Gastro-resistant Pellets of Didanosine obtained by Extrusion and Spheronization: Assessing the Production Process

The aim this work was develop gastro-resistant pellets of didanosine as well as study the impact on the pellets properties, regarding the way as the binder was added and drying process used. The pellets formation was accompanied by analysis of morphological parameters and didanosine dissolution. In the most cases, pellets showed diameter around 1.0 mm and shape parameters acceptable. The variations of the process did not interfere significantly in pellets size. In turn, drying in fluid bed favored the dissolution of didanosine, in contrast to binder addition on powder form that impaired. In another hand, this last resulted in the best aspect factor (about 1.1). Gastro-resistant pellets showed adequate dissolution, compatible with this type of dosage form. The variables of process studied enabled obtain pellets with characteristics of shape and dissolution just slightly different, indicating flexibility of the formulation for production of gastro-resistant pellets of didanosine.

Development and In Vitro Evaluation of Propranolol Hydrochloride Controlled Release Tablets Using HPMC as Matrix Former

The purpose of this paper was to produce controlled-release matrices with 120 mg of propranolol hydrochloride (PHCl) employing hydroxypropyl methylcellulose (HPMC, Methocel (R) K100) as the gel forming barrier. Although this class of polymers has been commonly used for direct compression, with the intent of use reduced polymer concentrations to achieve controlled drug release, in this study tablets were produced by the wet granulation process. HPMC percentages ranged from 15-34 % and both soluble and non soluble diluents were tested in the 10 proposed tablet compositions. Dissolution testing of matrices was performed over a 12 h period in 1.2 pH medium (the first 2 h) and in pH 6.8 (10 h). Dissolution kinetic analysis was performed by applying Zero-order, First-order and Higuchi models with the aim of elucidating the drug release mechanism. All physical-chemical characteristics such as average weight, friability, hardness, diameter, height, and drug content were in accordance to the pharmacopeial specifications. Taking into account that PHCl is a very soluble drug, low concentrations (15 %) of HPMC were sufficient to reduce the drug release and to promote controlled release of PHCl, presenting good dissolution efficiencies, between 50 % and 63 %. The Higuchi model has presented the best fit to the 15 % HPMC formulations, indicating that the main release mechanism was diffusion. It could be concluded that the application of the wet granulation method reduced matrices erosion and promoted controlled release of the drug at low HPMC percentages.

In Vitro Simultaneous Transfer of Lipids to HDL in Coronary Artery Disease and in Statin Treatment

The exchange of lipids with cells and other lipoproteins is a crucial process in HDL metabolism and for HDL antiatherogenic function. Here, we tested a practical method to quantify the simultaneous transfer to HDL of phospholipids, free-cholesterol, esterified cholesterol and triacylglycerols and to verify the lipid transfer in patients with coronary artery disease (CAD) or undergoing statin treatment. Twenty-eight control subjects without CAD, 27 with CAD and 25 CAD patients under simvastatin treatment were studied. Plasma samples were incubated with a donor nanoemulsion prepared by ultrasonication of the constituent lipids and labeled with radioactive lipids; % lipids transferred to HDL were quantified in the HDL-containing supernatant after chemical precipitation of non-HDL fractions and the nanoemulsion. The assay was precise and reproducible. Increase of temperature (4-37 A degrees C), of incubation period (5 min to 2 h), of HDL-cholesterol concentration (33-244 mg/dL) and of mass of nanoemulsion lipids (0.075-0.3 mg/mu L) resulted in increased lipid transfer from the nanoemulsion to HDL. In contrast, increasing pH (6.5-8.5) and albumin concentration (3.5-7.0 g/dL) did not affect lipid transfer. There was no difference between CAD and control non-CAD with regard to the lipid transfer, but statin treatment reduced the transfer to HDL of all four lipids. The test herein described is a valid and practical tool for exploring an important aspect of HDL metabolism.

