Chloroquine-resistant Isolates Research Articles

Mechanistic basis for multidrug resistance and collateral drug sensitivity conferred to the malaria parasite by polymorphisms in PfMDR1 and PfCRT.

Polymorphisms in the Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene and the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene alter the malaria parasite’s susceptibility to most of the current antimalarial drugs. However, the precise mechanisms by which PfMDR1 contributes to multidrug resistance have not yet been fully elucidated, nor is it understood why polymorphisms in pfmdr1 and pfcrt that cause chloroquine resistance simultaneously increase the parasite’s susceptibility to lumefantrine and mefloquine—a phenomenon known as collateral drug sensitivity. Here, we present a robust expression system for PfMDR1 in Xenopus oocytes that enables direct and high-resolution biochemical characterizations of the protein. We show that wild-type PfMDR1 transports diverse pharmacons, including lumefantrine, mefloquine, dihydroartemisinin, piperaquine, amodiaquine, methylene blue, and chloroquine (but not the antiviral drug amantadine). Field-derived mutant isoforms of PfMDR1 differ from the wild-type protein, and each other, in their capacities to transport these drugs, indicating that PfMDR1-induced changes in the distribution of drugs between the parasite’s digestive vacuole (DV) and the cytosol are a key driver of both antimalarial resistance and the variability between multidrug resistance phenotypes. Of note, the PfMDR1 isoforms prevalent in chloroquine-resistant isolates exhibit reduced capacities for chloroquine, lumefantrine, and mefloquine transport. We observe the opposite relationship between chloroquine resistance-conferring mutations in PfCRT and drug transport activity. Using our established assays for characterizing PfCRT in the Xenopus oocyte system and in live parasite assays, we demonstrate that these PfCRT isoforms transport all 3 drugs, whereas wild-type PfCRT does not. We present a mechanistic model for collateral drug sensitivity in which mutant isoforms of PfMDR1 and PfCRT cause chloroquine, lumefantrine, and mefloquine to remain in the cytosol instead of sequestering within the DV. This change in drug distribution increases the access of lumefantrine and mefloquine to their primary targets (thought to be located outside of the DV), while simultaneously decreasing chloroquine’s access to its target within the DV. The mechanistic insights presented here provide a basis for developing approaches that extend the useful life span of antimalarials by exploiting the opposing selection forces they exert upon PfCRT and PfMDR1.

Discovery of 5-Chlorobenzimidazole-based as Promising Inhibitors of Chloroquine-Resistant Plasmodium Strains: Synthesis, Biological Evaluation, Molecular Docking and Computational Studies

Background: To overcome drug resistance to current antimalarial drugs, we propose the synthesis and in vitro evaluation of the antiplasmodial activity of a series of 5-chlorobenzimidazolyl-chalcones against chloroquino sensitive (CQ-S) and chloroquino resistant (CQ-R) strains of P. falciparum.
 Objective: This study aimed to establish through structure-activity relationship studies and docking, the structural elements essential for antiplasmodial activities.
 Methods: The antiplasmodial activity of these benzimidazolylchalcones was carried out according to the Rieckmann microtest technique, followed by the determination of the concentrations inhibiting 50% of the production of parasitic HRP2 antigens (IC50) by ELISA. Chloroquine was used as a reference molecule with a sensitivity threshold set at 100 µM. Molecular docking was performed using sensitive (PDB ID: 1J3I) and resistant (PDB ID: 4DP3) dihydrofolate reductase-thymidylate synthase proteins (PfDHFR-TS).
 Results: All benzimidazolylchalcones tested expressed antiplasmodial activities especially against chloroquine resistant isolates (IC50 = 0.32-44.38 µM). The best profile against both isolates was the methoxylated derivative (3e) with an IC50 ranging from 0.32 to 1.96 µM. This compound had the best antimalarial activity against CQ-S isolates. On CQ-R isolates, the unsubstituted 5-chlorobenzimidazole derivative (3b) had exalted activity (IC50 = 0.78 µM). We selected a weakly active non-chlorinated derivative 3a and chlorinated derivatives 3b, 3d, 3e and 3f) with IC50< 3µM against the chloroquine-resistant strain to perform docking studies. These revealed that the pyrrolic nitrogen of benzimidazole and the ketone of propenone are the main chemical entities involved in the interaction at the receptor. Moreover, ADMET studies showed favorable pharmacokinetic properties.
 Conclusion: Molecular docking studies confirmed the experimental findings and revealed the possible interactions pattern. Derivatives 3b and 3e, which showed promising binding affinities against PfDHFR-TS, can be proposed as lead compounds for the development of antimalarial drug candidates.

