Novel saturated 1,2,4-trioxanes and their antimalarial activity against multidrug resistant Plasmodium yoelii nigeriensis in Swiss mice model via oral route.

Novel saturated 6-(4'-aryloxy phenyl) vinyl 1,2,4-trioxanes 12a(1-3)-12d(1-3) and 13a(1-3)-13d(1-3) have been designed and synthesized, in one single step from diimide reduction of 11a(1-3)-11d(1-3). All the newly synthesized trioxanes were evaluated for their antimalarial activity against multi-drug resistant Plasmodium yoelii nigeriensis via oral route. Cyclopentane-based trioxanes 12b1, 12c1 and 12d1, provided 100 % protection to the infected mice at 24 mg/kg × 4 days. The most active compound of the series, trioxane 12b1, provided 100 % protection even at 12 mg/kg × 4 days and 60 % protection at 6 mg/kg × 4 days. The currently used drug, ß-arteether provides only 20 % protection at 24 mg/kg × 4 days.

An Alternative Autophagy-Related Mechanism of Chloroquine Drug Resistance in the Malaria Parasite.

We generated highly chloroquine (CQ)-resistant (ResCQ) Plasmodium yoelii parasites by stepwise exposure to increasing concentrations of CQ and CQ-sensitive parasites (SenCQ) by parallel mock treatments. No mutations in genes that are associated with drug resistance were detected in ResCQ clones. Autophagy-related genes were highly upregulated in SenCQ compared to ResCQ parasites during CQ treatment. This indicates that CQ resistance can be developed in the malaria parasite by the inhibition of autophagy as an alternative drug resistance mechanism.

Novel hydrazone derivatives of N-amino-11-azaartemisinin with high order of antimalarial activity against multidrug-resistant Plasmodium yoelii nigeriensis in Swiss mice via intramuscular route.

Novel hydrazone derivatives 10a-m were prepared from N-Amino-11-azaartemisinin (9) and screened for their antimalarial activity by oral and intramuscular (i.m.) routes against multidrug-resistant Plasmodium yoelii in Swiss mice model. Several of the hydrazone derivatives showed higher order of antimalarial activity. Compounds 10b, 10g, 10m provided 100% protection to the infected mice at the dose of 24 mg/kg × 4 days via oral route. Fluorenone based hydrazone 10m the most active compound of the series, provided 100% protection at the dose of 6 mg/kg × 4 days via intramuscular route and also provided 100% protection at the dose of 12 mg/kg × 4 days via oral route. While artemisinin gave 100% protection at 48 mg/kg × 4 days and only 60% protection at 24 mg/kg × 4 days via intramuscular (i.m.) route. Compound 10m found to be four-fold more active than artemisinin via intramuscular route.

Identification of first-in-class plasmodium OTU inhibitors with potent anti-malarial activity.

OTU proteases antagonize the cellular defense in the host cells and involve in pathogenesis. Intriguingly, P. falciparum, P. vivax, and P. yoelii have an uncharacterized and highly conserved viral OTU-like proteins. However, their structure, function or inhibitors have not been previously reported. To this end, we have performed structural modeling, small molecule screening, deconjugation assays to characterize and develop first-in-class inhibitors of P. falciparum, P. vivax, and P. yoelii OTU-like proteins. These Plasmodium OTU-like proteins have highly conserved residues in the catalytic and inhibition pockets similar to viral OTU proteins. Plasmodium OTU proteins demonstrated Ubiquitin and ISG15 deconjugation activities as evident by intracellular ubiquitinated protein content analyzed by western blot and flow cytometry. We screened a library of small molecules to determine plasmodium OTU inhibitors with potent anti-malarial activity. Enrichment and correlation studies identified structurally similar molecules. We have identified two small molecules that inhibit P. falciparum, P. vivax, and P. yoelii OTU proteins (IC50 values as low as 30 nM) with potent anti-malarial activity (IC50 of 4.1-6.5 µM). We also established enzyme kinetics, druglikeness, ADME, and QSAR model. MD simulations allowed us to resolve how inhibitors interacted with plasmodium OTU proteins. These findings suggest that targeting malarial OTU-like proteases is a plausible strategy to develop new anti-malarial therapies.

