Planejamento, síntese e avaliação farmacológica de compostos híbridos potencialmente ativos para o tratamento da anemia falciforme

Sickle cell disease (SCD) is a hereditary hemolytic anemia caused by the inheritance of one S hemoglobin gene from each ancestor. Patients with SCD present increased circulating levels of cytokines, including TNF-alpha (TNF-α). Hydroxyurea (HU) is the available therapeutically strategy for treatment; it acts as a source of nitric oxide and benefits patients by increasing the levels of fetal hemoglobin (HbF). Thus, within one research line that aims at finding new drugs, a series of compounds with TNF-α inhibition and nitric oxide donation properties have been synthesized in order to explore possible synergism of actions beneficial in the treatment of the disease. Six compounds were synthesized: five derivatives of organic nitrates and one of sulfonamide. The compounds, (1,3-dioxo-1,3-dihydro-2Hisoindol-2-yl) methyl nitrate (compound I); (1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) ethyl nitrate (compound II); 3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) benzyl nitrate (compound III);4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N-hydroxybenzenesulfonamide (compound IV); 4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) benzyl nitrate (compound V) and 2-[4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) phenyl]ethyl nitrate (compound VI), were synthesized using linear synthetic methodology, with excellent overall yields. All compounds showed anti-inflammatory and analgesic effects with a reduction in 43%-65% of ear edema in mice and a reduction of 25%-42% of writhing induced by acetic acid. All compounds showed comparable reductions in the leukocyte infiltration capacity and ability to generate nitric oxide. The aryl compounds (III, IV and V) presented less mutagenic activity compared to compounds I, II and VI according to the salmonella mutagenicity assay (Ames test). Compounds IV and VI showed activity in K562 culture cells, with increases in gamma globin gene expression to levels higher than with hydroxyurea suggesting a potential to increase fetal hemoglobin. This data set characterizes new potentially useful drug candidates for the treatment of symptoms of sickle cell anemia.

Development and validation of a microbiological agar assay for determination of orbifloxacin in pharmaceutical preparations

Orbifloxacin is a fluoroquinolone with broad-spectrum antimicrobial activity, and belongs to the third generation of quinolones. Regarding the quality control of medicines, a validated microbiological assay for determination of orbifloxacin in pharmaceutical formulations has not as yet been reported. For this purpose, this paper reports the development and validation of a simple, sensitive, accurate and reproducible agar diffusion method to quantify orbifloxacin in tablet formulations. The assay is based on the inhibitory effect of orbifloxacin upon the strain of Staphylococcus aureus ATCC 25923 used as test microorganism. The results were treated statistically by analysis of variance and were found to be linear (r = 0.9992) in the selected range of 16.0-64.0 μg/mL, precise with relative standard deviation (RSD) of repeatability intraday = 2.88%, intermediate precision RSD = 3.33%, and accurate (100.31%). The results demonstrated the validity of the proposed bioassay, which allows reliable orbifloxacin quantitation in pharmaceutical samples and therefore can be used as a useful alternative methodology for the routine quality control of this medicine. © 2011 by the authors; licensee MDPI, Basel, Switzerland.

Validation of microbiological assay for determination of cefuroxime in injectable preparations

The validation of a microbiological assay, applying agar diffusion method for determination of the active of cefuroxime in power for injection, is described. Using a strain of Micrococcus luteus ATCC 9341 as the test organism, cefuroxime was measured in concentrations ranging from 30.0 to 120.0 μg/mL. The method validation showed that it is linear (r = 0.9999), precise (relative standard deviation = 0.37%) and accurate (it measured the added quantities). Microbiological assay is satisfactory for quantitation of cefuroxime in powder for injection and the validity of the proposed bioassay, which is a simple and a useful alternative methodology for cefuroxime determination in routine quality control.

Development and validation of a successful microbiological agar assay for determination of ceftriaxone sodium in powder for injectable solution

Ceftriaxone sodium is a cephalosporin with broad-spectrum antimicrobial activity and belongs to the third generation of cephalosporins. Regarding the quality control of medicines, a validated microbiological assay for the determination of ceftriaxone sodium in powder for injectable solution has not been reported yet. This paper reports the development and validation of a simple, accurate and reproducible agar diffusion method to quantify ceftriaxone sodium in powder for injectable solution. The assay is based on the inhibitory effect of ceftriaxone sodium on the strain of Bacillus subtilis ATCC 9371 IAL 1027 used as test microorganism. The results were treated statistically by analysis of variance and were found to be linear (r = 0.999) in the selected range of 15.0-60.0 μg/mL, precise with a relative standard deviation (RSD) of repeatability intraday = 1.40%, accurate (100.46%) and robust with a RSD lower than 1.28%. The results demonstrated the validity of the proposed bioassay, which allows reliable ceftriaxone sodium quantitation in pharmaceutical samples and therefore can be used as a useful alternative methodology for the routine quality control of this medicine. © 2012 by the authors; licensee MDPI, Basel, Switzerland.