Global assessment of genetic paradigms of Pvmdr1 mutations in chloroquine-resistant Plasmodium vivax isolates.

Chloroquine (CQ) is generally prescribed as the front-line antimalarial drug of choice to treat Plasmodium vivax infections; however, some clinical CQ-resistant P. vivax isolates have been indigenously reported around the world during the last decade. In this study, P. vivax isolates (n=52) were obtained from autochthonous samples in southeast Iran during 2015-2017. The genomic DNA of samples was extracted, amplified (nested PCR) and sequenced by targeting the multidrug-resistance 1 gene. To verify the global genetic diversity of CQ-resistant P. vivax strains, the sequences of Pvmdr1 originating from Asia and the Americas were retrieved. A total of 46 haplotypes were grouped into three distinct geographical haplogroups. The haplotype diversity and occurrence rates of Pvmdr1 976F/1076L mutations indicate that the efficacy of CQ is being compromised in Mexico, China, Nicaragua, Thailand, Brazil (2016), Ethiopia, Mauritania (2012) and southwest India in the near future. The cladistic phylogenetic tree showed that Pvmdr1 sequences isolated from the southeast Asian clade has a partial sister relationship with the American clade. The current findings will serve as a basis to develop appropriate malaria control strategies and public health policies in symptomatic imported malaria cases or plausible CQ-resistant P. vivax strains.

Antiplasmodial activities of dyes against Plasmodium falciparum asexual and sexual stages: Contrasted uptakes of triarylmethanes Brilliant green, Green S (E142), and Patent Blue V (E131) by erythrocytes

The search for safe antimalarial compounds acting against asexual symptom-responsible stages and sexual transmission-responsible forms of Plasmodium species is one of the major challenges in malaria elimination programs. So far, among current drugs approved for human use, only primaquine has transmission-blocking activity. The discovery of small molecules targeting different Plasmodium falciparum life stages remains a priority in antimalarial drug research. In this context, several independent studies have recently reported antiplasmodial and transmission-blocking activities of commonly used stains, dyes and fluorescent probes against P. falciparum including chloroquine-resistant isolates. Herein we have studied the antimalarial activities of dyes with different scaffold and we report that the triarylmethane dye (TRAM) Brilliant green inhibits the growth of asexual stages (IC50 ≤ 2 μM) and has exflagellation-blocking activity (IC50 ≤ 800 nM) against P. falciparum reference strains (3D7, 7G8) and chloroquine-resistant clinical isolate (Q206). In a second step we have investigated the antiplasmodial activities of two polysulfonated triarylmethane food dyes. Green S (E142) is weakly active against P. falciparum asexual stage (IC50 ≃ 17 μM) whereas Patent Blue V (E131) is inactive in both antimalarial assays. By applying liquid chromatography techniques for the culture supernatant analysis after cell washings and lysis, we report the detection of Brilliant green in erythrocytes, the selective uptake of Green S (E142) by infected erythrocytes, whereas Patent Blue V (E131) could not be detected within non-infected and 3D7-infected erythrocytes. Overall, our results suggest that two polysulfonated food dyes might display different affinity with transporters or channels on infected RBC membrane.

Correlation of molecular markers, Pfmdr1-N86Y and Pfcrt-K76T, with in vitro chloroquine resistant Plasmodium falciparum, isolated in the malaria endemic states of Assam and Arunachal Pradesh, Northeast India.

