Synthesis of metronidazole based thiazolidinone analogs as promising antiamoebic agents.

Metronidazole and its derivatives are widely used for the treatment of amoebiasis. However, metronidazole is considered as the standard drug but it has many side effects. The present study describes the synthesis of a series of metronidazole based thiazolidinone analogs via Knoevenagel condensation of 4-[2-(2-methyl-5-nitro-1H-imidazole-1-yl)ethoxy]benzaldehyde 1 with various thiazolidinone derivatives 2-14 to get the new scaffold (15-27) having better activity and lesser toxicity. Six compounds have shown better efficacy and lesser cytotoxicity than the standard drug metronidazole towards HM1: IMSS strain of Entamoeba histolytica. These compounds may combat the problem of drug resistance and might be effective in identifying potential alternatives for future drug discovery against EhOASS.

Antiamoebic activity of synthetic tetrazoles against Acanthamoeba castellanii belonging to T4 genotype and effects of conjugation with silver nanoparticles.

Acanthamoeba causes diseases such as Acanthamoeba keratitis (AK) which leads to permanent blindness and granulomatous Acanthamoeba encephalitis (GAE) where there is formation of granulomas in the brain. Current treatments such as chlorhexidine, diamidines, and azoles either exhibit undesirable side effects or require immediate and prolonged treatment for the drug to be effective or prevent relapse. Previously, antifungal drugs amphotericin B, nystatin, and fluconazole-conjugated silver with nanoparticles have shown significantly increased activity against Acanthamoeba castellanii. In this study, two functionally diverse tetrazoles were synthesized, namely 5-(3-4-dimethoxyphenyl)-1H-tetrazole and 1-(3-methoxyphenyl)-5-phenoxy-1H-tetrazole, denoted by T1 and T2 respectively. These compounds were evaluated for anti-Acanthamoeba effects at different concentrations ranging from 5 to 50 µM. Furthermore, these compounds were conjugated with silver nanoparticles (AgNPs) to enhance their efficacy. Particle size analysis showed that T1-AgNPs and T2-AgNPs had an average size of 52 and 70 nm respectively. After the successful synthesis and characterization of tetrazoles and tetrazole-conjugated AgNPs, they were subjected to anti-Acanthamoeba studies. Amoebicidal assay showed that at concentration 10 µM and above, T2 showed promising antiamoebic activities between the two compounds while encystation and excystation assays reveal that both T1 and T2 have inhibited differentiation activity against Acanthamoeba castellanii. Conjugation of T1 and T2 to AgNP also increased efficacy of tetrazoles as anti-Acanthamoeba agents. This may be due to the increased bioavailability as AgNP allows better delivery of treatment compounds to A. castellanii. Human cell cytotoxicity assay revealed that tetrazoles and AgNPs are significantly less toxic towards human cells compared with chlorhexidine which is known to cause undesirable side effects. Cytopathogenicity assay also revealed that T2 conjugated with AgNPs significantly reduced cytopathogenicity of A. castellanii compared with T2 alone, suggesting that T2-conjugated AgNP is an effective and safe anti-Acanthamoeba agent. The use of a synthetic azole compound conjugated with AgNPs can be an alternative strategy for drug development against A. castellanii. However, mechanistic and in vivo studies are needed to explore further translational values.

Chloroquine as a possible disinfection adjunct of disinfection solutions against Acanthamoeba.

Acanthamoeba keratitis is commonly encountered by contact lens wearers. Contact lens solution plays an important role in the safe use of contact lenses. The most popular products for disinfecting lenses are multipurpose disinfecting solutions (MPDS). However, almost all MPDS retailed in Korea are ineffective in killing Acanthamoeba. The objective of this study was to determine the possibility of using autophagy inhibitor chloroquine as a disinfecting agent to improve the amoebicidal activity of MPDS against Acanthamoeba, especially the cyst. Amoebicidal effects of eight different MPDSs combined with chloroquine (CQ), an autophagy inhibitor, and their cytotoxicities to human corneal epithelium cells were determined. Almost all MPDS showed strong amoebicidal effect on trophozoites after 8 h of exposure. However, they showed inadequate amoebicidal effect on cysts even after 24 h of exposure. MPDSs combined with 100 µM CQ increased their amoebicidal effects on immature cyst by inhibiting formation of mature cysts. Incubation with 100 µM CQ for 30 min did not have cytotoxicity to human corneal epithelial cells.

