Targeting liver stage malaria with metformin.

Despite an unprecedented 2 decades of success, the combat against malaria - the mosquito-transmitted disease caused by Plasmodium parasites - is no longer progressing. Efforts toward eradication are threatened by the lack of an effective vaccine and a rise in antiparasite drug resistance. Alternative approaches are urgently needed. Repurposing of available, approved drugs with distinct modes of action are being considered as viable and immediate adjuncts to standard antimicrobial treatment. Such strategies may be well suited to the obligatory and clinically silent first phase of Plasmodium infection, where massive parasite replication occurs within hepatocytes in the liver. Here, we report that the widely used antidiabetic drug, metformin, impairs parasite liver stage development of both rodent-infecting Plasmodium berghei and human-infecting P. falciparum parasites. Prophylactic treatment with metformin curtails parasite intracellular growth in vitro. An additional effect was observed in mice with a decrease in the numbers of infected hepatocytes. Moreover, metformin provided in combination with conventional liver- or blood-acting antimalarial drugs further reduced the total burden of P. berghei infection and substantially lessened disease severity in mice. Together, our findings indicate that repurposing of metformin in a prophylactic regimen could be considered for malaria chemoprevention.

Aspidosperma pyrifolium, a medicinal plant from the Brazilian caatinga, displays a high antiplasmodial activity and low cytotoxicity.

BACKGROUND: Several species of Aspidosperma plants are referred to as remedies for the treatment of malaria, especially Aspidosperma nitidum. Aspidosperma pyrifolium, also a medicinal plant, is used as a natural anti-inflammatory. Its fractionated extracts were assayed in vitro for activity against malaria parasites and for cytotoxicity. METHODS: Aspidosperma pyrifolium activity was evaluated against Plasmodium falciparum using extracts in vitro. Toxicity towards human hepatoma cells, monkey kidney cells or human monocytes freshly isolated from peripheral blood was also assessed. Anti-malarial activity of selected extracts and fractions that presented in vitro activity were tested in mice with a Plasmodium berghei blood-induced infection. RESULTS: The crude stem bark extract and the alkaloid-rich and ethyl acetate fractions from stem extract showed in vitro activity. None of the crude extracts or fractions was cytotoxic to normal monkey kidney and to a human hepatoma cell lines, or human peripheral blood mononuclear cells; the MDL50 values of all the crude bark extracts and fractions were similar or better when tested on normal cells, with the exception of organic and alkaloidic-rich fractions from stem extract. Two extracts and two fractions tested in vivo caused a significant reduction of P. berghei parasitaemia in experimentally infected mice. CONCLUSION: Considering the high therapeutic index of the alkaloidic-rich fraction from stem extract of A. pyrifolium, it makes the species a candidate for further investigation aiming to produce a new anti-malarial, especially considering that the active extract has no toxicity, i.e., no mutagenic effects in the genototoxicity assays, and that it has an in vivo anti-malarial effect. In its UPLC-HRMS analysis this fraction was shown to have two major components compatible with the bisindole alkaloid Leucoridine B, and a novel compound, which is likely to be responsible for the activity against malaria parasites demonstrated in in vitro tests.

Evaluation of the hepatic effect of concomitant administration of ciprofloxacin and some antimalarial drugs in Plasmodium berghei infected mice: An in vivo study.

This study evaluated the hepatotoxic effects of artesunate (AS), artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ) co-administration with ciprofloxacin (CIP) using animal model. Chloroquine sensitive Plasmodium berghei NK65 strain infected albino mice (120) were utilized for this study, carried out in three phases. Phase 1 comprised eleven groups treated with different doses of either AS, AL, ASAQ or CIP alone. Phase 2 consisted of nine groups treated with 7mg/kg of CIP combined with different doses of AS, AL, ASAQ. Phase 3 comprised ten groups treated with 14mg/kg of CIP (CIP2) with different doses of AS, AL, ASAQ. Seventy-two hours after administration of drugs, toxicity was determined by evaluating the effect of drugs on liver enzymes using spectrophotometer. Statistical analysis revealed that CIP alone significantly (P<0.05) reduced the levels of Aspartate Transaminase (AST) and Serum Alanine Transaminase (ALT) compared to AS, AL and ASAQ alone. Combination of different doses of AS, AL and ASAQ with 7mg/kg CIP significantly increased the level of AST and ALT while combination of AS, AL and ASAQ with 14mg/kg CIP significantly decreased AST and ALT levels. Care should be taken during the co-administration of low dose ciprofloxacin with artesunate, artemether-lumefantrine or artesunate-amodiaquine.

