In vitro antimalarial activity of flavonoid derivatives dehydrosilybin and 8-(1;1)-DMA-kaempferide

Multidrug-resistant Plasmodium falciparum strains are an increasing problem in endemic areas and are partly responsible for the worsening malaria situation around the world. New cheap and effective compounds active in combination with available drug in the field are urgently needed. The aim of this work was to explore the potential antiplasmodial effect of flavonoid derivatives on parasites growth in vitro. In vitro antiplasmodial activity of dehydrosilybin and 8-(1;1)-DMA-kaempferide has been evaluated by real time PCR for five P. falciparum strains. Both revealed significative antimalarial activity against the different strains. Since this drug family has been largely used and well-tolerated in humans, flavonoid derivatives could be in the near future associated with already available drugs in order to delay the spread of P. falciparum resistance.     

Phytochemical licochalcone A enhances antimalarial activity of artemisinin in vitro

Resistance to synthetic first-line antimalarial drugs is considered to be a major cause of increased malaria morbidity and mortality. Use of artemisinin-based combination therapies (ACTs) is being encouraged to reduce the malaria mortality in areas of falciparum resistance. Artemisinin is a natural product at times in short supply. With projected rise in demand of artemisinin there is an unmet need for alternate ACTs. Novel compounds that reduce dependance on artemisinin are required. In vitro cultures of Plasmodium falciparum provide a screen system for identifying and evaluating new drug combinations. Interactions of two phytochemicals, artemisinin and licochalcone A, has been studied against synchronized erythrocytic stages of chloroquine-sensitive 3D7 and chloroquine-resistant RKL 303 strains of P. falciparum. These two compounds in combination show synergistic antiplasmodial activity in vitro on these strains. Artemisinin but not licochalcone A interferes with hemozoin formation. Neither of the phytochemicals alone or in combination obstructs sorbitol-induced hemolysis.     

Inhibition of the growth of Plasmodium falciparum and Plasmodium berghei in vitro by an extract of Cochlospermum angolense (Welw.)

An extract of Cochlospermum angolense (Welw.) is used in the traditional medicine of Angola for the therapy of icterus and for the prophylaxis of malaria. From the roots of this plant red crystalline substances have been isolated and tested for their effect on Plasmodium falciparum in vitro and on the DNA and protein synthesis of Plasmodium berghei. The multiplication of P. falciparum was decreased to 50% of the control in the presence of 10 μg/ml extracted material and there was a total inhibition at a concentration of 50 μ/ml. If mice erythrocytes infected by P. berghei were incubated for 6 h with 25 μg/ml of the extract DNA synthesis was depressed to nearly background level. And, even more important, this effect could be demonstrated immediately. On the contrary, protein synthesis continued for at least 90 min at a reduced rate and stopped then. The results obtained show the direct antiparasitic effect of the substances extracted from C. angolense. The activity seems to be directed against DNA synthesis.     

In vitro antimalarial activity of retinoids and the influence of selective retinoic acid receptor antagonists

Retinol (vitamin A alcohol) may have a beneficial role in the host response to malaria in humans and previously published data have suggested that it has a direct inhibitory effect on the growth of Plasmodium falciparum in vitro. To further investigate the role of retinoids as potential antimalarial agents, we assessed the effect of all-trans-retinoic acid (RA), 9-cis-RA and 13-cis-RA, as well as retinol itself and its ester, retinyl palmitate, on 3H-hypoxanthine uptake by the laboratory-adapted strains of P. falciparum 3D7 and K1. In addition, we examined the influence of three specific RA receptor antagonists, ER 27191, Ro 415253 and AGN 194301, on retinoid-induced growth inhibition of 3D7. All-trans-RA, 9-cis-RA and 13-cis-RA in concentrations ranging from 1 x 10(-4) to 5 x 10(-10) M each had antimalarial activity, but at IC50 values (5.9 x 10(-5) to 7.9 x 10(-5) M) that were less than those of retinol (2.5 x 10(-5) to 3.2 x 10(-5) M). Retinyl palmitate had minimal effect on 3H-hypoxanthine uptake. Each of the three specific antagonists inhibited growth of 3D7 (IC50 range 1.2 x 10(-5) to 3.0 x 10(-5) M) but, in isobolographic analysis, were antagonistic to retinol (dose factor potentiation, DFP 0.46-0.79) and, in the case of Ro 415253, to all-trans-RA (DFP=0.39). Although we did not attempt to quantify losses of retinoids from the system, these data suggest that retinol has greater antimalarial activity than its RA metabolites and especially retinyl palmitate. The specific RA receptor antagonists showed paradoxical antimalarial activity but consistently antagonised the effect of retinol and all-trans-RA in isobolographic experiments. We conclude that RA metabolites may be less suitable than retinol per se as antimalarial agents and that P. falciparum might possess or acquire a RA receptor-like moiety.     

