A Plant-Derived Morphinan as a Novel Lead Compound Active against Malaria Liver Stages

BACKGROUND: The global spread of multidrug–resistant malaria parasites has led to an urgent need for new chemotherapeutic agents. Drug discovery is primarily directed to the asexual blood stages, and few drugs that are effective against the obligatory liver stages, from which the pathogenic blood infection is initiated, have become available since primaquine was deployed in the 1950s. METHODS AND FINDINGS: Using bioassay-guided fractionation based on the parasite's hepatic stage, we have isolated a novel morphinan alkaloid, tazopsine, from a plant traditionally used against malaria in Madagascar. This compound and readily obtained semisynthetic derivatives were tested for inhibitory activity against liver stage development in vitro (P. falciparum and P. yoelii) and in vivo (P. yoelii). Tazopsine fully inhibited the development of P. yoelii (50% inhibitory concentration [IC(50)] 3.1 μM, therapeutic index [TI] 14) and P. falciparum (IC(50) 4.2 μM, TI 7) hepatic parasites in cultured primary hepatocytes, with inhibition being most pronounced during the early developmental stages. One derivative, N-cyclopentyl-tazopsine (NCP-tazopsine), with similar inhibitory activity was selected for its lower toxicity (IC(50) 3.3 μM, TI 46, and IC(50) 42.4 μM, TI 60, on P. yoelii and P. falciparum hepatic stages in vitro, respectively). Oral administration of NCP-tazopsine completely protected mice from a sporozoite challenge. Unlike the parent molecule, the derivative was uniquely active against Plasmodium hepatic stages. CONCLUSIONS: A readily obtained semisynthetic derivative of a plant-derived compound, tazopsine, has been shown to be specifically active against the liver stage, but inactive against the blood forms of the malaria parasite. This unique specificity in an antimalarial drug severely restricts the pressure for the selection of drug resistance to a parasite stage limited both in numbers and duration, thus allowing researchers to envisage the incorporation of a true causal prophylactic in malaria control programs

Plasmodium falciparum Clearance Is Rapid and Pitting Independent in Immune Malian Children Treated With Artesunate for Malaria

Background. In Plasmodium falciparum-infected patients treated with artemisinins, parasitemia declines through so-called pitting, an innate splenic process that transforms infected red blood cells (iRBCs) into onceinfected RBCs (O-iRBCs). Methods. We measured pitting in 83 French travelers and 42 Malian children treated for malaria with artesunate. Results. In travelers, O-iRBCs peaked at 107.7% initial parasitemia. In Malian children aged 1.5-4 years, OiRBCs peaked at higher concentrations than in children aged 9-13 years (91.60% vs 31.95%; P = .0097). The parasite clearance time in older children was shorter than in younger children (P = .0001), and the decline in parasitemia in children aged 1.5-4 years often started 6 hours after treatment initiation, a lag phase generally absent in infants and older children. A 6-hour lag phase in artificial pitting of artesunate-exposed iRBCs was also observed in vitro. The proportion of iRBCs recognized by autologous immunoglobulin G (IgG) correlated with the parasite clearance time (r = −0.501; P = .0006) and peak O-iRBC concentration (r = −0.420; P = .0033). Conclusions. Antimalarial immunity correlates with fast artemisinin-induced parasite clearance and low pitting rates. In nonimmune populations, artemisinin-induced P. falciparum clearance is related to pitting and starts after a 6-hour lag phase. In immune populations, passively and naturally acquired immune mechanisms operating faster than pitting may exist. This mechanism may mitigate the emergence of artemisinin-resistant P. falciparum in Africa

Reduced erythrocyte deformability associated with hypoargininemia during Plasmodium falciparum malaria.

International audienceThe mechanisms underlying reduced red blood cell (RBC) deformability during Plasmodium falciparum (Pf) malaria remain poorly understood. Here, we explore the possible involvement of the L-arginine and nitric oxide (NO) pathway on RBC deformability in Pf-infected patients and parasite cultures. RBC deformability was reduced during the acute attack (day0) and returned to normal values upon convalescence (day28). Day0 values correlated with plasma L-arginine levels (r = 0.69; p = 0.01) and weakly with parasitemia (r = -0.38; p = 0.006). In vitro, day0 patient's plasma incubated with ring-stage cultures at 41°C reduced RBC deformability, and this effect correlated strongly with plasma L-arginine levels (r = 0.89; p < 0.0001). Moreover, addition of exogenous L-arginine to the cultures increased deformability of both Pf-free and trophozoite-harboring RBCs. NO synthase activity, evidenced in Pf-infected RBCs, induced L-arginine-dependent NO production. These data show that hypoargininemia during P. falciparum malaria may altogether impair NO production and reduce RBC deformability, particularly at febrile temperature

In Vitro Activities of 25 Quinolones and Fluoroquinolones against Liver and Blood Stage Plasmodium spp.