WHO comparative evaluation of serologic assays for Chagas disease

Evaluation of commercially available test kits for Chagas disease for use in blood bank screening is difficult due to a lack of large and well-characterized specimen panels. This study presents a collaborative effort of Latin American blood centers and the World Health Organization (WHO) to establish such a panel. A total of 437 specimens, from 10 countries were collected and sent to the WHO Collaborating Center in Sao Paulo and used to evaluate 19 screening assays during 2001 through 2005. Specimens were assigned a positive or negative status based on concordant results in at least three of the four confirmatory assays (indirect immunofluorescence, Western blot, radioimmunoprecipitation assay, and recombinant immunoblot). Of the 437 specimens, 168 (39%) were characterized as positive, 262 (61%) were characterized as negative, and 7 (2%) were judged inconclusive and excluded from the analysis. Sensitivity and specificity varied considerably: 88 to 100 and 60 to 100 percent, respectively. Overall, enzyme immunoassays (EIAs) performed better than the other screening assays. Four EIAs had both parameters higher than 99 percent. Of the four confirmatory assays, only the RIPA gave a 100 percent agreement with the final serologic status of the specimens. The sensitivities and specificities of at least four of the commercially available EIAs for Chagas disease are probably high enough to justify their use for single-assay screening of blood donations. Our data suggest that the majority of commercially available indirect hemagglutination assays should not be used for blood donor screening and that the RIPA could be considered a gold standard for evaluating the performance of other assays.

A new acidic myotoxic, anti-platelet and prostaglandin I(2) inductor phospholipase A(2) isolated from Bothrops moojeni snake venom

Phospholipase A(2) (PLA(2), EC 3.1.1.4), a major component of snake venoms, specifically catalyzes the hydrolysis of fatty acid ester bonds at position 2 of 1,2-diacyl-sn-3-phosphoglycerides in the presence of calcium. This article reports the purification and biochemical/functional characterization of BmooTX-I, a new myotoxic acidic phospholipase A(2) from Bothrops moojeni snake venom. The purification of the enzyme was carried out through three chromatographic steps (ion-exchange on DEAE-Sepharose, molecular exclusion on Sephadex G-75 and hydrophobic chromatography on Phenyl-Sepharose). BmooTX-I was found to be a single-chain protein of 15,000 Da and pI 4.2. The N-terminal sequence revealed a high homology with other acidic Asp49 PLA(2)S from Bothrops snake venoms. It displayed a high phospholipase activity and platelet aggregation inhibition induced by collagen or ADP. Edema and myotoxicity in vivo were also induced by BmooTX-I. Analysis of myotoxic activity was carried out by optical and ultrastructural microscopy, demonstrating high levels of leukocytary infiltrate. Previous treatment of BmooTX-1 with BPB reduced its enzymatic and myotoxic activities, as well as the effect on platelet aggregation. Acidic myotoxic PLA(2)S from Bothrops snake venoms have been little explored and the knowledge of its structural and functional features will be able to contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities. (C) 2008 Elsevier Ltd. All rights reserved.

Aortas isolated from sinoaortic-denervated rats exhibit rhythmic contractions that are regulated by pharmacologically distinct calcium sources

Sinoaortic denervation is characterized by arterial pressure lability, without sustained hypertension. Aortas isolated from rats with sinoaortic denervation present rhythmic contractions. We studied the participation of distinct Ca2+ sources in the maintenance of the oscillations. Three days after the surgeries, aortic rings were placed in an organ chamber, and the incidence of aortas presenting rhythmic contractions was measured. Specific drugs were employed to analyse the participation of the Ca2+ released from the sarcoplasmic reticulum [2-APB (diphenylborinic acid 2-aminoethyl ester), thapsigargin and ryanodine] and external Ca2+ entry [Bay K 8644, verapamil and DMB (dimethylbenzyl amiloride)] on the rhythmic contractions. Additionally, we verified the effects of chloride channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)benzoic acid] on the maintenance of the rhythmic contractions. Under phenylephrine stimulus, sinoaortic-denervated rat aortas exhibited rhythmic contractions in the frequency of 4.5 +/- 0.50 cycles/min. and an amplitude of 0.465 +/- 0.05 g. 2-APB, thapsigargin and ryanodine inhibited the rhythmic contractions. Bay K 8644 increased the oscillations, reaching maximum values with a concentration of 50 nM (18.5 +/- 2.5 cycles/min.). The rhythmic contractions were inhibiting by verapamil and Ca2+-free solution. DMB and NPPB did not alter the oscillations. In conclusion, we observed that aorta isolated from sinoaortic-denervated rats present rhythmic contractions. Moreover, drugs that impaired intracellular Ca2+ release from sarcoplasmic reticulum interrupted the oscillations. The oscillations also depend on the extracellular Ca2+ entry through L-type Ca2+.