The mechanism of chloroquine (CQ) resistance in Plasmodium falciparum is not clearly understood. However, CQ resistance has been shown to be associated with point mutations in Pfcrt and Pfmdr1. These genes encode for digestive vacuole transmembrane proteins Pfcrt and Pgh1, respectively. The present study was carried out to analyze the association of Pfcrt-K76T and Pfmdr1-N86Y mutations with CQ resistance in Northeast Indian P. falciparum isolates. 115 P. falciparum isolates were subjected to in vitro CQ sensitivity testing and PCR-RFLP analysis for the Pfmdr1-N86Y and Pfcrt-K76T mutations. 100 isolates of P. falciparum were found to be resistant to CQ by the in vitro susceptibility test (geometric mean EC50 2.21 µM/L blood) while 15 were found to be CQ sensitive (geometric mean EC50 0.32 µM/L blood). All the CQ resistant isolates showed the presence of Pfmdr1 and Pfcrt mutations. CQ sensitive isolates were negative for these mutations. Strong linkage disequilibrium was observed between the alleles at these two loci (Pfmdr1-N86Y and Pfcrt-K76T). The results indicate that Pfmdr1-N86Y and Pfcrt-K76T mutations can be used as molecular markers to identify CQ resistance in P. falciparum. The result necessitates the evaluation of CQ in vivo therapeutic efficacy in endemic areas for more effective malaria control strategies.

Antimalarial activity of isoquine against Kenyan Plasmodium falciparum clinical isolates and association with polymorphisms in pfcrt and pfmdr1 genes

The use of amodiaquine in prophylaxis is associated with serious toxicity, resulting from its metabolic conversion into a reactive quinone-imine metabolite by the hepatic cytochrome P450. To circumvent this toxicity, several amodiaquine analogues that lack the potential to form a quinone-imine derivative, while retaining antimalarial activity, have been designed. Isoquine is one of these promising molecules that has already reached Phase I clinical trials in humans. We analysed the in vitro activity of isoquine against 62 Plasmodium falciparum isolates collected in Kenya and the association of this activity with polymorphisms in pfcrt and pfmdr1 genes. The median concentration of isoquine that inhibited 50% of parasite growth (IC50) was 9 nM, compared with 56 nM chloroquine, 8 nM amodiaquine, 10 nM desethylamodiaquine, 69 nM lumefantrine and 1 nM dihydroartemisinin. Isoquine activity was correlated with polymorphisms in pfcrt at codon 76, but not in pfmdr1 at codon 86. The high activity of isoquine against field isolates, including chloroquine-resistant isolates, with IC50 <10 nM, warrants its further development as an antimalarial.

Indolone-N-oxide derivatives: in vitro activity against fresh clinical isolates of Plasmodium falciparum, stage specificity and in vitro interactions with established antimalarial drugs

Indolone-N-oxides are characterized by the presence of a highly reactive pharmacophore, the nitrone moiety (C=N(+)-O(-)), which undergoes oxidation-reduction reactions. The aims of the present study were to: (i) evaluate the in vitro activity of the parent compound, designated as compound 1, against 34 fresh clinical isolates of Plasmodium falciparum; (ii) compare the activity of compound 1 with that of chloroquine and dihydroartemisinin to assess the potential for cross-resistance; (iii) investigate drug interactions of indolone-N-oxides with standard antimalarials; and (iv) determine the stage-dependent activity of indolone-N-oxides. In vitro antimalarial activity was evaluated against clinical isolates collected from Cameroonian patients by the [(3)H]hypoxanthine incorporation assay. In vitro interactions between compound 1 or another analogue, compound 4, and established antimalarial drugs were assessed by the fixed ratio method. Stage specificity was evaluated by light microscopy using highly synchronized P. falciparum cultures. The geometric mean 50% inhibitory concentration (IC(50)) of compound 1 was 48.6 nM. Its activity did not differ between the chloroquine-susceptible and the chloroquine-resistant isolates. There was no correlation between chloroquine and compound 1 responses (r = 0.015; P > 0.05), but the in vitro responses of compound 1 and dihydroartemisinin were significantly and positively correlated (r = 0.444; P < 0.05). No significant in vitro interaction was observed between indolone-N-oxide derivatives and established antimalarial drugs (artemisinin and its derivatives, chloroquine, amodiaquine, quinine and mefloquine). Compound 1 and compound 4, as well as artesunate, inhibited parasite maturation at the ring stage. These findings suggest that other indolone-N-oxide derivatives with more potent activity than the parent compound may hold promise as antimalarials in the future.

