Salinomycin and other ionophores as a new class of antimalarial drugs with transmission-blocking activity.

The drug target profile proposed by the Medicines for Malaria Venture for a malaria elimination/eradication policy focuses on molecules active on both asexual and sexual stages of Plasmodium, thus with both curative and transmission-blocking activities. The aim of the present work was to investigate whether the class of monovalent ionophores, which includes drugs used in veterinary medicine and that were recently proposed as human anticancer agents, meets these requirements. The activity of salinomycin, monensin, and nigericin on Plasmodium falciparum asexual and sexual erythrocytic stages and on the development of the Plasmodium berghei and P. falciparum mosquito stages is reported here. Gametocytogenesis of the P. falciparum strain 3D7 was induced in vitro, and gametocytes at stage II and III or stage IV and V of development were treated for different lengths of time with the ionophores and their viability measured with the parasite lactate dehydrogenase (pLDH) assay. The monovalent ionophores efficiently killed both asexual parasites and gametocytes with a nanomolar 50% inhibitory concentration (IC50). Salinomycin showed a fast speed of kill compared to that of standard drugs, and the potency was higher on stage IV and V than on stage II and III gametocytes. The ionophores inhibited ookinete development and subsequent oocyst formation in the mosquito midgut, confirming their transmission-blocking activity. Potential toxicity due to hemolysis was excluded, since only infected and not normal erythrocytes were damaged by ionophores. Our data strongly support the downstream exploration of monovalent ionophores for repositioning as new antimalarial and transmission-blocking leads.

Plasmodium transmission blocking activities of Vernonia amygdalina extracts and isolated compounds.

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.

Poly-Electrophilic Sesquiterpene Lactones from Vernonia amygdalina: New Members and Differences in Their Mechanism of Thiol Trapping and in Bioactivity.

In addition to known compounds, the leaves of Vernonia amygdalina afforded the new sesquiterpene lactones 14-O-methylvernolide (2), 3'-deoxyvernodalol (6), and vernomygdalin (8). These and related compounds were evaluated for modulation of a series of thiol trapping-sensitive transcription factors (NF-κB, STAT3, and Nrf2), involved in the maintenance of the chronic inflammatory condition typical of human degenerative diseases. Vernolide (1) emerged as a potent inhibitor of STAT3 and NF-κB and showed cytostatic activity toward the prostate cancer cell line DU45, arresting the cell cycle at the S phase. The exomethylene lactones are characterized by multiple Michael acceptor sites, as exemplified by vernolide (1) and vernodalol (5). By using the nuclear magnetic resonance-based cysteamine assay, the most reactive thiophilic site could be identified in both compounds, and competitive experiments qualified vernolide (1) as being more thiophilic than vernodalol (5), in agreement with the results of the pharmacological assays.

In vitro Anti-Leishmanial Activities of Methanol Extract of Brucea antidysenterica J.F. Mill Seeds and Its Solvent Fractions.

Introduction: Leishmaniasis is one of the neglected tropical diseases, threatening lives of about 350 million people globally. Brucea antidysenterica seeds are used for the treatment of cutaneous leishmaniasis in the traditional medicine in Ethiopia. Objective: This study aimed to evaluate Brucea antidysenterica seeds' anti-leishmanial activity in vitro. Methods: The crude (80% methanol) extract of Brucea antidysenterica seeds and its fractions were evaluated for their anti-leishmanial activities against promastigotes and intracellular amastigotes of Leishmania donovani and Leishmania aethiopica, and for their cytotoxic effects against mammalian cells. The quantitative estimations of total phenolic compounds (TPCs), flavonoids (TFCs) and alkaloids (TACs) were determined, spectrophotometrically. Median inhibitory concentration (IC50) and median cytotoxic concentration (CC50) of the extract and its solvent fractions were calculated using GraphPad Prism 9.1.0 computer software. Data was presented as mean ± standard error of the mean (SEM). Results: The crude extract and its hexane, ethyl acetate and butanol fractions showed anti-leishmanial activities, with IC50 values of 4.14-60.12 µg/mL against promastigotes, and 6.16-40.12 µg/mL against amastigotes of both Leishmania species. They showed moderate cytotoxicity against Vero cell lines and peritoneal mice macrophages, with CC50 values of 100-500 µg/mL, but >1600 µg/mL against red blood cells. Selectivity indices ranged from 7.97 to 30.97. The crude extract, and its ethyl acetate and hexane fractions possessed 54.78-127.72 mg of gallic acid equivalent TPC, 18.30-79.21 mg of quercetin equivalent TFC, and 27.62-97.22 mg of atropine equivalent TAC per gram of extracts. Conclusion: The seeds of the plant possessed anti-leishmanial activities against L. aethiopica and L. donovani that might provide a scientific justification for its use in the treatment of leishmaniasis by traditional healers. Future works are recommended to isolate, purify and identify the possible secondary metabolites attributed to the anti-leishmanial activity.

