to identify compounds that bind to the TgENR-NAD⁺ complex in the low femtomolar range. This shows how TSA data combined with enzyme inhibition, parasite growth inhibition data, and ADMET predictions allow for better discrimination between potent ENR inhibitors for the future development of medicine.

Novel diaryl ureas with efficacy in a mouse model of malaria.

Exploration of triclosan analogs has led to novel diaryl ureas with significant potency against in vitro cultures of drug-resistant and drug-sensitive strains of the human malaria parasite Plasmodium falciparum. Compound 18 demonstrated EC(50) values of 37 and 55 nM versus in vitro cultured parasite strains and promising in vivo efficacy in a Plasmodium berghei antimalarial mouse model, with >50% survival at day 31 post-treatment when administered subcutaneously at 256 mg/kg. This series of compounds provides a chemical scaffold of novel architecture, as validated by cheminformatics analysis, to pursue antimalarial drug discovery efforts.

Triclosan derivatives: towards potent inhibitors of drug-sensitive and drug-resistant Mycobacterium tuberculosis.

Triclosan has been previously shown to inhibit InhA, an essential enoyl acyl carrier protein reductase involved in mycolic acid biosynthesis, the inhibition of which leads to the lysis of Mycobacterium tuberculosis. Using a structure-based drug design approach, a series of 5-substituted triclosan derivatives was developed. Two groups of derivatives with alkyl and aryl substituents, respectively, were identified with dramatically enhanced potency against purified InhA. The most efficacious inhibitor displayed an IC(50) value of 21 nM, which was 50-fold more potent than triclosan. X-ray crystal structures of InhA in complex with four triclosan derivatives revealed the structural basis for the inhibitory activity. Six selected triclosan derivatives were tested against isoniazid-sensitive and resistant strains of M. tuberculosis. Among those, the best inhibitor had an MIC value of 4.7 microg mL(-1) (13 microM), which represents a tenfold improvement over the bacteriocidal activity of triclosan. A subset of these triclosan analogues was more potent than isoniazid against two isoniazid-resistant M. tuberculosis strains, demonstrating the significant potential for structure-based design in the development of next generation antitubercular drugs.

The fatty acid biosynthesis enzyme FabI plays a key role in the development of liver-stage malarial parasites.

The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.

Novel triazine JPC-2067-B inhibits Toxoplasma gondii in vitro and in vivo.

BACKGROUND AND METHODOLOGY: Toxoplasma gondii causes substantial morbidity, mortality, and costs for healthcare in the developed and developing world. Current medicines are not well tolerated and cause hypersensitivity reactions. The dihydrotriazine JPC-2067-B (4, 6-diamino-1, 2-dihydro-2, 2-dimethyl-1-(3'(2-chloro-, 4-trifluoromethoxyphenoxy)propyloxy)-1, 3, 5-triazine), which inhibits dihydrofolate reductase (DHFR), is highly effective against Plasmodium falciparum, Plasmodium vivax, and apicomplexans related to T. gondii. JPC-2067-B is the primary metabolite of the orally active biguanide JPC-2056 1-(3'-(2-chloro-4-trifluoromethoxyphenyloxy)propyl oxy)- 5-isopropylbiguanide, which is being advanced to clinical trials for malaria. Efficacy of the prodrug JPC-2056 and the active metabolite JPC-2067-B against T. gondii and T. gondii DHFR as well as toxicity toward mammalian cells were tested. PRINCIPAL FINDINGS AND CONCLUSIONS: Herein, we found that JPC-2067-B is highly effective against T. gondii. We demonstrate that JPC-2067-B inhibits T. gondii growth in culture (IC50 20 nM), inhibits the purified enzyme (IC50 6.5 nM), is more efficacious than pyrimethamine, and is cidal in vitro. JPC-2067-B administered parenterally and the orally administered pro-drug (JPC-2056) are also effective against T. gondii tachyzoites in vivo. A molecular model of T. gondii DHFR-TS complexed with JPC-2067-B was developed. We found that the three main parasite clonal types and isolates from South and Central America, the United States, Canada, China, and Sri Lanka have the same amino acid sequences preserving key binding sites for the triazine. SIGNIFICANCE: JPC-2056/JPC-2067-B have potential to be more effective and possibly less toxic treatments for toxoplasmosis than currently available medicines.

Multicenter randomized, double-blind, placebo-controlled, clinical trial of dapsone as a glucocorticoid-sparing agent in maintenance-phase pemphigus vulgaris.