Global gene expression profiling of Plasmodium falciparum in response to the anti-malarial drug pyronaridine.

BackgroundPyronaridine (PN) and chloroquine (CQ) are structurally related anti-malarial drugs with primarily the same mode of action. However, PN is effective against several multidrug-resistant lines of Plasmodium falciparum, including CQ resistant lines, suggestive of important operational differences between the two drugs.MethodsSynchronized trophozoite stage cultures of P. falciparum strain K1 (CQ resistant) were exposed to 50% inhibitory concentrations (IC50) of PN and CQ, and parasites were harvested from culture after 4 and 24 hours exposure. Global transcriptional changes effected by drug treatment were investigated using DNA microarrays.ResultsAfter a 4 h drug exposure, PN induced a greater degree of transcriptional perturbation (61 differentially expressed features) than CQ (10 features). More genes were found to respond to 24 h treatments with both drugs, and 461 features were found to be significantly responsive to one or both drugs across all treatment conditions.Filtering was employed to remove features unrelated to primary drug action, specifically features representing genes developmentally regulated, secondary stress/death related processes and sexual stage development. The only significant gene ontologies represented among the 46 remaining features after filtering relate to host exported proteins from multi-gene families.ConclusionsThe malaria parasite's molecular responses to PN and CQ treatment are similar in terms of the genes and pathways affected. However, PN appears to exert a more rapid response than CQ. The faster action of PN may explain why PN is more efficacious than CQ, particularly against CQ resistant isolates. In agreement with several other microarray studies of drug action on the parasite, it is not possible, however, to discern mechanism of drug action from the drug-responsive genes.

Dibenzylideneacetone analogues as novel Plasmodium falciparum inhibitors

A series of dibenzylideneacetones (A1-A12) and some of their pyrazolines (B1-B4) were synthesized and evaluated in vitro for blood stage antiplasmodial properties in Plasmodium falciparum culture using SYBR-green-I fluorescence assay. The compound (1E, 4E)-1,5-bis(3,4-dimethoxyphenyl)penta-1,4-dien-3-one (A9) was found to be the most active with IC(50) of 1.97 μM against chloroquine-sensitive strain (3D7) and 1.69 μM against chloroquine-resistant field isolate (RKL9). The MTT based cytotoxicity assay on HeLa cell line has confirmed that A9 is selective in its action against malaria parasite (with a therapeutic index of 166). Our results revealed that these compounds exhibited promising antiplasmodial activities which can be further explored as potential leads for the development of cheaper, safe, effective and potent drugs against chloroquine-resistant malarial parasites.

Is Chloroquine Making a Comeback?

Is Chloroquine Making a Comeback?

Synthesis of novel α-pyranochalcones and pyrazoline derivatives as Plasmodium falciparum growth inhibitors

Both the lack of a credible malaria vaccine and the emergence and spread of parasites resistant to most of the clinically used antimalarial drugs and drug combination have aroused an imperative need to develop new drugs against malaria. In present work, α-pyranochalcones and pyrazoline analogs were synthesized to discover chemically diverse antimalarial leads. Compounds were tested for antimalarial activity by evaluation of the growth of malaria parasite in culture using the microtiter plate based SYBR-Green-I assay. The (E)-3-(3-(2,3,4-trimethoxyphenyl)-acryloyl)-2H-chromen-2-one (Ga6) turned out to be the most potent analog of the series, showing IC50 of 3.1μg/ml against chloroquine-sensitive (3D7) strain and IC50 of 1.1μg/ml against chloroquine-resistant field isolate (RKL9) of Plasmodium falciparum. Cytotoxicity study of the most potent compounds was also performed against HeLa cell line using the MTT assay. All the tested compounds showed high therapeutic indices suggesting that they were selective in their action against the malaria parasite. Furthermore, docking of Ga6 into active site of falcipain enzyme revealed its predicted interactions with active site residues. This is the first instance wherein chromeno-pyrazolines have been found to be active antimalarial agents. Further exploration and optimization of this new lead could provide novel, antimalarial molecules which can ward off issues of cross-resistance to drugs like chloroquine.