In vitro antiplasmodial activity of Central American medicinal plants

The in vitro antiplasmodial activities of 14 plant species traditionally used in Central America for the treatment of malaria or fever were evaluated. Lipophilic extracts of Piper hispidum, Siparuna andina, S. pauciflora, S. tonduziana, and Xylopia cf. frutescens, proved to be active against both a chloroquine-sensitive and a resistant strain of Plasmodium falciparum. IC50 values ranged between 3.0 microg/ml and 21.9 microg/ml; however, moderate cytotoxicity of active extracts was observed. Bioactivity-guided fractionation of Piper hispidum yielded 2',4, 6'-trihydroxy-4'-methoxydihydrochalcone (asebogenin) as an active compound.     

Plasmodium falciparum GPI toxin: A common foe for man and mosquito

The glycosylphosphatidylinositol (GPI) anchor of the malaria parasite, Plasmodium falciparum, which can be regarded as an endotoxin, plays a role in the induced pathology associated with severe malaria in humans. However, it is unclear whether the main mosquito vector, Anopheles gambiae, can specifically recognize, and respond to GPI from the malaria parasite. Recent data suggests that the malaria vector does mount a specific response against malaria GPI. In addition, following the strong immune response, mosquito fecundity is severely affected, resulting in a significant reduction in viable eggs produced. In this mini-review we look at the increased interest in understanding the way that malaria antigens are recognized in the mosquito, and how this relates to a better understanding of the interactions between the malaria parasite and both human and vector.     

In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen

The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust high-throughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of approximately 1.7 million compounds, we identified a diverse collection of approximately 6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 microM). Most known antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities.     

In vitro antiplasmodial activity and cytotoxicity of extracts and fractions of Vitex madiensis, medicinal plant of Gabon

Vitex madiensis Oliv. (Lamiaceae) is traditionally used to treat malaria symptoms in Haut‐Ogooué, Gabon. Leaves and stem barks extracts were obtained using dichloromethane (CH₂Cl₂), ethyl acetate (EtOAc) and methanol (MeOH) as extraction solvents and fractionated on silica gel column. The in vitro antiplasmodial activity of CH₂Cl₂, EtOAc and MeOH extracts and fractions was evaluated against the chloroquine‐resistant FCB strain and field isolates of Plasmodium falciparum using the DELI test. The cytotoxicity of the extracts was tested on MRC‐5 and THP1 cells using the tetrazolium salt MTT colorimetric assay, and the selectivity index (SI) of each extract was calculated. CH₂Cl₂ extract, the EA1 fraction from EtOAc extract of stem barks and cyclohexane (L_cycl), dichloromethane (L_DM) and butanol (L_but) fractions from MeOH/H₂O extract of leaves exhibited the highest in vitro antiplasmodial activity on FCB strain and field isolates (IC₅₀ from 0.53 to 4.87 μg/ml) with high selectivity index (of 20.15–1800). These data support the use of V. madiensis in malaria treatment along with continued investigations within traditional medicines in the search of new antimalarial agents. The EA1, C₆H₁₂ and CH₂Cl₂ fractions could be selected for future investigation or/and for the treatment of malaria symptoms after standardization.     

