Development, physical-chemical stability and release studies of four alcohol-free spironolactone suspensions for use in pediatrics

Dissolution studies have become of great significance because, in most cases, drug dissolution is the rate-limiting step in the absorption process. As occurs with solid oral dosage forms, heterogeneous disperse systems (suspensions) could also have some problems with their in vitro dissolution. The objective of this study was to evaluate influence of the excipients on the release of spironolactone from four alcohol free suspensions (pharmaceutical compounding) of spironolactone 5 mg/mL suitable for pediatric use. Also the comparison of the physical and chemical stability of the suspensions stored at 4, 25 and 40 ºC over a 60- day period has been studied. Rheological behavior, particle size, a prediction of long-term physical stability, pH and assay of spironolactone by HPLC were assessed at prefixed times. The dissolution profile of each suspension was determined and compared with that of the commercial tablets. A microbiological study of the best formula was also performed. Chemically, the four spironolactone suspensions were stable for 60 days stored at three temperatures; Suspension IV had optimum pH values and the highest recovery percentage. In terms of physical stability, sedimentation occurred in Suspension IV and flotation of spironolactone in Suspensions I, II and III. Suspension III had the highest viscosity and the slowest drug release. Suspension IV was also microbiologically stable for 60 days. In conclusion, Suspension IV had the best properties and the least suitable form was Suspension III, as its high viscosity made it difficult to achieve homogeneous redispersion, and it had the slowest dissolution profile.

Conditional BDNF release under pathological conditions improves Huntington's disease pathology by delaying neuronal dysfunction

Background Brain-Derived Neurotrophic Factor (BDNF) is the main candidate for neuroprotective therapy for Huntington's disease (HD), but its conditional administration is one of its most challenging problems. Results Here we used transgenic mice that over-express BDNF under the control of the Glial Fibrillary Acidic Protein (GFAP) promoter (pGFAP-BDNF mice) to test whether up-regulation and release of BDNF, dependent on astrogliosis, could be protective in HD. Thus, we cross-mated pGFAP-BDNF mice with R6/2 mice to generate a double-mutant mouse with mutant huntingtin protein and with a conditional over-expression of BDNF, only under pathological conditions. In these R6/2:pGFAP-BDNF animals, the decrease in striatal BDNF levels induced by mutant huntingtin was prevented in comparison to R6/2 animals at 12 weeks of age. The recovery of the neurotrophin levels in R6/2:pGFAP-BDNF mice correlated with an improvement in several motor coordination tasks and with a significant delay in anxiety and clasping alterations. Therefore, we next examined a possible improvement in cortico-striatal connectivity in R62:pGFAP-BDNF mice. Interestingly, we found that the over-expression of BDNF prevented the decrease of cortico-striatal presynaptic (VGLUT1) and postsynaptic (PSD-95) markers in the R6/2:pGFAP-BDNF striatum. Electrophysiological studies also showed that basal synaptic transmission and synaptic fatigue both improved in R6/2:pGAP-BDNF mice. Conclusions These results indicate that the conditional administration of BDNF under the GFAP promoter could become a therapeutic strategy for HD due to its positive effects on synaptic plasticity.

Membrane-destabilizing activity of pH-responsive cationic lysine-based surfactants: role of charge position and alkyl chain length

Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Here, we report novel cationic lysine-based surfactants (hydrochloride salts of N¿- and N¿-acyl lysine methyl ester) that differ in the position of the positive charge and the length of the alkyl chain. Amino acid-based surfactants could be promising novel biomaterials in drug delivery systems, given their biocompatible properties and low cytotoxic potential. We examined their ability to disrupt the cell membrane in a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model of endosomal membranes. Furthermore, we addressed the mechanism of surfactant-mediated membrane destabilization, including the effects of each surfactant on erythrocyte morphology as a function of pH. We found that only surfactants with the positive charge on the ¿-amino group of lysine showed pH-sensitive hemolytic activity and improved kinetics within the endosomal pH range, indicating that the positive charge position is critical for pH-responsive behavior. Moreover, our results showed that an increase in the alkyl chain length from 14 to 16 carbon atoms was associated with a lower ability to disrupt cell membranes. Knowledge on modulating surfactant-lipid bilayer interactions may help us to develop more efficient biocompatible amino acid-based drug delivery devices.

Dexamethasone inhibition of leucocyte adhesion to rat mesenteric postcapillary venules: role of intercellular adhesion molecule 1 and KC