The in vitro activities of 25 quinolones and fluoroquinolones against erythrocytic stages of Plasmodium falciparum and against liver stages of Plasmodium yoelii yoelii and P. falciparum were studied. All compounds were inhibitory for chloroquine-sensitive and chloroquine-resistant P. falciparum grown in red blood cells. This inhibitory effect increased with prolonged incubation and according to the logarithm of the drug concentration. Grepafloxacin, trovafloxacin, and ciprofloxacin were the most effective drugs, with 50% inhibitory concentrations of <10 μg/ml against both strains. Only grepafloxacin, piromidic acid, and trovafloxacin had an inhibitory effect against hepatic stages of P. falciparum and P. yoelii yoelii; this effect combined reductions of the numbers and the sizes of schizonts in treated cultures. Thus, quinolones have a potential for treatment or prevention of malaria through their unique antiparasitic effect against erythrocytic and hepatic stages of Plasmodium

Plasmodium falciparum Clearance Is Rapid and Pitting Independent in Immune Malian Children Treated With Artesunate for Malaria

International audienceBackground. In Plasmodium falciparum-infected patients treated with artemisinins, parasitemia declines through so-called pitting, an innate splenic process that transforms infected red blood cells (iRBCs) into once-infected RBCs (O-iRBCs).Methods. We measured pitting in 83 French travelers and 42 Malian children treated for malaria with artesunate.Results. In travelers, O-iRBCs peaked at 107.7% initial parasitemia. In Malian children aged 1.5-4 years, O-iRBCs peaked at higher concentrations than in children aged 9-13 years (91.60% vs 31.95%; P = .0097). The parasite clearance time in older children was shorter than in younger children (P = .0001), and the decline in parasitemia in children aged 1.5-4 years often started 6 hours after treatment initiation, a lag phase generally absent in infants and older children. A 6-hour lag phase in artificial pitting of artesunate-exposed iRBCs was also observed in vitro. The proportion of iRBCs recognized by autologous immunoglobulin G (IgG) correlated with the parasite clearance time (r = −0.501; P = .0006) and peak O-iRBC concentration (r = −0.420; P = .0033).Conclusions. Antimalarial immunity correlates with fast artemisinin-induced parasite clearance and low pitting rates. In nonimmune populations, artemisinin-induced P. falciparum clearance is related to pitting and starts after a 6-hour lag phase. In immune populations, passively and naturally acquired immune mechanisms operating faster than pitting may exist. This mechanism may mitigate the emergence of artemisinin-resistant P. falciparum in Africa

Inhibitory Activity of NCP-Tazopsine on <i>Plasmodium</i> Hepatic Stages in vitro and Protective Activity of Tazopsine and NCP-Tazopsine for P. yoelii Infections in vivo

<div><p>(A) Representative curve of the dose-dependent inhibition of NCP-tazopsine (the number of parasite forms in test wells divided by the number of parasites in control wells) for (graph a) P. yoelii (filled squares [], average number in control wells = 152), and (graph b) P. falciparum (filled circles [•], average number in control wells = 384). Toxicity is presented for (graph c) mouse (open squares [□]) or (graph d) human (open circle [○]) primary hepatocytes. Results are means ± standard deviation within triplicates of one experiment.</p> <p>(B) Proportion of mice that failed to acquire a blood-stage infection following sporozoite challenge through treatment with tazopsine (filled diamonds [♦], 100 mg/kg), NCP-tazopsine (filled circles [•], 100 mg/kg, and open circles [○], 200 mg/kg), distilled water or 10% Tween in distilled water (asterisks [*] indicate control groups).</p> <p>(C) Dose-dependent reduction of hepatic parasite load by NCP-tazopsine following sporozoite challenge. Bar represents the average value. Significance was calculated in one-way ANOVA analysis for the whole dataset (<i>p</i> = 0.0012), coupled to a Tukey HSD test: double asterisks (**) indicate <i>p</i> < 0.01.</p></div

Inhibitory Activity of Tazopsine on <i>Plasmodium</i> Hepatic Stages in vitro

<div><p>(A) Representative curve of the dose-dependent inhibition of tazopsine (the number of parasite forms in test wells divided by the number of parasites in control wells) for graph a, P. yoelii (filled squares [], average number in control wells = 274), and graph b, P. falciparum (filled circles [•], average number in control wells = 83). Toxicity as assessed by the neutral red assay is presented for graph c, mouse (open squares [□]), or graph d, human (open circles [○]) primary hepatocytes. Results are means ± standard deviation within triplicates of one experiment. <i>R</i> represents the factor of regression calculated by SigmaPlot.</p> <p>(B) Dose-dependent influence on the size of surviving P. falciparum hepatic forms, (21 forms were counted for each point, and the bar represents the median value). Significance was calculated in one-way ANOVA analysis for the whole dataset (<i>p</i> < 0.0001), coupled to a Tukey HSD test: double asterisks (**) indicate <i>p</i> < 0.01.</p> <p>(C) P. falciparum hepatic forms (arrow) from cultures treated with tazopsine (15 μM) for 5 d, or untreated control cultures. DAPI staining is shown in the right images, and P. falciparum–specific staining (anti-PfHSP70 antibodies) in the left images. N, hepatocyte nucleus. The bar represents 5 μm.</p></div