Effect of α-tocopheryloxy acetic acid, a vitamin E derivative mitocan, on the experimental infection of mice with Plasmodium yoelii.

BACKGROUND: Malaria parasites are known to be vulnerable to oxidative stress. In this study, the effects of the administration of α-tocopheryloxy acetic acid (α-TEA), which is a vitamin E analogue mitocan, on Plasmodium yoelii infection in mice were examined. METHODS: Alpha-TEA was mixed with diet and fed to C57BL/6J mice before and/or after infection. For parasite infection, 4 × 104 red blood cells infected with P. yoelii (strain 17XL) were inoculated by intraperitoneal injection. In another series of experiment, the effect of the oral administration of α-TEA on P. yoelii 17XL infection in mice was examined. Finally, the combined effect of α-TEA and dihydroartemisinin or chloroquine on P. yoelii 17XL infection was examined. RESULTS: When 0.25% α-TEA was mixed with the diet for 7 days before infection and 14 days after infection (in total for 21 days), for 14 days after infection, and for 11 days from the third day after infection, all P. yoelii 17XL-infected mice survived during the observation period. However, all control mice died within 12 days after infection. These results indicated that α-TEA functions effectively even when administered post-infection. The oral administration of α-TEA for P. yoelii 17XL infection was also significant. Although the infected mice in the solvent control died within 10 days after infection, 90% of the mice infected with P. yoelii 17XL survived during the observation period when treated with 10 mg/head/day of α-TEA for 3 days from day 3 after infection. Although the combined effect of α-TEA and dihydroartemisinin (DHA) or chloroquine on P. yoelii 17XL infection was significant, no synergistic or additive effects were observed from the survival curve. CONCLUSIONS: This study showed the beneficial effects of α-TEA on the experimental infection of mice with P. yoelii 17XL. The stimulatory action of α-TEA on mitochondria and the accompanying reactions, such as reactive oxygen species production, and induction of apoptosis might have some effect on malarial infection.

Novel naphthyl based 1,2,4-trioxanes: Synthesis and in vivo efficacy in the Plasmodium yoelii nigeriensis in Swiss mice.

A new series of 1,2,4-trioxanes 9a1-a4, 9b1-b4, 10-13 and 9c1-c4 were synthesized and evaluated against multidrug-resistant Plasmodium yoelii nigeriensis in Swiss mice via oral and intramuscular (i.m.) routes. Adamantane-based trioxane 9b4, the most active compound of the series, provided 100% protection to the infected mice at the dose 48 mg/kg × 4 days and 100% clearance of parasitemia at the dose 24 mg/kg × 4 days via oral route. Adamantane-based trioxane 9b4, is twice active than artemisinin. We have also studied the photooxygenation behaviour of allylic alcohols 6a-b (3-(4-alkoxynaphthyl)-but-2-ene-1-ols) and 6c (3-[4-(tert-butyl-dimethyl-silanyloxy)-naphthalen-1-yl]-but-2-en-1-ol). Being behaving as dienes, they furnished corresponding endoperoxides, while behaving as allylic alcohols, they yielded ß-hydroxyhydroperoxides. All the endoperoxides (7a-c) and ß-hydroxyhydroperoxides (8a-c) have been separately elaborated to the corresponding 1,2,4-trioxanes, except from endoperoxide 7c. It is worthy to note that TBDMS protected naphthoyl endoperoxide 7c unable to deliver 1,2,4-trioxane, which demonstrated the strength of the O-Si bond is not easy to cleave under acidic condition.

Synthesis of novel 1,2,4-trioxanes and antimalarial evaluation against multidrug-resistant Plasmodium yoelii nigeriensis.

Malaria epidemics represent one of the life-threatening diseases to low-income lying countries which subsequently affect the economic and social condition of mankind. In continuation in the development of a novel series of 1,2,4-trioxanes 13a1-c1, 13a2-c2, and 13a3-c3 have been prepared and further converted into their hemisuccinate derivatives 14a1-c1, 14a2-c2, and 14a3-c3 respectively. All these new compounds were evaluated for their antimalarial activity against multidrug-resistant Plasmodium yoelii nigeriensis in mice by both oral and intramuscular (im) routes. Hydroxy-functionalized trioxane 13a1 showed 80% protection and its hemisuccinate derivative 14a1 showed 100% protection at a dose of 48 mg/kg × 4 days by both routes, which is twice active than artemisinin by oral route.

