A release mechanism for stored ATP in ocular ciliary epithelial cells

Purines can modify ciliary epithelial secretion of aqueous humor into the eye. The source of the purinergic agonists acting in the ciliary epithelium, as in many epithelial tissues, is unknown. We found that the fluorescent ATP marker quinacrine stained rabbit and bovine ciliary epithelia but not the nerve fibers in the ciliary bodies. Cultured bovine pigmented and nonpigmented ciliary epithelial cells also stained intensely when incubated with quinacrine. Hypotonic stimulation of cultured epithelial cells increased the extracellular ATP concentration by 3-fold; this measurement underestimates actual release as the cells also displayed ecto-ATPase activity. The hypotonically triggered increase in ATP was inhibited by the Cl−-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in both cell types. In contrast, the P-glycoprotein inhibitors tamoxifen and verapamil and the cystic fibrosis transmembrane conductance regulator (CFTR) blockers glybenclamide and diphenylamine-2-carboxylate did not affect ATP release from either cell type. This pharmacological profile suggests that ATP release is not restricted to P-glycoprotein or the cystic fibrosis transmembrane conductance regulator, but can proceed through a route sensitive to NPPB. ATP release also was triggered by ionomycin through a different NPPB-insensitive mechanism, inhibitable by the calcium/calmodulin-activated kinase II inhibitor KN-62. Thus, both layers of the ciliary epithelium store and release ATP, and purines likely modulate aqueous humor flow by paracrine and/or autocrine mechanisms within the two cell layers of this epithelium.

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.

Glucose release from GLUT2-null hepatocytes: characterization of a major and a minor pathway.

We previously reported that glucose can be released from GLUT2-null hepatocytes through a membrane traffic-based pathway issued from the endoplasmic reticulum. Here, we further characterized this glucose release mechanism using biosynthetic labeling protocols. In continuous pulse-labeling experiments, we determined that glucose secretion proceeded linearly and with the same kinetics in control and GLUT2-null hepatocytes. In GLUT2-deficient hepatocytes, however, a fraction of newly synthesized glucose accumulated intracellularly. The linear accumulation of glucose in the medium was inhibited in mutant, but not in control, hepatocytes by progesterone and low temperature, as previously reported, but, importantly, also by microtubule disruption. The intracellular pool of glucose was shown to be present in the cytosol, and, in pulse-chase experiments, it was shown to be released at a relatively slow rate. Release was not inhibited by S-4048 (an inhibitor of glucose-6-phosphate translocase), cytochalasin B, or progesterone. It was inhibited by phloretin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, and low temperature. We conclude that the major release pathway segregates glucose away from the cytosol by use of a membrane traffic-based, microtubule-dependent mechanism and that the release of the cytosolic pool of newly synthesized glucose, through an as yet unidentified plasma membrane transport system, cannot account for the bulk of glucose release.

Poly(ethylene-co-methyl acrylate)/poly(caprolactone) triol blends for drug delivery systems: characterization and drug release

Poly(ethylene-co-methyl acrylate) (EMA) and poly (caprolactone) triol (PCL-T) blends, a biodegradable aliphatic polyester with low molecular weight and moderate water solubility containing diltiazem hydrochloride (DZ) were studied in terms of the thermal and morphological properties, and drug release mechanism. An increase in the PCL-T content in the EMA/PCL-T/DZ films decreased the degree of DZ crystallinity. Drug release from these films is temperature-dependent, and it is possible to modify the drug release rate by adjusting the EMA/PCL-T composition of the blends. The mechanism of drug release is governed by PCL-T melting and PCL-T leaching from EMA matrix.

Polymeric electro-mechanic devices applied to antibiotic-controlled release

Polymeric electroactive blends formed by electropolymerized aniline inside a non-conductive polyacrylamide porous matrix were already shown as suitable materials for the electrocontrolled release of model compounds like safranin. In this paper the intermolecular interactions between the two components of the blend are put in evidence by Raman spectroscopy measurements. Also, in situ optical microscopy was used to follow changes occurring in the polyaniline/polyacrylamide blend during pyrocathecol violet release tests. These two sets of experiments show the possibility of controlling electrochemically the release of both, safranin (a cation) and pyrocathecol violet (an anion) and allow to infer a release mechanism based on the electromechanical properties of the blends explaining the dependence of the release kinetics on the applied potential. Tetracycline release curves for different potentials and pHs are shown and the obtained profiles are in agreement with those expected for a device acting as an electrochemically driven pump due to the artificial muscle properties of the conducting phase of the blends. (c) 2007 Elsevier B.V. All rights reserved.

