Antiprotozoal Activity Against Plasmodium falciparum. and Trypanosoma cruzi. of Aeroplysinin-1 Isolated from the New Sponge Aplysina chiriquensis.

ABSTRACT

Crude methanol extracts from the recently discovered sponge, Aplysina chiriquensis. and Aplysina gerardogreeni., collected at the southwest Pacific Coast of Panama, showed moderate antiplasmodial activity. Fractionation of the crude extracts from A.. chiriquensis. led to the isolation of the known dibromotyrosine derivatives 1–5. Compound 3, aeroplysinin-1, displayed antiprotozoal activity against chloroquine-resistant Plasmodium falciparum. and Trypanosoma cruzi.. Comparative analysis by HPLC of the five specimens collected showed the presence of the same metabolites but in different relative proportions. Compounds 1–5 are reported for the first time as constituents of A.. chiriquensis. and A.. gerardogreeni.. This is the first report of the antiprotozoal activity of aeroplysinin-1 (3).     

Activity of Senna villosa against Trypanosoma cruzi

Methanol, chloroform and aqueous extracts from Senna villosa were tested in vitro against epimastigote and trypomastigote forms of Trypanosoma cruzi. Methanol and chloroform extracts were found to possess significant activity against both forms of the parasite, while chloroform extract at doses of 1.65, 3.3, and 6.6 μg/mL demonstrated activity similar to gentian violet and allopurinol.     

Disposition of Nifurtimox and Metabolite Activity Against Trypanosoma cruzi using Rat Isolated Perfused Liver

Abstract— Nifurtimox disposition was investigated using the rat isolated perfused-liver method after administration of 25 μg mL^?1 nifurtimox, and its disappearance was monitored by analysing the perfusate sample at various times. Biliary excretion was also measured. The drug concentration profile underwent a biexponential decline over the 2-h study period, with a terminal half-life of 62·76 ± 17·56 min. Nifurtimox is a high clearance compound (15·23±5·53 mL min^?1). The extraction ratio was 0·621 ±0·159. Biliary excretion accounted for 0·05% of the dose, the remainder consisting of highly polar metabolites. By 2 h, a minimal fraction of unchanged nifurtimox was recovered from the perfusate. Nifurtimox activity against Trypanosoma cruzi (clone CA-1) during the perfusion was also determined. Epimastigotes isolated from continuous culture were exposed to the samples of perfusate at different perfusion times in a microtitre plate. After an incubation time of 72 h at 27°C, the parasite number in each well was counted under a microscope. From 0 to 75 min after the perfusion, the anti-trypanosomal activity decreased, but an increase in activity was observed at the later times. These findings show that active metabolites are formed during the perfusion.     

氢溴酸2，4—二氯—6—苯基苯氧乙基二乙基胺（Lilly 18947）抗克氏锥虫的作用

AIM: To study the effect of the inhibitor of cytochrome P450 known as Lilly 18947 (2,4 dichloro-6 phenyl-phenoxy ethyl diethylamine) on Trypanosoma cruzi. METHODS: Trypanosoma cruzi epimastigotes wen grown in culture, in absence or in presence of drug. The inhibition of its growth was followed by daily counting using a Neubauer chamber. The effect of Lilly 18947 on the parasite ultrastructure was examined by electron microscopy. To test the effect of different concentrations of drug on the parasite cycle, Vero cells were inoculated with trypomastigotes ( RA strain ) and after 72 h the percentage of infected cells and the number of intracellular parasites were estimated and expressed as the endocytic index. RESULTS: Growth of epimastigotes was inhibited by Lilly 18947. Concentrations as low as 50 μmol/L resulted in a complete disappearance of the parasites in culture by the fourth day. With lower concentrations, little growth was observed and total (25 μmol/L) or partial lysis (10μmol/L) were register     

Detection and treatment of Trypanosoma cruzi: a patent review (2011-2015)

Introduction: Trypanosoma cruzi is the etiologic agent of American trypanosomiasis (Chagas disease), which is one of the important parasitic diseases worldwide. The number of infected people with T. cruzi diminished from 18 million in 1991 to 6 million in 2010, but it is still the most prevalent parasitic disease in the Americas. The existing chemotherapy is still deficient and based on two drugs: nifurtimox and benznidazole, which are not FDA-approved in the United States.

