Desenvolvimento de blendas de dextranos de pesos moleculares 70 e 148 KDA para a obtenção de hidrogéis contendo praziquantel

Pós-graduação em Ciências Farmacêuticas - FCFAR

In Vitro Antiparasitic Activity and Chemical Composition of the Essential Oil Obtained from the Fruits of Piper cubeba

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação de sistemas lipidicos nanoestruturados utilizados como estratégia para melhorar as propriedades biofarmaceuticas do praziquantel no tratamento da esquistossomose

Pós-graduação em Nanotecnologia Farmacêutica - FCFAR

Nanostructured Lipid Carriers as a Strategy to Improve the In Vitro Schistosomiasis Activity of Praziquantel

Praziquantel (PZQ) is a pyrazinoisoquinoline anthelmintic that was discovered in 1972 by Bayer Germany. Currently, due to its efficacy, PZQ is the drug of choice against all species of Schistosoma. Although widely used, PZQ exhibits low and erratic bioavailability because of its poor water solubility. Nanostructured lipid carriers (NLC), second-generation solid lipid nanoparticles, were developed in the 1990s to improve the bioavailability of poorly water soluble drugs. The aim of this study was to investigate nanostructured lipid carriers as a strategy to improve the efficacy. of PZQ in S. mansoni treatment. We prepared NLC2 and NLC4 by adding seventy percent glycerol monostearate (GMS) as the solid lipid, 30% oleic acid (OA) as the liquid lipid and two surfactant systems containing either soybean phosphatidylcholine/poloxamer (PC/P-407) or phosphatidylcholine/Tween 60 (PC/T60), respectively. The carriers were characterized by nuclear magnetic resonance, differential scanning calorimetry, thermogravimetric analysis and Fourier transform-infrared spectroscopy. The safety profile was evaluated using red cell hemolysis and in vitro cytotoxicity assays. The results showed that the encapsulation of PZQ in NLC2 or NLC4 improved the safety profile of the drug. Treatment efficacy was evaluated on the S. mansoni BH strain. PZQ-NLC2 and PZQ-NLC4 demonstrated an improved efficacy in comparison with free PZQ. The results showed that the intestinal transport of free PZQ and PZQ-NLC2 was similar. However, we observed that the concentration of PZQ absorbed was smaller when PZQ was loaded in NLC4. The difference between the amounts of absorbed PZQ could indicate that the presence of T60 in the nanoparticles (NLC4) increased the rigid lipid matrix, prolonging release of the drug. Both systems showed considerable in vitro activity against S. mansoni, suggesting that these systems may be a promising platform for the administration of PZQ for treating schistosomiasis.

Effectiveness of hyperbaric oxygen for experimental treatment of schistosomiasis mansoni using praziquantel-free and encapsulated into liposomes: Assay in adult worms and oviposition

The treatment of schistosomiasis depends on a single drug: praziquantel (PZQ). However, this treatment presents limitations such as low and/or erratic bioavailability that can contribute to cases of tolerance. Improvements to the available drug are urgently needed and studies with a controlled system of drug release, like liposomes, have been gaining prominence. The present study evaluated the activity and synergy between liposomal-praziquantel (lip.PZQ) and hyperbaric oxygen therapy (HBO). Mice received doses of 60 or 100mg/kg PZQ or lip.PZQ, 50 days post-infection, and after the treatment, were exposed to HBO (3 atmosphere absolute - ATA) for 1h. The viability of adult worms and oviposition were analyzed, by necropsy and Kato-Katz examination performed after 15 days of treatment. A concentration of 100mg/kg of lip.PZQ+HBO was more effective (48.0% reduction of worms, 83.3% reduction of eggs/gram of feces) and 100% of the mice had altered of oograms (indicating interruption of oviposition) compared to other treatments and to the Control group (infected and untreated). It is known that PZQ requires participation of the host immune system to complete its antischistosomal activity and that HBO is able to stimulate the immune system. The drug became more available in the body when incorporated into liposomes and, used with HBO, the HBO worked as an adjuvant. This explains the decreases of oviposition and worms recovered form hepatic portal system.