Polyhexamethylene biguanide and chloroquine induce programmed cell death in Acanthamoeba castellanii.

Several chemotherapeutic drugs have been described as amoebicidal agents acting against Acanthamoeba trophozoites and cysts. However, the underlying mechanism of action is poorly characterized. Here, we describe programmed cell death (PCD) in A. castellanii induced by polyhexamethylene biguanide (PHMB) and chloroquine. We used four types of amoebicidal agents including 0.02% PHMB, 0.02% chlorhexidine digluconate, 100 µM chloroquine, and 100 µM 2,6-dichlorobenzonitrile to kill Acanthamoeba trophozoites and cysts. Exposure to PHMB and chloroquine induced cell shrinkage and membrane blebbing in Acanthamoeba, observed microscopically. Externalization of phosphatidyl serine on the membranes of Acanthamoeba was detected by annexin V staining. Apoptotic cell death of Acanthamoeba by PHMB and chloroquine was confirmed by FACS analysis. Nuclear fragmentation of Acanthamoeba was demonstrated by DAPI staining. PHMB induced PCD in trophozoites and cysts, and chloroquine induced PCD in cysts. These findings are discussed to establish the most effective treatment for Acanthamoeba-induced keratitis.

In vitro activity of 1H-phenalen-1-one derivatives against Acanthamoeba castellanii Neff and their mechanisms of cell death.

Acanthamoeba is an opportunistic pathogen which is the causal agent of a sight-threatening ulceration of the cornea known as Acanthamoeba keratitis (AK) and, more rarely, an infection of the central nervous system called "granulomatous amoebic encephalitis" (GAE). The symptoms of AK are non-specific, and so it can be misdiagnosed as a viral, bacterial, or fungal keratitis. Furthermore, current therapeutic measures against AK are arduous, and show limited efficacy against the cyst stage of Acanthamoeba. 1H-Phenalen-1-one (PH) containing compounds have been isolated from plants and fungi, where they play a crucial role in the defense mechanism of plants. Natural as well as synthetic PHs exhibit a diverse range of biological activities against fungi, protozoan parasites or human cancer cells. New synthetic PHs have been tested in this study and they show a potential activity against this protozoa.

Reduction in maternal Polycomb levels contributes to transgenerational inheritance of a response to toxic stress in flies.

Transgenerational persistence of parental responses to environmental stimuli has been reported in various organisms, but the underlying mechanisms remain underexplored. In one of these reported examples, we have shown that exposure of fly larvae to G418 antibiotic leads to non-Mendelian inheritance of ectopic induction of certain developmental genes. Here we investigate if this inheritance involves changes in mRNA composition within the early, maternal-stage offspring embryos of exposed flies. Exposure to G418 in F1 modified the maternal RNA levels of many genes in their early (F2) embryos. This includes reduction of maternal Polycomb group genes which persisted in the following generation of embryos (F3). To investigate the functional meaning of this reduction, we compared genetically normal embryos of Polycomb mutant females to normal embryos of normal females. Analysis with two different alleles of Polycomb, Pc1 and Pc3, revealed that maternal reduction in Polycomb gene dosage has a positive influence on the inheritance of induced expression. Together, this shows that exposure to G418 stress reduces the maternal levels of Polycomb in the offspring embryos and this reduction contributes to the inheritance of induced expression.

Amoebicidal Activity of Poly-Epsilon-Lysine Functionalized Hydrogels.