History and current status of Plasmodium falciparum antimalarial drug resistance in Madagascar

Malaria remains a major health problem in Madagascar. Over past decades, the burden of malarial disease has fluctuated over time, partly in line with the successes and failures of antimalarial policy. In the 1950s and 1960s, a sharp decline in malaria transmission was observed in the central highlands due to indoor spraying with DDT and to the massive use of chloroquine by the population. Following this, the discontinuation of the 'nivaquinization' policy was followed by devastating outbreaks in the central highlands in the 1980s. Currently, the rate of in vitro chloroquine-resistant Plasmodium falciparum isolates does not exceed 5%. This figure appears disconnected from the high level of clinical treatment failure (near 40%). pfcrt mutant isolates are found in less than 1% of isolates on the Island. Conversely, pfmdr1 mutant isolates are found in more than 60% of isolates and may be responsible for the bulk of resistance to chloroquine in Madagascar. Other antimalarials remain generally effective in Madagascar. Recent clinical and in vitro data support the complete efficacy of the combination artesunate-amodiaquine in Madagascar. As such, this artemisinin combination therapy should play a central role in the control and possible elimination of P. falciparum malaria in Madagascar

Analysis of Single-Nucleotide Polymorphisms in thecrt-oandmdr1Genes ofPlasmodium vivaxamong Chloroquine-Resistant Isolates from the Brazilian Amazon Region

Plasmodium vivax parasites with chloroquine resistance (CQR) are already circulating in the Brazilian Amazon. Complete single-nucleotide polymorphism (SNP) analyses of coding and noncoding sequences of the pvmdr1 and pvcrt-o genes revealed no associations with CQR, even if some mutations had not been randomly selected. In addition, striking differences in the topologies and numbers of SNPs in these transporter genes between P. vivax and P. falciparum reinforce the idea that mechanisms other than mutations may explain this virulent phenotype in P. vivax.

Influence of the asexual parasite biomass on in vitro susceptibility of Plasmodium falciparum to antimalarial drugs in Abidjan

The in vitro activities of artemisinin, dihydroartemisinin (the biologically active metabolite of artemisinin derivatives), chloroquine and pyronaridine were assessed in 32 isolates of Plasmodium falciparum from Abobo in the northern of Abidjan district (Cote dfIvoire) using a test based on the standard microtechnique recommended by the World Health Organization (WHO). The parasites densities were ranged between 8,000 and 540,000 rings/ƒEl of blood. The geometric means 50% inhibitory concentration (GMIC50) values for chloroquine, pyronaridine and artemisinin were 145.5 nM (95% confidence interval (CI) =65-226 nM), 17.69 nM (95% CI=9.1-26.3 nM) and 5.72 nM (95% CI=2.3-9.1 nM), respectively. Dihydroartemisinin was the most potent drug against chloroquine-sensitive and chloroquine-resistant isolates with a geometric mean of 2.72 nM. There was no correlation between the parasite densities and the responses to chloroquine (r2=0.01, p<0.5), pyronaridine (r2=0.13, p<0.05), artemisinin (r2=0.13, p<0.05) and dihydroartemisinin (r2=0.07, p<0.1).

Polymorphisms in the pfcrt and pfmdr1 Genes of Plasmodium falciparum and in Vitro Susceptibility to Amodiaquine and Desethylamodiaquine

The potential role of polymorphisms in the pfcrt and pfmdr1 genes and in vitro susceptibility to amodiaquine and desethylamodiaquine were explored in 15 chloroquine-resistant Colombian Plasmodium falciparum isolates. Single nucleotide polymorphisms in the pfcrt gene, including a newly reported mutation (S334N), were seen in isolates with decreased susceptibility to amodiaquine and desethylamodiaquine. The lowest susceptibility found to amodiaquine was observed in an isolate carrying a pfcrt and pfmdr1 Dd2-like haplotype, whereas a pfcrt haplotype related to the 7G8 Brazilian strain was found in a Colombian isolate with the lowest susceptibility to desethylamodiaquine. This exploratory study suggests that polymorphisms in the pfcrt and pfmdr1 genes play a role in amodiaquine and desethylamodiaquine resistance and warrants further study.