OBJECTIVE: To determine the efficacy of dapsone as a glucocorticoid-sparing agent in maintenance-phase pemphigus vulgaris (PV). DESIGN: A randomized, double-blind, placebo-controlled study with a crossover arm for those who failed treatment. SETTING: A US multicenter outpatient study. Patients A total of 19 subjects enrolled among 5 centers, 9 randomized to receive dapsone and 10 to receive placebo. Inclusion criteria were biopsy and direct immunofluorescence-proven PV controlled with glucocorticoids and/or cytotoxic agents, disease in maintenance phase, and aged 18 to 80 years. Physicians had tried at least 2 tapers of glucocorticoids unsuccessfully and had 30 days of stable steroid dosage. Treatment for any patient unable to taper glucocorticoids by more than 25% within 4 months was declared a failure, and the patient was allowed to switch to the opposite medication while maintaining the double-blind. Main Outcome Measure The ability of patients to taper to 7.5 mg/d or less within 1 year of reaching the maximum dosage of the study drug. RESULTS: Of the 9 patients receiving dapsone, 5 were successfully treated, 3 failed treatment, and 1 dropped out of the study. Of the 10 patients receiving placebo, 3 were successfully treated, and 7 failed treatment. This primary end point favored the dapsone-treated group but was not statistically significant (P = .37). Four patients who failed treatment while receiving placebo were switched to treatment with dapsone. Of these, 3 were successfully treated after switching to dapsone treatment, and 1 failed treatment. We found that, overall, 8 of 11 patients (73%) receiving dapsone vs 3 of 10 (30%) receiving placebo reached the primary outcome of a prednisone dosage of 7.5 mg/d or less. CONCLUSION: This trial demonstrates a trend to efficacy of dapsone as a steroid-sparing drug in maintenance-phase PV.

Role of specific cytochrome P450 isoforms in the conversion of phenoxypropoxybiguanide analogs in human liver microsomes to potent antimalarial dihydrotriazines.

Phenoxypropoxybiguanides, such as PS-15, are antimalarial prodrugs analogous to the relationship of proguanil and its active metabolite cycloguanil. Unlike cycloguanil, however, WR99210, the active metabolite of PS-15, has retained in vitro potency against newly emerging antifolate-resistant malaria parasites. Recently, in vitro metabolism of a new series of phenoxypropoxybiguanide analogs has examined the production of the active triazine metabolites by human liver microsomes. The purpose of this investigation was to elucidate the primary cytochrome P450 isoforms involved in the production of active metabolites in the current lead candidate. By using expressed human recombinant isoform preparations, specific chemical inhibitors, and isoform-specific inhibitory antibodies, the primary cytochrome P450 isoforms involved in the in vitro metabolic activation of JPC-2056 were elucidated. Unlike proguanil, which is metabolized primarily by CYP2C19, the results indicate that CYP3A4 plays a more important role in the metabolism of both PS-15 and JPC-2056. Whereas CYP2D6 appears to play a major role in the metabolism of PS-15 to WR99210, it appears less important in the conversion of JPC-2056 to JPC-2067. These results are encouraging, considering the prominence of CYP2C19 and CYP2D6 polymorphisms in certain populations at risk for contracting malaria, because the current clinical prodrug candidate from this series may be less dependent on these enzymes for metabolic activation.

Antimalarial pharmacodynamics and pharmacokinetics of a third-generation antifolate--JPC2056--in cynomolgus monkeys using an in vivo in vitro model.

OBJECTIVES: To assess the antimalarial pharmacodynamics and pharmacokinetics of the novel dihydrofolate reductase (DHFR) inhibitor, JPC2056 and its principal active metabolite JPC2067 in cynomolgus monkeys using an in vivo-in vitro model. METHODS: In a two-phase crossover design, five cynomolgus monkeys were administered a single dose (20 mg/kg) and multiple doses (20 mg/kg daily for 3 days) of JPC2056. Plasma samples collected from treated monkeys were assessed for in vitro antimalarial activity against Plasmodium falciparum lines having wild-type (D6), double-mutant (K1) and quadruple-mutant (TM90-C2A) DHFR-thymidylate synthase (TS) and a P. falciparum line transformed with a Plasmodium vivax dhfr-ts quadruple-mutant allele (D6-PvDHFR). Plasma JPC2056 and JPC2067 concentrations were measured by LC-mass spectrometry. RESULTS: The mean inhibitory dilution (ID(90)) of monkey plasma at 3 h after drug administration against D6, K1 and TM90-C2A was, respectively, 1253, 585 and 869 after the single-dose regimen and 1613, 1120 and 1396 following the multiple-dose regimen. Less activity was observed with the same monkey plasma samples against the D6-PvDHFR line, with a mean ID(90) of 53 after multiple dosing. Geometric mean plasma concentrations of JPC2056 and JPC2067 at 3 h after drug administration were, respectively, 113 and 12 ng/mL after the single dose and 150 and 17 ng/mL after multiple dosing. The mean elimination half-life of JPC2056 was shorter than its metabolite after both regimens (single dose, 7.3 versus 11.8 h; multiple doses, 6.6 versus 11.1 h). CONCLUSIONS: The high potency of JPC2056 against P. falciparum DHFR-TS quadruple-mutant lines provides optimism for the future development of JPC2056 for the treatment of malaria infections.