Anti-malarial activity of traditional Kampo medicine Coptis rhizome extract and its major active compounds.

BACKGROUND: Herbal medicine has been a rich source of new drugs exemplified by quinine and artemisinin. In this study, a variety of Japanese traditional herbal medicine ('Kampo') were examined for their potential anti-malarial activities. METHODS: A comprehensive screening methods were designed to identify novel anti-malarial drugs from a library of Kampo herbal extracts (n = 120) and related compounds (n = 96). The anti-malarial activity was initially evaluated in vitro against chloroquine/mefloquine-sensitive (3D7) and-resistant (Dd2) strains of Plasmodium falciparum. The cytotoxicity was also evaluated using primary adult mouse brain cells. After being selected through the first in vitro assay, positive extracts and compounds were examined for possible in vivo anti-malarial activity. RESULTS: Out of 120 herbal extracts, Coptis rhizome showed the highest anti-malarial activity (IC50 1.9 µg/mL of 3D7 and 4.85 µg/mL of Dd2) with a high selectivity index (SI) > 263 (3D7) and > 103 (Dd2). Three major chlorinated compounds (coptisine, berberine, and palmatine) related to Coptis rhizome also showed anti-malarial activities with IC50 1.1, 2.6, and 6.0 µM (against 3D7) and 3.1, 6.3, and 11.8 µM (against Dd2), respectively. Among them, coptisine chloride exhibited the highest anti-malarial activity (IC50 1.1 µM against 3D7 and 3.1 µM against Dd2) with SI of 37.8 and 13.2, respectively. Finally, the herbal extract of Coptis rhizome and its major active compound coptisine chloride exhibited significant anti-malarial activity in mice infected with Plasmodium yoelii 17X strain with respect to its activity on parasite suppression consistently from day 3 to day 7 post-challenge. The effect ranged from 50.38 to 72.13% (P < 0.05) for Coptis rhizome and from 81 to 89% (P < 0.01) for coptisine chloride. CONCLUSION: Coptis rhizome and its major active compound coptisine chloride showed promising anti-malarial activity against chloroquine-sensitive (3D7) and -resistant (Dd2) strains in vitro as well as in vivo mouse malaria model. Thus, Kampo herbal medicine is a potential natural resource for novel anti-malarial agents.

The gender-related variability in the pharmacokinetics and antiplasmodial activity of naphthoquine in rodents.

BACKGROUND: Naphthoquine (NQ) is a suitable partner anti-malarial for the artemisinin-based combination therapy (ACT), which is recommended to be taken orally as a single-dose regimen. The metabolism of NQ was mainly mediated by CYP2D6, which is well-known to show gender-specific differences in its expression. In spite of its clinical use, there is limited information on the pharmacokinetics of NQ, and no data are available for females. In this study, the effect of gender on the pharmacokinetics and antiplasmodial efficacy of NQ in rodents was evaluated. The underlying factors leading to the potential gender difference, i.e., plasma protein binding and metabolic clearance, were also evaluated. METHODS: The pharmacokinetic profiles of NQ were investigated in healthy male or female rats after a single oral administration of NQ. The antiplasmodial efficacy of NQ was studied in male or female mice infected with Plasmodium yoelii. The recrudescence and survival time of infected mice were also recorded after drug treatment. Plasma protein binding of NQ was determined in pooled plasma collected from male or female mice, rat or human. In vitro metabolism experiments were performed in the liver microsomes of male or female mice, rat or human. RESULTS: The results showed that the gender of rats did not affect NQ exposure (AUC0-t and Cmax) significantly (P > 0.05). However, a significant (P < 0.05) longer t1/2 was found for NQ in male rats (192.1 ± 47.7), compared with female rats (143.9 ± 27.1). Slightly higher but not significant (P > 0.05) antiplasmodial activity was found for NQ in male mice (ED90, 1.10 mg/kg) infected with P. yoelii, compared with female mice (ED90, 1.67 mg/kg). The binding rates of NQ to plasma protein were similar in males and females. There was no metabolic difference for NQ in male and female mice, rat or human liver microsomes. CONCLUSIONS: These results indicated that the pharmacokinetic profiles of NQ were similar between male and female rats, except for a longer t1/2 in male rats. The difference was not associated with plasma protein binding or hepatic metabolic clearance. Equivalent antiplasmodial activity was found for NQ in male and female mice infected with P. yoelii. This study will be helpful for the rational design of clinical trials for NQ.