Anti-plasmodial and anti-inflammatory activities of cyclotide-rich extract and fraction of Oldenlandia affinis (R. & S.) D.C. (Rubiaceae).

BACKGROUND: Oldenlandia affinis, commonly called 'kalata-kalata', a versatile plant used locally to treat malaria fever in some parts of sub-Saharan Africa was investigated for anti-plasmodial and anti-inflammatory activities. OBJECTIVE: The study was designed to evaluate the antiplasmodial as well as anti-inflammatory activities of whole extract and cyclotide-rich fraction of Oldenlandia affinis. METHOD: The dichloromethane-methanol extract (ODE) of the plant, O. affinis was investigated for suppressive and curative antiplasmodial activities against Plasmodium berghei in mice. ODE and the cyclotide-rich fraction (CRF) was investigated for chronic and acute anti-inflammatory activities in rat models of inflammation. Inhibition of pro-inflammatory mediators was studied in RAW264.7 macrophages. RESULTS: ODE exhibited significant (p<0.05) reduction in mean parasitaemia in both the suppressive and curative models of Plasmodium berghei infection in mice.Administration of ODE(100, 200, or 400 mg/kg) and CRF (100, 200, or 400 mg/kg) produced significant inhibition of rodent models of acute and chronic inflammation . This observation is supported by the significant (P<0.05) inhibition of pro-inflammatory mediators, inducible nitric oxide (iNO) and tumour necrosis factor-alpha (TNF-α), and the reactive radical scavenging activities in RAW264.7 macrophages. CONCLUSION: These findings could explain, at least in part, the successes reported in the use of the herb, Oldenlandia affinis in the traditional treatment of malaria fever.

A novel model fitted to multiple life stages of malaria for assessing efficacy of transmission-blocking interventions.

BACKGROUND: Transmission-blocking interventions (TBIs) aim to eliminate malaria by reducing transmission of the parasite between the host and the invertebrate vector. TBIs include transmission-blocking drugs and vaccines that, when given to humans, are taken up by mosquitoes and inhibit parasitic development within the vector. Accurate methodologies are key to assess TBI efficacy to ensure that only the most potent candidates progress to expensive and time-consuming clinical trials. Measuring intervention efficacy can be problematic because there is substantial variation in the number of parasites in both the host and vector populations, which can impact transmission even in laboratory settings. METHODS: A statistically robust empirical method is introduced for estimating intervention efficacy from standardised population assay experiments. This method will be more reliable than simple summary statistics as it captures changes in parasite density in different life-stages. It also allows efficacy estimates at a finer resolution than previous methods enabling the impact of the intervention over successive generations to be tracked. A major advantage of the new methodology is that it makes no assumptions on the population dynamics of infection. This enables both host-to-vector and vector-to-host transmission to be density-dependent (or other) processes and generates easy-to-understand estimates of intervention efficacy. RESULTS: This method increases the precision of intervention efficacy estimates and demonstrates that relying on changes in infection prevalence (the proportion of infected hosts) alone may be insufficient to capture the impact of TBIs, which also suppress parasite density in secondarily infected hosts. CONCLUSIONS: The method indicates that potentially useful, partially effective TBIs may require multiple infection cycles before substantial reductions in prevalence are observed, despite more rapidly suppressing parasite density. Accurate models to quantify efficacy will have important implications for understanding how TBI candidates might perform in field situations and how they should be evaluated in clinical trials.

Dynamic control of hepatic Plasmodium numbers by hepcidin despite elevated liver iron during iron supplementation.