BACKGROUND A previous study showed that the glucocorticoid dexamethasone, at doses of 100 ¿g/kg and above, inhibited leucocyte adhesion to rat mesenteric postcapillary venules activated with interleukin 1ß (IL-1ß), as assessed by videomicroscopy. AIMS To identify whether the adhesion molecule, intercellular adhesion molecule 1 (ICAM-1), or the chemokine KC could be targeted by the steroid to mediate its antiadhesive effect. METHODS Rat mesenteries were treated with IL-1ß (20 ng intraperitoneally) and the extent of leucocyte adhesion measured at two and four hours using intravital microscopy. Rats were treated with dexamethasone, and passively immunised against ICAM-1 or KC. Endogenous expression of these two mediators was validated by immunohistochemistry, ELISA, and the injection of specific radiolabelled antibodies. RESULTS Dexamethasone greatly reduced IL-1ß induced leucocyte adhesion, endothelial expression of ICAM-1 in the postcapillary venule, and release of the mast cell derived chemokine KC. Injection of specific antibodies to the latter mediators was also extremely effective in downregulating (>80%) IL-1ß induced leucocyte adhesion. CONCLUSIONS Induction by IL-1ß of endogenous ICAM-1 and KC contributes to leucocyte adhesion to inflamed mesenteric vessels. Without excluding other possible mediators, these data clearly show that dexamethasone interferes with ICAM-1 expression and KC release from mast cells, resulting in suppression of leucocyte accumulation in the bowel wall, which is a prominent feature of several gastrointestinal pathologies.

Structure and dynamics of the membrane attaching nitric oxide transporter nitrophorin 7

Nitrophorins represent a unique class of heme proteins that are able to perform the delicate transportation and release of the free-radical gaseous messenger nitric oxide (NO) in a pH-triggered manner. Besides its ability to bind to phospholipid membranes, the N-terminus contains an additional Leu-Pro-Gly stretch, which is a unique sequence trait, and the heme cavity is significantly altered with respect to other nitrophorins. These distinctive features encouraged us to solve the X-ray crystallographic structures of NP7 at low and high pH and bound with different heme ligands (nitric oxide, histamine, imidazole). The overall fold of the lipocalin motif is well preserved in the different X-ray structures and resembles the fold of other nitrophorins. However, a chain-like arrangement in the crystal lattice due to a number of head-to-tail electrostatic stabilizing interactions is found in NP7. Furthermore, the X-ray structures also reveal ligand-dependent changes in the orientation of the heme, as well as in specific interactions between the A-B and G-H loops, which are considered to be relevant for the biological function of nitrophorins. Fast and ultrafast laser triggered ligand rebinding experiments demonstrate the pH-dependent ligand migration within the cavities and the exit route. Finally, the topological distribution of pockets located around the heme as well as from inner cavities present at the rear of the protein provides a distinctive feature in NP7, so that while a loop gated exit mechanism to the solvent has been proposed for most nitrophorins, a more complex mechanism that involves several interconnected gas hosting cavities is proposed for NP7.

The effects of feeding Karlodinium veneficum (PLY # 103; Gymnodinium veneficum Ballantine) to the blue mussel Mytilus edulis

The effects of exposure to the type species for Karlodinium veneficum (PLY # 103) on immune function and histopathology in the blue mussel Mytilus edulis were investigated. Mussels from Whitsand Bay, Cornwall (UK) were exposed to K. veneficum (PLY # 103) for 3 and 6 days. Assays for immune function included total and differential cells counts, phagocytosis and release of extra cellular reactive oxygen species. Histology was carried out on digestive gland and mantle tissues. The toxin cell quota for K. veneficum (PLY #103) was measured by liquid chromatography-mass spectrometry detecting two separable toxins KvTx1 (11.6 ± 5.4 ng/ml) and KvTx2 (47.7 ± 4.2 ng/ml). There were significant effects of K. veneficum exposure with increasing phagocytosis and release of reactive oxygen species following 6 days exposure. There were no significant effects on total cell counts. However, differential cell counts did show significant effects after 3 days exposure to the toxic alga. All mussels produced faeces but not pseudofaeces indicating that algae were not rejected prior to ingestion. Digestive glands showed ingestion of the algae and hemocyte infiltration after 3 days of exposure, whereas mantle tissue did not show differences between treatments. As the effects of K. veneficum were not observed in the mantle tissue it can be hypothesized that the algal concentration was not high enough, or exposure long enough, to affect all the tissues. Despite being in culture for more than 50 years the original K. veneficum isolate obtained by Mary Parke still showed toxic effects on mussels.

MACSIM : diseño, implementación y liberación

En el present projecte s'exposa el disseny, la implementació i l'alliberament d'una aplicació de programari lliure denominada MACsim. Aquesta eina permet simular el funcionament dels protocols MAC (Medium Acces Control) de les xarxes de comunicacions mòbils de tercera generació. En concret, s'implementa el protocol WISPER.

Examining the sustainability issues in UKOER projects : Developing a sustainable OER ecosystem in HE

The development of open educational resources (OERs) is becoming a strategic priority for governments and education institutions around the world, in response to funding cuts and rising costs in educational provision. In the United Kingdom, a government-sponsored Pilot Programme on Open Educational Recourses (JISC/HEA, 2009) was launched in 2009 with an initial budget of £5.7m. This paper reviews the key sustainability issues identified by the projects including the different approaches and models that have been adopted in order to sustain the continuing development and release of OER once funding has ended. The analysis also considers the challenges relating to the development and implementation of policies and processes for sustainable OER practice within institutions and among academics. The paper concludes by drawing on the experiences from the wider United Kingdom and international OER communities to develop a sustainable OER ecosystem model that can facilitate discussions on future development of OER initiatives.