The semi-synthetic kaurane ent-16 alpha-methoxykauran-19-oic acid induces vascular relaxation and hypotension in rats

The present work investigates the mechanisms involved in the vasorelaxant effect of ent-16 alpha-methoxykauran-19-oic acid (KA-OCH(3)), a semi-synthetic derivative obtained from the kaurane-type diterpene ent-kaur-16-en-19-oic acid (kaurenoic acid). Vascular reactivity experiments were performed in aortic rings isolated from male Wistar rats using standard muscle bath procedures. The cytosolic calcium concentration ([Ca(2+)]c) was measured by confocal microscopy using the fluorescent probe Fluo-3 AM. Blood pressure measurements were performed in conscious rats. KA-OCH(3) (10,50 and 100 mu mol/l) inhibited phenylephrine-induced contraction in either endothelium-intact or endothelium-denuded rat aortic rings. KA-OCH(3) also reduced CaCl(2)-induced contraction in a Ca(2+)-free solution containing KCl (30 mmol/l) or phenylephrine (0.1 mu mol/l). KA-OCH(3) (0.1-300 mu mol/l) concentration-dependently relaxed endothelium-intact and endothelium-denuded aortas pre-contracted with either phenylephrine or KCl, to a greater extent than kaurenoic acid. Moreover, a Ca(2+) mobilisation study showed that KA-OCH(3) (100 mu mol/l) inhibited the increase in Ca(2+) concentration in smooth muscle and endothelial cells induced by phenylephrine or KCl. Pre-incubation of intact or denuded aortic rings with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mu mol/l), 7-nitroindazole (100 mu mol/l), wortmannin (0.5 mu mol/l) and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ 1 mu mol/l) produced a rightward displacement of the KA-OCH(3) concentration-response curve. Intravenous administration of KA-OCH(3) (1-10 mg/kg) reduced mean arterial blood pressure in normotensive rats. Collectively, our results show that KA-OCH(3) induces vascular relaxation and hypotension. The mechanisms underlying the cardiovascular actions of KA-OCH(3) involve blockade of Ca(2+) influx and activation of the NO-cGMP pathway. (C) 2011 Elsevier B.V. All rights reserved.

Extracellular alkalinization induces endothelium-derived nitric oxide dependent relaxation in rat thoracic aorta

Aim: To investigate the mechanism through which the extracellular alkalinization promotes relaxation in rat thoracic aorta. Methods: The relaxation response to NaOH-induced extracellular alkalinization (7.4-8.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10(-6) M). The vascular reactivity experiments were performed in endothelium-intact and -denuded rings, in the presence or and absence of indomethacin (10(-5) M), NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide/HCl (W-7, 10(-7) M), 2,5-dimethylbenzimidazole (DMB, 2 x 10(-5) M) and methyl-B-cyclodextrin (10(-2) M). In addition, the effects of NaOH-induced extracellular alkalinization (pH 8.0 and 8.5) on the intracellular nitric oxide (NO) concentration was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 mu M), in the presence and absence of DMB (2 x 10(-5) M). Results: The extracellular alkalinization failed to induce any change in vascular tone in aortic rings pre-contracted with KCl. In rings pre-contracted with Phe, the extracellular alkalinization caused relaxation in the endothelium-intact rings only, and this relaxation was maintained after cyclooxygenase inhibition; completely abolished by the inhibition of nitric oxide synthase (NOS), Ca(2+)/calmodulin and Na(+)/Ca(2+). exchanger (NCX), and partially blunted by the caveolae disassembly. Conclusions: These results suggest that, in rat thoracic aorta, that extracellular alkalinization with NaOH activates the NCX reverse mode of endothelial cells in rat thoracic aorta, thereby the intracellular Ca(2+) concentration and activating the Ca(2+)/calmodulin-dependent NOS. In turn, NO is released promoting relaxation. (C) 2010 Elsevier Inc. All rights reserved.