Treatment of iron deficiency anemia in malaria endemic areas is complicated as iron supplementation increases malaria risk while malaria decreases iron absorption. Here we measured the influence of hepcidin expression and non-heme iron during iron supplementation on hepatic Plasmodium berghei numbers in anemic and non-anemic mice. Despite elevated hepatic non-heme iron on the high iron diet, elevated hepcidin expression is associated with less parasite bioavailable iron and lower hepatic parasite loads in anemic, iron deficient mice after both two and six weeks of supplementation. A marginal trend to lower parasite hepatic numbers was seen in non-anemic, iron replete mice. In a transgenic model of severe anemia, mice with a deletion in Sec15l1, which reportedly have normal liver iron and normal hepcidin expression, there were no changes in liver parasite numbers or blood stage numbers or outcome in the lethal Plasmodium yoelii model. In summary during iron supplementation the lower hepatic malaria numbers are regulated more by hepcidin than the absolute level of non-heme hepatic iron.

Antiplasmodial activity of aqueous extract of Berberis aristata roots against Plasmodium berghei-infected BALB/c mice.

CONTEXT: The rising problem of resistance to present antimalarial drugs stresses the need to look for newer antiplasmodial components with effective modes of action. The roots of Berberis aristata DC. (Berberidaceae) are used in the traditional medicine for malaria in various parts of India. OBJECTIVE: The objective of this study was to evaluate antiplasmodial activity of B. aristata roots extract for the validation of its traditional medicinal use. MATERIAL AND METHODS: Aqueous root extract of Berberis aristata (AREBA) was screened for its in vitro as well as in vivo antiplasmodial activity against lethal rodent malaria parasite Plasmodium berghei NK65. In vitro activity was evaluated against schizont maturation of P. berghei using various concentrations ranging from 1 to 100 µg/mL. For in vivo studies, AREBA at the doses of 150, 250, 350, and 650 mg/kg/d was administered to P. berghei infected BALB/c mice orally for 4 consecutive days (D0-D3). RESULTS: AREBA showed in vitro antiplasmodial activity with an IC50 value of 40 µg/mL. In vivo studies demonstrated a variable dose-dependent chemosuppression with higher efficacy at lower doses. At a dose of 350 mg/kg/d, the suppressive and preventive activities were found to be 67.1% and 53.9%, respectively, followed by enhancing mean survival period up to 12.8 d for the curative assay versus 7.5 d for the untreated mice. DISCUSSION AND CONCLUSION: These results provide relevant scientific evidences for the traditional medicinal use of this plant as malaria remedy and further advocates the isolation and characterization of active antiplasmodial principle from this plant.

Efficacy and pharmacokinetic evaluation of a novel anti-malarial compound (NP046) in a mouse model.

BACKGROUND: Even though malaria is a completely preventable and treatable disease, it remains a threat to human life and a burden to the global economy due to the emergence of multiple-drug resistant malaria parasites. According to the World Malaria Report 2013, in 2012 there were an estimated 207 million malaria cases and 627,000 deaths. Thus, the discovery and development of new, effective anti-malarial drugs are required. To achieve this goal, the Department of Chemistry at the University of the Free State has synthesized a number of novel amino-alkylated chalcones and analogues, which showed in vitro anti-malarial activity against both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains. The lead compound (NP046) was selected for a comprehensive pharmacokinetic (PK) and in vivo efficacy evaluation in a mouse model. METHODS: In vivo efficacy: Water solutions of NP046 were administered orally at 50 and 10 mg/kg using oral gavage and IV at 5 and 1 mg/kg via the dorsal penile vein to Plasmodium berghei (ANKA strain) infected male C57BL/6 mice (n = 5), once a day for four days. Blood samples were collected via tail bleeding in tubes containing phosphate buffer saline (PBS) on day five to determine the % parasitaemia by flow cytometry.In vivo PK: NP046 solutions in water were administered orally (50 and 10 mg/kg) and IV (5 mg/kg) to male C57BL/6 mice (n = 5). Blood samples were collected via tail bleeding into heparinized tubes and analysed using a validated LC-MS/MS assay. Data obtained from the concentration-time profile was evaluated using Summit PK software to determine the PK parameters of NP046. RESULTS: NP046 inhibited parasite growth for the oral and IV groups. Better parasite growth inhibition was observed for the IV group. The PK evaluation of NP046 showed low oral bioavailability (3.2% and 6% at 50 mg/kg and 10 mg/kg dose, respectively and a moderate mean half-life ranging from 3.1 to 4.4 hours. CONCLUSION: Even though the oral bioavailability of NP046 is low, its percentage parasite growth inhibition is promising, but in order to improve the oral bioavailability, structure-activity-relationship (SAR) optimization studies are currently being conducted.