Areas covered: This review covers the current and future directions of Chagas disease chemotherapy based on drugs that interfere with relevant metabolic pathways. This article also illustrates the challenges of diagnosis, which in recent infections, is only detected when the parasitemia is high (direct detection); whereas, in the chronic phase is reached after multiple serological tests.

Expert opinion: The current chemotherapy is associated with long term treatments and severe side effects. Nifurtimox and benznidazole are able to cure at least 50% of recent infections. Nevertheless, they suffer from major drawbacks: selective drug sensitivity on different T. cruzi strains and serious side effects. The aim of this review is focused on presenting an up-to-date status of the chemotherapy and diagnosis.     

Structure-based design,optimization of lead,synthesis, and biological evaluation of compounds active against Trypanosoma cruzi

Chagas' disease affects approximately eight million people throughout the world, especially the poorest individuals. The protozoan that causes this disease–Trypanosoma cruzi–has the enzyme cruzipain, which is the main therapeutic target. As no available medications have satisfactory effectiveness and safety, it is of fundamental importance to design and synthesize novel analogues that are more active and selective. In the present study, molecular docking and the in silico prediction of ADMET properties were used as strategies to optimize the trypanocidal activity of the pyrimidine compound ZN3F based on interactions with the target site in cruzipain. From the computational results, eight 4-amino-5-carbonitrile-pyrimidine analogues were proposed, synthesized ( 5a-f and 7g-h) and, tested in vitro on the trypomastigote form of the Tulahuen strain of T. cruzi. The in silico study showed that the designed analogues bond favorably to important amino acid residues of the active site in cruzipain. An in vitro evaluation of cytotoxicity was performed on L929 mammal cell lines. All derivatives inhibited the Tulahuen strain of T. cruzi and also exhibited lower toxicity to L929 cells. The 5e product, in particular, proved to be a potent, selective (IC₅₀ = 2.79 ± 0.00 μM, selectivity index = 31.3) inhibitor of T. cruzi. The present results indicated the effectiveness of drugs based on the structure of the receptor, revealing the potential trypanocidal of pyrimidines. This study also provides information on molecular aspects for the inhibition of cruzipain.     

Antifolate Agents Against Wild and Mutant Strains of Plasmodium falciparum

Plasmodium falciparum dihydrofolate reductase is an important target for antimalarial chemotherapy. The emergence of resistance has significantly reduced the efficacy of the classic antifolate drugs cycloguanil and pyrimethamine. In this paper we report new dihydrofolate reductase inhibitors identified using molecular modelling principles with the goal of designing new antifolate agents active against both wild and tetramutant dihydrofolate reductase strains three series of trimethoprim analogues were designed, synthesised and tested for biological activity. Pyrimethamine and cycloguanil have been reported to loose efficacy because of steric repulsion in the active site pocket produced due to mutation in Plasmodium falciparum dihydrofolate reductase. The synthesised molecules have sufficient flexibility to withstand this steric repulsion to counteract the resistance. The molecules have been synthesised by conventional techniques and fully characterised by spectroscopic methods. The potency of these molecules was evaluated by in vitro enzyme specific assays. Some of the molecules were active in micromolar concentrations and can easily be optimised to improve binding and activity.     

Antiprotozoal activity of extracts and isolated triterpenoids of ‘carnauba’ (Copernicia prunifera) wax from Brazil

Context: ‘Carnauba’ wax is a natural product obtained from the processing of the powder exuded from Copernicia prunifera (Miller) H. E. Moore (Arecaceae). This material is widely used in the Brazilian folk medicine, including the treatment of rheumatism and syphilis.

Objective: To investigate the antiprotozoal activity of hexane and EtOH extracts from the ‘carnauba’ wax as well as from the isolated compounds from the bioactive extracts.

