Characterisation of the in vitro activity of a Nitazoxanide-N-methyl-1H-benzimidazole hybrid molecule against albendazole and nitazoxanide susceptible and resistant strains of Giardia intestinalis and its in vivo giardicidal activity

BACKGROUND It was previously demonstrated that CMC-20, a nitazoxanide and N-methyl-1H-benzimidazole hybrid molecule, had higher in vitro activity against Giardia intestinalis WB strain than metronidazole and albendazole and similar to nitazoxanide. OBJETIVES To evaluate the in vitro activity of CMC-20 against G. intestinalis strains with different susceptibility/resistance to albendazole and nitazoxanide and evaluate its effect on the distribution of parasite cytoskeletal proteins and its in vivo giardicidal activity. METHODS CMC-20 activity was tested against two isolates from patients with chronic and acute giardiasis, an experimentally induced albendazole resistant strain and a nitazoxanide resistant clinical isolate. CMC-20 effect on the distribution of parasite cytoskeletal proteins was analysed by indirect immunofluorescence and its activity was evaluated in a murine model of giardiasis. FINDINGS CMC-20 showed broad activity against susceptible and resistant strains to albendazole and nitaxozanide. It affected the parasite microtubule reservoir and triggered the parasite encystation. In this process, alpha-7.2 giardin co-localised with CWP-1 protein. CMC-20 reduced the infection time and cyst load in feces of G. muris infected mice similar to albendazole. MAIN CONCLUSIONS The in vitro and in vivo giardicidal activity of CMC-20 suggests its potential use in the treatment of giardiasis.

Characterisation of the in vitro activity of a Nitazoxanide-N-methyl-1H-benzimidazole hybrid molecule against albendazole and nitazoxanide susceptible and resistant strains of Giardia intestinalis and its in vivo giardicidal activity

BACKGROUND It was previously demonstrated that CMC-20, a nitazoxanide and N-methyl-1H-benzimidazole hybrid molecule, had higher in vitro activity against Giardia intestinalis WB strain than metronidazole and albendazole and similar to nitazoxanide. OBJETIVES To evaluate the in vitro activity of CMC-20 against G. intestinalis strains with different susceptibility/resistance to albendazole and nitazoxanide and evaluate its effect on the distribution of parasite cytoskeletal proteins and its in vivo giardicidal activity. METHODS CMC-20 activity was tested against two isolates from patients with chronic and acute giardiasis, an experimentally induced albendazole resistant strain and a nitazoxanide resistant clinical isolate. CMC-20 effect on the distribution of parasite cytoskeletal proteins was analysed by indirect immunofluorescence and its activity was evaluated in a murine model of giardiasis. FINDINGS CMC-20 showed broad activity against susceptible and resistant strains to albendazole and nitaxozanide. It affected the parasite microtubule reservoir and triggered the parasite encystation. In this process, alpha-7.2 giardin co-localised with CWP-1 protein. CMC-20 reduced the infection time and cyst load in feces of G. muris infected mice similar to albendazole. MAIN CONCLUSIONS The in vitro and in vivo giardicidal activity of CMC-20 suggests its potential use in the treatment of giardiasis.

[Evalution of antileishmanial activity of the hydroalcoholic extract of medicago lupulina' leaves against leishmania major [MHOM/IR/75/ER] promastigotes by using MTT and microscopy method and GC/MS for hydroalcoholic extract of this plant' leaves]

Background: Leishmaniasis, has created enormous global health problem. Side effects, drug resistance and the lack of effective vaccines and to make the new compounds effective due to plant

Objective: The traditional medical plants such as black alfalfa can be a valuable source of new pharmaceutical agents against leishmaniasis

Methods: Alcoholic extracts were prepared by maceration method. L. major promastigotes [Leishmania major] in Schneider and then were cultured in RPMI- 1640. Then, using MTT [Methyl Thiazole Tetrazolium], the IC50 [Inhibitory Concentrations 50%] for extract and Glucantime was determined. MTT assay did for each sample, 3 times

