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Background: The spread of antimalarial drug resistance parasites is a major obstacle in eliminating malaria in endemic areas. This increases the urgency for developing novel antimalarial drugs with improved profiles to eliminate both sensitive and resistant parasites in populations. The invention of the drug candidates needs a model for sensitive and resistant parasites on a laboratory scale., Methods: Repeated Incomplete Treatment (RIcT) method was followed in raising the rodent malaria parasite, Plasmodium berghei, resistant to sulfadoxine. Plasmodium berghei were exposed to an adequate therapeutic dose of sulfadoxine without finishing the treatment to let the parasite recover. Cycles of drug treatment and parasite recovery were repeated until phenotypic resistance appeared., Results: After undergoing 3-4 cycles, phenotypic resistance was not yet found in mice treated with sulfadoxine. Nevertheless, the molecular biology of dhps gene (the target of sulfadoxine) was analyzed at the end of the RIcT cycle. There was no mutations found in the gene target. Interestingly, the appearance of gametocytes at the end of every cycle of drug treatment and parasite recovery was observed. These gametocytes later on would no longer extend their life in the RBC stage, unless mosquitoes bite the infected host. This phenomenon is similar to the case in human malaria infections treated with sulfadoxine-pyrimethamine (SP)., Conclusions: In this study, the antimalarial drug sulfadoxine induced gametocytogenesis in P. berghei, which could raise the risk factor for malaria transmission., (© 2024. The Author(s).)

Background: Malaria is a global health problem for which novel therapeutic compounds are needed. To this end, a recently published novel family of antiplasmodial macrolides, strasseriolides A-D, was herein subjected to in vivo efficacy studies and preclinical evaluation in order to identify the most promising candidate(s) for further development., Methods: Preclinical evaluation of strasseriolides A-D was performed by MTT-based cytotoxicity assay in THLE-2 (CRL-2706) liver cells, cardiotoxicity screening using the FluxOR™ potassium assay in hERG expressed HEK cells, LC-MS-based analysis of drug-drug interaction involving CYP3A4, CYP2D6 and CYP2C9 isoforms inhibition and metabolic stability assays in human liver microsomes. Mice in vivo toxicity studies were also accomplished by i.v. administration of the compounds (vehicle: 0.5% HPMC, 0.5% Tween 80, 0.5% Benzyl alcohol) in mice at 25 mg/kg dosage. Plasma were prepared from mice blood samples obtained at different time points (over a 24-h period), and analysed by LC-MS to quantify compounds. The most promising compounds, strasseriolides C and D, were subjected to a preliminary in vivo efficacy study in which transgenic GFP-luciferase expressing Plasmodium berghei strain ANKA-infected Swiss Webster female mice (n = 4-5) were treated 48 h post-infection with an i.p. dosage of strasseriolide C at 50 mg/kg and strasseriolide D at 22 mg/kg for four days after which luciferase activity was quantified on day 5 in an IVIS ® Lumina II imager., Results: Strasseriolides A-D showed no cytotoxicity, no carditoxicity and no drug-drug interaction problems in vitro with varying intrinsic clearance (CLint). Only strasseriolide B was highly toxic to mice in vivo (even at 1 mg/kg i.v. dosage) and, therefore, discontinued in further in vivo studies. Strasseriolide D showed statistically significant activity in vivo giving rise to lower parasitaemia levels (70% lower) compared to the controls treated with vehicle., Conclusions: Animal efficacy and preclinical evaluation of the recently discovered potent antiplasmodial macrolides, strasseriolides A-D, led to the identification of strasseriolide D as the most promising compound for further development. Future studies dealing on structure optimization, formulation and establishment of optimal in vivo dosage explorations of this novel compound class could enhance their clinical potency and allow for progress to later stages of the developmental pipeline., (© 2021. The Author(s).)

Background: Evidence of Plasmodium resistance to some of the current anti-malarial agents makes it imperative to search for newer and effective drugs to combat malaria. Therefore, this study evaluated whether the co-administrations of xylopic acid-amodiaquine and xylopic acid-artesunate combinations will produce a synergistic anti-malarial effect., Methods: Antiplasmodial effect of xylopic acid (XA: 3, 10, 30, 100, 150 mg kg -1 ), artesunate (ART: 1, 2, 4, 8, 16 mg kg -1 ), and amodiaquine (AQ: 1.25, 2.5, 5, 10, 20 mg kg -1 ) were evaluated in Plasmodium berghei (strain ANKA)-infected mice to determine respective ED 50 s. Artemether/lumefantrine was used as the positive control. XA/ART and XA/AQ were subsequently administered in a fixed-dose combination of their ED 50 s (1:1) and the combination fractions of their ED 50 s (1/2, 1/4, 1/8, 1/16, and 1/32) to determine the experimental ED 50 s (Z exp ). An isobologram was constructed to determine the nature of the interaction between XA/ART, and XA/AQ combinations by comparing Z exp with the theoretical ED 50 (Z add ). Bodyweight and 30-day survival post-treatment were additionally recorded., Results: ED 50 s for XA, ART, and AQ were 9.0 ± 3.2, 1.61 ± 0.6, and 3.1 ± 0.8 mg kg -1 , respectively. The Z add , Z exp, and interaction index for XA/ART co-administration was 5.3 ± 2.61, 1.98 ± 0.25, and 0.37, respectively while that of XA/AQ were 6.05 ± 2.0, 1.69 ± 0.42, and 0.28, respectively. The Z exp for both combination therapies lay significantly (p < 0.001) below the additive isoboles showing XA acts synergistically with both ART and AQ in clearing the parasites. High doses of XA/ART combination significantly (p < 0.05) increased the survival days of infected mice with a mean hazard ratio of 0.40 while all the XA/AQ combination doses showed a significant (p < 0.05) increase in the survival days of infected mice with a mean hazard ratio of 0.27 similar to AL. Both XA/ART and XA/AQ combined treatments significantly (p < 0.05) reduced weight loss., Conclusion: Xylopic acid co-administration with either artesunate or amodiaquine produces a synergistic anti-plasmodial effect in mice infected with P. berghei.

Background: Kniphofia foliosa is a flamboyant robust perennial herb which has dense clumps and tick upright rhizomes with leaves at the base. In Ethiopia, it has several vernacular names including Abelbila, Ashenda, Amelmela, Yeznjero Ageda, Shemetmetie and Yezinjero Ageda. The plant is endemic to Ethiopian highlands, where its rhizomes are traditionally used for the treatment of malaria, abdominal cramps and wound healing. In the present study, the 80% methanol extract of K. foliosa rhizomes and its constituents are tested against Plasmodium berghei in mice., Methods: Isolation was carried out using column and preparative thin layer chromatography (PTLC). The chemical structures of the compounds were elucidated by spectroscopic methods (ESI-MS, 1D and 2D-NMR). Peters' 4-day suppressive test against P. berghei in mice was utilized for in vivo anti-malarial evaluation of the test substances., Results: Two compounds, namely knipholone and dianellin were isolated from the 80% methanolic extract of K. foliosa rhizomes, and characterized. The hydroalcoholic extract (400 mg/kg) and knipholone (200 mg/kg) showed the highest activity with chemosuppression values of 61.52 and 60.16%, respectively. From the dose-response plot, the median effective (ED 50 ) doses of knipholone and dianellin were determined to be 81.25 and 92.31 mg/kg, respectively. Molecular docking study revealed that knipholone had a strong binding affinity to Plasmodium falciparum l-lactate dehydrogenase (pfLDH) target., Conclusion: Results of the current study support the traditional use of the plant for the treatment of malaria.

Background: The menace of resistance to anti-malarial drugs is a great challenge to malaria control, necessitating the search for new anti-malarial agents. This search has led to the exploration of natural products for efficacy in malaria therapy. Omidun is the supernatant of fermenting maize (ogi) slurry that has been widely investigated and reported to possess several health benefits and it is used traditionally as solvent for preparing anti-malarial herbs. However, there is no information on the anti-malarial activity of omidun itself. This study was conducted to investigate the prophylactic, curative and suppressive anti-malarial potential of omidun., Methods: Experimental mice in the curative group were infected with 1 × 10 6 cells of Plasmodium berghei strain ANKA and treated with either 0.2 ml of omidun containing 3 × 10 9  cfu/ml of viable lactic acid bacteria or 0.2 ml of 5 mg/kg of chloroquine (positive control) or 0.2 ml of saline (negative control) for 4 days from day 3 post infection. The prophylactic group of mice were pre-treated with either omidun, chloroquine or saline for 4 days before infection with P. berghei, while the suppressive group was treated with omidun or chloroquine or saline and infected with P. berghei simultaneously. A group of mice were uninfected but treated (with omidun and control samples), while a final group was uninfected and untreated (controls). Parasitaemia and histopathology analysis were done in all groups., Results: The curative and suppressive groups showed a significant difference between the omidun-treated mice (100% parasitaemia reduction) and the untreated mice (54.5% parasitaemia increase). There was no significance difference between the omidun treatment and chloroquine (positive control) treatment in suppressive group as both treatment had 100% parasitaemia reduction. The omidun prophylactic treatment however did not show any parasitaemia suppression, but a significant difference was observed between the omidun treatment (85% increase) and the chloroquine (positive control) treatment (100% reduction) in the group. Omidun treatment is non-toxic to the kidney., Conclusion: This study provides scientific evidence supporting omidun usage in the treatment of malaria. Consequently, further work may yield the specific component of omidun responsible for the anti-malarial activity.

Background: Resistance of Plasmodium falciparum against common anti-malarial drugs emphasizes the need of alternative and more effective drugs. Synthetic derivatives of 1-(heteroaryl)-2-((5-nitroheteroaryl)methylene) hydrazine have showed in vitro anti-plasmodial activities. The present study aimed to evaluate the molecular binding and anti-plasmodial activity of synthetic compounds in vivo., Methods: The molecular docking was used to study the binding of compounds to haem and Plasmodium falciparum lactate dehydrogenase (PfLDH). Acute toxicity of the synthetic compounds was evaluated based on the modified up & down method. The anti-plasmodial activity of the compounds was conducted by the two standard tests of Peters' and of Rane, using chloroquine-sensitive Plasmodium berghei in mice. Also, the toxicity to the internal organs of mice was evaluated on the seventh day after the treatment in addition to the histopathology of their liver. Compound 3 that showed high activity in the lowest dose was selected for further pharmacodynamic studies., Results: According to the docking studies, the active site of PfLDH had at least four common residues, including Ala98, Ile54, Gly29, and Tyr97 to bind the compounds with the affinity, ranging from - 8.0 to - 8.4 kcal/mol. The binding mode of ligands to haem revealed an effective binding affinity, ranging from - 5.1 to - 5.5 kcal/mol. Compound 2 showed the highest  % suppression of parasitaemia (99.09%) at the dose of 125 mg/kg/day in Peters' test. Compound 3, with 79.42% suppression, was the best in Rane's test at the lowest dose (31 mg/kg/day). Compound 3 was confirmed by the pharmacodynamic study to have faster initial parasite elimination in the lowest concentration. The histopathology of the livers of mice did not reveal any focal necrosis of hepatocytes in the studied compounds., Conclusions: The docking studies verified Pf LDH inhibition and the inhibitory effect on the haemozoin formation for the studied compounds. Accordingly, some compounds may provide new avenues for the development of anti-malarial drugs without liver toxicity, although further studies are required to optimize their anti-plasmodial activity.