The hexose transporter of Plasmodium falciparum is a worthy drug target

Despite substantial efforts at control over several decades, malaria is still a major global health problem as chemotherapy of malaria parasites is limited by established drug resistance and lack of novel treatment options. Intraerythrocytic stages of these parasites are wholly dependent upon host glucose for energy and malarial proteins involved in hexose permeation are therefore attractive new drug targets. For Plasmodium falciparum, the causative agent of severe malaria, a facilitative hexose transporter (PfHT), encoded by a single-copy gene mediates glucose uptake. We first established heterologous expression in Xenopus laevis to allow functional characterisation of PfHT. This review describes the value of using Xenopus oocytes in heterologous studies of P. falciparum-encoded proteins and summarises the properties of PfHT. Comparisons between Gluts (mammalian facilitative hexose transporters) and PfHT using this expression system have highlighted important mechanistic and structural differences between parasite and host proteins. Certain O-methyl derivatives of glucose proved particularly useful discriminators between mammalian transporters and PfHT. We exploited this selectivity and synthesised a long-chain O-3-hexose derivative (compound 3361) that potently inhibits PfHT expressed in oocytes and also kills P. falciparum when it is cultured in medium containing either glucose or fructose as a carbon source. To extend our observations to the second most important human malarial pathogen, we have cloned and expressed the Plasmodium vivax orthologue of PfHT, and demonstrate inhibition of glucose uptake by compound 3361. These findings validate malarial hexose transporters as a novel target. We now aim to design a new class of antimalarials by the discovery of highly specific inhibitors which could act with a broad spectrum of action on different Plasmodium spp. infections.     

Multiple synergistic interactions between atovaquone and antifolates against Plasmodium falciparum in vitro: a rational basis for combination therapy

The use of synergistic drug combinations for the treatment of drug-resistant malaria is a major strategy to slow the selection and spread of Plasmodium falciparum resistant strains. In order to investigate synergistic compounds, with different modes of action, as alternative candidates for combination therapy, we used standard in vitro P. falciparum cultures and an established synergy testing method to define interactions among dapsone (DDS), atovaquone (ATQ), chlorproguanil (CPG) and its triazine metabolite chlorcycloguanil (CCG). Strong synergy was observed in the combinations DDS/CCG and ATQ/CPG. Multiple combination of these drugs, DDS/CCG/CPG/ATQ also exhibited high synergy although not higher than that of either of the two drug combinations separately. The use of this triple combination DDS/CPG/ATQ, even without an increase in synergy over their double combinations, ATQ/CPG and DDS/CCG, would contribute towards slowing the selection pressure since these drugs act against different targets and would delay the selection of parasites resistant to the three drugs, extending the useful therapeutic life of these valuable compounds.     

Development of cellular immune responses to Plasmodium falciparum blood stage antigens from birth to 36 months of age in Cameroon

Naturally acquired immunity to Plasmodium falciparum is related to immune system that changes during normal development and ageing. The effects of repeated infections during the early life on the maturation of the immune system are still unknown. Elucidation of these effects is of considerable interest given that malaria originates high mortality, especially during the first years of life. We conducted a cohort study to identify naturally acquired immune responses to P. falciparum. Cellular responses of Cameroonian neonates from birth to 36 months of age were evaluated every 6 months by cell proliferation and cytokines (IFN-gamma, IL-2 and IL-4) production after in vitro culture in the presence of schizont extract and Pf155/RESA peptides. Data were analyzed by a multiple correspondence analysis (MCA) exhibiting three main findings. Firstly, the lack of time-dependant evolution of specific immune pathways recruitment in the response to a given antigen, no antigen inducing a specific mode of response at a given time-point. Secondly, most of the data variability was expressed by IFN-gamma and IL-4 productions, and the major variation of the immune response with age involved this change in IFN-gamma production. Thirdly, the age-related immune response evolution is characterized by the acquisition of the capacity to mount a IFN-gamma response, a transient phase during which children produce a high IL-4 response, and the fast vanishing of the dominance of the IL-2 response. These results suggest that P. falciparum specific immune responses are first oriented towards a Th2-type of response, and later switch to Th1-type of response.     