Results: IC50 for alcoholic extract of alfalfa black against L. major promastigotes in vitro after 24, 48 and 72 hours, respectively 165, 98 and 45 micrograms per ml and for Glucantime also equal to 27, 12 and 8 mg l respectively. IC50 between Extract and Glucantime after 24, 48 and 72 hours there was a significant difference [P <0.05]. Morphological changes after challenge with meglumine and alcoholic extracts including cell shrinkage, round, dense cytoplasm and the cell was smaller. Presence of alkaloids and flavonoids in alcoholic extracts have been proved

Conclusion: As regards, plant extract had anti- leishmanial effects in vitro, further works are required to appraise the exact effect on Leishmania agent in animal models

Synthesis and cytotoxicity evaluation of thiosemicarbazones and their thiazole derivatives

ABSTRACT The aims of this study were to synthesize a series of thiosemicarbazones and their thiazole derivatives, to investigate their cytotoxic activity against three human cancers and normal (Vero cells) cell lines, and to evaluate the pro-apoptotic potential of the most active compounds. Materials and Methods: The thiosemicarbazones were obtained by reacting an aromatic aldehyde with thiosemicarbazide (yield 71-96%), which were subjected to a cyclization with α-bromoacetophenone to yield the required thiazole heterocycles (yield 63-100%). All the synthesized compounds were screened at 50 µM concentration against three cell lines representing HL60 (promyelocytic leukemia), Jurkat (acute lymphoblastic leukemia), and MCF-7 (breast cancer). The pro-apoptotic effect was measured by flow cytometry as the percentage of cells with hypodiploid DNA. Results: Three thiazole compounds showed activity against at least one tumor cell line (IC50 = 43-76 µM) and low cytotoxicity against Vero cells (IC50 > 100 M). The most active compound of this series induced 91% and 51% DNA fragmentation in HL60 and MCF-7 cell lines, respectively, suggesting that this compound triggered apoptosis in these cells. Conclusion: Among the synthesized compounds, one in particular was found to exert antiproliferative and pro-apoptotic activity on tumor cells and can be considered promising as a lead molecule for the design of new analogues with improved activity.

RESUMO O estudo teve como objetivo a síntese de uma série de tiossemicarbazonas e seus derivados tiazólicos e a avaliação da atividade citotóxica contra três linhagens de células tumorais humanas e células normais (Vero), a fim de se avaliar o potencial pró-apoptótico dos compostos mais ativos. As tiossemicarbazonas foram obtidas por reação entre um aldeído aromático e tiossemicarbazida (rend. 71-96%), as quais foram submetidas à ciclização com α-bromoacetofenona, fornecendo os heterociclos tiazólicos desejados (rend. 63-100%). Todos os compostos sintetizados foram testados na concentração de 50 µM contra três linhagens de células tumorais: HL60 (leucemia promielocítica), Jurkat (leucemia linfoblástica aguda) e MCF-7 (câncer de mama). O efeito pró-apoptótico foi avaliado por citometria de fluxo como porcentagem de células com DNA hipodiplóide. Três compostos tiazólicos foram ativos contra, pelo menos, uma linhagem tumoral (CI50=43-76 µM), com baixa citotoxicidade contra células Vero (CI50 > 100 M). O composto mais ativo dessa série induziu fragmentação do DNA de 91% e 51% nas linhagens HL60 e MCF-7, respectivamente, sugerindo que este composto ativou a apoptose nessas células. Dentre os compostos sintetizados, um em particular apresentou atividade antiproliferativa e pró-apoptótica em células tumorais e pode ser considerado composto protótipo promissor na busca por novos análogos com atividade melhorada.

Cytotoxicity assays as tools to assess water quality in the Sinos River basin / Ensaios de citotoxicidade como ferramentas para avaliar a qualidade da água da bacia do Rio do Sinos