IN VITRO SUSCEPTIBILITY OF P. FALCIPARUM POPULATIONS FROM COLOMBIA AND TANZANIA TO A NEW SYNTHETIC PEROXIDE (OZ277)

Sensitivity of Plasmodium falciparum populations from Colombia (N = 38) and Tanzania (N = 45) to the newly developed, fully synthetic peroxide OZ277 was investigated using a standard isotopic microtest. OZ277 showed excellent activity against chloroquine-resistant isolates in Colombia with median IC(50) [range] values of 2.5 ng/mL [0.34-8] (4.4 nM [0.6-14]) and Tanzania with 1.5 ng/mL [0.22-10] (2.65 nM [0.4-17.7]). The potency of OZ277 was similar to artesunate, showing median IC(50) values of 1.5 ng/mL [0.42-8.6] (3.8 nM [1.1-22.3]) and 1.8 ng/mL [0.2-10] (4.7 nM [0.5-26.04]) in Colombia and Tanzania, respectively. These results support the development of this new antimalarial compound.

MOLECULAR EPIDEMIOLOGY OF MALARIA IN CAMEROON. XXIV. TRENDS OF IN VITRO ANTIMALARIAL DRUG RESPONSES IN YAOUNDE, CAMEROON

In vitro response to chloroquine, monodesethylamodiaquine, mefloquine, lumefantrine, and dihydroartemisinin was assessed by the radioisotopic microtest in Yaoundé, Cameroon, during 2000-2004 and compared with our previous data obtained during 1996-1999. Based on the cut-off value of 100 nmol/L, 36.3% of isolates were chloroquine-susceptible (N = 175; geometric mean IC(50), 40.3 nmol/L) and 63.7% were chloroquine-resistant (N = 307; geometric mean IC(50), 211 nmol/L). There was no significant difference (P > 0.05) in the mean IC(50)s from 1996 to 2004, but a significant linear trend (P < 0.05) toward an increased proportion of chloroquine-resistant isolates was observed from 1996 (49%) to 2004 (69%). All chloroquine-susceptible isolates and most chloroquine-resistant isolates were susceptible to monodesethylamodiaquine (i.e., IC(50) < 60 nmol/L). Despite the positive correlation between chloroquine and monodesethylamodiaquine (r = 0.739, P < 0.05), the IC(50)s for monodesethylamodiaquine remained stable during 1997-2004, with no increase in the proportion of monodesethylamodiaquine-resistant isolates. Mefloquine, lumefantrine, and dihydroartemisinin were equally active against the chloroquine-susceptible and chloroquine-resistant parasites. The responses to these three drugs were positively correlated, and a significant decrease (P < 0.05) in the mean IC(50)s was observed during the study period compared with our earlier data in 1997-1999, probably because of their inverse relationship with chloroquine response. The in vitro results were in general agreement with the in vivo response to chloroquine and amodiaquine. In vitro drug susceptibility assay is a useful, complementary laboratory tool for determining the trend of response to drugs for which there is still no established molecular marker and may serve as an early warning system for emerging drug resistance.

Role of pfmdr1 mutations on chloroquine resistance in Plasmodium falciparum isolates with pfcrt K76T from Papua New Guinea