Purpose: To determine the amoebicidal activity of functionalized poly-epsilon-lysine hydrogels (pɛK+) against Acanthamoeba castellanii. Methods: A. castellanii trophozoites and cysts were grown in the presence of pɛK solution (0-2.17 mM), pɛK or pɛK+ hydrogels, or commercial hydrogel contact lens (CL) for 24 hours or 7 days in PBS or Peptone-Yeast-Glucose (PYG) media (nutrient-deplete or nutrient-replete cultures, respectively). Toxicity was determined using propidium iodide and imaged using fluorescence microscopy. Ex vivo porcine corneas were inoculated with A. castellanii trophozoites ± pɛK, pɛK+ hydrogels or commercial hydrogel CL for 7 days. Corneal infection was assessed by periodic acid-Schiff staining and histologic analysis. Regrowth of A. castellanii from hydrogel lenses and corneal discs at 7 days was assessed using microscopy and enumeration. Results: The toxicity of pɛK+ hydrogels resulted in the death of 98.52% or 83.31% of the trophozoites at 24 hours or 7 days, respectively. The toxicity of pɛK+ hydrogels resulted in the death of 70.59% or 82.32% of the cysts in PBS at 24 hours or 7 days, respectively. Cysts exposed to pɛK+ hydrogels in PYG medium resulted in 75.37% and 87.14% death at 24 hours and 7 days. Ex vivo corneas infected with trophozoites and incubated with pɛK+ hydrogels showed the absence of A. castellanii in the stroma, with no regrowth from corneas or pɛK+ hydrogel, compared with infected-only corneas and those incubated in presence of commercial hydrogel CL. Conclusions: pɛK+ hydrogels demonstrated pronounced amoebicidal and cysticidal activity against A. castellanii. pɛK+ hydrogels have the potential for use as CLs that could minimize the risk of CL-associated Acanthamoeba keratitis.

Structure-activity relationships of mononuclear metal-thiosemicarbazone complexes endowed with potent antiplasmodial and antiamoebic activities.

A useful concept for the rational design of antiparasitic drug candidates is the complexation of bioactive ligands with transition metals. In view of this, an investigation was conducted into a new set of metal complexes as potential antiplasmodium and antiamoebic agents, in order to examine the importance of metallic atoms, as well as the kind of sphere of co-ordination, in these biological properties. Four functionalized furyl-thiosemicarbazones (NT1-4) treated with divalent metals (Cu, Co, Pt, and Pd) to form the mononuclear metallic complexes of formula [M(L)2Cl2] or [M(L)Cl2] were examined. The pharmacological characterization, including assays against Plasmodium falciparum and Entamoeba histolytica, cytotoxicity to mammalian cells, and interaction with pBR 322 plasmid DNA was performed. Structure-activity relationship data revealed that the metallic complexation plays an essential role in antiprotozoal activity, rather than the simple presence of the ligand or metal alone. Important steps towards identification of novel antiplasmodium (NT1Cu, IC50 of 4.6 microM) and antiamoebic (NT2Pd, IC50 of 0.6 microM) drug prototypes were achieved. Of particular relevance to this work, these prototypes were able to reduce the proliferation of these parasites at concentrations that are not cytotoxic to mammalian cells.

Amoebicidal and Amoebistatic Effects of Artemisia argyi Methanolic Extracts on Acanthamoeba castellanii Trophozoites and Cysts.

PURPOSE : The present study aimed to investigate the amoebicidal and amoebistatic efects of Artemisia argyi leaf methanolic extract by testing the effects on trophozoites and on cysts. We also determined cytotoxic effect, enzymatic and non-enzymatic antioxidant activities, total phenolic, lavonoid and antioxidative contents of A. argyi. METHODS: A. argyi was harvested from various geographic sites in Ordu province in Turkey. The fresh leaves were subjected to methanolic extraction. In 100 µl culture, different concentrations of A. argyi methanolic extract (in quantities from 1.2, 2.3, 4.7, 9.4, 18.7, 37.4, 74.8 mg/ml) and the same volume of trophozoite/cyst suspension were mixed for the determination of the amoebicidal activity of the plant extract. Human bronchial epithelial cells were treated with the same concentrations of Artemisia extracts to determine cytotoxic potential. RESULTS: Total phenolic and lavonoid contents of the extract were calculated as 261 mg gallic acid/g dry extract and 29 mg quercetin/g dry extract, respectively. Total antioxidant activity was also calculated as 367 mg ascorbic acid/g dry extract. The growth of trophozoites stopped in A. argyi methanolic extract with 50% inhibitory concentrations (IC50)/8 h for 37.4 mg/ ml and 74.8 mg/ml extract solution and had stronger amoebicidal activity on the cysts with IC50/72 h. Artemisia showed stronger inhibitory effects on bronchial epithelial cells at the concentrations of 9.4, 18.7, 37.4 and 74.8 mg/ml. CONCLUSION: The study indicated that A. argyi leaf extract has cytotoxic and anti-amoebic activities.