In vitro and in vivo antimalarial activity and cytotoxicity of extracts and fractions from the leaves, root-bark and stem-bark of Triclisia gilletii.

ETHNOPHARMACOLOGICAL RELEVANCE: To evaluate the in vitro antiplasmodial activity and cytotoxicity, and the in vivo activity of extracts and fractions from the leaves, root-bark and stem-bark of Triclisia gilletii (De Wild) Staner (Menispermaceae), used in traditional medicine against malaria. MATERIALS AND METHODS: The aqueous and 80% MeOH extracts, and a series of fractions and subfractions from the leaves, stem and root-bark of Triclisia gilletii were tested in vitro for their antiplasmodial activity against a Congolese-sensitive strain of Plasmodium falciparum, against the chloroquine and pyrimethamine-resistant K1 strain of Plasmodium falciparum, for cytotoxicity against MRC-5 cells, and in vivo in mice infected with Plasmodium berghei berghei. RESULTS: Many samples from the three plant parts exhibited pronounced activity against the Congolese chloroquine-sensitive strain of Plasmodium falciparum with some IC50 values <0.02 µg/ml, and against the K1 strain, with some IC50 <0.25; the selectivity was higher against the Congolese strain. At oral doses of 200 and 400mg/kg body weight in infected mice, the aqueous, 80% methanol and total alkaloid extracts from the three plant parts produced more than 65% and 75% chemosuppression, respectively. The antiplasmodial activity of these three plant parts of Triclisia gilletii can at least in part be attributed to bisbenzylisoquinoline alkaloids, and supports its use for the treatment of uncomplicated malaria in traditional medicine.

Liver-stage malaria parasites vulnerable to diverse chemical scaffolds.

Human malaria infection begins with a one-time asymptomatic liver stage followed by a cyclic symptomatic blood stage. All high-throughput malaria drug discovery efforts have focused on the cyclic blood stage, which has limited potential for the prophylaxis, transmission blocking, and eradication efforts that will be needed in the future. To address these unmet needs, a high-throughput phenotypic liver-stage Plasmodium parasite screen was developed to systematically identify molecules with liver-stage efficacy. The screen recapitulates liver-stage infection by isolating luciferase-expressing Plasmodium berghei parasites directly from the salivary glands of infected mosquitoes, adding them to confluent human liver cells in 384-well plates, and measuring luciferase activity after a suitable incubation period. Screening 5,375 known bioactive compounds identified 37 liver-stage malaria inhibitors with diverse modes of action, as shown by inhibition time course experiments. Further analysis of the hits in the Food and Drug Administration-approved drug subset revealed compounds that seem to act specifically on the liver stage of infection, suggesting that this phase of the parasite's life cycle presents a promising area for new drug discovery. Notably, many active compounds in this screen have molecular structures and putative targets distinctly different from those of known antimalarial agents.

The effect of vitamin A supplementation and diphtheria-tetanus-pertussis vaccination on parasitaemia in an experimental murine malaria model.