Nanocellulose/Fe3O4/Ag Nanozyme with Robust Peroxidase Activity for Enhanced Antibacterial and Wound Healing Applications.

Peroxidase memetic nanozymes with their free radical-mediated catalytic actions proved as efficacious antibacterial agents for combating bacterial resistance. Herein, nanocellulose (NC) extracted from Eragrostis teff straw was used to prepare NC/Fe3O4/Ag peroxidase nanozyme as an antibacterial and wound healing agent. Characterization of the nanozyme with XRD, FTIR, SEM-EDX, and XPS confirmed the presence of silver NPs and the magnetite phase of iron oxide dispersed on nanocellulose. The peroxidase activity of the prepared nanozyme was examined using TMB and H2O2 as substrates which turned blue in acidic pH (λmax = 652 nm). With a lower Km (0.387 mM), the nanozyme showed a comparable affinity for TMB with that reported for the HRP enzyme. Furthermore, the nanozyme remained efficient over a broader temperature range while maintaining 61.53% of its activity after the fourth cycle. In vitro, antibacterial tests against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacterial strains showed that NC/Fe3O4/Ag exhibits concentration-dependent and enhanced antibacterial effect for Escherichia coli compared to NC and NC-Fe3O4 and negative control. Furthermore, the wound-healing performance of the NC-Fe3O4-Ag nanozyme was investigated in vivo using an animal model (mice). The nanozyme showed 30% higher wound healing performance compared to the control base ointment and is comparable with the commercial nitrofurazone ointment. The results show the potential of the prepared nanozyme for wound-healing purposes.

In vitro growth inhibitory activity of Medicines for Malaria Venture pathogen box compounds against Leishmania aethiopica.

INTRODUCTION: Leishmania aethiopica (L. aethiopica) is responsible for different forms of cutaneous leishmaniasis (CL) in Ethiopia. Treatment heavily depends on limited drugs, together with drawbacks like toxicity and microbial resistance. The current research aimed to investigate in vitro growth inhibitory activity of Medicines for Malaria Ventures - Pathogen Box (MMV - PB) compounds against L. aethiopica clinical isolate. METHODOLOGY: Four hundred MMV - PB compounds were screened against L. aethiopica using resazurin based colourimetric assay. Compounds with > 70% inhibition were further tested using macrophage based intracellular amastigote assay. Cytotoxic and hemolytic activity of candidate hits were assessed on THP1- cells and sheep red blood cells (RBCs), respectively. In vitro drug interaction study was also conducted for the most potent hit using the combination index method. RESULTS: At the test concentration of 1 µM, twenty-three compounds showed > 50% inhibition of promastigotes parasite growth, of which 11 compounds showed > 70% inhibition. The 50% growth inhibition (IC50) of the 11 compounds was ranged from 0.024 to 0.483 µM in anti-promastigote assay and from 0.064 to 0.899 µM in intracellular amastigote assay. Candidate compounds demonstrated good safety on sheep RBCs and THP-1 cell lines. MMV688415 demonstrated a slight hemolytic activity on sheep RBC (5.3% at 25 µM) and THP-1 cell line (CC20 = 25 µM) while MMV690102 inhibited half of THP-1 cells at 36.5 µM (selectivity index = 478). No synergistic activity was observed from the combinations of MMV690102 and amphotericin B (CI > 1), and MMV690102 and Pentamidine (CI > 1) at lower and higher combination points. CONCLUSION: The present study identified a panel of compounds that can be used as a novel starting point for lead optimization. MMV690102 appears to be the most potent inhibitor against L. aethiopica promastigotes and amastigotes. Future works should investigate the antileishmanial mechanism of action and in vivo antileishmanial activities of identified hits.

N-Acetyl Cysteine as an Adjunct in the Treatment of Tuberculosis.