Background: Artemisinin-based combination therapy (ACT) is used as the first-line treatment of uncomplicated malaria caused by the Plasmodium falciparum parasite and chloroquine-resistant Plasmodium vivax parasites. Evidence of resistance to ACT has been reported in Cambodia, and without new and effective anti-malarial agents, malaria burden and mortality will rise., Methods: The used MolPrint 2D fingerprints and the Tanimoto similarity index were used to perform a structural similarity search within the Malaria Box collection to select diverse molecular scaffolds that are different from artesunate. Next, the inhibitory potency against the P. falciparum 3D7 strain (SYBR Green I inhibition assay) and the cytotoxicity against HepG2 cells (MTT and neutral red assays) were evaluated. Then, the speed of action, the combination profile of selected inhibitors with artesunate, and the P. berghei in vivo activity of the best compounds were assessed., Results: A set of 11 structurally diverse compounds from the Malaria Box with a similarity threshold of less than 0.05 was selected and compared with artesunate. The in vitro inhibitory activity of each compound confirmed the reported potencies (IC 50 values ranging from 0.005 to 1 µM). The cytotoxicity of each selected compound was evaluated and used to calculate the selectivity index (SI values ranging from 15.1 to 6100). Next, both the speed of action and the combination profile of each compound with artesunate was assessed. Acridine, thiazolopyrimidine, quinoxaline, benzimidazole, thiophene, benzodiazepine, isoxazole and pyrimidoindole derivatives showed fast in vitro inhibitory activity of parasite growth, whereas hydrazinobenzimidazole, indenopyridazinone and naphthalenone derivatives were slow-acting in vitro inhibitors. Combinatory profile evaluation indicated that thiazolopyrimidinone and benzodiazepine derivatives have an additive profile, suggesting that the combination of these inhibitors with artesunate is favourable for in vitro inhibitory activity. The remaining compounds showed an antagonistic combinatory profile with artesunate. The collected data indicated that the indenopyridazinone derivative, a bc 1 complex inhibitor, had a similar association profile in combination with proguanil when compared to atovaquone combined with proguanil, thereby corroborating the correlation between the molecular target and the combination profile. Lastly, the in vivo activity of the thiazolopyrimidinone and benzodiazepine derivatives were assessed. Both compounds showed oral efficacy at 50 mg/kg in a mouse model of Plasmodium berghei malaria (64% and 40% reduction in parasitaemia on day 5 post-infection, respectively)., Conclusions: The findings in this paper shed light on the relationship among the speed of action, molecular target and combinatory profile and identified new hits with in vivo activity as candidates for anti-malarial combination therapy.

Background: The immune modulating potential of IL-35 in multiple human disorders has been reported. Consequent upon the recognition of inflammatory cytokine activation and its preponderance for mediating pathology during malaria infection, the study aimed to characterize the expression and functional contribution(s) of IL-35 in Plasmodium berghei (strain ANKA) infected mice., Methods: Plasmodium berghei infection in male ICR mice was used as the rodent model of choice. The time course of IL-35 expression in the systemic circulation and tissues of P. berghei infected mice as well as their healthy control counterparts was assessed by enzyme linked immunosorbent assay and immunohistochemistry respectively. The effect of modulating IL-35 by recombinant IL-35 protein or neutralizing anti-Epstein-Barr virus-induced gene 3 antibody on the cytokine environment during P. berghei infection was assessed by flow cytometry. Furthermore, the influence of modulating IL-35 on histopathological hallmarks of malaria and disease progression was evaluated., Results: Interleukin-35 was significantly up regulated in serum and tissues of P. berghei infected mice and correlated with parasitaemia. Neutralization of IL-35 significantly enhanced the release of IFN-γ, decreased the expression of IL-6 and decreased parasitaemia patency. Neutralization of IL-35 was also associated with a tendency towards increased survival as well as the absence of pathological features associated with malaria infection unlike recombinant IL-35 protein administration which sustained a normal course of infection and unfavourable malaria associated histological outcomes in P. berghei infected mice., Conclusion: These results indicate the involvement of IL-35 in P. berghei induced malaria infection. IL-35 neutralization strategies may represent viable therapeutic modalities beneficial for the resolution of malaria infection.

Background: The development of Plasmodium resistance to the last effective anti-malarial drugs necessitates the urgent development of new anti-malarial therapeutic strategies. To this end, plants are an important source of new molecules. The objective of this study was to evaluate the anti-malarial effects of Terminalia albida, a plant used in Guinean traditional medicine, as well as its anti-inflammatory and antioxidant properties, which may be useful in treating cases of severe malaria., Methods: In vitro antiplasmodial activity was evaluated on a chloroquine-resistant strain of Plasmodium falciparum (K-1). In vivo efficacy of the plant extract was measured in the experimental cerebral malaria model based on Plasmodium berghei (strain ANKA) infection. Mice brains were harvested on Day 7-8 post-infection, and T cells recruitment to the brain, expression levels of pro- and anti-inflammatory markers were measured by flow cytometry, RT-qPCR and ELISA. Non-malarial in vitro models of inflammation and oxidative response were used to confirm Terminalia albida effects. Constituents of Terminalia albida extract were characterized by ultra-high performance liquid chromatography coupled with high resolution mass spectrometry. Top ranked compounds were putatively identified using plant databases and in silico fragmentation patterns., Results: In vitro antiplasmodial activity of Terminalia albida was confirmed with an IC50 of 1.5 μg/mL. In vivo, Terminalia albida treatment greatly increased survival rates in P. berghei-infected mice. Treated mice were all alive until Day 12, and the survival rate was 50% on Day 20. Terminalia albida treatment also significantly decreased parasitaemia by 100% on Day 4 and 89% on Day 7 post-infection. In vivo anti-malarial activity was related to anti-inflammatory properties, as Terminalia albida treatment decreased T lymphocyte recruitment and expression of pro-inflammatory markers in brains of treated mice. These properties were confirmed in vitro in the non-malarial model. In vitro, Terminalia albida also demonstrated a remarkable dose-dependent neutralization activity of reactive oxygen species. Twelve compounds were putatively identified in Terminalia albida stem bark. Among them, several molecules already identified may be responsible for the different biological activities observed, especially tannins and triterpenoids., Conclusion: The traditional use of Terminalia albida in the treatment of malaria was validated through the combination of in vitro and in vivo studies.

Background: Tamoxifen is an oestrogen receptor modulator that is widely used for the treatment of early stage breast cancer and reduction of recurrences. Tamoxifen is also used as a powerful research tool for controlling gene expression in the context of the Cre/loxP site-specific recombination system in conditional mutant mice., Methods: To determine whether the administration of tamoxifen affects Plasmodium growth and/or disease outcome in malaria, in vitro studies assessing the effect of tamoxifen and its active metabolite 4-hydroxytamoxifen on Plasmodium falciparum blood stages were performed. Tamoxifen effects were also evaluated in vivo treating C57/B6 mice infected with Plasmodium berghei (ANKA strain), which is the standard animal model for the study of cerebral malaria., Results: Tamoxifen and its active metabolite, 4-hydroxytamoxifen, show activity in vitro against P. falciparum (16.7 to 5.8 µM IC50, respectively). This activity was also confirmed in tamoxifen-treated mice infected with P. berghei, which show lower levels of parasitaemia and do not develop signs of cerebral malaria, compared to control mice. Mice treated with tamoxifen for 1 week and left untreated for an additional week before infection showed similar parasitaemia levels and signs of cerebral malaria as control untreated mice., Conclusions: Tamoxifen and its active metabolite, 4-hydroxytamoxifen, have significant activity against the human parasite P. falciparum in vitro and the rodent parasite P. berghei in vivo. This activity may be useful for prevention of malaria in patients taking this drug chronically, but also represents a major problem for scientists using the conditional mutagenic Cre/LoxP system in the setting of rodent malaria. Allowing mice to clear tamoxifen before starting a Plasmodium infection allows the use the Cre/LoxP conditional mutagenic system to investigate gene function in specific tissues.

Background: More than 200 medicinal plants including Euphorbia abyssinica are utilized for treatment of malaria in Ethiopian traditional medical practices. However, the safety, efficacy and quality of these medicinal plants are largely unknown. Pharmacological and toxicological investigations of these plants are among the prioritized issues in every country. The aim of this study was, therefore, to evaluate the anti-malarial activity of Euphorbia abyssinica root extract against Plasmodium berghei infection in mice., Methods: The fresh roots of Euphorbia abyssinica were identified and collected. They were dried and extracted by 80% methanol using maceration. Acute toxicity of the extract was done using female Swiss albino mice. Anti-malarial activity of the extract was done by a standard 4-day suppressive test using chloroquine-sensitive Plasmodium berghei. Twenty-five male Swiss albino mice were randomly grouped into 5 groups of 5 mice each. Group I was treated with distilled water (10 ml/kg), group II, III, and IV were treated with 200, 400, and 600 mg/kg of extract, respectively and group V was treated with chloroquine (25 mg/kg). The level of parasitaemia, survival time, and variation in weight were utilized to determine the anti-malarial activity of the extract. Data was analysed using ANOVA followed by Tukey test., Results: The plant extract did not show any sign of toxicity and mortality at 2000 mg/kg. The 4-day chemosuppressive anti-malarial activities produced by the crude extract were 66.87% (P < 0.001), 84.94% (P < 0.001) and 93.69% (P < 0.001) at 200, 400 and 600 mg/kg extract, respectively, compared to distilled water treated group. Mice treated with 400 mg/kg (P < 0.01), and 600 mg/kg extract (P < 0.001) showed significant chemosuppressive anti-malarial activity variations as compared to mice treated with 200 mg/kg extract. Mice treated with 600 mg/kg extract significantly (P < 0.001) lived longer than distilled water treated mice. However, the crude extract did not cause any significant change on body weights of mice., Conclusions: From this study, it can be concluded that the root of Euphorbia abyssinica showed very good 4-day chemosuppressive anti-malarial activity. The plant might contain biologically active compounds which are relevant for treatment of malaria. Further phytochemical, toxicological and pharmacological investigations are, therefore, required to evaluate its anti-malarial potential.

Background: Reverse genetics approaches have become powerful tools to dissect the biology of malaria parasites. In a previous study, development of an in vitro drug selection method for generating transgenic parasite of Plasmodium berghei was reported. Using this method, two novel and independent selection markers using the P. berghei heat shock protein 70 promoter was previously established. While the approach permits the easy and flexible genetic manipulation of P. berghei, shortcomings include a low variety in promoter options to drive marker gene expression and increased complexity of the selection procedure. In this study, addressing these issues was attempted., Methods: To secure a variety of promoters, the use of a P. berghei elongation factor-1α promoter for marker gene expression was attempted. To simplify the procedure of in vitro selection, the establishment of a two cell-cycle culture method and its application for drug selection were attempted., Results: The P. berghei elongation factor-1α (pbef-1α) promoter, which is commonly used to drive marker gene expression, was successfully applied as an alternative promoter model for marker gene expression, using the parasite's codon-optimized marker sequence. To simplify the in vitro selection method, a two cell-cycle culture method in which the merozoite was released by filtration of the culture containing matured schizont-infected erythrocytes was also developed and successfully applied for drug selection., Conclusion: The pbef-1α promoter was successfully applied in an in vitro selection system. The in vitro selection procedure also could be simplified for practical use using a two cell-cycle culture method. These improvements provide a more versatile platform for the genetic manipulation of P. berghei.