In Vitro Schistosomicidal Activity of Balsaminol F and Karavilagenin C

Five cucurbitane-type triterpenes (1-5), previously isolated from the African medicinal plant Momordica balsamina, along with five ester derivatives (6-10) of karavilagenin C (2), were evaluated for their potential schistosomicidal activity against Schistosoma mansoni adult worms. The natural compounds were isolated from the ethyl acetate-soluble fraction of the methanol extract of the aerial parts of M. balsamina. In a preliminary study, a significant schistosomicidal activity was observed for both the crude methanol extract and the ethyl acetate fraction. The compounds responsible for the activity were found to be balsaminol F (1) and karavilagenin C (2) with LC50 values of 14.7 +/- 1.5 and 28.9 +/- 1.8 mu M, respectively, after 24 h of incubation (positive control praziquantel, LC50 = 1.2 +/- 0.1 mu M). Both compounds (1, 2), at 10-50 mu M, induced significant reductions in the motor activity of the worms and significantly decreased the egg production. Furthermore, they were able (at 10-100 mu M) to separate the adult worm pairs into male and female after 24 h. Compounds 3-5, bearing a sugar moiety as a substituent, and the acylated derivatives of karavilagenin C (6-10) were inactive, suggesting that the presence of free hydroxyl groups in the tetracyclic skeleton might be important for the activity. A correlation between activity and the molecular volume/weight of compounds was also found.

In Vitro Schistosomicidal Activity of Some Brazilian Cerrado Species and Their Isolated Compounds

Miconia langsdorffii Cogn. (Melastomataceae), Roupala montana Aubl. (Proteaceae), Struthanthus syringifolius (Mart.) (Loranthaceae), and Schefflera vinosa (Cham. & Schltdl.) Frodin (Araliaceae) are plant species from the Brazilian Cerrado whose schistosomicidal potential has not yet been described. The crude extracts, fractions, the triterpenes betulin, oleanolic acid, ursolic acid and the flavonoids quercetin 3-O-beta-D-rhamnoside, quercetin 3-O-beta-D-glucoside, quercetin 3-O-beta-D-glucopyranosyl-(1-2)-alpha-L-rhamnopyranoside and isorhamnetin 3-O-beta-D-glucopyranosyl-(1-2)-alpha-L-rhamnopyranoside were evaluated in vitro against Schistosoma mansoni adult worms and the bioactive n-hexane fractions of the mentioned species were also analyzed by GC-MS. Betulin was able to cause worm death percentage values of 25% after 120 h (at 100 mu M), and 25% and 50% after 24 and 120 h (at 200 mu M), respectively; besides the flavonoid quercetin 3-O-beta-D-rhamnoside promoted 25% of death of the parasites at 100 mu M. Farther the flavonoids quercetin 3-O-beta-D-glucoside and quercetin 3-O-beta-D-rhamnoside at 100 mu M exhibited significantly reduction in motor activity, 75% and 87.5%, respectively. Biological results indicated that crude extracts of R. montana, S. vinosa, and M. langsdorffii and some n-hexane and EtOAc fractions of this species were able to induce worm death to some extent. The results suggest that lupane-type triterpenes and flavonoid monoglycosides should be considered for further antiparasites studies.

Fungal Transformation and Schistosomicidal Effects of Pimaradienoic Acid

The schistosomicidal effects of pimaradienoic acid (PA) and two derivatives, obtained by fungal transformation in the presence of Aspergillus ochraceus, were investigated. PA was the only compound with antischistosomal activity among the three diterpenes studied, with the ability to significantly reduce the viability of the parasites at concentrations ranging from 25 to 100 mu M. PA also promoted morphological alterations of the tegument of Schistosoma mansoni, separated all the worm couples, and affected the production and development of eggs. Moreover, this compound was devoid of toxicity toward human fibroblasts. In a preliminary in vivo experiment, PA at a dose of 100 mg/kg significantly diminished the number of parasites in infected Balb/c mice. Taken together, these results show that PA may be potentially employed in the discovery of novel schistosomicidal agents, and that diterpenes are an important class of natural compounds for the investigation of agents capable of fighting the parasite responsible for human schistosomiasis.