Application of micronucleus test and comet assay to evaluate BTEX biodegradation

The BTEX (benzene, toluene, ethylbenzene and xylene) mixture is an environmental pollutant that has a high potential to contaminate water resources, especially groundwater. The bioremediation process by microorganisms has often been used as a tool for removing BTEX from contaminated sites. The application of biological assays is useful in evaluating the efficiency of bioremediation processes, besides identifying the toxicity of the original contaminants. It also allows identifying the effects of possible metabolites formed during the biodegradation process on test organisms. In this study, we evaluated the genotoxic and mutagenic potential of five different BTEX concentrations in rat hepatoma tissue culture (HTC) cells, using comet and micronucleus assays, before and after biodegradation. A mutagenic effect was observed for the highest concentration tested and for its respective non-biodegraded concentration. Genotoxicity was significant for all non-biodegraded concentrations and not significant for the biodegraded ones. According to our results, we can state that BTEX is mutagenic at concentrations close to its water solubility, and genotoxic even at lower concentrations, differing from some described results reported for the mixture components, when tested individually. Our results suggest a synergistic effect for the mixture and that the biodegradation process is a safe and efficient methodology to be applied at BTEX-contaminated sites. © 2012 Elsevier Ltd.

Development and validation of a microbiological assay for determination of chlorhexidine digluconate in aqueous solution

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Assessment of the chemopreventive effect of casearin B, a clerodane diterpene extracted from Casearia sylvestris (Salicaceae)

Studies have shown that Casearia sylvestris compounds protect DNA from damage both in vitro and in vivo. Complementarily, the aim of the present study was to assess the chemopreventive effect of casearin B (CASB) against DNA damage using the Ames test, the comet assay and the DCFDA antioxidant assay. The genotoxicity was assessed by the comet assay in HepG2 cells. CASB was genotoxic at concentrations higher than 0.30μM when incubated with the FPG (formamidopyrimidine-DNA glycosylase) enzyme. For the antigenotoxicity comet assay, CASB protected the DNA from damage caused by H2O2 in the HepG2 cell line in concentrations above 0.04μM with post-treatment, and above 0.08μM with pre-treatment. CASB was not mutagenic (Ames test) in TA 98 and TA 102. In the antimutagenicity assays, the compound was a strong inhibitor against aflatoxin B1 (AFB) in TA 98 (>88.8%), whereas it was moderate (42.7-59.4%) inhibitor against mytomicin C (MMC) in TA 102. Additionally, in the antioxidant assay using DCFDA, CASB reduced reactive oxygen species (ROS) generated by H2O2. In conclusion, CASB was genotoxic to HepG2 cells at high concentrations; was protective of DNA at low concentrations, as shown by the Ames test and comet assay; and was also antioxidant. © 2012 Elsevier Ltd.

Assessment of the genotoxicity of two agricultural residues after processing by diplopods using the Allium cepa assay

Agroindustrial by-products and residues from treatment of sewage sludge have been recently recycled as soil amendments. This study was aimed at assessing toxic potential of biosolid, obtained from a sewage treatment plant (STP), vinasse, a by-product of the sugar cane industry, and a combination of both residues using Allium cepa assay. Bioprocessing of these samples by a terrestrial invertebrate (diplopod Rhinocricus padbergi) was also examined. Bioassay assembly followed standards of the Brazilian legislation for disposal of these residues. After adding residues, 20 diplopods were placed in each terrarium, where they remained for 30 days. Chemical analysis and the A. cepa assay were conducted before and after bioprocessing by diplopods. At the end of the bioassay, there was a decrease in arsenic and mercury. For the remaining metals, accumulation and/or bioavailability varied in all samples but suggested bioprocessing by animals. The A. cepa test revealed genotoxic effects characterized by different chromosome aberrations. Micronuclei and chromosome breaks on meristematic cells and F1 cells with micronuclei were examined to assess mutagenicity of samples. After 30 days, the genotoxic effects were significantly reduced in the soil + biosolid and soil + biosolid + vinasse groups as well as the mutagenic effects in the soil + biosolid + vinasse group. Similar to vermicomposting, bioprocessing of residues by diplopods can be a feasible alternative and used prior to application in crops to improve degraded soils and/or city dumps. Based on our findings, further studies are needed to adequately dispose of these residues in the environment. © 2013 Springer Science+Business Media Dordrecht.