Anti-malarial efficacy of pyronaridine and artesunate in combination in vitro and in vivo

Pyronaridine is a Mannich base anti-malarial with demonstrated efficacy against drug resistant Plasmodium falciparum, P. vivax, P. ovale and P. malariae. However, resistance to pyronaridine can develop quickly when it is used alone but can be considerably delayed when it is administered with artesunate in rodent malaria models. The aim of this study was to evaluate the efficacy of pyronaridine in combination with artesunate against P. falciparum in vitro and in rodent malaria models in vivo to support its clinical application. Pyronaridine showed consistently high levels of in vitro activity against a panel of six P. falciparum drug-sensitive and resistant strains (Geometric Mean IC50=2.24 nM, 95% CI=1.20-3.27). In vitro interactions between pyronaridine and artesunate showed a slight antagonistic trend, but in vivo compared to pyronaridine and artesunate administered alone, the 3:1 ratio of the combination, reduced the ED90 of artesunate by approximately 15.6-fold in a pyronaridine-resistant P. berghei line and by approximately 200-fold in an artesunate-resistant line of P. berghei. Complete cure rates were achieved with doses of the combination above or equal to 8 mg/kg per day against P. chabaudi AS. These results indicate that the combination had an enhanced effect over monotherapy and lower daily doses of artesunate could be used to obtain a curative effect. The data suggest that the combination of pyronaridine and artesunate should have potential in areas of multi-drug resistant malaria.     

Plasma and in vitro levels of cytokines during and after a Plasmodium falciparum malaria attack in Gabon

Fifty subjects living in a malaria endemic area were studied at diagnosis of a Plasmodium falciparum attack and 3 weeks later. Absolute numbers of CD3(+), CD4(+) and CD8(+) lymphocytes as well as plasma cytokines and secreted cytokines after in vitro mitogenic stimulation were measured. At enrollment, lymphopenia was observed, lending support to the reallocation hypothesis during the acute phase. A significant elevation of the number of CD8(+) cells was present in the peripheral blood during the recovery phase. During the acute phase, plasma IL-6 levels peaked while in vitro production capacity was high at both phases. Plasma IL-6 concentrations were positively related to blood parasite density at D0, as IL-4 and IFN-gamma, suggesting an early intervention of these cytokines. Plasma IL-2 levels were low at diagnosis although cells retained their ability to produce IL-2, which was found more frequently in plasma after cure. Acquisition of immunity with age was in relation with greater secretion abilities of cells for type 1 and type 2 cytokines during the parasite clearance phase. We conclude to an early implication of type 2 cytokines and IFN-gamma, with particularly high levels of IL-6.     

In vitro monitoring of Plasmodium falciparum susceptibility to artesunate, mefloquine, quinine and chloroquine in Cambodia: 2001-2002

We used a classical isotopic microtest to assess the in vitro sensitivity of 352 Plasmodium falciparum isolates collected in Cambodia in 2001 and 2002 to chloroquine, mefloquine, quinine and artesunate. Our results confirm conclusions drawn from earlier studies conducted by the Cambodian national malaria centre. Chloroquine-resistant phenotypes were highly prevalent in Cambodia. Similarly, a high proportion of isolates displayed elevated IC50 to mefloquine. In contrast, only 0.67 and 1.7% of isolates presented decreased susceptibility to quinine and artesunate, respectively. Distributions of mean IC50 according to drug and geographic origin indicated that the parasites circulating to the west of Cambodia largely account for the global situation of drug resistances in Cambodia. Isolates with decreased susceptibility to chloroquine and mefloquine were common along the border with Thailand. In contrast, most of the isolates from eastern Cambodia were susceptible to these compounds. Isolates collected at the western and eastern borders did not respond differently to artesunate. No major differences in responses to antimalarial drugs were observed between 2001 and 2002, suggesting that the situation of drug resistance is now stabilized and under control in Cambodia. However, the decreased susceptibility of isolates collected in the western provinces of Cambodia to mefloquine and the correlation between susceptibility to artesunate and susceptibility to mefloquine and quinine justify the need for an improved international surveillance program for malaria drug resistance in the Mekong sub region.     