The toxicology of Clioquinol.

5-Chloro-7-iodo-quinolin-8-ol (Clioquinol) is a halogenated 8-hydroxyquinoline that was used in 1950-1970s as an oral anti-parasitic agent for the treatment and prevention of intestinal amebiasis. However in the 1970s oral Clioquinol was withdrawn from the market due to reports of neurotoxicity in Japanese patients. Recently, reports have demonstrated that Clioquinol has activities beyond its use as an antimicrobial. For example, Clioquinol inhibits the function of the proteasome and displays preclinical efficacy in the treatment of malignancy. In addition, due to its ability to bind copper and dissolve beta-amyloid plaques in the brain, Clioquinol has been investigated for the treatment of Alzheimer's disease. As such, efforts are underway to repurpose Clioquinol. In light of the reemergence of oral Clioquinol, we review the toxicology of this compound in animals and humans with an emphasis on its neurotoxicity. Such information will aid in the design of clinical trials of oral Clioquinol for new indications such as cancer therapy.

Cysticidal effect on acanthamoeba and toxicity on human keratocytes by polyhexamethylene biguanide and chlorhexidine.

PURPOSE: To evaluate the cysticidal effect of polyhexamethylene biguanide (PHMB) and chlorhexidine on Acanthamoeba and its toxic effect on cultured human keratocytes. METHODS: Each well of a twofold-diluted Acanthamoeba cyst-containing suspension of 5 x 10(4) cysts/mL was treated with PHMB and chlorhexidine for 8, 24, and 48 hours to determine the minimal cysticidal concentration (MCC) of each disinfectant. Human corneal keratocytes (5 x 10(4) cells/mL) were exposed to PHMB and chlorhexidine for the same time to determine the survival rate of keratocytes. Inverted phase-contrast and electron microscopy were used to observe the morphologic changes. RESULTS: The mean MCC of PHMB for 8, 24, and 48 hours was 9.42, 5.62, and 2.37 microg/mL, respectively. The mean MCC of chlorhexidine for 8, 24, and 48 hours was 24.32, 10.02, and 7.02 microg/mL, respectively. The respective survival rate of keratocytes at the MCC was 91.7%, 64.6%, and 49.7% for PHMB and 95.7%, 90.6%, and 78.1% for chlorhexidine, respectively. The cysts and keratocytes showed more damaged appearances after treatment with PHMB than chlorhexidine. CONCLUSIONS: PHMB and chlorhexidine showed a similar amoebicidal efficacy. However, PHMB seemed to be more toxic to keratocytes than chlorhexidine.

Synthesis, characterization and antiamoebic activity of new indole-3-carboxaldehyde thiosemicarbazones and their Pd(II) complexes.

In continuation of our research on thiosemicarbazones and their metal complexes as antiamoebic agents, a new series of indole-3-carboxaldehyde thiosemicarbazones (TSC) 1-7 were prepared by condensing indole-3-carboxaldehyde with cycloalkylaminothiocarbonyl hydrazines. Their palladium(II) complexes of the [Pd(TSC)Cl2] type, were synthesized upon coordination with [Pd(DMSO)2Cl2]. The chemical structures of all the compounds were established by elemental analyses, electronic, IR, (1)H NMR and (13)C NMR spectral data. The structure of the complexes was further established by thermogravimetric analysis and FAB MS. Spectroscopic data revealed that thiosemicarbazones act as bidentate ligands, making use of thione sulphur and azomethine nitrogen atom for coordination to the Pd(II) ion. Among all the compounds evaluated for antiamoebic activity using HM1:IMSS strain of Entamoeba histolytica, all palladium complexes were found to be more active than their respective ligands. Moreover, ligand 5 and complexes 1a-3a, 5a and 7a showed antiamoebic activity, at lower IC(50) doses when compared to the reference drug metronidazole with IC(50)=1.81 microM.