Polymerase chain reaction assay based on ratA gene allows differentiation between Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum

Salmonella Pullorum and Salmonella Gallinarum are classified as biovars of Salmonella enterica subsp. enterica serovar Gallinarum. These salmonellae are the causative agents of Pullorum disease and fowl typhoid, respectively, and are widely distributed throughout the world. Although many developed countries have eradicated these diseases from commercial poultry, they are still the cause of significant economic loss in developing countries. When serovar Gallinarum is isolated, it is difficult to immediately differentiate between biovars because they are antigenically identical by serotyping. However, they cause distinct diseases with different epidemiology, and therefore it is important to differentiate them. This may be done biochemically but takes 2 to 3 days. In the present study, S. Pullorum and S. Gallinarum whole genomes were compared, and 1 genomic region of difference, which is part of the ratA gene, was chosen as a molecular marker for a polymerase chain reaction assay to differentiate rapidly between these organisms. In all, 26 strains of S. Gallinarum and 17 S. Pullorum strains were tested and successfully differentiated by the assay. © 2013 The Author(s).

Microplate alamarblue assay for paracoccidioides susceptibility testing

CLSI method M27-A3 is not available for use with dimorphic fungi, such as those of the Paracoccidioides genus. In this study, we developed a microdilution method and added the alamarBlue reagent to test the responses of Paracoccidioides brasiliensis and Paracoccidioides lutzii against amphotericin B and itraconazole antifungals. The test proved to be sensitive, practical, and inexpensive and can be used to monitor the activity of low-growth microorganisms and their response to various drugs. © 2013, American Society for Microbiology.

Development and validation of a D-loop mtDNA SNP assay for the screening of specimens in forensic casework

Mitochondrial DNA (mtDNA) analysis is usually a last resort in routine forensic DNA casework. However, it has become a powerful tool for the analysis of highly degraded samples or samples containing too little or no nuclear DNA, such as old bones and hair shafts. The gold standard methodology still constitutes the direct sequencing of polymerase chain reaction (PCR) products or cloned amplicons from the HVS-1 and HVS-2 (hypervariable segment) control region segments. Identifications using mtDNA are time consuming, expensive and can be very complex, depending on the amount and nature of the material being tested. The main goal of this work is to develop a less labour-intensive and less expensive screening method for mtDNA analysis, in order to aid in the exclusion of non-matching samples and as a presumptive test prior to final confirmatory DNA sequencing. We have selected 14 highly discriminatory single nucleotide polymorphisms (SNPs) based on simulations performed by Salas and Amigo (2010) [1] to be typed using SNaPShotTM (Applied Biosystems, Foster City, CA, USA). The assay was validated by typing more than 100 HVS-1/HVS-2 sequenced samples. No differences were observed between the SNP typing and DNA sequencing when results were compared, with the exception of allelic dropouts observed in a few haplotypes. Haplotype diversity simulations were performed using 172 mtDNA sequences representative of the Brazilian population and a score of 0.9794 was obtained when the 14 SNPs were used, showing that the theoretical prediction approach for the selection of highly discriminatory SNPs suggested by Salas and Amigo (2010) [1] was confirmed in the population studied. As the main goal of the work is to develop a screening assay to skip the sequencing of all samples in a particular case, a pair-wise comparison of the sequences was done using the selected SNPs. When both HVS-1/HVS-2 SNPs were used for simulations, at least two differences were observed in 93.2% of the comparisons performed. The assay was validated with casework samples. Results show that the method is straightforward and can be used for exclusionary purposes, saving time and laboratory resources. The assay confirms the theoretic prediction suggested by Salas and Amigo (2010) [1]. All forensic advantages, such as high sensitivity and power of discrimination, as also the disadvantages, such as the occurrence of allele dropouts, are discussed throughout the article. © 2013 Elsevier B.V.

Genotoxicological assessment of two reactive dyes extracted from cotton fibres using artificial sweat

Human eyes have a remarkable ability to recognize hundreds of colour shades, which has stimulated the use of colorants, especially for clothing, but toxicological studies have shown that some textile dyes can be hazardous to human health. Under conditions of intense perspiration, dyes can migrate from coloured clothes and penetrate into human skin. Garments made from cotton fabrics are the most common clothing in tropical countries, due to their high temperatures. Aiming to identify safe textile dyes for dyeing cotton fabrics, the genotoxicity [in vitro Comet assay with normal human dermal fibroblasts (NHDF), Tail Intensity] and mutagenicity [Salmonella/microsome preincubation assay (30 min), tester strains TA98, TA100, YG1041 and YG1042] of Reactive Blue 2 (RB2, CAS No. 12236-82-7, C.I. 61211) and Reactive Green 19 (RG19, CAS No. 61931-49-5, C.I. 205075) were evaluated both in the formulated form and as extracted from cotton fibres using different artificial sweats. Both the dyes could migrate from cotton fibres to sweat solutions, the sweat composition and pH being important factors during this extraction. However, the dye sweat solutions showed no genotoxic/mutagenic effects, whereas a weak mutagenic potential was detected by the Ames test for both dyes in their formulated form. These findings emphasize the relevance of textile dyes assessment under conditions that more closely resemble human exposure, in order to recognize any hazard. © 2013 Elsevier Ltd. All rights reserved.