The N86Y mutation in pfmdr1 is reported to play an additional role for the chloroquine resistance in Plasmodium falciparum isolates. However, not much has been done to clarify whether this mutation augments the level of chloroquine resistance in the isolates harboring pfcrt K76T mutation. We compared the in vitro chloroquine efficacy between pfcrt K76T mutant parasites with or without N86Y mutation from Papua New Guinea. A total of 57 isolates (4% sensitive, 14% borderline, and 82% resistant) were successfully tested in vitro for chloroquine sensitivity. We found a slightly higher effective concentration of chloroquine needed to inhibit P. falciparum by 50% (mean EC 50 = 107 nM) in isolates with the pfcrt K76T + pfmdr1 N86Y than that in isolates with the pfcrt K76T + pfmdr1 N86 (EC 50 = 88 nM), but this difference was not statistically significant. A significant non-random association was observed between the pfcrt K76T and pfmdr1 N86Y alleles. Our results suggest that the pfmdr1 N86Y mutation plays a compensatory role to chloroquine-resistant isolates under a chloroquine pressure while it may also augment the level of chloroquine resistance in the K76T parasites to a small extent.

In vitro activity of iron-binding compounds against Senegalese isolates of Plasmodium falciparum

The in vitro activities of FR160, a synthetic catecholate siderophore, and two iron-binding agents, desferrioxamine and doxycycline, were evaluated against Plasmodium falciparum isolates. Correlations between these compounds and standard antimalarial drugs (chloroquine, quinine, amodiaquine, pyronaridine, artemether, artesunate, atovaquone, cycloguanil and pyrimethamine) were assessed to determine any degree of cross-resistance. Between October 1997 and February 1998, and September and November 1998, 189 P. falciparum isolates were obtained in Dielmo and Ndiop (Dakar). Their susceptibilities were assessed using an isotopic, microwell format, drug susceptibility test. The 137 inhibitory concentrations (IC(50)) values of FR160 ranged from 0.1 to 10 microM and the geometric mean IC(50) was 1.48 microM (95% CI = 1.29-1.68 microM). The geometric mean IC(50) of doxycycline for 121 isolates was 18.9 microM (95% CI = 16.8-21.3 microM) and that of desferrioxamine for 73 isolates was 20.7 microM (95% CI = 17.3-24.8 microM). FR160 was significantly less active against the chloroquine-resistant isolates (P < 0.0001). The mean IC(50)s of doxycycline were significantly higher for the chloroquine-susceptible isolates than for the resistant parasites (P = 0.0447). There was a weak correlation between the responses to FR160, desferrioxamine or doxycycline and those to the other antimalarial compounds (r(2) < 0.22). The activities of FR160 and desferrioxamine, determined for P. falciparum clones, were confirmed against 137 isolates. The coefficients of determination between the responses to FR160, doxycycline or desferrioxamine and those to all the antimalarial drugs tested are too weak to suggest cross-resistance. FR160 could be a rationale partner to use in combination with doxycycline.

Fractions of an antimalarial neem-leaf extract have activities superior to chloroquine, and are gametocytocidal

The antimalarial activities of two fractions (IRDN-A and IRDN-B) of an extract from the leaves of the neem tree (Azadirachta indica) were compared with those of chloroquine, in in-vitro assays against Plasmodium falciparum. The asexual stages of a chloroquine-sensitive clone (ITG2F6) and a chloroquine-resistant isolate (W2) and the gametocytes of the NF 54 (BD-7) isolate of P. falciparum were used as the drug targets. Activity against the asexual stages was generally evaluated as the concentrations inhibiting the parasitaemias recorded in the control cultures, after an incubation of 48-72 h, by 50% (IC50) or 100% (IC100). For the ITG2F6 strain, the IC50 and IC100 (in microg/ml) were, respectively, 10(-5) and 10(-4) for IRDN-A, 10(-3) and 10(-2) for IRDN-B, and 10(-2) and 1.0 for chloroquine. The corresponding values for the W2 strain were 10(-5) and 1.0 for IRDN-A, and 10.0 and >100 for chloroquine (even at 100 microg/ml, chloroquine only inhibited the parasitaemia by 85%). Each of the two neem-leaf fractions lysed 50% and 100% of developing gametocytes, at 10(-3) and 1.0 microg/ml, respectively; and 50% and 100% of mature gametocytes at 10(-3) and 10(2) microg/ml, respectively. If they are found safe and effective in vivo, the neem-leaf fractions may form the basis of new antimalarial drugs that not only cure chloroquine-sensitive and chloroquine-resistant malaria but also markedly reduce transmission.