Structure- and ligand-based drug design approaches for neglected tropical diseases

Drug discovery has moved toward more rational strategies based on our increasing understanding of the fundamental principles of protein-ligand interactions. Structure( SBDD) and ligand-based drug design (LBDD) approaches bring together the most powerful concepts in modern chemistry and biology, linking medicinal chemistry with structural biology. The definition and assessment of both chemical and biological space have revitalized the importance of exploring the intrinsic complementary nature of experimental and computational methods in drug design. Major challenges in this field include the identification of promising hits and the development of high-quality leads for further development into clinical candidates. It becomes particularly important in the case of neglected tropical diseases (NTDs) that affect disproportionately poor people living in rural and remote regions worldwide, and for which there is an insufficient number of new chemical entities being evaluated owing to the lack of innovation and R&D investment by the pharmaceutical industry. This perspective paper outlines the utility and applications of SBDD and LBDD approaches for the identification and design of new small-molecule agents for NTDs.

Synthesis, Biological Evaluation, And Molecular Modeling of Chalcone Derivatives As Potent Inhibitors of Mycobacterium tuberculosis Protein Tyrosine Phosphatases (PtpA and PtpB)

Tuberculosis (TB) is a major infectious disease caused by Mycobacterium tuberculosis (Mtb). According to the World Health Organization (WHO), about 1.8 million people die from TB and 10 million new cases are recorded each year. Recently, a new series of naphthylchalcones has been identified as inhibitors of Mtb protein tyrosine phosphatases (PTPs). In this work, 100 chalcones were designed, synthesized, and investigated for their inhibitory properties against MtbPtps. Structure-activity relationships (SAR) were developed, leading to the discovery of new potent inhibitors with IC50 values in the low-micromolar range. Kinetic studies revealed competitive inhibition and high selectivity toward the Mtb enzymes. Molecular modeling investigations were carried out with the aim of revealing the most relevant structural requirements underlying the binding affinity and selectivity of this series of inhibitors as potential anti-TB drugs.

Modulation of Lung Allergic Response by Renal Ischemia and Reperfusion Injury

The Th1/Th2 balance represents an important factor in the pathogenesis of renal ischemia-reperfusion injury (IRI). In addition, IRI causes a systemic inflammation that can affect other tissues, such as the lungs. To investigate the ability of renal IRI to modulate pulmonary function in a specific model of allergic inflammation, C57Bl/6 mice were immunized with ovalbumin/albumen on days 0 and 7 and challenged with an ovalbumin (OA) aerosol on days 14 and 21. After 24 h of the second antigen challenge, the animals were subjected to 45 minutes of ischemia. After 24 h of reperfusion, the bronchoalveolar lavage (BAL) fluid, blood and lung tissue were collected for analysis. Serum creatinine levels increased in both allergic and non-immunized animals subjected to IRI. However, BAL analysis showed a reduction in the total cells (46%) and neutrophils (58%) compared with control allergic animals not submitted to IRI. In addition, OA challenge induced the phosphorylation of ERK and Akt and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lung homogenates. After renal IRI, the phosphorylation of ERK and expression of COX-2 and iNOS were markedly reduced; however, there was no difference in the phosphorylation of Akt between sham and ischemic OA-challenged animals. Mucus production was also reduced in allergic mice after renal IRI. IL-4, IL-5 and IL-13 were markedly down-regulated in immunized/challenged mice subjected to IRI. These results suggest that renal IRI can modulate lung allergic inflammation, probably by altering the Th1/Th2 balance and, at least in part, by changing cellular signal transduction factors. Copyright (C) 2012 S. Karger AG, Basel

Evaluation of the schistosomicidal activity of the steroidal alkaloids from Solanum lycocarpum fruits

Solanum lycocarpum (Solanaceae), a Brazilian medicinal plant known as "wolf fruit," contains about 1.5% of glycoalkaloids in its dried fruits, consisting mainly of solamargine and solasonine. The present work reports the obtainment of the alkaloidic extract of the S. lycocarpum fruit by acid-base extraction and the isolation of the major alkaloid heterosides by chromatographic means, as well as the evaluation of their in vitro schistosomicidal activities. The in vitro schistosomicidal activities of the alkaloidic extract of S. lycocarpum fruits and its isolated steroidal alkaloids were undertaken against adult worms of Schistosoma mansoni. The alkaloidic extract (20, 32, and 50 mu g mL(-1)), solasonine (50 mu M), solamargine (32 and 50 mu M), and equimolar mixture of glycoalkaloids (20, 32, and 50 mu M) lead to the separation of all couple worms and extensive disruption on their teguments, such as sloughing, as well as their deaths within 24 h of incubation. In addition, the alkaloidic extract (10 and 15 mu g mL(-1)), solasonine (50 mu M), solamargine (10, 15, and 20 mu M), and equimolar mixtures of glycoalkaloids (10 and 15 mu M) reduced the development of eggs produced by the adult worms. Solamargine, containing the sugar chain moiety chacotriose, was more active than the solasonine, which contains solatriose sugar chain moiety. A synergistic effect was also observed for a mixture of solamargine and solasonine. Therefore, the alkaloidic extract of S. lycocarpum, and its major components, solamargine and solasonine, showed promising schistosomicidal activity.