Cytotoxicity assays using cell cultures may be an alternative to assess biological toxicity of surface waters and may help to improve the control of water quality. This study compared two methods to prepare culture media for the exposure of Hep-2 cells to water samples collected from the Rolante River, an important affluent of the Sinos River. The toxicity was evaluated using the MTT and neutral red assays. Two methods were used to prepare culture media. In method 1, the sample was diluted at 1:1, 1:10, 1:100, 1:1000, 1:10.000 (v/v, sample/medium) in a standard culture medium; in method 2, water samples were used as the solvent for the culture medium, which was prepared at concentrations of 100, 80, 60, 40 and 20%. Semi-confluent cultures were then exposed to the media test for 24 hours, and cytotoxicity was determined immediately using the MTT and NR assays. Mitochondrial activity (MTT) was significantly lower at all concentrations in both methods, except at 1:1000 in method 1. However, the lysosome viability (NR) results revealed cytotoxicity only in the 1:1 sample of method 1. Both culture preparation methods were efficient and sensitive to the MTT assay, but method 2 seemed to be more adequate for the NR assay. The Rolante River has cytotoxic contaminants to Hep-2 cells, which may be one of the explanations for the poor water quality of the Sinos River basin.

Os ensaios de citotoxicidade utilizando culturas de células constituem uma alternativa para avaliar a toxicidade biológica de águas de superfície e podem auxiliar no controle da qualidade da água. Este estudo comparou dois métodos de preparação dos meios de cultura com amostras de água coletadas no rio Rolante, um importante afluente do Rio dos Sinos, para a exposição de células Hep-2. A toxicidade foi avaliada usando os ensaios do MTT e do vermelho neutro (VN). Dois métodos foram utilizados para preparar os meios de cultura. No método 1, a amostra foi diluída a 1:1, 1:10, 1:100, 1:1000 e 1:10.000 (v/v, amostra/ meio de cultivo) em um meio de cultura padrão; no método 2, as amostras de água foram utilizados como solventes para o meio de cultura, o qual foi preparado em concentração de 100% e nas diluições de 80, 60, 40 e 20%. Culturas semi-confluentes foram então expostas aos meios teste durante 24 horas, e a citotoxicidade foi determinada imediatamente usando os ensaios MTT e VN. A atividade mitocondrial (MTT) foi significativamente menor em todas as concentrações em ambos os métodos, exceto na diluição 1:1000 do método 1. No entanto, os resultados de viabilidade lisossomal (VN) revelaram citotoxicidade apenas no na diluição 1:1 do método 1. Ambos os métodos de preparação do meio cultura foram eficientes e sensíveis para o ensaio do MTT, mas o método 2 foi mais adequado para o ensaio do VN. O rio Rolante possui contaminantes citotóxicos para as células Hep-2, o que pode ser uma das explicações para a baixa qualidade da água da Bacia do Rio dos Sinos.

Evaluation of protein undernourishment on the condylar process of the Wistar rat mandible correlation with insulin receptor expression

The mandible condylar process cartilage (CP) of Wistar rats is a secondary cartilage and acts as a mandibular growth site. This phenomenon depends on adequate proteins intake and hormone actions, including insulin. Objectives The present study evaluated the morphological aspects and the expression of the insulin receptor (IR) in the cartilage of the condylar process (CP) of rats subjected to protein undernourishment. Material and Methods The nourished group received a 20% casein diet, while the undernourished group (U) received a 5% casein diet. The re-nourished groups, R and RR, were used to assess the effects of re-nutrition during puberty and adulthood, respectively. CPs were processed and stained with picro-sirius red, safranin-O and azocarmine. Scanning electron microscopy and immunohistochemistry were also performed. Results The area of the CP cartilage and the number of cells in the chondroblastic layer decreased in the U group, as did the thickness of the CP layer in the joint and hypertrophic layer. Renourishment during the pubertal stage, but not during the adult phase, restored these parameters. The cell number was restored when re-nutrition occurred in the pubertal stage, but not in the adult phase. The extracellular matrix also decreased in the U group, but was restored by re-nutrition during the pubertal stage and further increased in the adult phase. IR expression was observed in all CPs, being higher in the chondroblastic and hypertrophic cartilage layers. The lowest expression was found in the U and RR groups. Conclusions Protein malnutrition altered the cellularity, the area, and the fibrous cartilage complex, as well as the expression of the IRs. .