Material and methods: Two different samples of ‘carnauba’ (C. prunifera) waxes – types 1 and 4 – were individually extracted using hexane (EH) and EtOH (EE). Aliquots of hexane (type 1 – EH-1 and EH-4) and EtOH (type 4 – EE-1 and EE-4) extracts were tested against promastigote (2–200?μg/mL in DMSO during 48?h at 24?°C) and amastigote (3–150?μg/mL in DMSO during 120?h at 37?°C) forms of Leishmania infantum as well as against trypomastigote (3–150?μg/mL in DMSO during 24?h at 37?°C) forms of Trypanosoma cruzi. Bioactive extracts EH-1 and EE-4 were subjected to a bioactivity-guided fractionation to afford three dammarane-type triterpenoids (1–3). The in vitro antiprotozoal activities of the obtained compounds were evaluated as described above. Additionally, the cytotoxicity activity of compounds 1–3 against mammalian conjunctive cells (NCTC – 2–200?μg/mL in DMSO during 48?h at 37?°C) was determined.

Results: From the bioactive hexane and EtOH extracts from the ‘carnauba’ (C. prunifera) wax, were isolated three dammarane-type triterpenoids: (24R*)-methyldammar-25-ene-3β,20-diol (carnaubadiol, 1), (24R*)-methyldammara-20,25-dien-3-one (2) and (24R*)-methyldammara-20,25-dien-3α-ol (3). These compounds were identified based on the analysis of NMR and MS spectroscopic data. Compounds 1–3 were effective against the intracellular amastigotes of L. infantum, with IC₅₀ values ranging from 8 to 52?μM, while compounds 1 and 3 displayed activity against trypomastigote forms of T. cruzi with IC₅₀ values of 15 and 35?μM, respectively. The mammalian cytotoxicity assay demonstrated no damage to NCTC conjunctive cells up to 200?μM, except for compound 1, which demonstrated a CC₅₀ value of 34?μM.

Conclusion: Based on the results, it was possible to conclude that the detected antiprotozoal bioactivity of ‘carnauba’ (C. prunifera) wax extracts could be related to the presence of the natural dammarane triterpenoid derivatives. The results suggested that these compounds could be used as promising scaffolds for drug design studies for leishmaniasis and Chagas disease.     

Activity against Plasmodium falciparum of Lactones Isolated from the Endophytic Fungus Xylaria sp.

Three lactones were isolated from the culture medium of the endophytic fungus Xylaria sp. Grev. (Xylariaceae). The major compound, which showed weak activity (13 μ g/mL) against a chloroquine-resistant strain of Plasmodium falciparum, was identified as (+)-phomalactone (1). The others were 6-(1-propenyl)-3,4,5,6-tetrahydro-5-hydroxy-4H-pyran-2-one (2) and 5-hydroxymellein (3). Compounds 1 and 2 are reported for the first time as constituents of Xylaria. Also, this is the first report of the activity of the compounds 1–3 against a chloroquine-resistant Plasmodium falciparum strain.     

Isolation and characterization of microcystins from a river nile strain of Oscillatoria tenuis Agardh ex Gomont.

The River Nile is the major source of drinking water in Egypt, however, increased eutrophication due to agricultural, municipal and industrial runoff has contributed to the growth of toxin producing cyanobacteria. This study describes the isolation and characterization of microcystins (MCYSTs), cyclic heptapeptide hepatotoxins, from a rare strain of Oscillatoria tenuis, isolated from the River Nile at Sohag province in July 1995. The MCYST concentration of laboratory-cultured O. tenuis strain E6 was found to be 0.3 mg/g freeze-dried weight determined by enzyme-linked immunosorbent assay (ELISA). Two microcystins, 1 and 2, were isolated from lyophilized cells using solid phase extraction and reversed-phase high performance liquid chromatography (HPLC). Structures were assigned based upon their amino acid analyses, electrospray ionization mass spectrometry (ESIMS, ESIMS-CID-MS), high resolution fast atom bombardment mass spectrometry, and nuclear magnetic resonance data (¹H and ¹H COSY NMR). Toxin 1 was identified as MCYST-LR, and toxin 2, a new MCYST, as MCYST-LHArg ([ -homoarginine⁴]). Previous studies indicate that Oscillatoria agardhii strains produce demethylated MCYSTs (containing -Asp and/or dehydroalanine). This is the first report of a toxic O. tenuis, strain E6, one which produces a fully methylated MCYST, MCYST-LR and a new -homoarginine containing MCYST, MCYST-LHArg.     