Background: The protective efficacy of the most promising malaria whole-parasite based vaccine candidates critically depends on the parasite's potential to migrate in the human host. Key components of the parasite motility machinery (e.g. adhesive proteins, actin/myosin-based motor, geometrical properties) have been identified, however the regulation of this machinery is an unknown process., Methods: In vitro microscopic live imaging of parasites in different formulations was performed and analysed, with the quantitative analysis software SMOOT In vitro , their motility; their adherence capacity, movement pattern and velocity during forward locomotion., Results: SMOOT In vitro enabled the detailed analysis of the regulation of the motility machinery of Plasmodium berghei in response to specific (macro)molecules in the formulation. Albumin acted as an essential supplement to induce parasite attachment and movement. Glucose, salts and other whole serum components further increased the attachment rate and regulated the velocity of the movement., Conclusions: Based on the findings can be concluded that a complex interplay of albumin, glucose and certain salts and amino acids regulates parasite motility. Insights in parasite motility regulation by supplements in solution potentially provide a way to optimize the whole-parasite malaria vaccine formulation.

Background: Traditional medicinal plants are one of the potential sources of anti-malarial drugs and there is an increasing interest in the use and development of traditional herbal remedies for the treatment of malaria and other ailments. This study was carried out with the aim to investigate the phytochemical screening, cytotoxic effect and antiplasmodial activities of Dichrostachys cinerea and Commiphora africana. Both plants are used by the Maasai in Tanzania in suspected malaria and other diseases. No previous work appears to have investigated the potential anti-malarial activity of the two plants., Methods: This study aimed to investigate the in vitro anti-malarial activity of methanol and dichloromethane extracts of the two plants against chloroquine sensitive (D6) and chloroquine resistant (Dd2) strains of Plasmodium falciparum. The anti-malarial property was assessed by the lactate dehydrogenase method (pLDH). The in vivo anti-malarial study was carried out using the Peters' 4-day suppressive test in Plasmodium berghei in Balb/c mice. Cytotoxic tests were carried out using monkey kidney epithelial cell line in [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. Qualitative phytochemical screening was carried out using standard methods of analysis., Results: The phytochemical screening of plant extracts revealed the presence of alkaloids, flavonoids, tannins, steroids, triterpenoids, glycosides and saponins. However, alkaloids were absent in most plant extracts. The dichloromethane extracts of C. africana (stem bark); D. cinerea (stem bark) and methanol extracts of D. cinerea (whole stem) all showed promising in vitro anti-malarial activities. All other extracts did not show any significant anti-malarial activity. The two most promising extracts based on in vitro studies, DCM extracts of C. africana (stem bark) and D. cinerea (stems bark), equally exhibited very significant anti-malarial activities in the mouse model. They exhibited parasite suppression rates of 64.24 and 53.12%, respectively, and considerable improvement in weight and survival rate. Most plant extracts were not cytotoxic except for DCM extract of D. cinerea (whole stem) CC 50 (29.44 µg/mL)., Conclusion: The findings of this study provide scientific evidence supporting the traditional use of the plants in the treatment of malaria by the Maasai in Arusha region, Tanzania. Consequently, further work including bioassay-guided fractionation and advanced toxicity testing may yield new anti-malarial drug candidates from the two plants.

Background: Medicinal plant research may contribute to develop new pharmacological control tools for vector borne diseases, such as malaria., Methods: The effects of methanol extracts (ME) obtained from seed kernel of ripe and unripe Azadirachta indica fruits were studied on erythrocytic proliferation of the rodent malaria parasite Plasmodium berghei strain ANKA and on mice pro-inflammatory response, as evaluated by measuring the matrix-metalloproteinase-9 (MMP-9) and tumour necrosis factor (TNF) plasma levels, in two mouse strains (C57BL/6 and BALB/c) which are considered as prototypical of Th1 and Th2 immune response, respectively., Results: ME obtained from seed kernel of unripe Azadirachta indica fruits decreased by about 30% the proportion of erythrocytes infected with the malaria parasite in C57BL/6 mice in the 4 days suppressive test. In this treatment group, MMP-9 and TNF levels were notably higher than those measured in the same mouse strain treated with the anti-malarial drug artesunate, Azadirachta indica kernel extracts from ripe fruits or solvent. In BALB/c mice, treatment with kernel extracts did not influence parasitaemia. MMP-9 and TNF levels measured in this mouse strain were notably lower than those recorded in C57BL/6 mice and did not vary among treatment groups., Conclusions: The effects of the ME on the parasite-host interactions appeared to be mouse strain-dependent, but also related to the ripening stage of the neem fruits, as only the unripe fruit seed kernel extracts displayed appreciable bioactivity.

Background: Anti-malarial compounds have not yet been identified that target the first obligatory step of infection in humans: the migration of Plasmodium sporozoites in the host dermis. This movement is essential to find and invade a blood vessel in order to be passively transported to the liver. Here, an imaging screening pipeline was established to screen for compounds capable of inhibiting extracellular sporozoites., Methods: Sporozoites expressing the green fluorescent protein were isolated from infected Anopheles mosquitoes, incubated with compounds from two libraries (MMV Malaria Box and a FDA-approved library) and imaged. Effects on in vitro motility or morphology were scored. In vivo efficacy of a candidate drug was investigated by treating mice ears with a gel prior to infectious mosquito bites. Motility was analysed by in vivo imaging and the progress of infection was monitored by daily blood smears., Results: Several compounds had a pronounced effect on in vitro sporozoite gliding or morphology. Notably, monensin sodium potently affected sporozoite movement while gramicidin S resulted in rounding up of sporozoites. However, pre-treatment of mice with a topical gel containing gramicidin did not reduce sporozoite motility and infection., Conclusions: This approach shows that it is possible to screen libraries for inhibitors of sporozoite motility and highlighted the paucity of compounds in currently available libraries that inhibit this initial step of a malaria infection. Screening of diverse libraries is suggested to identify more compounds that could serve as leads in developing 'skin-based' malaria prophylactics. Further, strategies need to be developed that will allow compounds to effectively penetrate the dermis and thereby prevent exit of sporozoites from the skin.

Background: Malaria continues to be a global burden as the efficacy of most commercial anti-malarial drugs has been compromised by the evolution of parasite resistance. With the urgent need created for the development of alternative and more efficient anti-malarial drugs, this study focused on the evaluation of anti-malarial agents of the Warburgia salutaris stem bark., Methods: The stem bark was extracted with dichloromethane followed by silica gel column chromatography that led to the isolation of iso-mukaadial acetate, a drimanoid sesquiterpene. This compound was characterized by spectroscopic analysis ( 1 H NMR, 13 C NMR, IR and MS), and its structure was confirmed by X-ray crystallography. In vitro anti-plasmodial activity was investigated using a chloroquine sensitive NF54 Plasmodium falciparum strain. Cytotoxicity was measured using the MTT assay on HEK239 and HEPG2 cell lines. Chloroquine-sensitive Plasmodium berghei was used to infect Sprague-Dawley rats for in vivo studies. The W. salutaris crude extract and iso-mukaadial acetate were administered orally at 0.5; 1.5, 2.5 and 5 mg/kg, chloroquine was used as the reference drug. Determination of percentage parasitaemia, haematological parameters, and rat body weights was done throughout the experimental study period., Results: The crude extract and iso-mukaadial acetate showed very good activity on the inhibition of parasite growth (IC 50 0.01 ± 0.30 µg/ml) and (IC 50 0.44 ± 0.10 µg/ml), respectively, with iso-mukaadial acetate having cytotoxicity activity of 36.7 ± 0.8 and 119.2 ± 8.8 (µg/ml) on HEK293 and HEPG2 cells, respectively. The crude extract and iso-mukaadial acetate reduced percentage parasitaemia in a dose-dependent manner in comparison to the control. There were no significant differences in the haematological parameters in all the experimental groups in comparison to control group. This study proves that W. salutaris contains components (including iso-mukaadial acetate) that exhibit anti-malarial activity. This study scientifically validates the use of this plant in folk medicine., Conclusions: This study proves that Warburgia salutaris contains components (including iso-mukaadial acetate) that exhibit anti-malarial activity and scientifically validates the use of this plant in folk medicine.

Background: Increasing resistance to current anti-malarial therapies requires a renewed effort in searching for alternative therapies to combat this challenge, and combination therapy is the preferred approach to address this. The present study confirms the anti-plasmodial effects of two compounds, cryptolepine and xylopic acid and the relationship that exists in their combined administration determined., Methods: Anti-plasmodial effect of cryptolepine (CYP) (3, 10, 30 mg kg -1 ) and xylopic acid (XA) (3, 10, 30 mg kg -1 ) was evaluated in Plasmodium berghei-infected male mice after a 6-day drug treatment. The respective doses which produced 50% chemosuppression (ED 50 ) was determined by iterative fitting of the log-dose responses of both drugs. CYP and XA were then co-administered in a fixed dose combination of their ED 50 s (1:1) as well as different fractions of these combinations (1/2, 1/4, 1/8, 1/16 and 1/32) to find the experimental ED 50 (Z exp ). The nature of interaction between cryptolepine and xylopic acid was determined by constructing an isobologram to compare the Z exp with the theoretical ED 50 (Z add ). Additionally, the effect of cryptolepine/xylopic acid co-administration on vital organs associated with malarial parasiticidal action was assessed., Results: The Z add and Z exp were determined to be 12.75 ± 0.33 and 2.60 ± 0.41, respectively, with an interaction index of 0.2041. The Z exp was significantly (P < 0.001) below the additive isobole indicating that co-administration of cryptolepine and xylopic acid yielded a synergistic anti-plasmodial effect. This observed synergistic antiplasmodial effect did not have any significant deleterious effect on the kidney, liver and spleen. However, the testis were affected at high doses., Conclusion: The co-administration of cryptolepine and xylopic acid produces synergistic anti-malarial effect with minimal toxicity.