A better resolution for integrating methods for monitoring Plasmodium falciparum resistance to antimalarial drugs

Effective chemotherapy is the mainstay of malaria control. However, resistance of falciparum malaria to antimalarial drugs compromised the efforts to eliminate the disease and led to the resurgence of malaria epidemics. Three main approaches are used to monitor antimalarial drug efficacy and drug resistance; namely, in vivo trials, in vitro/ex vivo assays and molecular markers of drug resistance. Each approach has its implications of use as well as its advantages and drawbacks. Therefore, there is a need to use an integrated approach that would give the utmost effect to detect resistance as early as its emergence and to track it once spread. Such integration becomes increasingly needed in the era of artemisinin-based combination therapy as a forward action to deter resistance. The existence of regional and global networks for the standardization of methodology, provision of high quality reagents for the assessment of antimalarial drug resistance and dissemination of open-access data would help in approaching an integrated resistance surveillance system on a global scale.     

Pharmacodynamic interactions of amodiaquine and its major metabolite desethylamodiaquine with artemisinin, quinine and atovaquone in Plasmodium falciparum in vitro

The antimalarial in vitro activities of amodiaquine and desethylamodiaquine in combination with atovaquone, quinine and artemisinin against Plasmodium falciparum were investigated in strains F-32, FCR-3 and K-1. These parasitic strains have different sensitivity profiles to the standard antimalarial chloroquine, but all can be considered sensitive to the test drugs, representing the recommended situation for introduction of two partner drugs in combination therapy. Amodiaquine showed marked synergism when combined with each of the three partner compounds at concentration ratios between 90 and 9x10(-7), including the therapeutically relevant range. The interaction profiles of desethylamodiaquine with quinine and artemisinin also showed predominantly synergism over a wide range of concentration ratios between 70 and 9x10(-7). The responses to all combinations exhibited signs of strain-specificity, but such phenomena were usually observed outside the therapeutic range of the concentration ratios. Synergism was generally more marked with increasing EC values, i.e. at concentrations expected to be therapeutically effective and thus clinically relevant. Even trace quantities of amodiaquine were able to potentiate the activity of structurally unrelated antimalarial drugs.     

Evidence for erythrocyte-binding antigen 175 as a component of a ligand-blocking blood-stage malaria vaccine

The ligands that pathogens use to invade their target cells have often proven to be good targets for vaccine development. However, Plasmodium falciparum has redundant ligands that mediate invasion of erythrocytes. The first requirement for the development of a successful ligand-blocking malaria vaccine is the demonstration that antibodies induced to each ligand can block the erythrocyte invasion of parasites with polymorphic sequences. Because of P. falciparum's redundancy in erythrocyte invasion, each ligand needs to be studied under artificial conditions in which parasite invasion is restricted in its use of alternative pathways. Here we investigate the role of erythrocyte-binding antigen 175 (EBA-175), a parasite ligand that binds to sialic acid on glycophorin A, in the invasion of erythrocytes by 10 P. falciparum clones under conditions in which invasion is partially limited to the EBA-175-glycophorin A pathway, using chymotrypsin-treated erythrocytes. We show that the ability to invade erythrocytes for both sialic acid-independent and sialic acid-dependent pathways requires the EBA-175-glycophorin A pathway for erythrocyte invasion. Importantly, antibodies against region II of EBA-175 from the 3D7 clone blocked invasion of chymotrypsin-treated erythrocytes by >50% by all parasite clones studied, including those with multiple different mutations described in the literature. The one exception was FCR3, which had a similar sequence to 3D7 but only 30% inhibition of invasion of chymotrypsin-treated erythrocytes, indicating alternative pathways for invasion of chymotrypsin-treated erythrocytes. Our findings suggest that antibodies to region II of EBA-175, as one component of a ligand-blocking malaria vaccine, are largely unaffected by polymorphism in EBA-175.     