In vivo antimalarial activity and pharmacokinetics of artelinic acid-choline derivative liposomes in rodents.

It is urgent to develop new antimalarial drugs with good therapeutic effects to address the emergence of drug resistance. Here, the artelinic acid-choline derivative (AD) was synthesized by dehydration reaction and esterification reaction, aimed to avoid the emergence of drug resistance by synergistic effect of artemisinins and choline derivative, which could compete with choline for rate-limiting enzymes in the phosphatidylcholine (PC) biosynthetic pathway. AD was formulated into liposomes (ADLs) by the thin-film hydration method. Efficacy of ADLs was evaluated by Peters 4-day suppression test. The suppression percentage against Plasmodium yoelii BY265 (PyBY265) in ADLs group was higher than those of positive control groups (dihydroartemisinin liposomes, P < 0.05) and other control groups (P ⩽ 0.05) at the doses of 4.4, 8.8, 17.6 µmol (kg·d)-1, respectively. The negative conversion fraction, recrudescence fraction and survival fraction of ADLs group were superior to other control groups. Pharmacokinetics in rats after intravenous injection suggested that ADLs exhibited higher exposure levels (indexed by area under concentration-time curve) than that of AD solution, artelinic acid liposomes or artelinic acid solution (P < 0.01). Taken together, ADLs exhibited promising antimalarial efficacy and pharmacokinetic characteristics.

Antimalarial Eudesmane Sesquiterpenoids from Dobinea delavayi.

Eleven new angeloylated eudesmane sesquiterpenoids, dobinins D-N (2, 3, 5, 6, 8, 9, and 11-15), and four known compounds (1, 4, 7, and 10) were isolated from the roots of Dobinea delavayi. A new oxidation product (8a) was also obtained from dobinin H (8). Their structures were elucidated by spectroscopic data and single-crystal X-ray diffraction analyses. Dobinins K-N (12-15) are the first examples of rearrangement noreudesmane analogue sesquiterpenoids with a unique 6/5-fused carbon skeleton. A putative biosynthetic pathway of compounds 12-15 is proposed. Compound 12 exhibited significant antimalarial activity superior to artemisinin with the inhibition ratio of 59.1%, and compounds 3, 5, and 15 exhibited moderate antimalarial activities against Plasmodium yoelii BY265RFP with inhibition ratios ranging from 14.5% to 18.5% at a dose of 30 mg/kg/day. In addition, the apoptosis of P. yoelii BY265RFP by the depolarization of mitochondrial membrane potential with striking ROS production, after parasitized erythrocyte lysis mediated by cytokines IL-12 and IFN-γ, may be a possible mechanism of antimalarial action of compound 12 against P. yoelii BY265RFP.

Dodelates A-E: Five dimeric eudesmane sesquiterpenoids from Dobinea delavayi.

Dodelates A-E (1-5), five angeloylated eudesmane sesquiterpenoid dimers were isolated from the roots of Dobinea delavayi. Their structures were elucidated by extensive spectroscopic data and single-crystal X-ray diffraction analyses. A possible biosynthetic pathway of these five compounds was proposed. Compounds 1 and 3 exhibited moderate antimalarial activities against Plasmodium yoelii BY265RFP.

ELQ-331 as a prototype for extremely durable chemoprotection against malaria.