Oxidative stress is a common feature of tuberculosis (TB), and persons with reduced antioxidants are at more risk of TB. TB patients with relatively severe oxidative stress had also more advanced disease as measured by the Karnofsky performance index. Since adverse effects from anti-TB drugs are also mediated by free radicals, TB patients are prone to side effects, such as hearing loss. In previous articles, researchers appealed for clinical trials aiming at evaluating N-acetyl cysteine (NAC) in attenuating the dreaded hearing loss during multidrug-resistant TB (MDR-TB) treatment. However, before embarking on such trials, considerations of NAC's overall impact on TB treatment are crucial. Unfortunately, such a comprehensive report on NAC is missing in the literature and this manuscript reviews the broader effect of NAC on TB treatment. This paper discusses NAC's effect on mycobacterial clearance, hearing loss, drug-induced liver injury, and its interaction with anti-TB drugs. Based on the evidence accrued to date, NAC appears to have various beneficial effects on TB treatment. However, despite the favorable interaction between NAC and first-line anti-TB drugs, the interaction between the antioxidant and some of the second-line anti-TB drugs needs further investigations.

Leishmania donovani Growth Inhibitors from Pathogen Box Compounds of Medicine for Malaria Venture.

INTRODUCTION: Leishmaniasis is a collective term used to describe various pathological conditions caused by an obligate intracellular protozoan of the genus Leishmania. It is one of the neglected diseases and has been given minimal attention by drug discovery and development stakeholders to narrow the safety and efficacy gaps of the drugs currently used to treat leishmaniasis. The challenge is further exacerbated by the emergence of drug resistance by the parasites. METHODS: Aiming to look for potential anti-leishmanial hits and leads, we screened Medicines for Malaria Venture (MMV) Pathogen Box compounds against clinically isolated Leishmania donovani strain. In this medium-throughput primary screening assay, the compounds were screened against promastigotes, and then against amastigote stages. RESULTS: From the total 400 compounds screened, 35 compounds showed >50% inhibitory activity on promastigotes in the initial screen (1 µM). Out of these compounds, nine showed >70% inhibition, with median inhibitory concentration (IC50) ranging from 12 to 491 nM using the anti-promastigote assay, and from 53 to 704 nM using the intracellular amastigote assay. Identified compounds demonstrated acceptable safety profiles on THP-1 cell lines and sheep red blood cells, and had appropriate physicochemical properties suitable for further drug development. Two compounds (MMV690102 and MMV688262) were identified as leads. The anti-tubercular agent MMV688262 (delamanid) showed a synergistic effect with amphotericin B, indicating the prospect of using this compound for combination therapy. CONCLUSION: The current study indicates the presence of additional hits which may hold promise as starting points for anti-leishmanial drug discovery and in-depth structure-activity relationship studies.

Acute and Subacute Toxicity of Methanol Extract of Syzygium guineense Leaves on the Histology of the Liver and Kidney and Biochemical Compositions of Blood in Rats.

Plant medicine is the oldest form of health care known to mankind. Syzygium guineense is one of the many species of Ethiopian medicinal plants which has a long history of use as remedies for various ailments such as dysentery, diarrhea, and hypertension. In many countries, herbal medicines and related products are introduced into the market without safety or toxicological evaluation. The aim of this study was to investigate the histopathological effect of 80% methanol extract of S. guineense on liver and kidney and blood parameters of rats. For acute toxicity study, rats were randomly divided into three groups (n=4). The control group received distilled water, while the experimental groups received a single dose of 2000 mg/kg and 5000 mg/kg 80% methanolic extract of S. guineense leaves per oral. For subacute toxicity study, the rats were randomly divided into three groups (n=6). The control group received distilled water, while the experimental groups received 500 mg/kg and 1500 mg/kg 80% methanol extract of S. guineense leaves orally for 28 days. At the end of the experiment, blood samples were collected for hematology and clinical chemistry evaluations. Gross pathology and histopathology of liver and kidneys were assessed. In the acute toxicity study, rats treated with 2000 mg/kg and 5000 mg/kg showed no toxicological signs observed on behavior, gross pathology, and body weight of rats. In the subacute toxicity study rats have showed no significant changes on behavior, gross pathology, body weight, and hematological and biochemical parameters, whereas both experimental groups had a lower blood glucose level compared with the control group (p < 0.05). There were no significant differences in the gross and histopathology of the liver and kidneys of experimental animals in extract exposed groups and their counterpart control. The 80% methanol extract of S. guineense does not produce adverse effects in rats after acute and subacute treatment. Before marketing a S. guineense leaf based remedy, subchronic and chronic toxicity evaluations need to be done.