Hepatoprotective Activity of Xanthones and Xanthonolignoids Against tert-Butylhydroperoxide-Induced Toxicity in Isolated Rat Hepatocytes—Comparison with Silybin

Purpose. Synthesize and evaluate the protective activity against tert-butylhydroperoxide-induced toxicity in freshly isolated rat hepatocytes of trans-kielcorin, trans-isokielcorin B, as well as their respective building blocks 3,4-dihydroxy-2-methoxyxanthone and 2,3-dihydroxy-4-methoxyxanthone. Methods. Wistar rats, weighing 200-250g were used. Hepatocyte isolation was performed by collagenase perfusion. Incubations were performed at 37°C, using 1 million cells per milliliter in modified Krebs—Henseleit buffer. The protective activity was evaluated by measuring reduced and oxidized glutathione, lipid peroxidation and cell viability after inducing toxicity with tert-butylhydroperoxide (1.0 mM, 30 min), with or without the studied compounds in the concentrations of 0.025, 0.050, 0.100 and 0.200 mM. Silybin was tested in the same experimental conditions to serve as a positive control. Results. Using these concentrations, the tested compounds prevented tert-butylhydroperoxide-induced lipid peroxidation and cell death in freshly isolated rat hepatocytes. All compounds were also effective in preventing perturbation of cell glutathione homeostasis in some extent. 3,4-Dihydroxy-2-methoxyxanthone and 2,3-dihydroxy-4-methoxyxanthone were more effective than trans-kielcorin and trans-isokielcorin B respectively. Silybin was less effective in protecting cells against lipid peroxidation and loss of cell viability than the four xanthonic derivatives. Conclusions. The tested compounds protected the freshly isolated rat hepatocytes against tert-butylhydroperoxide-induced toxicity.     

Ultrastructural alterations in Trypanosoma (Schizotrypanum) cruzi induced by Delta(24(25)) sterol methyl transferase inhibitors and their combinations with ketoconazole

We report the ultrastructural alterations induced on the proliferative stages of Trypanosoma cruzi, the causative agent of Chagas' disease, by two Δ²⁴⁽²⁵⁾ sterol methyl transferase (24(25)-SMT) inhibitors, 22,26-azasterol and 24(R,S),25-epiminolanosterol. Both compounds are sterol biosynthesis inhibitors which had previously been shown to be potent growth inhibitors and whose effects are potentiated by the C14a demethylase inhibitor, ketoconazole. Epimastigotes treated with the minimal growth inhibitory concentration of 22,26-azasterol (10 μM) for 144 h, which were completely depleted of endogenous 4-desmethyl sterols and accumulated 24-desalkyl sterols, showed the appearance of electron-dense granules, mitochondrial swelling and intense vacuolization. At high concentration (≥ 30 μM) the sterol analog induced gross alterations in the organization of chromatin and rapid cell lysis. The treatment of epimastigotes with 24-(R,S),25-epiminolanosterol induced, at low concentrations, (1 μM) alterations similar to those observed with 22,26-azasterol but additionally, modifications of the kinetoplast were observed. Higher concentrations (≥ 3 μM) induced total lysis. The combination of both sterol analogs with ketoconazole, at sub-optimal concentrations, induced the same alterations as 22,26-azasterol 10 μM or epiminolanosterol 1 μM. The results confirm the conclusions of previous studies which indicated that one important cytotoxic effects of sterol biosynthesis inhibitors in this organism is the alteration of the parasite's mitochondrial system.     

In vitro screening of traditional South African malaria remedies against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum

Three hundred extracts were prepared from plants traditionally used in South Africa to treat malaria and screened in vitro for activity against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum. For the 43 extracts which inhibited the growth of one or more parasites to more than 95?% at 9.7?μg/mL, the IC?? values against all four protozoal parasites and cytotoxic IC??s against rat myoblast L6 cells were determined. Amongst the most notable results are the activities of AGATHOSMA APICULATA (IC?? of 0.3?μg/mL) against Plasmodium falciparum, as well as Salvia repens and Maytenus undata against Leishmania donovani with IC??s of 5.4?μg/mL and 5.6?μg/mL, respectively. This screening is the starting point for a HPLC-based activity profiling project in antiprotozoal lead discovery.     