BACKGROUND: The potential benefits of long-acting injectable chemoprotection (LAI-C) against malaria have been recently recognized, prompting a call for suitable candidate drugs to help meet this need. On the basis of its known pharmacodynamic and pharmacokinetic profiles after oral dosing, ELQ-331, a prodrug of the parasite mitochondrial electron transport inhibitor ELQ-300, was selected for study of pharmacokinetics and efficacy as LAI-C in mice. METHODS: Four trials were conducted in which mice were injected with a single intramuscular dose of ELQ-331 or other ELQ-300 prodrugs in sesame oil with 1.2% benzyl alcohol; the ELQ-300 content of the doses ranged from 2.5 to 30 mg/kg. Initial blood stage challenges with Plasmodium yoelii were used to establish the model, but the definitive study measure of efficacy was outcome after sporozoite challenge with a luciferase-expressing P. yoelii, assessed by whole-body live animal imaging. Snapshot determinations of plasma ELQ-300 concentration ([ELQ-300]) were made after all prodrug injections; after the highest dose of ELQ-331 (equivalent to 30 mg/kg ELQ-300), both [ELQ-331] and [ELQ-300] were measured at a series of timepoints from 6 h to 5½ months after injection. RESULTS: A single intramuscular injection of ELQ-331 outperformed four other ELQ-300 prodrugs and, at a dose equivalent to 30 mg/kg ELQ-300, protected mice against challenge with P. yoelii sporozoites for at least 4½ months. Pharmacokinetic evaluation revealed rapid and essentially complete conversion of ELQ-331 to ELQ-300, a rapidly achieved (< 6 h) and sustained (4-5 months) effective plasma ELQ-300 concentration, maximum ELQ-300 concentrations far below the estimated threshold for toxicity, and a distinctive ELQ-300 concentration versus time profile. Pharmacokinetic modeling indicates a high-capacity, slow-exchange tissue compartment which serves to accumulate and then slowly redistribute ELQ-300 into blood, and this property facilitates an extremely long period during which ELQ-300 concentration is sustained above a minimum fully-protective threshold (60-80 nM). CONCLUSIONS: Extrapolation of these results to humans predicts that ELQ-331 should be capable of meeting and far-exceeding currently published duration-of-effect goals for anti-malarial LAI-C. Furthermore, the distinctive pharmacokinetic profile of ELQ-300 after treatment with ELQ-331 may facilitate durable protection and enable protection for far longer than 3 months. These findings suggest that ELQ-331 warrants consideration as a leading prototype for LAI-C.

Primaquine-quinoxaline 1,4-di-N-oxide hybrids with action on the exo-erythrocytic forms of Plasmodium induce their effect by the production of reactive oxygen species.

BACKGROUND: The challenge in anti-malarial chemotherapy is based on the emergence of resistance to drugs and the search for medicines against all stages of the life cycle of Plasmodium spp. as a therapeutic target. Nowadays, many molecules with anti-malarial activity are reported. However, few studies about the cellular and molecular mechanisms to understand their mode of action have been explored. Recently, new primaquine-based hybrids as new molecules with potential multi-acting anti-malarial activity were reported and two hybrids of primaquine linked to quinoxaline 1,4-di-N-oxide (PQ-QdNO) were identified as the most active against erythrocytic, exoerythrocytic and sporogonic stages. METHODS: To further understand the anti-malarial mode of action (MA) of these hybrids, hepg2-CD81 were infected with Plasmodium yoelii 17XNL and treated with PQ-QdNO hybrids during 48 h. After were evaluated the production of ROS, the mitochondrial depolarization, the total glutathione content, the DNA damage and proteins related to oxidative stress and death cell. RESULTS: In a preliminary analysis as tissue schizonticidals, these hybrids showed a mode of action dependent on peroxides production, but independent of the activation of transcription factor p53, mitochondrial depolarization and arrest cell cycle. CONCLUSIONS: Primaquine-quinoxaline 1,4-di-N-oxide hybrids exert their antiplasmodial activity in the exoerythrocytic phase by generating high levels of oxidative stress which promotes the increase of total glutathione levels, through oxidation stress sensor protein DJ-1. In addition, the role of HIF1a in the mode of action of quinoxaline 1,4-di-N-oxide is independent of biological activity.