Influence of phosphated cross-linked high amylose on in vitro release of different drugs

The influence of structural characteristics of high amylose cross-linked at different degrees on the release of drugs with important molecular differences, namely sodium diclophenac (SD) and nicotinamide (NI), was assessed in vitro from non-compacted systems. The release profiles were related with classical kinetic mathematical models for better understanding of the release mechanism. An increase in polymer cross-linking degree resulted in longer release time for both drugs, although SD generally was released slower than NI. SD release from samples cross-linked at 2% of basis was driven mainly by Fickian diffusion, while from samples cross-linked at 4% of basis follows anomalous mechanism. Inversely, anomalous mechanism was responsible for NI release from 2% samples and Fickian diffusion from 4% samples. Results suggest that the performance of cross-linked high amylose as excipient for controlled drug release not only depends on cross-linking degree but also is highly influenced by structural characteristics of the drug. (C) 2009 Elsevier Ltd. All rights reserved.

Poly(epsilon-caprolactone) nanocapsules carrying the herbicide atrazine: effect of chitosan-coating agent on physico-chemical stability and herbicide release profile

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Insights into the swelling process and drug release mechanisms from cross-linked pectin/high amylose starch matrices

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Microparticles as a Strategy for Low-Molecular-Weight Heparin Delivery

The aims of this work were preparation and physical-chemical characterization of a microparticulate release system for delivery of enoxaparin sodium (ENX), a low-molecular-weight heparin, as a potential vehicle for optimization of deep venous thrombosis therapy. Microparticles (MPs) containing ENX were prepared from polylactide-co-glycolic acid [PLGA; (50: 50)] by a double emulsification/solvent evaporation method. The preparation parameters, such as proportion ENX/PLGA, surfactant concentration, type, time, and speed of stirring, were evaluated. The encapsulation efficiency and yield process were determined and optimized, and the in vitro release profile was analysed at 35 days. The MPs showed a spherical shape with smooth and regular surfaces. The size distribution showed a unimodal profile with an average size of 2.0 +/- 0.9 mu m. The low encapsulation efficiency (< 30%), characteristic of hydrophilic macromolecules was improved, reaching 50.2% with a procedure yield of 71.3%. The in vitro profile of ENX release from the MPs was evaluated and showed pseudo-zero-order kinetics. This indicated that diffusion was the main drug release mechanism. (C) 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:1783-1792, 2011

Mécanismes moléculaires de la sécrétion d'insuline : implication de Slac2c/MyRIP, protéine effectrice de Rab27a, et rôle des phosphoinositides dans le processus d'exocytose