Investigating Potential Modes of Actions of Mimusops kummel Fruit Extract and Solvent Fractions for Their Antidiarrheal Activities in Mice.

BACKGROUND: Fruits of Mimusops kummel A. DC. (Sapotaceae) are traditionally used for the treatment of diarrhea. The present study aimed at investigating modes of actions of this fruits for antidiarrheal action to guide future drug development process. METHODS: Fractions of chloroform, n-butanol, and water were obtained from 80% methanol extract, which was prepared by maceration. Antidiarrheal activities and the modes of actions were investigated in mice. RESULTS: In castor oil induced diarrheal model, the extract delayed onset of diarrhea and reduced number and weight of feces at all tested doses significantly. In this model all fractions significantly delayed onset of diarrhea at all tested doses. Charcoal meal test showed that the extract and all the fractions produced a significant antimotility effect at all tested doses. Enteropooling test showed that the extract as well as n-butanol and aqueous fractions at all tested doses produced a significant decline in volume and weight of intestinal contents, whereas chloroform fraction had substantial effect only at high dose. CONCLUSION: This study demonstrated that the extract and solvent fractions produced antidiarrheal activities due to dual inhibitory effect, intestinal motility, and fluid secretion, with the aqueous fraction being the most active among fractions in three models.

Tafenoquine and its potential in the treatment and relapse prevention of Plasmodium vivax malaria: the evidence to date.

Despite declining global malaria incidence, the disease continues to be a threat to people living in endemic regions. In 2015, an estimated 214 million new malaria cases and 438,000 deaths due to malaria were recorded. Plasmodium vivax is the second most common cause of malaria next to Plasmodium falciparum. Vivax malaria is prevalent especially in Southeast Asia and the Horn of Africa, with enormous challenges in controlling the disease. Some of the challenges faced by vivax malaria-endemic countries include limited access to effective drugs treating liver stages of the parasite (schizonts and hypnozoites), emergence/spread of drug resistance, and misperception of vivax malaria as nonlethal. Primaquine, the only 8-aminoquinoline derivative approved by the US Food and Drug Administration, is intended to clear intrahepatic hypnozoites of P. vivax (radical cure). However, poor adherence to a prolonged treatment course, drug-induced hemolysis in patients with glucose-6-phosphate dehydrogenase deficiency, and the emergence of resistance make it imperative to look for alternative drugs. Therefore, this review focuses on data accrued to date on tafenoquine and gives insight on the potential role of the drug in preventing relapse and radical cure of patients with vivax malaria.

The Effect of Early Initiation of Antiretroviral Therapy in TB/HIV-Coinfected Patients: A Systematic Review and Meta-Analysis.

BACKGROUND: The importance of early initiation of antiretroviral therapy (ART) for tuberculosis (TB) and HIV-coinfected patients is controversial. We conducted a systematic review and meta-analysis to assess the effect of early initiation of ART (within 2-4 weeks of TB treatment) on several treatment outcomes among TB/HIV-coinfected patients. METHOD: A systematic search of clinical trials was performed in PubMed, Embase, Google Scholar, Science Direct, Medscape, and the Cochrane library. Clinical trials which were published in any language before the last date of search (March 31, 2015) were included. The qualities of the studies were assessed using criteria from the Cochrane Library. Heterogeneity test was conducted to assess the variations among study outcomes. For each study outcome, the risk ratio (RR) with 95% confidence interval (CI) was calculated as a measure of intervention effect. The Mantel-Haenszel method was used to estimate the RR using a fixed-effects model. FINDINGS: A total of 2272 study participants from 6 trials were included in the meta-analysis. Early ART initiation during TB treatment was associated with reduced all-cause mortality (RR = 0.78; 95% CI = 0.63-0.98) and increased rate of TB-associated immune reconstitution inflammatory syndrome (TB-IRIS; RR = 2.19; 95% CI = 1.77- 2.70) and death related to TB-IRIS (RR = 6.94; 95% CI = 1.26-38.22). However, the time of ART initiation has no association with TB cure rate (RR = 0.99; 95% CI = 0.81-1.07), rate of drug toxicity (RR = 1.00; 95% CI = 0.93-1.08), death associated with drug toxicity (RR = 0.40; 95% CI = 0.14- 1.16), rate of low viral load (less than 400 copies/mL; RR = 1.00; 95% CI = 0.96-1.04), and rate of new AIDS-defining illness (RR = 0.84; 95% CI = 0.60-1.18). Immunological response in early ART arms of study participant in different trials showed a greater or equal response compared with late ART arms. CONCLUSION: This systematic review presents conclusive evidence on the reduction of all-cause mortality as a result of early initiation of ART. However, this study also confirms the high rate of TB-IRIS and death associated with it. Operational and implementation research are required to maintain the benefit of early ART initiation and proper management of TB-IRIS. Studies on the timing of ART in extrapulmonary and multidrug-resistant TB are recommended.