Evidence for continued two-brood replication of Plasmodium falciparum in vivo during quinine treatment

To investigate the relationship between fever and parasite clearance in falciparum malaria, we studied 54 adults with Plasmodium falciparum infections who were all treated with quinine. The median oral temperature profile showed peaks at 24 h intervals during the first 3 days. Although there was no equivalent pattern evident in the median parasite clearance curve, we hypothesize that small numbers of two distinct parasite broods continued to develop in antiphase through schizogony despite quinine therapy. These data are consistent with previous reports of two dominant broods in untreated humans and monkeys infected with P. falciparum, and highlight the need for an adequate duration of quinine treatment.     

High prevalence of PfCRT K76T mutation in Plasmodium falciparum isolates in Ghana

Plasmodium falciparum has successfully developed resistance to almost all currently used antimalarials. A single nucleotide polymorphism in the P. falciparum chloroquine resistance transporter (Pfcrt) gene at position 76 resulting in a change in coding from lysine to threonine (K76T) has been implicated to be the corner stone of chloroquine resistance. Widespread resistance to chloroquine in endemic regions led to its replacement with other antimalarials. In some areas this replacement resulted in a reversion of the mutant T76 allele to the wild-type K76 allele. This study was conducted to determine the prevalence of the K76T mutation of the Pfcrt gene eight years after the ban on chloroquine sales and use. A cross-sectional study was conducted in 6 regional hospitals in Ghana. PCR-RFLP was used to analyse samples collected to determine the prevalence of Pfcrt K76T mutation. Of the 1318 participants recruited for this study, 246 were found to harbour the P. falciparum parasites, of which 60.98% (150/246) showed symptoms for malaria. The prevalence of the Pfcrt T76 mutant allele was 58.54% (144/246) and that of the K76 wild-type allele was 41.46% (102/246). No difference of statistical significance was observed in the distribution of the alleles in the symptomatic and asymptomatic participants (P = 0.632). No significant association was, again, observed between the alleles and parasite density (P = 0.314), as well as between the alleles and Hb levels of the participants (P = 0.254). Notwithstanding the decline in the prevalence of the Pfcrt T76 mutation since the antimalarial policy change in 2004, the 58.54% prevalence recorded in this study is considered high after eight years of the abolishment of chloroquine usage in Ghana. This is in contrast to findings from other endemic areas where the mutant allele significantly reduced in the population after a reduction chloroquine use.     

Changing patterns of forest malaria among the mobile adult male population in Chumkiri District, Cambodia

Forest malaria remains a major problem in many parts of Southeast Asia and South America. In Cambodia, where a significant reduction of malaria morbidity and mortality has been observed in the last 20 years, the forest malaria situation was studied in Chumkiri District by analysing the available passive case detection data and conducting malariometric (n=1018) and questionnaire surveys (n=374) in four forest-fringe villages. There has been a decreasing trend of malaria incidence from 2001. Plasmodium falciparum was highly predominant and P. vivax was rare. The nearby-forest villages showed significantly higher parasite rates than the far-from-forest villages (9.0% vs. 1.2%, p<0.01). Malaria was highly restricted to the male adults but was nearly non-existent in other accompanying family members, including small children and females. Low income and working in forests were strongly associated with the malaria risk. Our results suggest that transmission has greatly reduced in forest-fringe villages, but remains active in forests, which is primarily maintained between the forest vector Anopheles dirus and ethnic minority inhabitants. Specific interventions directed to these previously neglected in-forest inhabitants to protect themselves and male adult villagers during their forest activities are necessary to achieve an ultimate goal of malaria elimination from Cambodia.