Whole-Killed Blood-Stage Vaccine-Induced Immunity Suppresses the Development of Malaria Parasites in Mosquitoes.

As a malaria transmission-blocking vaccine alone does not confer a direct benefit to the recipient, it is necessary to develop a vaccine that not only blocks malaria transmission but also protects vaccinated individuals. In this study we observed that a whole-killed blood-stage vaccine (WKV) not only conferred protection against the blood-stage challenge but also markedly inhibited the transmission of different strains of the malaria parasite. Although the parasitemia is much lower in WKV-immunized mice challenged with malaria parasites, the gametocytemia is comparable between control and immunized mice during the early stages of infection. The depletion of CD4+ T cells prior to the adoptive transfer of parasites into WKV-immunized mice has no effect on the development of the malaria parasite in the mosquito, but the adoptive transfer of the serum from the immunized mice into the parasite-inoculated mice remarkably suppresses the development of malaria parasites in mosquitoes. Furthermore, immunized mice challenged with the malaria parasite generate higher levels of parasite-specific Abs and the inflammatory cytokines MCP-1 and IFN-γ. However, the adoptive transfer of parasite-specific IgG or the depletion of MCP-1, but not IFN-γ, to some extent is closely associated with the suppression of malaria parasite development in mosquitoes. These data strongly suggest that WKV-induced immune responses confer protection against the mosquito stage, which is largely dependent on malaria parasite-specific Abs and MCP-1. This finding sheds new light on blocking malaria transmission through the immunization of individuals with the WKV.

Metabolism of Piperaquine to Its Antiplasmodial Metabolites and Their Pharmacokinetic Profiles in Healthy Volunteers.

As a partner antimalarial for artemisinin drug-based combination therapy (ACT), piperaquine (PQ) can be metabolized into two major metabolites, including piperaquine N-oxide (M1) and piperaquine N,N-dioxide (M2). To better understand the antimalarial potency of PQ, the antimalarial activity of the PQ metabolites (M1 and M2) was studied in vitro (in Plasmodium falciparum strains Pf3D7 and PfDd2) and in vivo (in the murine species Plasmodium yoelii) in this study. The recrudescence and survival time of infected mice were also recorded after drug treatment. The pharmacokinetic profiles of PQ and its two metabolites (M1 and M2) were investigated in healthy subjects after oral doses of two widely used ACT regimens, i.e., dihydroartemisinin plus piperaquine phosphate (Duo-Cotecxin) and artemisinin plus piperaquine (Artequick). Remarkable antiplasmodial activity was found for PQ (50% growth-inhibitory concentration [IC50], 4.5 nM against Pf3D7 and 6.9 nM against PfDd2; 90% effective dose [ED90], 1.3 mg/kg of body weight), M1 (IC50, 25.5 nM against Pf3D7 and 38.7 nM against PfDd2; ED90, 1.3 mg/kg), and M2 (IC50, 31.2 nM against Pf3D7 and 33.8 nM against PfDd2; ED90, 2.9 mg/kg). Compared with PQ, M1 showed comparable efficacy in terms of recrudescence and survival time and M2 had relatively weaker antimalarial potency. PQ and its two metabolites displayed a long elimination half-life (∼11 days for PQ, ∼9 days for M1, and ∼4 days for M2), and they accumulated after repeated administrations. The contribution of the two PQ metabolites to the efficacy of piperaquine as a partner drug of ACT for the treatment of malaria should be considered for PQ dose optimization.

Synthesis, Biological Evaluation, and Molecular Modeling Studies of Chiral Chloroquine Analogues as Antimalarial Agents.

In a focused exploration, we designed, synthesized, and biologically evaluated chiral conjugated new chloroquine (CQ) analogues with substituted piperazines as antimalarial agents. In vitro as well as in vivo studies revealed that compound 7c showed potent activity (in vitro 50% inhibitory concentration, 56.98 nM for strain 3D7 and 97.76 nM for strain K1; selectivity index in vivo [up to at a dose of 12.5 mg/kg of body weight], 3,510) as a new lead antimalarial agent. Other compounds (compounds 6b, 6d, 7d, 7h, 8c, 8d, 9a, and 9c) also showed moderate activity against a CQ-sensitive strain (3D7) and superior activity against a CQ-resistant strain (K1) of Plasmodium falciparum Furthermore, we carried out docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of all in-house data sets (168 molecules) of chiral CQ analogues to explain the structure-activity relationships (SAR). Our new findings specify the significance of the H-bond interaction with the side chain of heme for biological activity. In addition, the 3D-QSAR study against the 3D7 strain indicated the favorable and unfavorable sites of CQ analogues for incorporating steric, hydrophobic, and electropositive groups to improve the antimalarial activity.