Mechanisms underlying the vasorelaxant action of the pimarane ent-8(14),15-pimaradien-3 beta-ol in the isolated rat aorta

Pimarane-type diterpenes were described to exert antispasmodic and relaxant activities. Based on this observation we hypothesized that the diterpene ent-8(14),15-pimaradien-3 beta-ol (PA-3 beta-ol) induced vascular relaxation. With this purpose, the present work investigates the mechanisms involved in the vasorelaxant effect of the pimarane-type diterpene PA-3 beta-ol. Vascular reactivity experiments, using standard muscle bath procedures, were performed in isolated aortic rings from male Wistar rats. Cytosolic calcium concentration ([Ca(2+)]c) was measured by confocal microscopy using the fluorescent probe Fluo-3AM. PA-3 beta-ol (10, 50 and 100 mu mol/l) inhibited phenylephrine and KCl-induced contraction in either endothelium-intact or denuded rat aortic rings. PA-3 beta-ol also reduced CaCl(2)-induced contraction in Ca(2+)-free solution containing KCl (30 mmol/l) or phenylephrine (0.1 mu mol/l). PA-3 beta-ol (1-300 mu mol/l) concentration dependently relaxed phenylephrine-pre-contracted rings with intact or denuded endothelium. The diterpene also relaxed KCl-pre-contracted rings with intact or denuded endothelium. Moreover, Ca(2+) mobilization study showed that PA-3 beta-ol (100 mu mol/l) and verapamil (1 mu mol/l) inhibited the increase in Ca(2+)-concentration in smooth muscle and endothelial cells induced by phenylephrine (10 mu mol/l) or KCl (60 mmol/l). Pre-incubation of intact or denuded aortic rings with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mu mol/l) and 1H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ 1 mu mol/l) produced a rightward displacement of the PA-3 beta-ol concentration-response curves. On the other hand, 7-nitroindazole (100 mu mol/l), 1400 W (1 mu mol/l), indomethacin (10 mu mol/l) and tetraethylammonium (1 mmol/l) did not affect PA-3 beta-ol-induced relaxation. Collectively, our results provide evidence that the effects elicited by PA-3 beta-ol involve extracellular Ca(2+) influx blockade. Its effects are also partly mediated by the activation of NO-cGMP pathway. (C) 2009 Elsevier B.V. All rights reserved.

Antimicrobial activity of kaurane diterpenes against oral pathogens

Two kaurane diterpenes, ent-kaur-16(17)-en-19-oic acid (KA) and 15-beta-isovaleryloxy-ent-kaur-16(17)-en-19-oic acid (KA-Ival), isolated from Aspilia foliacea, and the methyl ester derivative of KA (KA-Me) were evaluated against oral pathogens. KA was the most active compound, with MIC values of 10 mu g mL(-1) against the following microorganisms: Streptococcus sobrinus, Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, and Lactobacillus casei. However, KA did not show significant activity against Streptococcus salivarius and Enterococcus faecalis, with MIC values equal to 100 and 200 mu g mL(-1), respectively. Our results show that KA has potential to be used as a prototype for the discovery of new effective anti-infection agents against microorganisms responsible for caries and periodontal diseases. Moreover, these results allow to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to studies on the structure-activity relationship of this type of metabolites with respect to caries and periodontal diseases.

Partial characterization of proteins from baru (Dipteryx alata Vog) seeds

BACKGROUND: Baru (Dipteryx alata Vog.) is a fruit distributed throughout the Brazilian savanna and contains a seed with a high protein content, whose properties have been rarely explored. The purpose of this study was to characterize this protein, especially by isolation and quantifying its fractions and measuring some of its molecular properties. RESULTS: Baru seeds contain 244 g kg(-1) protein on a dry weight basis. Solubility profiles showed a preponderance of globulins. This fraction dominated the seed composition, with 61.7 wt% of the total soluble proteins. Albumins and glutelins accounted for 14 and 3.3 wt%, respectively. SDS-PAGE resolution of albumin and globulin showed main bands with molecular weights of 84 kDa and 64,66 and 73 kDa, respectively. The total protein of the flour and the globulin showed values of in vitro digestibility of 85.59% and 90.54%, relative to casein. Total globulin produced only one chromatographic peak, both on Sepharose CL-6B gel filtration and on DEAE-cellulose ion-exchange columns, eluted at a concentration of 0.12 mol L(-1) NaCl. CONCLUSION: The baru seed had high protein content with large quantities of storage proteins. The chromatographic and solubility profiles indicate the predominance of a fraction with characteristics of a legumin-type protein. (C) 2011 Society of Chemical Industry

In vitro Fluoride Release and Tensile Bond Strength of a Polymeric Intra-Buccal Bioadhesive