Trypanocidal and leishmanicidal activities of flavonoids isolated from Stevia satureiifolia var. satureiifolia

Context Chagas’ disease and leishmaniasis produce significant disability and mortality with great social and economic impact. The genus Stevia (Asteraceae) is a potential source of antiprotozoal compounds.

Objective Aerial parts of four Stevia species were screened on Trypanosoma cruzi. Stevia satureiifolia (Lam.) Sch. Bip. var. satureiifolia (Asteraceae) dichloromethane extract was selected for a bioassay-guided fractionation in order to isolate its active compounds. Additionally, the antileishmanial activity and the cytotoxicity of these compounds on mammalian cells were assessed.

Materials and methods The dichloromethane extract was fractionated by column chromatography. The isolated compounds were evaluated using concentrations of 0–100?μg/mL on T. cruzi epimastigotes and on Leishmania braziliensis promastigotes for 72?h, on trypomastigotes and amastigotes of T. cruzi for 24?h and 120?h, respectively. The compounds’ cytotoxicity (12.5–500?μg/mL) was assessed on Vero cells by the MTT assay. The structure elucidation of each compound was performed by spectroscopic methods and HPLC analysis.

Results The dichloromethane extracts of Stevia species showed significant activity on T. cruzi epimastigotes. The flavonoids eupatorin (1.3%), cirsimaritin (1.9%) and 5-desmethylsinensetin (1.5%) were isolated from S. satureiifolia var. satureiifolia extract. Eupatorin and 5-desmethylsinensetin showed IC₅₀ values of 0.2 and 0.4?μg/mL on T. cruzi epimastigotes and 61.8 and 75.1?μg/mL on trypomastigotes, respectively. The flavonoid 5-desmethylsinensetin showed moderate activity against T. cruzi amastigotes (IC_50? value?=_?78.7?μg/mL) and was the most active compound on L. braziliensis promastigotes (IC_50? value?=_?37.0?μg/mL). Neither of the flavonoids showed cytotoxicity on Vero cells, up to a concentration of 500?μg/mL.     

Synthetic,immunological and structural studies on repeat unit peptides of Plasmodium falciparum antigens

Using solid phase methodology, we have synthesized five peptides (16-18 residues long) corresponding to repeat sequences of four antigens of a human malarial parasite, Plasmodium falciparum. Three of these antigens (RESA, FIRA, and ABRA) are found in the asexual blood-stages of the parasite, while the remaining one (CSP) is found in the sporozoites. The synthetic peptides, conjugated to bovine serum albumin, elicited high levels of antibodies in rabbits, and these antibodies were found to cross-react with the heterologous peptides. The degree of cross-reactivity, as estimated in an ELISA, was quite remarkable among all the peptides. The peptide corresponding to the RESA tetrapeptide repeat was found to be the most immunogenic and highly cross-reactive. For this reason this tetrapeptide repeat unit, peptide 1, may be a suitable candidate for inclusion in a multiple epitope polypeptide vaccine design. Conformational studies using circular dichroism spectroscopy show that these peptides have similar conformational characteristics with a common feature OF ～30% and ～50% helical content in water and TFE respectively. Theoretical predictions regarding conformation using the Chou-Fasman method have also been presented.     

o-Nitroanilines as major metabolic products of anti-Trypanosoma cruzi 5-phenylethenylbenzofuroxans in microsomal and cytosolic fractions of rat hepatocytes and in whole parasitic cells

1. The metabolism of six anti-Trypanosoma cruzi 5-phenylethenylbenzofuroxans (PhEBfx) was studied in vitro using rat hepatic microsomal and cytosolic fractions as a mammalian model and whole cells of T. cruzi as a parasitic model.

2. Some of the expected metabolites were synthesized to provide authentic chromatographic standards.

3. The metabolites were identified using high-performance liquid chromatography (HPLC) in comparison with the authentic standards and their proportions were determined. Their structures were confirmed using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy.