Background: Malaria is an infectious disease caused by parasites of the genus Plasmodium, of which Plasmodium vivax and Plasmodium falciparum are the major species that cause the disease in humans. As there are relatively few alternatives for malaria treatment, it is necessary to search for new chemotherapeutic options. Colombia possesses a great diversity of plants, which are potential sources of new compounds of medical interest. Thus, in this study the antiplasmodial effect of extracts from two species of plants from the families Simaroubaceae and Picramniaceae (Picramnia latifolia and Picrolemma huberi) was evaluated in vitro and in vivo. These plants were chosen because they contain secondary metabolites with interesting medicinal effects., Results: The ethanolic extracts of both species were highly active with IC 50 : 1.2 ± 0.19 µg/mL for P. latifolia and IC 50 : 0.05 ± 0.005 µg/mL for P. huberi. The P. latifolia extract had a stage specific effect on trophozoites and inhibited parasite growth in vivo by 52.1 ± 3.4%, evaluated at 1000 mg/kg in Balb/c mice infected with Plasmodium berghei. On the other hand, evaluated at 150 mg/kg body weight in the same murine model, the ethanolic extract from P. huberi had an antiplasmodial effect in all the asexual intraerythrocytic stages of P. falciparum FCR3 and inhibited the parasitic growth in 93 ± 32.9%., Conclusions: This is the first report of anti-malarial activity for these two species of plants. Thus, P. latifolia and P. huberi are potential candidates for the development of new drugs for treating malaria.

Background: Plasmodium falciparum malaria is still one of the most deadly pathology worldwide. Efficient treatment is jeopardized by parasite resistance to artemisinin and its derivatives, and by poor access to treatment in endemic regions. Anti-malarial traditional remedies still offer new tracks for identifying promising antiplasmodial molecules, and a way to ensure that all people have access to care. The present study aims to validate the traditional use of Terminalia macroptera, a Malian plant used in traditional medicine., Methods: Terminalia macroptera was collected in Mali. Leaves (TML) and roots ethanolic extracts (TMR) were prepared and tested at 2000 mg/kg for in vivo acute toxicity in Albino Swiss mice. Antiplasmodial activity of the extracts was assessed against a chloroquine resistant strain P. falciparum (FcB1) in vitro. In vivo, anti-malarial efficacy was assessed by a 4-day suppressive test at 100 mg/kg in two malaria murine models of uncomplicated malaria (Plasmodium chabaudi chabaudi infection) and cerebral malaria (Plasmodium berghei strain ANKA infection). Constituents of TMR were characterized by ultra-high-performance liquid chromatography coupled to high resolution mass spectrometry. Top ranked compounds were putatively identified using plant databases and in silico fragmentation pattern., Results: Lethal dose of TML and TMR were greater than 2000 mg/kg in Albino Swiss mice. According to the OECD's Globally Harmonized System of Classification, both extracts are non-toxic orally. Antiplasmodial activity of T. macroptera extracts was confirmed in vitro against P. falciparum FcB1 strain with IC50 values of 1.2 and 1.6 µg/mL for TML and TMR, respectively. In vivo, oral administration of TML and TMR induced significant reduction of parasitaemia (37.2 and 46.4% respectively) in P. chabaudi chabaudi infected mice at the 7th day of infection compared to untreated mice. In the cerebral malaria experimental model, mice treated with TMR and TML presented respectively 50 and 66.7% survival rates at day 9 post-infection when all untreated mice died. Eleven major compounds were found in TMR. Among them, several molecules already known could be responsible for the antiplasmodial activity of the roots extract of T. macroptera., Conclusions: This study confirms both safety and anti-malarial activity of T. macroptera, thus validating its traditional use.

Background: Considering the need for new anti-malarial drugs, further investigations on Keetia leucantha (Rubiaceae), an in vitro antiplasmodial plant traditionally used to treat malaria, were carried out. This paper aimed to assess the in vivo anti-malarial efficacy of K. leucantha triterpenic esters previously identified as the most in vitro active components against Plasmodium falciparum and their potential toxicity as well as those of anti-malarial extracts., Results: These eight triterpenic esters and the major antiplasmodial triterpenic acids, ursolic and oleanolic acids, were quantified in the twigs dichloromethane extract by validated HPLC-UV methods. They account for about 19% of this extract (16.9% for acids and 1.8% for esters). These compounds were also identified in trace in the twigs decoction by HPLC-HRMS. Results also showed that extracts and esters did not produce any haemolysis, and were devoid of any acute toxicity at a total cumulative dose of 800 and 150 mg/kg respectively. Moreover, esters given intraperitoneally at 50 mg/kg/day to Plasmodium berghei-infected mice showed a very significant (p < 0.01) parasitaemia inhibition (27.8 ± 5.4%) on day 4 post-infection compared to vehicle-treated mice., Conclusions: These results bring out new information on the safety of K. leucantha use and on the identification of anti-malarial compounds from its dichloromethane extract. Its activity can be explained by the presence of triterpenic acids and esters which in vivo activity and safety were demonstrated for the first time.

Background: The emergence of multidrug-resistant strains of Plasmodium falciparum poses a great threat of increased fatalities in cases of cerebral and other forms of severe malaria infections in which parenteral artesunate monotherapy is the current drug of choice. The study aimed to investigate in a mouse model of human cerebral malaria whether a trioxaquine chemically synthesized by covalent linking of a 4,7-dichloroquinoline pharmacophore to artesunate through a recent drug development approach termed 'covalent bitherapy' could improve the curative outcomes in cerebral malaria infections., Methods: Human cerebral malaria rodent model, the C57BL/6 male mice were infected intraperitoneally (ip) with Plasmodium berghei ANKA and intravenously (iv) treated with the trioxaquine from day 8 post-infection (pi) at 12.5 and 25 mg/kg, respectively, twice a day for 3 days. Treatments with the trioxaquine precursors (artesunate and 4,7-dichloroquine), and quinine were also included as controls. In vivo safety evaluation for the trioxaquine was done according to Organization for Economic Co-operation and Development (OECD) guidelines 423, where female Swiss albino mice were orally administered with either 300 or 2000 mg/kg of the trioxaquine and monitored for signs of severity, and or mortality for 14 days post-treatment., Results: The trioxaquine showed a potent and a rapid antiplasmodial activity with 80% parasite clearance in the first 24 h for the two dosages used. Long-term parasitaemia monitoring showed a total parasite clearance as the treated mice survived beyond 60 days post-treatment, with no recrudescence observed. Artesunate treated mice showed recrudescence 8 days post-treatment, with all mice in this group succumbing to the infection. Also, 4,7-dichloroquinoline and quinine did not show any significant parasitaemia suppression in the first 24 h post-treatment, with the animals succumbing to the infection., Conclusion: Covalent bitherapy proves to be a viable source of urgently needed new anti-malarials for management of cerebral malaria, and this polypharmacology approach could be a potential strategy to protect artesunate from parasite resistance and in potentially improving clinical outcomes in severe forms of malaria infections.

Background: In addition to pharmacovigilance and pharmaco-economic concerns, resistance to anti-malarial medicines has been documented in all classes of anti-malarials and this is further worsened by resistance to common insecticides by malaria vector, which is a major threat to malaria control. As a means of facing the challenges of searching for new anti-malarial agents, the current study focused on evaluation of anti-malarial activity of root extract of Indigofera spicata., Methods: Chloroquine-sensitive rodent malaria parasite, Plasmodium berghei (ANKA strain) was used to infect the Swiss Albino mice in 4-day suppressive and curative models. The crude hydromethanolic root extract of I. spicata at 200, 400 and 600 mg/kg doses was administered to a group of five mice. Important parameters, such as level of parasitaemia, packed cell volume (PCV), survival time, and body weight were determined and the significance of the differences between mean values of the five groups was analysed by one-way ANOVA followed by post hoc Tukey's Multiple Comparison test., Results: In both the suppressive and curative models, 400 and 600 mg/kg doses of the extract suppressed the level of parasitaemia significantly (p < 0.001) compared to the vehicle-treated groups, 34.93 and 53.42%, respectively. However, only the mice which were treated with the 600 mg/kg dose of the extract had significant difference in their mean survival time. In other parameters, namely PCV and mean body weight, there was no statistically significant difference between the extract-treated groups when compared to the negative control., Conclusions: This study revealed that the root extract of I. spicata possesses anti-malarial activity and necessitates further scientific validation.

Background: Primaquine is an anti-malarial used to prevent Plasmodium vivax relapses and malaria transmission. However, PQ metabolites cause haemolysis in patients deficient in the enzyme glucose-6-phosphate dehydrogenase (G6PD). Fifteen PQ-thiazolidinone derivatives, synthesized through one-post reactions from primaquine, arenealdehydes and mercaptoacetic acid, were evaluated in parallel in several biological assays, including ability to block malaria transmission to mosquitoes., Results: All primaquine derivatives (PQ-TZs) exhibited lower cell toxicity than primaquine; none caused haemolysis to normal or G6PD-deficient human erythrocytes in vitro. Sera from mice pretreated with the test compounds thus assumed to have drug metabolites, caused no in vitro haemolysis of human erythrocytes, whereas sera from mice pretreated with primaquine did cause haemolysis. The ability of the PQ-TZs to block malaria transmission was evaluated based on the oocyst production and percentage of mosquitoes infected after a blood meal in drug pre-treated animals with experimental malaria caused by either Plasmodium gallinaceum or Plasmodium berghei; four and five PQ-TZs significantly inhibited sporogony in avian and in rodent malaria, respectively. Selected PQ-TZs were tested for their inhibitory activity on P. berghei liver stage development, in mice and in vitro, one compound (4m) caused a 3-day delay in the malaria pre-patent period., Conclusions: The compound 4m was the most promising, blocking malaria transmissions and reducing the number of exoerythrocytic forms of P. berghei (EEFs) in hepatoma cells in vitro and in mice in vivo. The same compound also caused a 3-day delay in the malaria pre-patent period.

Background: Anti-malarial herbal preparations (HPs) continue to enjoy high patronage in Ghana despite reports that the artemisinin-based combination therapy (ACT), the recommended first choice for treatment of uncomplicated malaria in the country, remains efficacious. A major issue with the use of these preparations is inadequate or unreliable data on their efficacy and quality. An assessment of the potency and quality of the most popular commercial anti-malarial HPs in Ghana was, therefore, carried out. The outcome of this investigation is herein discussed preceded by a short literature review of herbal medicines in Ghana., Methods: Using a questionnaire survey of 344 individuals in parts of Ghana, five of the most frequently used HPs were identified and selected for test of their efficacy and quality. The effect of the selected compounds on Plasmodium berghei in vivo was assessed using standard methods., Results: All five tested HPs (HP-A, HP-B, HP-C, HP-D and HP-E) showed chemo-suppressive activity against P. berghei in vivo. However the degree of parasites inhibition is significantly lower compared to the WHO-recommended artemether-lumefantrine combination (p < 0.05, 99.9% chemosuppression/activity, 28 days survival). Using the Solomon Saker's Test, two of the preparations were found to contain chloroquine or compounds with chemical properties like that of chloroquine., Conclusion: Popular anti-malarial HPs used in southern Ghana were found to have chemo-suppressive properties. Intentional addition of chloroquine or SCs to these preparations in order to enhance their effectiveness has serious public health concerns as it may induce cross resistance to amodiaquine, one of the partner drugs in the recommended ACT for use in Ghana.