Emetine and/or its metabolites are genotoxic in somatic cells of Drosophila melanogaster.

Emetine is one of the two active ingredients of syrup of ipecac which is used medicinally as antiparasitic and emetic, however little is known about its genotoxic activity. The goal of this study was to determine whether and how emetine and/or its metabolites might produce mitotic recombination using the in vivo Drosophila w/w+ eye somatic assay. A standard strain (which expresses basal levels of cytochrome P450 enzymes) and an insecticide-resistant strain (which constitutively over-expresses P450 genes) were employed. The results showed that emetine and/or its metabolites are active in the assay and that the genotoxic potential is significantly influenced in the presence of higher than normal concentrations of P450.

Potential Value of Cellulose Synthesis Inhibitors Combined With PHMB in the Treatment of Acanthamoeba Keratitis.

PURPOSE: The aim of this study was to improve the cytopathic effect (CPE) of antiamebic agents by combining with cellulose synthesis inhibitor as an encystation inhibitor. METHODS: Cellulose synthesis inhibitors, 2,6-dichlorobenzonitrile (DCB) and isoxaben were used to block encystation of Acanthamoeba during cultivation. Cultured human corneal epithelial (HCE) cells and Acanthamoeba were treated with polyhexamethylene biguanide (PHMB) combined with cellulose synthesis inhibitors to evaluate the CPE as an antiamebic agent. RESULTS: 0.02% PHMB showed a 51.9% CPE on HCE cells within 30 minutes but exhibited significant toxic effects on Acanthamoeba. At a level of 0.00125%, PHMB had no significant CPEs on HCE cells, whereas 100 µM DCB and 10 µM isoxaben significantly inhibited the formation of the inner cyst wall of Acanthamoeba during encystation, and Acanthamoeba trophozoites failed to convert into mature cysts. Although a low concentration (0.00125%) of PHMB was used, the novel combinations with 100 µM DCB or 10 µM isoxaben had 23.4% or 18.7% additional amebicidal effects on Acanthamoeba. However, 100 µM DCB and 10 µM isoxaben had no CPEs on HCE cells. CONCLUSIONS: The combination of cellulose synthesis inhibitors with low concentrations of PHMB reduced the CPE on HCE cells and improved the amebicidal effect on Acanthamoeba by inhibition of encystation.

Evaluation of the teratogenic potential in Ciba-Geigy Go 10213, a new nitroimidazole derivative: 1-methane-sulphonyl-3-(1-methyl-5-nitro-1H-imidazole-2-yl)-2- imidazolidinone, an amoebicide, trichomonicide and giardicide, in giardicide, in rats.

With a view to evaluate the possible teratogenic potential of 1-methane-sulphonyl-3-(1-methyl-5-nitro-1H-imidazole-2-yl)-2-imidazolidi none (Go 10213), groups of pregnant rats were medicated orally with: 0, 100, 300 and 600 mg/kg of the compound daily from day 6 to day 15 of gestation. Foetuses were removed on day 20 of gestation, and were examined for their mortality, size, weight and any gross malformation. One third of the foetuses were fixed in Bouin's Fluid and sectioned by Wilson's technique for histological evaluation. The remaining pups were examined for the skeletal anomalies. CIBA-GEIGY Go 10213 appears to be neither embryotoxic nor teratogenic to the rat-foetuses, when the mothers were medicated at the aforesaid doses, during their critical phase of the pregnancy.

In vitro amoebicidal activity of propamidine and pentamidine isethionate against Acanthamoeba species and toxicity to corneal tissues.