4. The behaviour of the six PhEBfx in the three different systems was similar. The main metabolites, formed by reductive processes, were the corresponding o-nitroanilines.

5. Two of the test compounds were studied for extended time periods in the rat liver preparations and their terminal metabolites were identified as o-phenylendiamine derivatives.

     

Parasitological cure of acute and chronic experimental Chagas disease using the long-acting experimental triazole TAK-187. Activity against drug-resistant Trypanosoma cruzi strains

We investigated the activity of TAK-187, an experimental antifungal triazole with a long terminal half-life in several experimental animals, against Trypanosoma cruzi. In vitro studies showed that the minimal inhibitory concentration (MIC) against the (extracellular) epimastigote form was 0.3-1 microM, while the corresponding concentration against clinically relevant intracellular amastigotes was 1 nM. At the MIC the endogenous epimastigote C4,14-desmethyl sterols were replaced by di- and tri-methylated sterols, supporting the notion that the primary target of TAK-187 is the parasite's sterol C14alpha demethylase. We investigated the in vivo activity of the compound in a murine model of acute Chagas disease, using T. cruzi strains with different susceptibilities to the drugs currently used clinically (nitrofurans and nitroimidazoles). It was found that TAK-187 given orally at 20 mg/kg induced complete protection against death and high levels (60-100%) of parasitological cures, independently of the infecting strain and even when administered every other day (e.o.d.), consistent with its long terminal half-life in mice. Other experiments, using longer treatment periods were carried out in both acute and chronic models of the disease and showed that TAK-187 given at 10-20 mg/kg e.o.d. induced 80-100% survival with 80-100% of parasitological cures of survivors in both models. No toxic side effects were observed in any of the experimental protocols. TAK-187 is a potent anti-T. cruzi compound with trypanocidal activity in vivo and should be considered for further studies as a potential specific treatment of human Chagas disease.     

Spermine isolated and identified as the major trypanocidal compound from the snake venom of Eristocophis macmahoni causes autophagy in Trypanosoma brucei.

The snake venom from the leaf-nosed viper Eristocophis macmahoni was analyzed regarding its toxic effects on the bloodstream form of Trypanosoma brucei. A considerable trypanocidal effect was measured with an IC5 value of 186 ng/ml in bloodstream form parasites. Following several high performance liquid chromatography (HPLC) separation steps, the major trypanocidal activity was assigned to a single fraction by in vitro toxicity assays. Analysis by off-line ESI-MS(n) revealed an m/z value of 202.2 for the precursor ion and fragment ions of m/z=129.1 (MS2) and 112.1 (MS3), respectively, clearly corresponding to the molecular mass and the fragmentation pattern of the polyamine spermine. Quantification of spermine within the viper venom using an on-line hydrophilic interaction chromatography (HILIC) ESI-MS method revealed that this compound constituted approximately 1% of the dry venom mass. The polyamine oxidase activity in the fetal calf serum used for cultivation was responsible for a trypanocidal effect of pure spermine in the low micromolar range, whereas the antitrypanosomal activity of crude snake venom was virtually independent from serum, suggesting the oxidation of spermine by intrinsic venom components. Using fetal calf serum, spermine was shown to induce autophagy in the parasites using transmission electron microscopy (TEM).     

4‐Aminoquinoline‐Triazine‐Based Hybrids with Improved In Vitro Antimalarial Activity Against CQ‐Sensitive and CQ‐Resistant Strains of Plasmodium falciparum

A systematic chemical modification in the triazine moiety covalently attached via suitable linkers to 4‐amino‐7‐chloroquinolines yielded a series of new 7‐chloro‐4‐aminoquinoline‐triazine hybrids exhibiting high in vitro activity against W2 (chloroquine‐resistant) and D6 (chloroquine‐sensitive) strains of Plasmodium falciparum without any toxicity against mammalian cell lines (Vero, LLC‐PK11, HepG2). Many of the compounds ( 6, 8, 10, 11, 13, 14, 16, 27, 29 and 33 ) showed excellent potency against chloroquine sensitive and resistant strains. In particular, compounds 6, 8 , 14 , 16 and 29 were found to be significantly more active than chloroquine against the chloroquine‐resistant strains (W2 clone) of P. falciparum.