Impact of repeated NeemAzal-treated blood meals on the fitness of Anopheles stephensi mosquitoes.

BACKGROUND: Herbal remedies are widely used in many malaria endemic countries to treat patients, in particular in the absence of anti-malarial drugs and in some settings to prevent the disease. Herbal medicines may be specifically designed for prophylaxis and/or for blocking malaria transmission to benefit both, the individual consumer and the community at large. Neem represents a good candidate for this purpose due to its inhibitory effects on the parasite stages that cause the clinical manifestations of malaria and on those responsible for infection in the vector. Furthermore, neem secondary metabolites have been shown to interfere with various physiological processes in insect vectors. This study was undertaken to assess the impact of the standardised neem extract NeemAzal on the fitness of the malaria vector Anopheles stephensi following repeated exposure to the product through consecutive blood meals on treated mice. METHODS: Batches of An. stephensi mosquitoes were offered 5 consecutive blood meals on female BALB/c mice treated with NeemAzal at an azadirachtin A concentration of 60, 105 or 150 mg/kg. The blood feeding capacity was estimated by measuring the haematin content of the rectal fluid excreted by the mosquitoes during feeding. The number of eggs laid was estimated by image analysis and their hatchability assessed by direct observations. RESULTS: A dose and frequency dependent impact of NeemAzal treatment on the mosquito feeding capacity, oviposition and egg hatchability was demonstrated. In the 150 mg/kg treatment group, the mosquito feeding capacity was reduced by 50% already at the second blood meal and by 50 to 80% in all treatment groups at the fifth blood meal. Consequently, a 50 - 65% reduction in the number of eggs laid per female mosquito was observed after the fifth blood meal in all treatment groups. Similarly, after the fifth treated blood meal exposure, hatchability was found to be reduced by 62% and 70% in the 105 and 150 mg/kg group respectively. CONCLUSIONS: The findings of this study, taken together with the accumulated knowledge on neem open the challenging prospects of designing neem-based formulations as multi-target phytomedicines exhibiting preventive, parasite transmission-blocking as well as anti-vectorial properties.

Plasmodium falciparum and Schistosoma mansoni coinfection and the side benefit of artemether-lumefantrine in malaria patients.

INTRODUCTION: The distribution of both malaria and schistosomiasis exhibits a large geographical overlap in tropical environments, particularly in sub-Saharan Africa. This part of the world currently harbours more than 85% of the estimated global burden of these diseases. Studies showed that artemisinin derivatives used for the treatment of malaria also have an antischistosomal effect. This study aimed to investigate the extent of malaria-schistosomiasis co-infection and the antischistosomal effect of artemether-lumefantrine when administered to treat falciparum malaria in Kemise, Northeast Ethiopia. METHODOLOGY: Stool samples were collected from 152 microscopically confirmed malaria patients and diagnosed for schistosomiasis using the Kato-Katz technique before treatment. The schistosomiasis cure rate and egg reduction were determined in co-infected patients, who were treated with artemether-lumefantrine,. RESULTS: Twenty-eight out of 152 malaria patients were co-infected (18.4%, n = 152) with schistosomiasis. All 28 co-infected patients were found stool-negative for Schistosoma mansoni eggs four weeks after treatment. The extent of co-infection was associated with age, sex and educational level. Cure rate and egg reduction rate following the treatment of artemether-lumefantrine were 100%. CONCLUSION: The co-infection rate was associated with patient characteristics. Artemether-lumefantrine was effective against S. mansoni in co-infected patient. Multicenter and randomized trials, however, are needed for a better understanding of the efficacy of artemether-lumefantrine against schistosome infection with ranges of intensity.