The intra-buccal polymeric bioadhesive systems that can stay adhered to the oral soft tissues for drug programmed release, with the preventive and/or therapeutic purpose has been employed for large clinical situations. A system based on hydroxypropyl methyl cellulose/Carbopol 934`/magnesium stearate (HPMC/Cp/StMg) was developed having the sodium fluoride as active principle. This kind of system was evaluated according to its resistance to the removal by means of physical test of tensile strength. Swine buccal mucosa extracted immediately after animals` sacrifice was employed as substrate for the physical trials, to obtain 16 test bodies. Artificial saliva with or without mucin was used to involve the substrate/bioadhesive system sets during the trials. Artificial salivas viscosity was determined by means of Brookfield viscometer, showing the artificial saliva with mucin 10.0 cP, and the artificial saliva without mucin 7.5 cP. The tensile strength assays showed the following averages: for the group ""artificial saliva with mucin"" - 12.89 Pa, and for the group ""without mucin"" - 12.35 Pa. Statistical analysis showed no significant difference between the assays for both artificial salivas, and it was possible to conclude that the variable mucin did not interfered with the bioadhesion process for the polymeric devices. The device was able to release fluoride in a safe, efficient and constant way up to 8 hours.

Effects of dehydroepiandrosterone-sulfate (DHEA-S) and benznidazole treatments during acute infection of two different Trypanosoma cruzi strains

A significant role for hormones in regulating the balance of Th1- and Th2-associated cytokines with a role in modulating diseases has been accumulating. Previously, we reported that dehydroepiandrosterone (DHEA), the most abundant steroid hormone synthesized by the adrenal cortex, markedly reduced the blood and tissue parasites in experimentally Trypanosoma cruzi-infected rats. Based on these findings, the main purpose of this study was to investigate the effect of dehydroepiandrosterone-sulfate ester (DHEA-S) therapy alone or in combination with benznidazole (BNZ) (recommended in Brazil for the treatment of T. cruzi infection) will be effective during the acute phase of two different lineages of T. cruzi strains: type I (Y strain) and type II (Bolivia strain) of T. cruzi. Administration of either DHEA-S or BNZ alone or in combination significantly reduced the Y strain parasite load as compared with untreated. Furthermore treatment with DHEA-S resulted in Bolivia strain clearance. This protective effect of DHEA-S was associated with the host`s immune response, as evidence by enhanced levels of interferon-gamma and interleukin-2. DHEA-S treatment also increased peritoneal macrophages levels and nitrite production. DHEA-S treatment was effective in reducing the mortality rate as compared to BNZ alone or to combiner DHEA-S+BNZ treatment of T. cruzi Bolivia strain infected animals. These findings suggest that hormonal therapy may have a protective effect in the treatment of T. cruzi infection. (C) 2009 Elsevier GmbH. All rights reserved.

Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus Paecilomyces variotii

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 degrees C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 degrees C, with a t(50) of 45 min at 60 degrees C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl alpha-D-maltoside, methyl-alpha-D-glucopyranoside, pullulan, alpha- and beta-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in alpha-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-alpha-D-glucan glucohydrolase).

Purification and Biochemical Characterization of Thermostable Alkaline Phosphatases Produced by Rhizopus microsporus var. rhizopodiformis

The biochemical properties of the alkaline phosphatases (AIPs) produced by Rhizopus micro-sporus are described. High enzymic levels were produced within 1-2 d in agitated cultures with 1% wheat bran. Intra- and extracellular AlPs were purified 5.0 and 9.3x, respectively, by DEAE-cellulose and ConA-sepharose chromatography. Molar mass of 118 and 120 kDa was estimated by gel filtration for both forms of phosphatases. SDS-PAGE indicated dimeric structures of 57 kDa for both forms. Mn(2+), Na(+) and Mg(2+) Stimulated the activity, while Al(3+) and Zn(2+) activated only the extracellular form. Optimum temperature and pH for both phosphatases were 65 degrees C and pH 8.0, respectively. The enzymes were stable at 50 degrees C for at least 15 min. Hydrolysis of 4-nitrophenyl phosphate exhibited a K(m) 0.28 and 0.22 mmol/L, with upsilon(lim) 5.89 and 4.84 U/mg, for intra- and extracellular phosphatases, respectively. The properties of the reported AlPs may be suitable for biotechnological application.