BACKGROUND: Vitamin A supplementation (VAS) decreases overall child mortality in low-income countries. For logistical reasons, VAS has been linked to routine childhood immunizations. However, several recent studies have indicated that VAS may increase mortality and morbidity from infectious diseases when given with the diphtheria-tetanus-pertussis (DTP) vaccine. The immunological effects of combining the 2 treatments are unknown. METHODS: We studied the effect of treating C57BL/6 mice with VAS and DTP, 1 week prior to infection with Plasmodium berghei ANKA. The progression of disease was monitored through parasite load and time to death. RESULTS: We found significantly higher levels of parasitaemia in VAS/DTP-treated mice than in control mice (crude geometric mean parasitaemia ratio 2.02 (1.08-3.76), p = 0.03). There was no effect of administering either VAS or DTP alone, indicating that the increase in parasitaemia was due to a synergistic effect of VAS and DTP (p for interaction = 0.02). The effect of VAS/DTP on levels of parasitaemia was modified by the specific parasite variant used. No effect was observed on time to death. CONCLUSION: Our results indicate that VAS/DTP can negatively influence the outcome of malaria infection in mice, adding to the concerns about simultaneous VAS and DTP administration to children in low-income, malaria endemic countries.

In vitro and in vivo anti-malarial activity of Boerhavia elegans and Solanum surattense.

BACKGROUND: There is an urgent need to identify new anti-malarial drug targets for both prophylaxis and chemotherapy, due to the increasing problem of drug resistance to malaria parasites. In the present study, the aim was to discover novel, effective plant-based extracts for the activity against malaria. METHODS: Ten plants found in Iran were selected by ethnobotanical survey of medicinal plants. The crude ethanolic extracts were tested for in vitro anti-plasmodial activity against two strains of Plasmodium falciparum: K1 (chloroquine-resistant strain) and CY27 (chloroquine-sensitive strain), using the parasite lactate dehydrogenase (pLDH) assay. The anti-plasmodial activity of the extracts was also assessed in the 4-day suppressive anti-malarial assay in mice inoculated with Plasmodium berghei (ANKA strain). Crude ethanolic extracts showed good anti-plasmodial activity were further fractionated by partitioning in water and dichloromethane. RESULTS: Of 10 plant species assayed, three species: Boerhavia elegans (Choisy), Solanum surattense (Burm.f.) and Prosopis juliflora (Sw.) showed promising anti-plasmodial activity in vitro (IC50 < or = 50 microg/ml) and in vivo with no toxicity. The dichloromethane fraction of three extracts revealed stronger anti-plasmodial activity than the total extracts. CONCLUSION: Anti-plasmodial activities of extracts of B. elegans and S. surattense are reported for the first time.

Ethanolic extract of Clerodendrum violaceum Gürke leaves enhances kidney function in mouse model of malaria.

Evaluation of the effects of daily oral administration of ethanolic extract of C. violaceum leaves (13 mg/kg body weight) for 5 days on some kidney function indices of uninfected and Plasmodium berghei-infected mice was done on days 3, 8 and 14 post-infection. The indices studied include serum urea and creatinine concentrations with the specific activities of alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase in the kidney. Treatment of P. berghei-infected mice with ethanolic extract of C. violaceum leaves (13 mg/kg body weight) for 5 days was able to ameliorate significantly the alterations in the various parameters observed in infected untreated mice, comparing favourably with chloroquine treatment in most cases. Administration of extract to uninfected mice had no significant effect on both serum and kidney parameters compared to the uninfected control. The results suggest that the ethanolic extract of C. violaceum leaves does not adversely affect kidney function at the dose used in traditional medicine for the treatment of malaria but rather enhances it.

Development of a pharmacodynamic model of murine malaria and antimalarial treatment with dihydroartemisinin.