Anti-parasitic action and elimination of intracellular Toxoplasma gondii in the presence of novel thiosemicarbazone and its 4-thiazolidinone derivatives

Toxoplasma, which infects all eukaryotic cells, is considered to be a good system for the study of drug action and of the behavior of infected host cells. In the present study, we asked if thiosemicarbazone derivatives can be effective against tachyzoites and which morphological and ultrastructural features of host cells and parasites are associated with the destruction of Toxoplasma. The compounds were tested in infected Vero cell culture using concentration screens (0.1 to 20 mM). The final concentration of 1 mM was chosen for biological assay. The following results were obtained: 1) These new derivatives decreased T. gondii infection with an in vitro parasite IC50 percent of 0.2-0.7 mM, without a significant effect on host cells and the more efficient compounds were 2, 3 (thiosemicarbazone derivatives) and 4 (thiazolidinone derivative); 2) The main feature observed during parasite elimination was continuous morphological disorganization of the tachyzoite secretory system, progressive organelle vesiculation, and then complete disruption; 3) Ultrastructural assays also revealed that progressive vesiculation in the cytoplasm of treated parasites did not occur in the host cell; 4) Vesiculation inside the parasite resulted in death, but this feature occurred asynchronously in different intracellular tachyzoites; 5) The death and elimination of T. gondii was associated with features such as apoptosis-like stage, acidification and digestion of parasites into parasitophorous vacuoles. Our results suggest that these new chemical compounds are promising for the elimination of intracellular parasites by mainly affecting tachyzoite development at 1 mM concentration for 24 h of treatment.

Synthesis of novel coumarin derivatives bearing heterocyclic substituents for chemoprophylactic evaluation

A series of coumarin derivatives bearing heterocyclic substituents was synthesized. Bromination of the key compound 8-acetyl-7-hydroxy-4-methylcoumarin [I] under varied conditions gave the brominated derivatives II, III and IV, Conversions of III by several cyclocondensations gave the corresponding thiazole V, quinoxaline VI, quinoxalinone VIII and furanone IX derivatives. Also, methylation and cyclocondensation of the methylene-active acetyl groups of II with several aldehydes gave the corresponding pyridone various amines gave the corresponding amino side chains. Some of these newly synthesized products were studied for their chemoprophylatic effect on Schistosoma mansoni infected mice. Compound Xc a showed moderate effect

The use of MTT [3-(4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl -2H- tetrazolium bromide]-reduction as an indicator of the effects of strain-specific, polyclonal rabbit antisera on Candida albicans and C. krusei.

There is only scanty data on the effects of specific antibody, with or without complement, on Candida albicans or Candida krusei in cell-free systems in vitro, although previously published work has shown that specific antibody mediates anti- Candida immunity in vivo by inhibition of adherence to host cells or surfaces and by the promotion of phagocytosis and intra-phagocytic killing. The MTT (3-[4, 5-dimethyl-2-thiazolyl] -2, 5-diphenyl -2H- tetrazolium bromide)-reduction method as a test of the viability of fungi was used to investigate the effect of complement, normal serum and immune serum on these two species of Candida that are of increasing importance as opportunistic pathogens. We report that normal rabbit serum or strain-specific, polyclonal anti- Candida rabbit antibody, with or without guinea pig complement, did not cause the reduction of total cell-mass or of the viability of either C. albicans or C. krusei, in vitro as determined by the MTT-reduction test. Complement alone without specific antibody, also, had no such effect on these two Candida species.

BOXTO as a real-time thermal cycling reporter dye.

The unsymmetrical cyanine dyes BOXTO (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2- methylidene)]-1-methyl-quinolinium chloride)and its positive divalent derivative BOXTO-PRO (4-[(3-methyl-6-(benzoxazole-2-yl)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-(3-trimethylammonium-propyl)-quinolinium dibromide) were studied as real-time PCR reporting fluorescent dyes and compared to SYBR GREEN I (SG)(2-[N- (3-dimethylaminopropyl)-N-propylamino] -4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl- quinolinium).Unmodified BOXTO showed no inhibitory effects on real-time PCR,while BOXTO-PRO showed complete inhibition. Sufficient fluorescent signal was acquired when 0.5-1.0 meu M BOXTO was used with RotorGene and iCycler platforms.Statistical analysis showed that there is no significant difference between the efficiency and dynamic range of BOXTO and SG.BOXTO stock solution (1.5 mM) was stable at -20 degree C for more than one year and 40 meu M BOXTO solution was more stable than 5x SG when both were stored at 4 degree C for 45 days.