Background: The widespread emergence of anti-malarial drug resistance has necessitated the discovery of novel anti-malarial drug candidates. In this study, chloroquine derivatives were evaluated for the improved anti-malarial activity., Results: Novel two derivatives (SKM13 and SKM14) were synthesized based on the chloroquine (CQ) template containing modified side chains such as α,β-unsaturated amides and phenylmethyl group. The selective index indicated that SKM13 was 1.28-fold more effective than CQ against the CQ-resistant strain Plasmodium falciparum. An in vivo mouse study demonstrated that SKM13 (20 mg/kg) could completely inhibit Plasmodium berghei growth in blood and increased the survival rate from 40 to 100% at 12 days after infection. Haematological parameters [red blood cell (RBC) count, haemoglobin level, and haematocrit level] were observed as an indication of clinical malarial anaemia during an evaluation of the efficacy of SKM13 in a 4-day suppression test. An in vivo study showed a decrease of greater than 70% in the number of RBC in P. berghei-infected mice over 12 days, but the SKM13 (20 mg/kg)-treated group showed no loss of RBC., Conclusions: CQ derivatives with substituents such as α,β-unsaturated amides and phenylmethyl group have enhanced anti-malarial activity against the CQ-resistant strain P. falciparum, and SKM13 is an excellent anti-malarial drug candidate in mice model.

Background: Malaria is one of the most life-threatening health problems worldwide and treatment has been compromised by drug resistance. Identifying lead molecules from natural products might help to find better anti-malarial drugs, since those obtained from natural sources are still effective against malarial parasites. This study aimed at investigating the in vivo antiplasmodial activity of crude extract of the leaves of Ajuga remota together with its safety in mice models., Methods: In vivo parasite growth inhibitory effect of crude extract was assessed in mice inoculated with Plasmodium berghei (ANKA strain). The in vivo antiplasmodial activity of the test extract was performed against early infection (4-day suppressive test), curative effect against established infection and prophylactic effect against residual infection. Acute and sub-acute toxicity were carried out according to OECD guidelines., Results: In vivo parasite growth inhibition effect of hydroethanolic crude extract of A. remota was evaluated at 30, 50 and 100 mg/kg dose levels. It suppressed parasitaemia by 77.34% at 100 mg/kg dose level in the 4-day test. In curative and prophylactic potential tests, it suppressed parasitaemia by 66.67 and 59.66% at 100 mg/kg dose level, respectively. In vivo toxicity tests revealed no toxicity. All parasitaemia suppressions were statistically significant at P < 0.05 as compared to the vehicle-treated group. The crude extract also prolonged survival time in a dose dependent manner., Conclusions: The investigation results suggest that the leave extract of Ajuga remota possesses antimalarial activity.

Background: Due to the ability of the 8-aminoquinolines (8AQs) to kill different stages of the malaria parasite, primaquine (PQ) and tafenoquine (TQ) are vital for causal prophylaxis and the eradication of erythrocytic Plasmodium sp. parasites. Recognizing the potential role of cytochrome (CYP) 450 2D6 in the metabolism and subsequent hepatic efficacy of 8-aminoquinolines, studies were designed to explore whether CYP2D-mediated metabolism was related to the ability of single-dose PQ and TQ to eliminate the asexual and sexual erythrocytic stages of Plasmodium berghei., Methods: An IV P. berghei sporozoite murine challenge model was utilized to directly compare causal prophylactic and erythrocytic activity (asexual and sexual parasite stages) dose-response relationships in C57BL/6 wild-type (WT) mice and subsequently compare the erythrocytic activity of PQ and TQ in WT and CYP2D knock-out (KO) mice., Results: Single-dose administration of either 25 mg/kg TQ or 40 mg/kg PQ eradicated the erythrocytic stages (asexual and sexual) of P. berghei in C57BL WT and CYP2D KO mice. In WT animals, the apparent elimination of hepatic infections occurs at lower doses of PQ than are required to eliminate erythrocytic infections. In contrast, the minimally effective dose of TQ needed to achieve causal prophylaxis and to eradicate erythrocytic parasites was analogous., Conclusion: The genetic deletion of the CYP2D cluster does not affect the ability of PQ or TQ to eradicate the blood stages (asexual and sexual) of P. berghei after single-dose administration.

Background: The emergence of artemisinin-resistant Plasmodium falciparum strains poses a serious challenge to the control of malaria. This necessitates the development of new anti-malarial drugs. Previous studies have shown that the natural beta-carboline alkaloid harmine is a promising anti-malarial agent targeting the P. falciparum heat-shock protein 90 (PfHsp90). The aim of this study was to test the anti-malarial activity of harmine analogues., Methods: Forty-two harmine analogues were synthesized and the binding of these analogues to P. falciparum heat shock protein 90 was investigated. The in vitro anti-malarial activity of two of the analogues, 17A and 21A, was evaluated using a 72-h growth inhibition assay. The in vivo anti-malarial activity was tested in Plasmodium berghei infection of BALB/c mice. The potential of 21A for a combination treatment with artemisinin was evaluated using in vivo combination study with dihydro-artemisinin in BALB/c mice. Cytotoxicity of the harmine analogues was tested in vitro using HepG2 and HeLa cell lines., Results: 17A and 21A bound to PfHsp90 with average IC 50 values of 12.2 ± 2.3 and 23.1 ± 8.8 µM, respectively. They also inhibited the P. falciparum W2 strain with average IC 50 values of 4.2 ± 1.3 and 5.7 ± 1.7 µM, respectively. In vivo, three daily injections of P. berghei-infected BALB/c mice with 100 mg/kg of either 17A or 21A showed significant reduction in parasitaemia with a 51.5 and 56.1% reduction, respectively. Mice treated with 17A and 21A showed a median survival time of 11 and 14 days, respectively, while the vehicle control mice survived a median of only 8.5 days. A dose-ranging experiment with 21A showed that the compound has a dose-dependent anti-malarial effect. Furthermore, treatment of infected mice with a combination of 21A and dihydroartemisinin (DHA) showed a dramatic reduction in parasitaemia compared to treatment with DHA alone., Conclusion: A novel and non-toxic harmine analogue has been synthesized which binds to PfHsp90 protein, inhibits P. falciparum in vitro at micromolar concentration, reduces parasitaemia and prolongs survival of P. berghei-infected mice with an additive anti-malarial effect when combined with DHA.

Background: Zanthoxylum heitzii (Rutaceae) (olon) is used in traditional medicine in Central and West Africa to treat malaria. To identify novel compounds with anti-parasitic activity and validate medicinal usage, extracts and compounds isolated from this tree were tested against the erythrocytic stages of the human malaria parasite Plasmodium falciparum and for inhibition of transmission in rodent malaria parasite Plasmodium berghei., Results: Hexane bark extract showed activity against P. falciparum (IC50 0.050 μg/ml), while leaf and seed extracts were inactive. Fractionation of the hexane bark extract led to the identification of three active constituents; dihydronitidine, pellitorine and heitziquinone. Dihydronitidine was the most active compound with an IC50 value of 0.0089 µg/ml (25 nM). This compound was slow acting, requiring 50 % longer exposure time than standard anti-malarials to reach full efficacy. Heitziquinone and pellitorine were less potent, with IC50 values of 3.55 μg/ml and 1.96 µg/ml, but were fast-acting. Plasmodium berghei ookinete conversion was also inhibited by the hexane extract (IC50 1.75 µg/ml), dihydronitidine (0.59 µg/ml) and heitziquinone (6.2 µg/ml). Water extracts of Z. heitzii bark contain only low levels of dihydronitidine and show modest anti-parasitic activity., Conclusions: Three compounds with anti-parasitic activity were identified in Z. heitzii bark extract. The alkaloid dihydronitidine is the most effective of these, accounting for the bulk of activity in both erythrocytic and transmission-blocking assays. These compounds may present good leads for development of novel anti-malarials and add to the understanding of the chemical basis of the anti-parasitic activity in these classes of natural product.

Background: The potential emergence and spread of resistance to artemisinins in the Plasmodium falciparum malaria parasite constitutes a major global health threat. Hence, improving the efficacy of artemisinins and of artemisinin-based combination therapy (ACT) represents a major short-term goal in the global fight against malaria. Mice defective in the enzyme pantetheinase (Vnn3) show increased susceptibility to blood-stage malaria (increased parasitaemia, reduced survival), and supplementation of Vnn3 mutants with the reaction product of pantetheinase, cysteamine, corrects in part the malaria-susceptibility phenotype of the mutants. Cysteamine (Cys) is a small, naturally occurring amino-thiol that has very low toxicity in vivo and is approved for clinical use in the life-long treatment of the kidney disorder nephropathic cystinosis., Methods: The ability of Cys to improve the anti-plasmodial activity of different clinically used artemisinins was tested. The effect of different CYS/ART combinations on malarial phenotypes (parasite blood-stage replication, overall and survival from lethal infection) was assessed in a series of in vivo experiments using Plasmodium strains that induce either blood-stage (Plasmodium chabaudi AS) or cerebral disease (Plasmodium berghei ANKA). This was also evaluated in an ex vivo experimental protocol that directly assesses the effect of such drug combinations on the viability of Plasmodium parasites, as measured by the ability of tested parasites to induce a productive infection in vivo in otherwise naïve animals., Results: Cys is found to potentiate the anti-plasmodial activity of artesunate, artemether, and arteether, towards the blood-stage malaria parasite P. chabaudi AS. Ex vivo experiments, indicate that potentiation of the anti-plasmodial activity of artemisinins by Cys is direct and does not require the presence of host factors. In addition, potentiation occurs at sub-optimal concentrations of artemisinins and Cys that on their own have little or no effect on parasite growth. Cys also dramatically enhances the efficacy and protective effect of artemisinins against cerebral malaria induced by infection with the P. berghei ANKA parasite., Conclusion: These findings indicate that inclusion of Cys in current formulations of ACT, or its use as adjunct therapy could improve the anti-plasmodial activity of artemisinin, decrease mortality in cerebral malaria patients, and prevent or delay the development and spread of artemisinin resistance.

Background: Malaria prevention has remained a critical area in the absence of efficacious vaccines against malaria. Drugs currently used as chemotherapeutics are also used in chemoprophylaxis increasing possible drug resistance. Asiatic acid is a natural phytochemical with oxidant, antioxidant and anti-inflammatory properties with emerging anti-malarial potential. The influence of asiatic acid administration prior to Plasmodium berghei infection of Sprague-Dawley rats on parasitaemia induction is here reported., Methods: Sprague-Dawley rats (90-120 g) were administered with asiatic acid (10 mg/kg) 48 h before intraperitoneal infection with P. berghei. Parasitaemia induction and progression, food and water intake as well as weight were compared to 30 mg/kg chloroquine-treated and infected control rats during sub-chronic studies (21 days)., Results: Asiatic acid pre-infection administration preserved food and water intake as well as increase in percentage weight gain of infected animals. In pre-infection treated animals, the pre-patent period was extended to day 6 from 72 h. Asiatic acid suppressed parasitaemia while oral chloroquine (30 mg/kg) did not influence malaria induction., Conclusions: Per-oral, pre-infection, asiatic acid administration influenced parasitaemia patency and parasitaemia progression, food, water, and weight gain percentage. This may suggest possible chemoprophylaxis effects of asiatic acid in malaria.