Résumé : La sécrétion de l'insuline en réponse au glucose circulant dans le sang est la fonction principale de la cellule β. La perte de cette fonction est une des caractéristiques du diabète de type 2. L'exocytose est une fonction cellulaire indispensable au renouvellement des composants lipidiques et protéiques de la membrane cellulaire, à la communication entre les cellules et au maintien d'un environnement adéquat. On peut distinguer deux types d'exocytose : l'exocytose constitutive et l'exocytose régulée. Cette dernière est déclenchée par des stimuli externes. L'exocytose régulée est contrôlée au niveau de la fusion des vésicules de sécrétion avec la membrane plasmique. Certains composants moléculaires impliqués dans ce processus font partie de la famille des GTPases Rab. Les deux membres de cette famille impliqués sont Rab3 et Rab27. Nous avons étudié le rôle de la GTPase Rab27 dans les cellules INS-1E, une lignée cellulaire pancréatique β qui sécrète de l'insuline de façon régulée. Nous avons trouvé que la diminution d'expression de la protéine en utilisant le technique de « RNA interference » diminue la sécrétion stimulée, mais que la distribution des granules n'est nullement affectées par ce changement d'activité intrinsèque. Un des effecteurs identifiés de cette GTPase est Slac2c/MyRIP. Cette protéine possède plusieurs domaines fonctionnels dont un qui lui permet de se lier à l'actine, constituant du cytosquelette cellulaire. L'ensemble de nos résultats suggèrent que Rab27 et MyRIP font partie d'un complexe permettant l'interaction de la granule de sécrétion avec le cytosquelette d'actine corticale et participent à la régulation des dernières étapes de l'exocytose d'insuline. Ensuite, nous avons étudié les phosphoinositides (PI). Les phosphoinositides sont d'importantes molécules impliquées dans le régulation du trafic vésiculaire. Nous avons trouvé que le phosphatidylinosito1-4-phosphate (PI4P) et le phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) augmentent la sécrétion sous l'action de 10µM de Ca2+ dans les cellules INS-1E perméabilisées avec la streptolysine-O. En plus, nous avons démontré que l'exocytose est diminuée dans les cellules intactes exprimant une protéine qui séquestre le PI(4,5)P2. Une diminution similaire est observée en diminuant l'expression de deux enzymes impliquées dans la production du PI(4,5)P2, la PI4Kinase β type III et la PIP5Kinase γ type I. Pour clarifier le mécanisme d'action des PI, nous avons investigué l'implication de trois cibles potentielles des PI, la PLD1, CAPS1 et Mint1. Pour ce faire, nous avons réduit le niveau d'expression endogène de ces protéines, ce qui inhibe la libération d'hormones provoquée par le glucose. Tout ceci indique donc que la production du PI(4,5)P2 est nécessaire pour le contrôle de la sécrétion et suggère qu'une partie de l'effet du PI sur la sécrétion pourrait être exercé par l'activation de la PLD1, CAPS1 et Mint1. Abstract Insulin release from pancreatic β-cells plays an essential role in the achievement of blood glucose homeostasis and defects in the regulation of this process lead to profound metabolic disorders and hyperglycaemia (eg. type 2 diabetes). Almost every cell in our organism releases proteins and other biological compounds using a fundamental cellular process known as constitutive exocytosis. In exocrine and endocrine glands, the cells are endowed with an additional and more refined release mechanism directly tuned by extracellular signals. This process, referred to as regulated exocytosis, ensures the timely delivery of molecules such as peptide hormones and digestive enzymes to match the moment¬-to-moment requirements of the organism. Some of the molecular components involved in this process have been identified, including Rab3 and Rab27, two GTPases that regulate the final steps of secretion in many cells. We investigated the involvement of Rab27 GTPase in the secretory process of the insulin-secreting cell line INS-1E. We found that selective reduction of Rab27 expression by RNA interference did not alter granule distribution but impaired exocytosis triggered by insulin secretagogues. Screening for potential effectors revealed that Slac2c/MyRIP is associated with granules and attenuation of Slac2c expression severely impaired hormone release. This protein contains several functional domains, including, a binding domain for the cellular cytoskeleton constituent actin. Taken together our data suggest the Rab27 and MyRIP are part of a complex mediating the interaction of secretory granules with cortical cytoskeleton and participate to the regulation of the final steps in insulin exoctytosis. In the second part of the thesis, we studied phosphoinositides (PI). Phosphoinositides are important molecules involved in the regulation of vesicular trafficking. We found that phosphatidylinosito1-4-phosphate (PI4P) and phosphatidylinosito1-4,5-biphosphate (PI(4,5)P2) increase the secretory response triggered by 10µM Ca2+ in streptolysin-O permeabilized insulin-secreting INS-1E cells. In addition, nutrient-induced exocytosis was diminished in intact cells expressing constructs that sequester PI(4,5)P2. A similar decrease was observed after silencing of two enzymes involved in PI(4,5)P2 production, type III PI4Kinase β and type I PIP5Kinase γ, by RNA interference. To clarify the mechanism of action of PI, we investigated the involvement in the regulation of exocytosis of three potential PI targets, PLD1, CAPS1 and Mint1. Transfection of cells with silencers capable of reducing the endogenous levels of these proteins inhibited hormone release elicited by glucose. Our data indicate that the production PI(4,5)P2 is necessary for proper control of p-cell secretion and suggest that at least part of the effects of PI on insulin exocytosis could be exerted through the activation of PLD1, CAPS1 and Mint1.

β-Adrenergic modulation of skeletal muscle contraction: key role of excitation-contraction coupling.

Our aim is to describe the acute effects of catecholamines/β-adrenergic agonists on contraction of non-fatigued skeletal muscle in animals and humans, and explain the mechanisms involved. Adrenaline/β-agonists (0.1-30 μm) generally augment peak force across animal species (positive inotropic effect) and abbreviate relaxation of slow-twitch muscles (positive lusitropic effect). A peak force reduction also occurs in slow-twitch muscles in some conditions. β2 -Adrenoceptor stimulation activates distinct cyclic AMP-dependent protein kinases to phosphorylate multiple target proteins. β-Agonists modulate sarcolemmal processes (increased resting membrane potential and action potential amplitude) via enhanced Na(+) -K(+) pump and Na(+) -K(+) -2Cl(-) cotransporter function, but this does not increase force. Myofibrillar Ca(2+) sensitivity and maximum Ca(2+) -activated force are unchanged. All force potentiation involves amplified myoplasmic Ca(2+) transients consequent to increased Ca(2+) release from sarcoplasmic reticulum (SR). This unequivocally requires phosphorylation of SR Ca(2+) release channels/ryanodine receptors (RyR1) which sensitize the Ca(2+) -induced Ca(2+) release mechanism. Enhanced trans-sarcolemmal Ca(2+) influx through phosphorylated voltage-activated Ca(2+) channels contributes to force potentiation in diaphragm and amphibian muscle, but not mammalian limb muscle. Phosphorylation of phospholamban increases SR Ca(2+) pump activity in slow-twitch fibres but does not augment force; this process accelerates relaxation and may depress force. Greater Ca(2+) loading of SR may assist force potentiation in fast-twitch muscle. Some human studies show no significant force potentiation which appears to be related to the β-agonist concentration used. Indeed high-dose β-agonists (∼0.1 μm) enhance SR Ca(2+) -release rates, maximum voluntary contraction strength and peak Wingate power in trained humans. The combined findings can explain how adrenaline/β-agonists influence muscle performance during exercise/stress in humans.