Estrogenic and chemopreventive activities of xanthones and flavones of Syngonanthus (Eriocaulaceae)

The possible benefits of some bioactive flavones and xanthones present in plants of the genus Syngonanthus prompted us to screen them for estrogenic activity. However, scientific research has shown that such substances may have undesirable properties, such as mutagenicity, carcinogenicity and toxicity, which restrict their use as therapeutic agents. Hence, the aim of this study was to assess the estrogenicity and mutagenic and antimutagenic properties. We used recombinant yeast assay (RYA), with the strain BY4741 of Saccharomyces cerevisiae, and Ames test, with strains TA100, TA98, TA97a and TA102 of Salmonella typhimirium, to evaluate estrogenicity, mutagenicity and antimutagenicity of methanolic extracts of Syngonanthus dealbatus (S.d.), Syngonanthus macrolepsis (S.m.), Syngonanthus nitens (S.n.) and Syngonanthus suberosus (S.s.), and of 9 compounds isolated from them (1 = luteolin, 2 = mix of A-1,3,6-trihydroxy-2-methoxyxanthone and B-1,3,6-trihydroxy-2,5- dimethoxyxanthone, 3 = 1,5,7-trihydroxy-3,6-dimethoxyxanthone, 4 = 1,3,6,8-tetrahydroxy-2,5-dimethoxyxanthone, 5 = 1,3,6,8-tetrahydroxy-5- methoxyxanthone, 6 = 7-methoxyluteolin-8-C-β-glucopyranoside, 7 = 7-methoxyluteolin-6-C-β-glucopyranoside, 8 = 7,3′-dimethoxyluteolin- 6-C-β-glucopyranoside and 9 = 6-hydroxyluteolin). The results indicated the estrogenic potential of the S. nitens methanol extract and four of its isolated xanthones, which exhibited, respectively, 14.74 ± 1.63 nM; 19.54 ± 6.61; 7.20 ± 0.37; 6.71 ± 1.02 e 10.01 ± 4.26 nM of estradiol-equivalents (EEQ). None of the extracts or isolated compounds showed mutagenicity in any of the test strains and all of them showed antimutagenic potential, in particular preventing mutations caused by aflatoxin B1 (AFB1) and benzo[a]pyrene (B[a]P). The results show that the xanthones, only isolated from the methanol extract of S. nitens capitula, probably were the responsible for its estrogenic activity and could be useful as phytoestrogens, providing a new opportunity to develop hormonal agents. In addition, flavones and xanthones could also be used as a new antimutagenic agent. Since, the mutagens are involved in the initiation and promotion of several human diseases, including cancer, the significance of novel bioactive phytocompounds in counteracting these pro-mutagenic and carcinogenic effects is now gaining credence. © 2013 Elsevier Inc. All rights reserved.

Antitumour and anti-inflammatory effects of palladium(II) complexes on Ehrlich tumour

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Liposomal-praziquantel: Efficacy against Schistosoma mansoni in a preclinical assay

Currently, schistosomiasis mansoni is treated clinically with praziquantel (PZQ). Nevertheless, cases of tolerance and resistance to this drug have been reported, creating the need to develop new drugs or to improve existing drugs. Considering the small number of new drugs against Schistosoma mansoni, the design of nanotechnology-based drug delivery systems is an important strategy in combating this disease. The aim of this study was to evaluate the activity of PZQ containing liposome (lip.PZQ) on S. mansoni, BH strain. Mice were treated orally with different concentrations of PZQ and lip.PZQ 30 and 45 days following infection. The number of worms, recovered by perfusion of the hepatic portal system, and the number of eggs found in the intestine and liver were analysed. Parasite egg counts were also performed. The most active formulation for all parameters was 300. mg/kg of lip.PZQ, since as it decreased the total number of worms by 68.8%, the number of eggs in the intestine by 79%, and the number of hepatic granulomas by 98.4% compared to untreated controls. In addition, this concentration decreased egg counts by 55.5%. The improved efficacy of the treatment with lip.PZQ, especially when administered 45 days following infection, compared with the positive-control group (untreated) and the groups that received free PZQ, can be explained by greater bioavailability in the host organism; the preferred target of lip.PZQ is the liver, and lip.PZQ is better absorbed by the tegument of S. mansoni, which has an affinity for phospholipids. © 2013 Elsevier B.V.