Background: Cryptolepine (CPE) is the major indoloquinoline isolated from the popular West African anti-malarial plant, Cryptolepis sanguinolenta. CPE possesses various pharmacological activities with potent anti-malarial activity against both chloroquine (CQ)-resistant and -sensitive strains. The search for safe and novel anti-malarial agents and combinations to delay resistance development to Plasmodium falciparum directed this work aimed at evaluating the anti-malarial interaction and safety of CPE in combination with some artemisinin derivatives., Methods: The in vitro SYBR Green I, fluorescent-based, drug sensitivity assay using a fixed ratio method was carried out on the CQ-sensitive plasmodial strain 3D7 to develop isobolograms from three CPE-based combinations with some artemisinin derivatives. CPE and artesunate (ART) combinations were also evaluated using the Rane's test in ICR mice infected with Plasmodium berghei NK-65 strains in a fixed ratio combination (1:1) and fractions of their ED50s in order to determine the experimental ED50 (Zexp) of the co-administered compounds. Isobolograms were constructed to compare the Zexp to the Zadd., Results: CPE exhibited promising synergistic interactions in vitro with ART, artemether and dihydroartemisinin. In vivo, CPE combination with ART again showed synergy as the Zexp was 1.02 ± 0.02, which was significantly less than the Zadd of 8.3 ± 0.31. The haematological, biochemical, organ/body weight ratio and histopathology indices in the rats treated with CPE at all doses (25, 50, 100 mg kg(-1) po) and in combination with ART (4 mg kg(-1)) showed no significant difference compared to the control group., Conclusion: The combination of CPE with the artemisinin derivatives were safe in the rodent model and showed a synergistic anti-malarial activity in vivo and in vitro. This study supports the basis for the selection of CPE as a prospective lead compound as the search for new anti-malarial combinations continues.

Background: The anti-malarials quinine and artemisinin were isolated from traditionally used plants (Cinchona spp. and Artemisia annua, respectively). The synthetic quinoline anti-malarials (e.g. chloroquine) and semi-synthetic artemisinin derivatives (e.g. artesunate) were developed based on these natural products. Malaria is endemic to the Amazon region where Plasmodium falciparum and Plasmodium vivax drug-resistance is of concern. There is an urgent need for new anti-malarials. Traditionally used Amazonian plants may provide new treatments for drug-resistant P. vivax and P. falciparum. Herein, the in vitro and in vivo antiplasmodial activity and cytotoxicity of medicinal plant extracts were investigated., Methods: Sixty-nine extracts from 11 plant species were prepared and screened for in vitro activity against P. falciparum K1 strain and for cytotoxicity against human fibroblasts and two melanoma cell lines. Median inhibitory concentrations (IC50) were established against chloroquine-resistant P. falciparum W2 clone using monoclonal anti-HRPII (histidine-rich protein II) antibodies in an enzyme-linked immunosorbent assay. Extracts were evaluated for toxicity against murine macrophages (IC50) and selectivity indices (SI) were determined. Three extracts were also evaluated orally in Plasmodium berghei-infected mice., Results: High in vitro antiplasmodial activity (IC50 = 6.4-9.9 µg/mL) was observed for Andropogon leucostachyus aerial part methanol extracts, Croton cajucara red variety leaf chloroform extracts, Miconia nervosa leaf methanol extracts, and Xylopia amazonica leaf chloroform and branch ethanol extracts. Paullinia cupana branch chloroform extracts and Croton cajucara red variety leaf ethanol extracts were toxic to fibroblasts and or melanoma cells. Xylopia amazonica branch ethanol extracts and Zanthoxylum djalma-batistae branch chloroform extracts were toxic to macrophages (IC50 = 6.9 and 24.7 µg/mL, respectively). Andropogon leucostachyus extracts were the most selective (SI >28.2) and the most active in vivo (at doses of 250 mg/kg, 71% suppression of P. berghei parasitaemia versus untreated controls)., Conclusions: Ethnobotanical or ethnopharmacological reports describe the anti-malarial use of these plants or the antiplasmodial activity of congeneric species. No antiplasmodial activity has been demonstrated previously for the extracts of these plants. Seven plants exhibit in vivo and or in vitro anti-malarial potential. Future work should aim to discover the anti-malarial substances present.

Background: During the erythrocytic stage in humans, malaria parasites digest haemoglobin of the host cell, and the toxic haem moiety crystallizes into haemozoin. Chloroquine acts by forming toxic complexes with haem molecules and interfering with their crystallization. In chloroquine-resistant strains, the drug is excluded from the site of action, which causes the parasites to accumulate less chloroquine in their acid food vacuoles than chloroquine-sensitive parasites. 3-Hydroxylpyridin-4-ones are known to chelate iron; hydroxypyridone-chloroquine (HPO-CQ) hybrids were synthesized in order to determine whether they can inhibit parasites proliferation in the parasitic digestive vacuole by withholding iron from plasmodial parasite metabolic pathway., Methods: Two HPO-CQ hybrids were tested against Plasmodium falciparum chloroquine-sensitive (D10 and 3D7) and -resistant strains (Dd2 and K1). The pharmacokinetic properties of active compounds were determined using a mouse model and blood samples were collected at different time intervals and analysed using LC-MS/MS. For in vivo efficacy the mice were infected with Plasmodium berghei in a 4-day Peters' test. The parasitaemia was determined from day 3 and the course of the infection was followed by microscopic examination of stained blood films every 2-3 days until a rise in parasitaemia was observed in all test subjects., Results: IC50 values of the two compounds for sensitive and resistant strains were 0.064 and 0.047 µM (compound 1), 0.041 and 0.122 µM (compound 2) and 0.505 and 0.463 µM (compound 1), 0.089 and 0.076 µM (compound 2), respectively. Pharmacokinetic evaluation of these compounds showed low oral bioavailability and this affected in vivo efficacy when compounds were dosed orally. However, when dosed intravenously compound 1 showed a clearance rate of 28 ml/min/kg, an apparent volume of distribution of 20 l/kg and a half-life of 4.3 h. A reduction in parasitaemia was observed when compound 1 was dosed intravenously for four consecutive days in P. berghei-infected mice. However, a rise in parasitaemia levels was observed on day 6 and on day 9 for chloroquine-treated mice., Conclusion: The hybrid compounds that were tested were able to reduce parasitaemia levels in P. berghei-infected mice when dosed intravenously, but parasites recrudesced 24 h after the administration of the least dose. Despite low oral bioavailability, the IV data obtained suggests that further structural modifications may lead to the identification of more HPO-CQ hybrids with improved pharmacokinetic properties and in vivo efficacy.

Background: Artemisinin (ART) is an efficacious and safe anti-malarial drugs but has low oral bioavailability and auto-induction profiles during multiple dosing. The pharmacokinetic disadvantages have been found to partially depend on the induction of cytochrome P-450 enzymes by ART and resulted in the therapeutic failure due to insufficient drug levels. The present study, therefore, investigated the impacts of chrysosplenetin (CHR), a polymethoxylated flavonoid from Artemisia annua, on the pharmacokinetics and the anti-malarial efficacy of ART against Plasmodium berghei. The inhibition of CHR on enzymatic activity of CYP1A2, CYP2A, CYP2C19, CYP2D6, CYP2E1, and CYP3A in rat liver microsome was also investigated. IC50, Km, Ki, and inhibitory type of CHR were respectively calculated., Methods: Twenty rats were randomly divided into four groups and received three-day oral doses of ART in absence or presence of CHR (in ratio of 1:0, 1:1, 1:2, and 1:4, respectively). Plasma samples were separately harvested for ART pharmacokinetics analysis using a valid liquid chromatography tandem mass spectrometric (LC-MS/MS) method. Female Kunming mice were inoculated by P. berghei K173 strain and pre-exposed to three-day oral administration of ART with or without CHR as pharmacokinetics protocol. Giemsa staining method was applied to calculate percent parasitaemia (%) and inhibition (%). In vitro rat liver microsomal model was employed to elucidate the inhibitory effect of CHR on CYP1A2, CYP2A, CYP2C19, CYP2D6, CYP2E1, and CYP3A., Results: The AUC0-t, Cmax, and t 1/2 of ART increased significantly (P < 0.05 or P < 0.01) as well as declined CLz (P < 0.05 or P < 0.01) after three-day oral doses of ART in presence of CHR (1:2) when compared with ART alone. Also, parasitaemia (%) remarkably attenuated 1.59 folds with 1.63-fold augmented inhibition (%) when the ratio between ART and CHR reached 1:2. CHR itself had no anti-malarial efficacy (P > 0.05). CHR inhibited in vitro activity of CYP1A2 and CYP2C19 (P < 0.01, IC50 = 4.61 and 6.23 μM) in a concentration-response manner. The inhibition did not emerge on CYP2E1 and CYP3A until the CHR concentration exceeded 4.0 μM (P < 0.01, IC50 = 28.17 and 3.38 µM). CHR has no impact on CYP 2A and CYP2D6 (P > 0.05). The inhibition types of CHR on CYP1A2 and CYP3A belonged to noncompetitive and uncompetitive, respectively., Conclusions: Co-administration of ART with CHR in ratio of 1:2 achieved a synergic anti-malarial effect partly because of the noncompetitive or uncompetitive inhibition of CHR of drug-metabolism enzymes, especially CYP3A which is closely related to the auto-induction of ART.

Background: Medicinal plants are a validated source for discovery of new leads and standardized herbal medicines. The aim of this study was to assess the activity of Vernonia amygdalina leaf extracts and isolated compounds against gametocytes and sporogonic stages of Plasmodium berghei and to validate the findings on field isolates of Plasmodium falciparum., Methods: Aqueous (Ver-H2O) and ethanolic (Ver-EtOH) leaf extracts were tested in vivo for activity against sexual and asexual blood stage P. berghei parasites. In vivo transmission blocking effects of Ver-EtOH and Ver-H2O were estimated by assessing P. berghei oocyst prevalence and density in Anopheles stephensi mosquitoes. Activity targeting early sporogonic stages (ESS), namely gametes, zygotes and ookinetes was assessed in vitro using P. berghei CTRPp.GFP strain. Bioassay guided fractionation was performed to characterize V. amygdalina fractions and molecules for anti-ESS activity. Fractions active against ESS of the murine parasite were tested for ex vivo transmission blocking activity on P. falciparum field isolates. Cytotoxic effects of extracts and isolated compounds vernolide and vernodalol were evaluated on the human cell lines HCT116 and EA.hy926., Results: Ver-H2O reduced the P. berghei macrogametocyte density in mice by about 50% and Ver-EtOH reduced P. berghei oocyst prevalence and density by 27 and 90%, respectively, in An. stephensi mosquitoes. Ver-EtOH inhibited almost completely (>90%) ESS development in vitro at 50 μg/mL. At this concentration, four fractions obtained from the ethylacetate phase of the methanol extract displayed inhibitory activity >90% against ESS. Three tested fractions were also found active against field isolates of the human parasite P. falciparum, reducing oocyst prevalence in Anopheles coluzzii mosquitoes to one-half and oocyst density to one-fourth of controls. The molecules and fractions displayed considerable cytotoxicity on the two tested cell-lines., Conclusions: Vernonia amygdalina leaves contain molecules affecting multiple stages of Plasmodium, evidencing its potential for drug discovery. Chemical modification of the identified hit molecules, in particular vernodalol, could generate a library of druggable sesquiterpene lactones. The development of a multistage phytomedicine designed as preventive treatment to complement existing malaria control tools appears a challenging but feasible goal.