Effective chemotherapy for Acanthamoeba keratitis has been hampered because of the marked resistance of the parasites to a variety of antimicrobial agents. In view of the fact that topical Brolene (propamidine isethionate) and neosporin are currently considered to be the medical treatment of choice in Europe, we sought to determine whether pentamidine may be equally effective, because the drug is more readily available to ophthalmologists in the United States. In this study, we compared the amoebicidal activity of the Brolene (commercial product), propamidine isethionate and pentamidine isethionate (Pentam) in vitro against three different species of Acanthamoeba, and the drugs' corresponding biocompatibility with rabbit corneal epithelial and endothelial cell cultures. The results indicated that there were significant species differences in drug sensitivity. Propamidine (> 1,000 micrograms/ml) was clearly less effective than pentamidine (> 125 micrograms/ml) against A. castellanii, although equivalent potency (> 250 micrograms/ml) was observed against A. polyphaga. On the other hand, propamidine (> 31.25 micrograms/ml) was slightly more effective than pentamidine (> 62.5 micrograms/ml) against A. hatchetti. Both drugs were also relatively nontoxic after short-term contact with cell cultures, even though the highest concentration of pentamidine caused low-grade injury to the superficial epithelium and reversible membrane damage to the endothelium. Steady-state levels of propamidine at effective amoebicidal concentrations, however, were much more toxic than pentamidine, which indicated that the drug has a much lower therapeutic index. Our data suggest that pentamidine may be an effective therapeutic option because of its potency and low toxicity.

Mutagenicity of antiamebic and anthelmintic drugs in the Salmonella typhimurium microsomal test system.

4 amebicides (chloroquine diphosphate, diiodohydroxyquin, iodochlorohydroxyquin and dehydroemetine) and 6 anthelmintics (bephenium hydroxynaphthoate, 4-hexylresorcinol, mebendazole, niclosamide, pyrantel pamoate and pyrvinium pamoate) were tested for mutagenicity in the Salmonella typhimurium microsomal test system. Frameshift mutations were induced by dehydroemetine and niclosamide following activation by microsomal enzymes, while pyrvinium pamoate induced both frameshift and base-pair substitution mutations with or without metabolic activation. The urine of mice treated with dehydroemetine or pyrvinium pamoate showed no mutagenic activity. However, urine obtained from mice treated with niclosamide was mutagenic in strains TA98 and TA1538. The fluctuation assay showed chloroquine diphosphate to be mutagenic in TA1537, a strain which detects frameshift mutations.

Influence of the Uvr repair system on the mutagenicity of antiparasitic drugs.

One amebicide (chloroquine diphosphate) and 2 anthelmintic compounds (niclosamide and pyrvinium pamoate) were found to be mutagenic for Salmonella typhimurium TA1537, TA1538, TA100 and TA98 Uvr- strains respectively. Drugs tested on homologous Uvr+ strains (TA1977, TA1978, UTH8414 and UTH8413) showed decreased mutagenic activity of the compounds. This indicates that premutational damage induced by the drugs was totally or partially repaired. Furthermore, results obtained in the present study suggest that niclosamide and pyrvinium pamoate induce premutational lesions by adduct formation, and that chloroquine diphosphate, known as an intercalating agent, behaves as an adduct-forming compound as regards its effects on Uvr- and Uvr+ S. typhimurium strains.

Anti-amoebic activity of plant compounds from Virgilia oroboides and Chlorophora excelsa.

The anti-amoebic activity of four plant extracts: maackiain and formononetin from Virgilia oroboides and chlorophorin and Iroko from Chlorophora excelsa, were evaluated. Anti-protozoal tests conducted on trophozoites of Entamoeba histolytica established that all four compounds had an affect on the trophozoites to some degree. Chlorophorin showed the highest anti-protozoal activity with an MIC of 0.25 microg/ml followed by maackiain and Iroko with MICs of 1 microg/ml. Chlorophorin and Iroko induced the release of acid phosphatase. Chlorophorin reduced alpha amylase levels by 89%. Formononetin and maackiain had a minimal effect on the enzyme levels. Ultrastructural changes occurred in trophozoites treated with plant compounds. The degree of destruction of the trophozoites increased with an increase in compound concentration. Trophozoite destruction was initiated by the disintegration of the nucleus and culminated with the rupture of the cytoplasmic membrane. Maackiain was the only compound that showed some level of mutagenicity. Formononetin and Iroko were very slightly mutagenic, while chlorophorin was non-mutagenic. In addition, none of the compounds tested showed cytopathic effects on any of the cell lines tested. Chlorophorin and Iroko exhibit the potential to be exploited as natural multi-functional safe control agents in the treatment of bacterial, fungal and protozoal infections.