Antimalarial treatment strategies based on in vitro studies are limited by the paucity of pharmacodynamic information for dosage regimen design. We postulated that a murine model could be used for pre-clinical stages of drug development, especially in dose-response studies and evaluation of combination therapies. Swiss mice infected with Plasmodium berghei parasites (2-5% starting parasitaemia) were given dihydroartemisinin (0-100 mg/kg single dose). Parasite density was regularly determined from thin blood films. A parasite population growth model comprising parasite multiplication, decline in erythrocyte count with increasing parasitaemia and parasite clearance after drug administration was developed. This model described the rise in parasitaemia following inoculation, the nadir following dihydroartemisinin administration, and the subsequent resurgence of parasitaemia (analogous to 'recrudescence'). At doses of 10, 30 and 100 mg/kg dihydroartemisinin, there was a graded response with 2.5+/-1, 5+/-1 and 12+/-4-fold decreases in parasitaemia, respectively. The nadir parasitaemia (at 21-27 h) was also dose-dependent. This study demonstrates that a murine malaria pharmacodynamic model is a valuable tool for understanding how single drugs and their dosing schedules alter the time course and level of infection.

Retinol supplementation in murine Plasmodium berghei malaria: effects on tissue levels, parasitaemia and lipid peroxidation.

Reduced plasma retinol concentrations occur in human malaria but the benefits of supplementation remain uncertain. We assessed the in vivo efficacy of retinol administration, and its effect on lipid peroxidation, in a Plasmodium berghei murine model. Animals received vehicle (n=17) or retinol (i) before P. berghei inoculation (four doses), (ii) at parasitaemia 10-15% (three to four doses) or (iii) before and after inoculation (six to seven doses; n=15 in each group), with euthanasia on day 8 post-inoculation or when the parasitaemia exceeded 50%. Multiple-dose pre-inoculation retinol reduced endpoint parasitaemia by 24% (P=0.001 versus controls). A reduction of 18% (P=0.042) was observed when retinol was given to parasitaemic animals. Retinol was ineffective when given both before and after infection (11% reduction; P=0.47). Although retinol supplementation did not change plasma retinol concentrations, liver retinol content increased and correlated inversely with endpoint parasitaemia (r=-0.45, P=0.001). Malaria infection augmented concentrations of the free radical lipid peroxidation end-product F(2)-isoprostanes in plasma, erythrocytes and liver by 1.8-, 2.8- and 4.9-fold, respectively, but retinol supplementation had no effect on these increases. Consistent with some human malaria studies, prophylactic retinol reduces P. berghei parasitaemia. This effect relates to augmentation of tissue retinol stores rather than to retinol-associated changes in oxidant status.

Effects of homeopathic medications Eupatorium perfoliatum and Arsenicum album on parasitemia of Plasmodium berghei-infected mice.

Malaria is one of the most important parasitic diseases in the world and a major public health problem because of emerging drug-resistant strains of Plasmodium. A number of synthetic and natural compounds are now being analysed to develop more effective antimalarial drugs. We investigated the effect of homeopathic preparations of Eupatorium perfoliatum and Arsenicum album on parasitemia using a rodent malaria model. We found significant inhibitory effect on parasite multiplication with both medications with a level of 60% for Eupatorium perfoliatum at a 30 CH potency. Arsenicum album 0/6 gave 70% inhibition but this was less stable than Eupatorium perfoliatum. The number of schizonts was higher in animals treated with homeopathic medications. Although the mechanism of action is unknown, these agents would be good candidates as alternative or complementary medications in the treatment of malaria.

Studies on the use of Cassia singueana in malaria ethnopharmacy.

Cassia singueana (Family: Fabaceae) is used in northern Nigeria for the treatment of acute malaria attack. We investigated the activities of the methanol extract of the root bark of this plant against rodent plasmodia infection, nociception, pyrexia and inflammation in mice and rats. The studies were carried out using acetic acid-induced writhing, hot plate algesia, rodent plasmodia (Plasmodium berghei) in mice; formalin test, yeast-induced pyrexia and egg-albumin-induced inflammation in rats. The results showed that the extract exhibited significant antinociceptive, antipyretic and antiplasmodial activity in all the models used. Phytochemical screening of the extract revealed the presence of phenols, saponins, tannins and some traces of anthraquinones. The LD50 of the extract was established to be 847+/-30 mg/kg, i.p. in mice. The observed pharmacological activities might be the scientific basis for the folkloric use of the plant in treating acute malaria attack. The study also paves way for the possible development of it, as a phytodrug against malaria.