Background: The survival of malaria parasites, under substantial haem-induced oxidative stress in the red blood cells (RBCs) is dependent on the pentose phosphate pathway (PPP). The PPP is the only source of NADPH in the RBC, essential for the production of reduced glutathione (GSH) and for protection from oxidative stress. Glucose-6-phosphate dehydrogenase (G6PD) deficiency, therefore, increases the vulnerability of erythrocytes to oxidative stress. In Plasmodium, G6PD is combined with the second enzyme of the PPP to create a unique bifunctional enzyme, named glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (G6PD-6PGL). RRx-001 is a novel, systemically non-toxic, epigenetic anticancer agent currently in Phase 2 clinical development for multiple tumour types, with activity mediated through increased nitric oxide (NO) production and PPP inhibition. The inhibition of G6PD and NO overproduction induced by RRx-001 suggested its application in cerebral malaria (CM)., Methods: Plasmodium berghei ANKA (PbA) infection in C57BL/6 mice is an experimental model of cerebral malaria (ECM) with several similar pathological features to human CM. This study uses intravital microscopy methods with a closed cranial window model to quantify cerebral haemodynamic changes and leukocyte adhesion to endothelial cells in ECM., Results: RRx-001 had both single agent anti-parasitic activity and significantly increased the efficacy of artemether. In addition, RRx-001 preserved cerebral perfusion and reduced inflammation alone or combined with artemether. RRx-001's effects were associated with inhibition of PPP (G6PD and G6PD-6PGL) and by improvements in microcirculatory flow, which may be related to the NO donating properties of RRx-001., Conclusion: The results indicate that RRx-001 could be used to potentiate the anti-malarial action of artemisinin, particularly on resistant strains, and to prevent infection.

Background: Medicinal plants have contributed significantly to current malaria treatment. Emergence of resistance to currently available drugs has necessitated the search for new plant-based anti-malarial agents and several plant-based, pharmacologically active anti-malarial compounds have been isolated. This study was conducted to validate the traditional usage of Echinops kebericho for treating malaria in the traditional health care system of Ethiopia., Methods: The roots of E. kebericho were collected from Masha Woreda, Sheka Zone. After collection, the plant materials were identified by a taxonomist, dried under shade and crushed to powder for extraction. The powdered roots were extracted by maceration using 70 % ethanol. Acute toxicity study of the crude extract was carried out in Swiss albino mice. The in vivo anti-malarial activity of plant extract (200, 350 and 500 mg/kg) of E. kebericho roots against a chloroquine (CQ) sensitive strain of Plasmodium berghei strain ANKA was assessed using the four-day suppressive test procedure. Parameters such as parasitaemia, packed cell volume, body weight and survival time were then determined using standard tests., Results: Oral administration of the ethanol extract showed significant (P<0.001) parasitaemia suppression at dose levels of 350 and 500 mg/kg in dose-related manner compared with the negative control. Five hundred mg/kg showed the highest (57.29±1.76 %) parasitaemia suppression. The survival times of P. berghei-infected mice were also increased in a dose-dependent manner but the test material did not prevent weight loss associated with increased parasitaemia. The result also showed the plant material prevented the loss in packed cell volume associated with increased parasitaemia. Its oral LD50 was found to be greater than 5,000 mg/kg, indicating its wider safety margin in mice., Conclusion: The result revealed the ethanol extract of E. kebericho roots has anti-malarial activity against P. berghei in an animal model and lends support to the use of the plant to combat malaria in Ethiopian folk medicine. Further work is necessary to isolate, identify and characterize the active principles from the plant material.

Background: Plasmodium falciparum has become resistant to some of the available drugs. Several plant species are used for the treatment of malaria, such as Himatanthus articulatus in parts of Brazil. The present paper reports the phyto-chemistry, the anti-plasmodial and anti-malarial activity, as well as the toxicity of H. articulatus., Methods: Ethanol and dichloromethane extracts were obtained from the powder of stem barks of H. articulatus and later fractionated and analysed. The anti-plasmodial activity was assessed against a chloroquine resistant strain P. falciparum (W2) in vitro, whilst in vivo anti-malarial activity against Plasmodium berghei (ANKA strain) was tested in mice, evaluating the role of oxidative stress (total antioxidant capacity--TEAC; lipid peroxidation--TBARS, and nitrites and nitrates--NN). In addition, cytotoxicity was evaluated using the HepG2 A16 cell-line. The acute oral and sub-chronic toxicity of the ethanol extract were evaluated in both male and female mice., Results: Plumieride was isolated from the ethyl acetate fraction of ethanol extract, Only the dichloromethane extract was active against clone W2. Nevertheless, both extracts reduced parasitaemia in P. berghei-infected mice. Besides, a significant reduction in pulmonary and cerebral levels of NN (nitrites and nitrates) was found, as well as in pulmonary TBARS, indicating a reduced oxidative damage to these organs. The ethanol extract showed low cytotoxicity to HepG2 A16 cells in the concentrations used. No significant changes were observed in the in vivo toxicity studies., Conclusions: The ethanol extract of H. articulatus proved to be promising as anti-malarial medicine and showed low toxicity.

Background: In Tanzania and elsewhere, medicinal plants, including Maytenus senegalensis, are still widely used in the treatment of malaria and other ailments. The aim of the present study was to investigate the in vivo antiplasmodial and toxic effects in mice., Methods: Oral antiplasmodial and acute toxicity of the ethanolic root extract of M. senegalensis was evaluated in mice. The Peters 4-day in vivo antiplasmodial effect against early rodent malaria infection in chloroquine-sensitive Plasmodium berghei NK 65 strain in mice., Results: The M. senegalensis extract was found non-toxic and the oral median lethal dose in mice was determined to be greater than 1,600 mg/kg body weight. The findings revealed a significant (P = 0.001) daily increase in the level of parasitaemia in the parasitized untreated groups and a significant (P < 0.001) dose dependent decrease in parasitaemia in the parasitized groups treated with varying doses ranging from 25 to 100 mg/kg body weight of M. senegalensis extract and the standard drug sulphadoxine/pyrimethamine at 25/1.25 mg/kg body weight. Overall, the dose dependent parasitaemia suppression effects were in the order of: 25/1.25 mg/kg body weight of sulphadoxine/pyrimethamine > 100 mg/kg > 75 mg/kg > 50 mg/kg > 25 mg/kg body weight of M. senegalensis extract., Conclusion: The implications of these findings is that M. senegalensis ethanolic root bark extract possess potent antiplasmodial effect and may, therefore, serve as potential sources of safe, effective and affordable anti-malarial drugs. The displayed high in vivo antiplasmodial activity and lack of toxic effect render M. senegalensis a candidate for the bioassay-guided isolation of compounds which could develop into new lead structures and candidates for drug development programmes against human malaria.

Background: Emergence of drug-resistant parasite strains has surfaced as a major obstacle in attempts to ameliorate malaria. Current treatment regimen of malaria relies on the concept of artemisinin-based combination therapy (ACT)., Methods: Fluoroquinolone analogues, compounds 10, 12 and 18 were investigated for their anti-malarial interaction in combination with artemisinin in vitro, against Plasmodium falciparum 3D7 strain, employing fixed-ratio combination isobologram method. In addition, the efficacy of these compounds was evaluated intraperitoneally in BALB/c mice infected with chloroquine-resistant Plasmodium berghei ANKA strain in the Peters' four-day suppressive test., Results: Promising results were obtained in the form of synergistic or additive interactions. Compounds 10 and 12 were found to have highly synergistic interactions with artemisinin. Antiplasmodial effect was further verified by the convincing ED50 values of these compounds, which ranged between 2.31 and 3.09 (mg/kg BW)., Conclusions: In vivo studies substantiated the potential of the fluoroquinolone derivatives to be developed as synergistic partners for anti-malarial drug combinations.

Background: The evolution of drug-resistant parasites is a major hindrance to malaria control, and thus understanding the behaviour of drug-resistant mutants is of clinical relevance. The study aimed to investigate how resistance against lumefantrine (LU) and piperaquine (PQ), anti-malarials used as partner drugs in artemisinin-based combination therapy (ACT), impacts parasite fitness. This is important since resistance to ACT, the first-line anti-malarial regimen is increasingly being reported., Methods: The stability of Plasmodium berghei ANKA strain that was previously selected for LU and PQ resistance was evaluated using the 4-day assay and established infection test in mice. Fitness cost of resistance was determined by comparing parasites proliferation rates in absence of drug pressure for the drug-exposed parasites between day 4 and 7 post-infection (pi), relative to the wild-type. Statistical analysis of data to compare mean parasitaemia and growth rates of respective parasite lines was carried out using student's t-test and one-way analysis of variance, with significance level set at p<0.05., Results: During serial passaging in the absence of the drug, the PQ-resistant parasite maintained low growth rates at day 7 pi (mean parasitaemia, 5.6% ± 2.3) relative to the wild-type (28.4% ± 6.6), translating into a fitness cost of resistance of 80.3%. Whilst resistance phenotype for PQ was stable, that of LU was transient since after several serial passages in the absence of drug, the LU-exposed line assumed the growth patterns of the wild-type., Conclusions: The contrasting behaviour of PQ- and LU-resistance phenotypes support similar findings which indicate that even for drugs within the same chemical class, resistance-conferred traits may vary on how they influence parasite fitness and virulence. Resistance-mediating polymorphisms have been associated with less fit malaria parasites. In the absence of drug pressure in the field, it is therefore likely that the wild-type parasite will out-compete the mutant form. This implies the possibility of reintroducing a drug previously lost to resistance, after a period of suspended use. Considering the recent reports of high failure rates associated with ACT, high fitness cost of resistance to PQ is therefore of clinical relevance as the drug is a partner in ACT.

Background: There is an urgent need to develop and test novel compounds against malaria infection. Carrageenans, sulphated polysaccharides derived from seaweeds, have been previously shown to inhibit Plasmodium falciparum in vitro. However, they are inflammatory and alter the permeability of the blood-brain barrier, raising concerns that their use as a treatment for malaria could lead to cerebral malaria (CM), a severe complication of the disease. In this work, the authors look into the effects of the administration of λ-carrageenan to the development and severity of CM in BALB/c mice, a relatively non-susceptible model, during infection with the ANKA strain of Plasmodium berghei., Methods: Five-week-old female BALB/c mice were infected with P. berghei intraperitoneally. One group was treated with λ-carrageenan (PbCGN) following the 4-day suppressive test protocol, whereas the other group was not treated (PbN). Another group of healthy BALB/c mice was similarly given λ-carrageenan (CGN) for comparison. The following parameters were assessed: parasitaemia, clinical signs of CM, and mortality. Brain and other vital organs were collected and examined for gross and histopathological lesions. Evans blue dye assays were employed to assess blood-brain barrier integrity., Results: Plasmodium berghei ANKA-infected BALB/c mice treated with λ-carrageenan died earlier than those that received no treatment. Histopathological examination revealed that intracerebral haemorrhages related to CM were present in both groups of infected BALB/c mice, but were more numerous in those treated with λ-carrageenan than in mock-treated animals. Inflammatory lesions were also observed only in the λ-carrageenan-treated mice. These observations are consistent with the clinical signs associated with CM, such as head tilt, convulsions, and coma, which were observed only in this group, and may account for the earlier death of the mice., Conclusion: The results of this study indicate that the administration of λ-carrageenan exacerbates the severe brain lesions and clinical signs associated with CM in BALB/c mice infected with P. berghei ANKA.