Molluscicidal activity of some marine substances against the snail Biomphalaria glabrata (Mollusca, Planorbidae)

Freshwater snails of the genus Biomphalaria play a major role as intermediate hosts of Schistosoma mansoni, the etiologic agent of schistosomiasis. While Biomphalaria spp. control by molluscicides is one of the main strategies to reduce the snail population in infected areas, there are few effective molluscicides commercially available. Natural products may be considered as potentially useful and safe molluscicides. We have evaluated the molluscicidal activity of 12 extracts from ten marine organisms on adult and embryonic stages of Biomphalaria glabrata. Only extracts of the red algae Liagora farinosa and of the sponge Amphimedon viridis presented molluscicidal activity. Lethal concentration (LC)(50) values obtained were 120 mu g/mL for L. farinosa CH2Cl2 extract (apolar fraction) and 20 mu g/mL for A. viridis extract and halitoxin. The polar alga fraction and halitoxin had no effect on B. glabrata embryos. The algae apolar fraction was active on B. glabrata in all embryonic development stages, with LC50 values for blastulae at 42 mu g/mL, gastrulae at 124 mu g/mL, trochophore at 180 mu g/mL, and veliger at 222 mu g/mL. This is the first report of extracts from marine organisms which presented molluscicidal activity.

Curcumin-loaded into PLGA nanoparticles

The incorporation of the curcumin into poly(lactic-co-glycolic)acid (PLGA) nanospheres by the nanoprecipitation technique, the characterization of the nanoparticles and the schistosomicidal activity of the curcumin-loaded into PLGA nanospheres were reported. The incorporation process occurred with high efficiency and the images of field-emission scanning electron microscopy (FESEM) revealed the production of spherically shaped particles. According to the dynamic light scattering measurements, the particles are nanometric and monodisperse. The curcumin-loaded PLGA nanoparticles (50 and 100 mu M) caused the death of all worms and a separation between 50% and 100% of Schistosoma mansoni couples at concentrations from 30 mu M. Moreover, the curcumin-loaded PLGA nanoparticles also decreased the motor activity and caused partial alterations in the tegument of adult worms. This study marks the first time that schistosomicidal activity has been reported for curcumin-loaded PLGA nanoparticles.

IgG Antibody responses in mice coinfected with Toxocara canis and other helminths or protozoan parasites

The immune response expressed by IgG antibodies in BALB/c mice experimentally infected with Toxocara canis, was studied with the aim of verifying the possible in vivo cross-reactivity between antigens of T. canis and other parasites (Ascaris suum, Taenia crassiceps, Schistosoma mansoni, Strongyloides venezuelensis and Toxoplasma gondii). Experiments included three groups of mice: one infected only by T. canis, another with one of the other species of parasites and a third concomitantly infected with T. canis and the other species in question. Animals were bled by orbital plexus at 23, 38 and 70 days post infection (p.i.). Sera were analyzed for anti-Toxocara antibodies by ELISA and Immunoblotting, using excretion-secretion antigens (ES), obtained from culture of third-stage larvae of T. canis. For all experiments a control group comprised by ten non-infected mice was used. Only in the case of A. suum infection, in these experimental conditions, the occurrence of cross-reactivity with T. canis was observed. However, in the case of co-infection of T. canis - S. mansoni, T. canis - S. venezuelensis and T. canis - T. crassiceps the production of anti-Toxocara antibodies was found at levels significantly lower than those found in mice infected with T. canis only. Co-infection with S. mansoni or S. venezuelensis showed lower mortality rates compared to what occurred in the animals with single infections. Results obtained in mice infected with T. canis and T. gondii showed significant differences between the mean levels of the optical densities of animals infected with T. canis and concomitantly infected with the protozoan only in the 23rd day p.i.