Adaptive immunity is essential in preventing recrudescence of Plasmodium yoelii malaria parasites after artesunate treatment.

Artemisinin-based antimalarials, such as artesunate (ART), alone or in combination, are the mainstay of the therapy against malaria caused by Plasmodium falciparum. However, the emergence and spread of artemisinin resistance threatens the future success of its global malaria eradication. Although much of the reported artemisinin resistance can be attributed to mutations intrinsic to the parasite, a significant proportion of treatment failures are thought to be due to other factors such as the host's immune system. Exactly how the immune system participates in the clearance and elimination of malaria parasites during ART treatment is unknown. Here, we show that a developing primary immune response, involving both B and CD4+ T cells, is necessary for the complete elimination but not initial clearance, of Plasmodium yoelii YM parasites in mice treated with ART. Our study uncovers a dynamic interplay between ART and host adaptive immunity in Plasmodium sp. elimination.

Differential activity of methylene blue against erythrocytic and hepatic stages of Plasmodium.

BACKGROUND: In the context of malaria elimination/eradication, drugs that are effective against the different developmental stages of the parasite are highly desirable. The oldest synthetic anti-malarial drug, the thiazine dye methylene blue (MB), is known for its activity against Plasmodium blood stages, including gametocytes. The aim of the present study was to investigate a possible effect of MB against malaria parasite liver stages. METHODS: MB activity was investigated using both in vitro and in vivo models. In vitro assays consisted of testing MB activity on Plasmodium falciparum, Plasmodium cynomolgi and Plasmodium yoelii parasites in human, simian or murine primary hepatocytes, respectively. MB in vivo activity was evaluated using intravital imaging in BALB/c mice infected with a transgenic bioluminescent P. yoelii parasite line. The transmission-blocking activity of MB was also addressed using mosquitoes fed on MB-treated mice. RESULTS: MB shows no activity on Plasmodium liver stages, including hypnozoites, in vitro in primary hepatocytes. In BALB/c mice, MB has moderate effect on P. yoelii hepatic development but is highly effective against blood stage growth. MB is active against gametocytes and abrogates parasite transmission from mice to mosquitoes. CONCLUSION: While confirming activity of MB against both sexual and asexual blood stages, the results indicate that MB has only little activity on the development of the hepatic stages of malaria parasites.

Antimalarial activity and metabolism of dihydroartemisinin-derived dimer.

The dihydroartemisinin-derived dimer (DHA dimer) was synthesized, and its antimalarial activities were evaluated both in vitro and in vivo. The dimer IC50 value of 0.51 ± 0.12 nM in vitro was significantly lower than that of DHA at 1.81 ± 0.70 nM. The dimer ED50 values were 0.44 ± 0.03 and 0.18 ± 0.03 mg/(kg·day) in vivo for intragastric (i.g.) and intravenous (i.v.) groups, respectively, to Plasmodium yoelii rodent malaria. It also performed better relative to those of DHA which had ED50 values of 0.76 ± 0.03 mg/(kg·day) (i.g.) and 0.32 ± 0.03 mg/(kg·day) (i.v.). Moreover, the recrudescence rate, negative conversion rate, and cure rate of the dimer showed superior performance. Furthermore, the metabolites and major metabolic pathways of the dimer in rats were preliminarily investigated using the HPLC-HRMSn method. Twenty-seven metabolites, including DHA, 11 metabolites of DHA, and 15 other novel metabolites, were detected in rats after i.g. administration of dimer. The metabolic pathways of the 15 novel metabolites were inferred: deoxygenation, hydroxylation, and hydroxylation with dehydration.