Background: Several antibiotics have shown promising anti-malarial effects and have been useful for malarial chemotherapy, particularly in combination with standard anti-malarial drugs. Tigecycline, a semi-synthetic derivative of minocycline with a unique and novel mechanism of action, is the first clinically available drug in a new class of glycylcycline antibiotics., Methods: Tigecycline was tested in vitro against chloroquine (CQ)-sensitive (D6) and resistant strains (W2) of Plasmodium falciparum alone and in combination with CQ. Tigecycline was also tested in vivo in combination with CQ in Plasmodium berghei-mouse malaria model for parasitaemia suppression, survival and cure of the malaria infection., Results: Tigecycline was significantly more active against CQ-resistant (W2) than CQ-susceptible (D6) strain of P. falciparum. Tigecycline potentiated the anti-malarial action of CQ against the CQ-resistant strain of P. falciparum by more than seven-fold. Further, treatment of mice infected with P. berghei with tigecycline (ip) produced significant suppression in parasitaemia development and also prolonged the mean survival time. Treatment with as low as 3.7 mg/kg dose of tigecycline, once daily for four days, produced 77-91% suppression in parasitaemia. In vivo treatment with tigecycline in combination with subcurative doses of CQ produced complete cure in P. berghei-infected mice., Conclusion: Results indicate prominent anti-malarial action of tigecycline in vitro and in vivo in combination with CQ and support further evaluation of tigecycline as a potential combination candidate for treatment of drug-resistant cases of malaria.

Background: Carapa guianensis is a cultivable tree used by traditional health practitioners in the Amazon region to treat several diseases and particularly symptoms related to malaria. Abundant residual pressed seed material (RPSM) results as a by-product of carapa or andiroba oil production. The objective of this study was to evaluate the in vitro and in vivo anti-malarial activity and cytotoxicity of limonoids isolated from C. guaianensis RPSM., Methods: 6α-acetoxyepoxyazadiradione (1), andirobin (2), 6α-acetoxygedunin (3) and 7-deacetoxy-7-oxogedunin (4) (all isolated from RPSM using extraction and chromatography techniques) and 6α-hydroxy-deacetylgedunin (5) (prepared from 3) were evaluated using the micro test on the multi-drug-resistant Plasmodium falciparum K1 strain. The efficacy of limonoids 3 and 4 was then evaluated orally and subcutaneously in BALB/c mice infected with chloroquine-sensitive Plasmodium berghei NK65 strain in the 4-day suppressive test., Results: In vitro, limonoids 1-5 exhibited median inhibition concentrations (IC50) of 20.7-5.0 μM, respectively. In general, these limonoids were not toxic to normal cells (MRC-5 human fibroblasts). In vivo, 3 was more active than 4. At oral doses of 50 and 100 mg/kg/day, 3 suppressed parasitaemia versus untreated controls by 40 and 66%, respectively, evidencing a clear dose-response., Conclusion: 6α-acetoxygedunin is an abundant natural product present in C. guianensis residual seed materials that exhibits significant in vivo anti-malarial properties.

Background: Repositioning of existing drugs has been suggested as a fast track for developing new anti-malarial agents. The compound libraries of GlaxoSmithKline (GSK), Pfizer and AstraZeneca (AZ) comprising drugs that have undergone clinical studies in other therapeutic areas, but not achieved approval, and a set of US Food and Drug Administration (FDA)-approved drugs and other bio-actives were tested against Plasmodium falciparum blood stages., Methods: Molecules were tested initially against erythrocytic co-cultures of P. falciparum to measure proliferation inhibition using one of the following methods: SYBR®I dye DNA staining assay (3D7, K1 or NF54 strains); [(3)H] hypoxanthine radioisotope incorporation assay (3D7 and 3D7A strain); or 4',6-diamidino-2-phenylindole (DAPI) DNA imaging assay (3D7 and Dd2 strains). After review of the available clinical pharmacokinetic and safety data, selected compounds with low μM activity and a suitable clinical profile were tested in vivo either in a Plasmodium berghei four-day test or in the P. falciparum Pf3D7(0087/N9) huSCID 'humanized' mouse model., Results: Of the compounds included in the GSK and Pfizer sets, 3.8% (9/238) had relevant in vitro anti-malarial activity while 6/100 compounds from the AZ candidate drug library were active. In comparison, around 0.6% (24/3,800) of the FDA-approved drugs and other bio-actives were active. After evaluation of available clinical data, four investigational drugs, active in vitro were tested in the P. falciparum humanized mouse model: UK-112,214 (PAF-H1 inhibitor), CEP-701 (protein kinase inhibitor), CEP-1347 (protein kinase inhibitor), and PSC-833 (p-glycoprotein inhibitor). Only UK-112,214 showed significant efficacy against P. falciparum in vivo, although at high doses (ED90 131.3 mg/kg [95% CI 112.3, 156.7]), and parasitaemia was still present 96 hours after treatment commencement. Of the six actives from the AZ library, two compounds (AZ-1 and AZ-3) were marginally efficacious in vivo in a P. berghei model., Conclusions: Repositioning of existing therapeutics in malaria is an attractive proposal. Compounds active in vitro at μM concentrations were identified. However, therapeutic concentrations may not be effectively achieved in mice or humans because of poor bio-availability and/or safety concerns. Stringent safety requirements for anti-malarial drugs, given their widespread use in children, make this a challenging area in which to reposition therapy.

Background: As anti-malarial drug resistance escalates, new safe and effective medications are necessary to prevent and treat malaria infections. The US Army is developing tafenoquine (TQ), an analogue of primaquine (PQ), which is expected to be more effective in preventing malaria in deployed military personnel., Methods: To compare the prophylactic efficacy of TQ and PQ, a transgenic Plasmodium berghei parasite expressing the bioluminescent reporter protein luciferase was utilized to visualize and quantify parasite development in C57BL/6 albino mice treated with PQ and TQ in single or multiple regimens using a real-time in vivo imaging system (IVIS). As an additional endpoint, blood stage parasitaemia was monitored by flow cytometry. Comparative pharmacokinetic (PK) and liver distribution studies of oral and intravenous PQ and TQ were also performed., Results: Mice treated orally with three doses of TQ at 5 mg/kg three doses of PQ at 25 mg/kg demonstrated no bioluminescence liver signal and no blood stage parasitaemia was observed suggesting both drugs showed 100% causal activity at the doses tested. Single dose oral treatment with 5 mg TQ or 25 mg of PQ, however, yielded different results as only TQ treatment resulted in causal prophylaxis in P. berghei sporozoite-infected mice. TQ is highly effective for causal prophylaxis in mice at a minimal curative single oral dose of 5 mg/kg, which is a five-fold improvement in potency versus PQ. PK studies of the two drugs administered orally to mice showed that the absolute bioavailability of oral TQ was 3.5-fold higher than PQ, and the AUC of oral TQ was 94-fold higher than oral PQ. The elimination half-life of oral TQ in mice was 28 times longer than PQ, and the liver tissue distribution of TQ revealed an AUC that was 188-fold higher than PQ., Conclusions: The increased drug exposure levels and longer exposure time of oral TQ in the plasma and livers of mice highlight the lead quality attributes that explain the much improved efficacy of TQ when compared to PQ.

Background: Stephania rotunda is used by traditional health practitioners in Southeast Asia to treat a wide range of diseases and particularly symptoms related to malaria. Cepharanthine (CEP) is an alkaloid isolated from this plant with potential innovative antiplasmodial activity. The analysis of interactions between antiplasmodial drugs is necessary to develop new drugs combinations to prevent de novo emergence of resistance. The objective of this study was to evaluate the anti-malarial activity of CEP in combination with usual anti-malarial compounds, both in vitro and in vivo., Methods: A fixed ratio method using the isotopic micro test was performed on the chloroquine-resistant plasmodial strain W2 to build isobolograms from eight CEP-based combinations with standard anti-malarial drugs. The efficacy of two combinations was then evaluated in the BALB/c mouse infected with Plasmodium berghei ANKA strain., Results: In vitro, efficiency gains were observed when CEP was combined with chloroquine (CQ), lumefantrine (LUM), atovaquone (ATO), piperaquine (PPQ) and particularly monodesethylamodiaquine (MdAQ), whereas an antagonistic interaction was observed with dihydroartemisinin (DHA) and mefloquine (MQ). In vivo, the combination of CEP with CQ or amodiaquine (AQ) improved significantly the survival of mice and extended the delay for parasitic recrudescence., Conclusion: All these observations suggest that CEP could be an interesting lead compound in the development of a combination therapy against malaria.

Background: Tafenoquine (TQ) is an 8-aminoquinoline (8AQ) that has been tested in several Phase II and Phase III clinical studies and is currently in late stage development as an anti-malarial prophylactic agent. NPC-1161B is a promising 8AQ in late preclinical development. It has recently been reported that the 8AQ drug primaquine requires metabolic activation by CYP 2D6 for efficacy in humans and in mice, highlighting the importance of pharmacogenomics in the target population when administering primaquine. A logical follow-up study was to determine whether CYP 2D activation is required for other compounds in the 8AQ structural class., Methods: In the present study, the anti-malarial activities of NPC-1161B and TQ were assessed against luciferase expressing Plasmodium berghei in CYP 2D knock-out mice in comparison with normal C57BL/6 mice (WT) and with humanized/CYP 2D6 knock-in mice by monitoring luminescence with an in vivo imaging system. These experiments were designed to determine the direct effects of CYP 2D metabolic activation on the anti-malarial efficacy of NPC-1161B and TQ., Results: NPC-1161B and TQ exhibited no anti-malarial activity in CYP 2D knock-out mice when dosed at their ED100 values (1 mg/kg and 3 mg/kg, respectively) established in WT mice. TQ anti-malarial activity was partially restored in humanized/CYP 2D6 knock-in mice when tested at two times its ED100., Conclusions: The results reported here strongly suggest that metabolism of NPC-1161B and TQ by the CYP 2D enzyme class is essential for their anti-malarial activity. Furthermore, these results may provide a possible explanation for therapeutic failures for patients who do not respond to 8AQ treatment for relapsing malaria. Because CYP 2D6 is highly polymorphic, variable expression of this enzyme in humans represents a significant pharmacogenomic liability for 8AQs which require CYP 2D metabolic activation for efficacy, particularly for large-scale prophylaxis and eradication campaigns.