Thermoanalytical Characterization and Dissolution Profiles Studies of Metronidazole Tablets

In the present study dissolution tests and thermoanalytical (TA) techniques were applied to metronidazole tablets from five laboratories (R, G, SA, SB, SC) available on the Brazilian market. The TA profiles indicated that in some formulations interactions between components led to eutectic products with lower melting points than metronidazole. The formulations SB and SC showed dissolution profiles that did not agree with published standards, confirming the TA results. All dissolution data were mathematically compared with kinetic models of release, demonstrating the main release mechanism was first order in all the tablets. The formulations were statistically compared by ANOVA and post-hoc tests (Tukey and Newman-Keuls), reveling significant differences in dissolution efficiency (DE).

Micropartículas de poli (ácido lático-co-ácido glicólico) obtidas por spray drying para a liberação prolongada de metotrexato

Methotrexate (MTX) is a drug used in the chemotherapy of some kind of cancers, autoimmune diseases and non inflammatory resistant to corticosteroids uveits. However, the rapid plasmatic elimination limits its therapeutic success, which leads to administration of high doses to maintain the therapeutic levels in the target tissues, occurring potential side effects. The aim of this study was to obtain spray dried biodegradable poly-lactic acid co-glycolic acid (PLGA) microparticles containing MTX. Thus, suitable amounts of MTX and PLGA were dissolved in appropriate solvent system to obtain solutions at different ratios drug/polymer (10, 20, 30 and 50% m/m). The physicochemical characterizing included the quantitative analysis of the drug using a validate UV-VIS spectrophotometry method, scanning electron microscopy (SEM), infrared spectrophotometry (IR), thermal analyses and X-ray diffraction analysis. The in vitro release studies were carried out in a thermostatized phosphate buffer pH 7.4 (0.05 M KH2PO4) medium at 37°C ± 0.2 °C. The in vitro release date was subjected to different kinetics release models. The MTX-loaded PLGA microparticles showed a spherical shape with smooth surface and high level of entrapped drug. The encapsulation efficiency was greater then 80%. IR spectroscopy showed that there was no chemical bond between the compounds, suggesting just the possible occurrence of hydrogen bound interactions. The thermal analyses and X-ray diffraction analysis shown that MTX is homogeneously dispersed inside polymeric matrix, with a prevalent amorphous state or in a stable molecular dispersion. The in vitro release studies confirmed the sustained release for distinct MTX-loaded PLGA microparticles. The involved drug release mechanism was non Fickian diffusion, which was confirmed by Kornmeyer-Peppas kinetic model. The experimental results demonstrated that the MTX-loaded PLGA microparticles were successfully obtained by spray drying and its potential as prolonged drug release system.

In vitro characterization of coevaporates containing chitosan for colonic drug delivery

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Preparation and characterization of poly(ε-caprolactone) nanospheres containing the local anesthetic lidocaine

The objective of this work was to develop a modified release system for the local anesthetic lidocaine (LDC), using poly(ε-caprolactone) (PCL) nanospheres (NSs), to improve the pharmacological properties of the drug when administered by the infiltration route. In vitro experiments were used to characterize the system and investigate the release mechanism. The NSs presented a polydispersion index of 0.072, an average diameter of 449.6nm, a zeta potential of -20.1mV, and an association efficiency of 93.3%. The release profiles showed that the release of associated LDC was slower than that of the free drug. Atomic force microscopy analyses showed that the spherical structure of the particles was preserved as a function of time, as well as after the release experiments. Cytotoxicity and pharmacological tests confirmed that association with the NSs reduced the toxicity of LDC, and prolonged its anesthetic action. This new formulation could potentially be used in applications requiring gradual anesthetic release, especially dental procedures. © 2012 Wiley Periodicals, Inc.