A pyrazolyl-thiazole derivative causes antinociception in mice

The present study investigates the antinociceptive effect of the pyrazolyl-thiazole derivative 2-(5-trichloromethyl-5-hydroxy-3-phenyl-4,5-dihydro-1 H-pyrazol-1-yl)-4-(4-bromophenyl)-5-methylthiazole (B50) in mice. Male albino Swiss mice (30-40 g) were used in the acetic acid-induced abdominal writhes and tail-immersion tests. B50 caused dose-dependent antinociception (8, 23 and 80 µmol/kg, sc) in the acetic acid writhing assay (number of writhes: vehicle: 27.69 ± 6.15; B50 (8 µmol/kg): 16.92 ± 3.84; B50 (23 µmol/kg): 13.85 ± 3.84; B50 (80 µmol/kg): 9.54 ± 3.08; data are reported as means ± SEM for 9 animals per group). On the other hand, B50 did not cause antinociception in the tail immersion assay. Naloxone (2.75 µmol/kg, sc) prevented B50-induced antinociception (number of writhes: vehicle-saline: 31.11 ± 3.15; vehicle-naloxone: 27.41 ± 3.70; B50 (80 µmol/kg)-saline: 8.70 ± 3.33; B50 (80 µmol/kg)-naloxone: 31.84 ± 4.26; morphine-saline: 2.04 ± 3.52; morphine-naloxone: 21.11 ± 4.26; 8-9 animals per group). The removal of the methyl group of the thiazole ring of B50 or substitution of the bromo substituent with the methyl at position 4 of the phenyl group, which is attached to the thiazole ring of B50, resulted in loss of activity, suggesting that these substituents are important for antinociceptive activity. B50 had no effect on spontaneous locomotion or rotarod performance, indicating that the antinociceptive effect of B50 is not related to nonspecific motor effects. The antinociceptive profile of B50 seems to be closer to nonsteroidal anti-inflammatory drugs than to classic opioid agents, since it had no analgesic effect in a thermally motivated test.

Synthesis of some thiazolidinone and triazole dderivatives of expected antimicrobial activity

New derivatives of semicarbazide [5] and thiosemicarbazide [6] had been synthesized together with new substituted alkyl and aryl 1, 2, 4-triazoles [7] and thiazolidinone [8] obtained by cyclizing the appropriate thiosemicarbazide [6]. Screening for antibacterial and antifungal activities using preliminary scan by the agar plate inhibition zone method, followed by MIC versus ampicillin and clotrimazole revealed high activity for compounds [5-c] and [7-a]. The partition coefficient of the most and least biologically active compounds was determined and the results were reported

New antimicrobial quinazolinone derivatives

A series of quinazolin-4-ones incorporated with pyridones, iminopyridines, thiazole, semicarbazone and thiosemicarbazone moieties were synthesized. Some tested compounds showed considerable biological activity against some microorganisms

Potential anticancer drugs: synthesis of some fused arylhydrazonoindeno [1,2-d] thiazolo and thiazinopyrimidinones

4-aryl-1,2,3,4-tetrahydro-5-oxoindeno [1,2-d] pyrimidine-2-thiols [I] were synthesized in the laboratories and their corresponding hydrazono derivatives [II] were submitted to react with chloroacetic acid, 2-bromopropionic acid or 3-bromopropionic acid in the presence of fused sodium acetate and acetic anhydride to give 5-aryl-2,3- dihydro-5H, 6-arylhydrazonoindeno [1,2-d] thiazolo [3,2-a] pyrimidin-3-ones [III], its methyl derivatives [IV] and 6-aryl-6H, 7-arylhydrazonoindeno [1,2-d]-pyrimidino-[2,1-b]-1,3-thiazin-4-ones [V], respectively. The indenothiazolopyrimidones [III] condensed with aromatic aldehydes in the presence of acetic anhydride to yield 2-arylmethylene-5 aryl-2,3-dihydro-5H-6-arylhydrazonoimdeno [1,2-d] thiazolo [3,2-a] pyrimidin-3-ones [VI], which was obtained directly from their arylhydrazono derivatives [II] by the reaction of chloroacetic acid in the presence of the corresponding aromatic aldehyde and in refluxing acetic acid/acetic anhydride mixture