What do academic libraries have to do with Open Educational Resources? Theme : Long term sustainability of open education projects first steps to start up

This paper will discuss the possible roles of academic libraries in promoting, supporting, and sustaining institutional Open Educational Resource initiatives. It will note areas in which libraries or librarians have skills and knowledge that intersect with some of the needs of academic staff and students as they use and release OERs. It will also present the results of a brief survey of the views of some OER initiatives on the current and potential role of academic libraries.

An investigation of the resolution of inflammation (catabasis) in COPD.

Background: Chronic Obstructive Pulmonary Disease (COPD) is characterized by an enhanced inflammatory response to smoking that persists despite quitting. The resolution of inflammation (catabasis) is a complex and highly regulated process where tissue resident macrophages play a key role since they phagocytose apoptotic cells (efferocytosis), preventing their secondary necrosis and the spill-over of their pro-inflammatory cytoplasmic content, and release pro-resolution and tissue repair molecules, such as TGFβ, VEGF and HGF. Because inflammation does not resolve in COPD, we hypothesized that catabasis may be abnormal in these patients. Methods: To explore this hypothesis, we studied lung tissue samples obtained at surgery from 21 COPD patients,22 smokers with normal spirometry and 13 non-smokers controls. In these samples we used: (1)immunohistochemistry to assess the expression of CD44, CD36, VEGF and TGFβ in lung macrophages; (2) real time PCR to determine HGF, PPARγ, TGFβ, VEGF and MMP-9 gene expression; and, (3) ELISA to quantify lipoxin A4, a lipid mediator of catabasis. Results: We found that current and former smokers with COPD showed: (1) more inflammation (higher MMP-9 expression); (2) reduced macrophage surface expression of CD44, a key efferocytosis receptor; and, (3) similar levels of TGFβ, VEGF, HGF, PPARγ, and lipoxin A4 than smokers with normal spirometry, despite the presence of inflammation and disease. Conclusions: These results identify several potential abnormalities of catabasis in patients with COPD.

The first SEPServer event catalogue 68-MeV solar proton events observed at 1 AU in 1996-2010

SEPServer is a three-year collaborative project funded by the seventh framework programme (FP7-SPACE) of the European Union. The objective of the project is to provide access to state-of-the-art observations and analysis tools for the scientific community on solar energetic particle (SEP) events and related electromagnetic (EM) emissions. The project will eventually lead to better understanding of the particle acceleration and transport processes at the Sun and in the inner heliosphere. These processes lead to SEP events that form one of the key elements of space weather. In this paper we present the first results from the systematic analysis work performed on the following datasets: SOHO/ERNE, SOHO/EPHIN, ACE/EPAM, Wind/WAVES and GOES X-rays. A catalogue of SEP events at 1 AU, with complete coverage over solar cycle 23, based on high-energy (~68-MeV) protons from SOHO/ERNE and electron recordings of the events by SOHO/EPHIN and ACE/EPAM are presented. A total of 115 energetic particle events have been identified and analysed using velocity dispersion analysis (VDA) for protons and time-shifting analysis (TSA) for electrons and protons in order to infer the SEP release times at the Sun. EM observations during the times of the SEP event onset have been gathered and compared to the release time estimates of particles. Data from those events that occurred during the European day-time, i.e., those that also have observations from ground-based observatories included in SEPServer, are listed and a preliminary analysis of their associations is presented. We find that VDA results for protons can be a useful tool for the analysis of proton release times, but if the derived proton path length is out of a range of 1 AU < s[3 AU, the result of the analysis may be compromised, as indicated by the anti-correlation of the derived path length and release time delay from the asso ciated X-ray flare. The average path length derived from VDA is about 1.9 times the nominal length of the spiral magnetic field line. This implies that the path length of first-arriving MeV to deka-MeV protons is affected by interplanetary scattering. TSA of near-relativistic electrons results in a release time that shows significant scatter with respect to the EM emissions but with a trend of being delayed more with increasing distance between the flare and the nominal footpoint of the Earth-connected field line.

Mutant huntingtin impairs the post-Golgi trafficking of brain-derived neurotrophic factor but not its Val66Met polymorphism.

Brain-derived neurotrophic factor (BDNF) polymorphism is associated with the pathophysiology of several neurodegenerative disorders, including Huntington"s disease. In view ofthese data andthe involvement of huntingtin in intracellular trafficking, we examined the intracellular transport and release of Val66Val BDNF (Val-BDNF) and Val66Met BDNF (Met-BDNF) in transfected striatal knock-in cells expressing wild-type or mutant full-length huntingtin. Colocalization studies with specific markers for endoplasmic reticulum showed no differences between the Val-BDNF and Met-BDNF and were not modified by mutant huntingtin. However, post-Golgi trafficking was altered by mutant huntingtin dependent on the BDNF form. Thus, fluorescence recovery after photobleaching (FRAP) and inverse FRAP analysis showed retention of Met-BDNF inthe Golgi apparatus with respectto Val-BDNF in wild-type cells. Strikingly, mutant huntingtin diminished post-Golgi trafficking of Val-BDNF, whereas Met-BDNF was not modified. Accordingly, a reduction in the number of transport vesicles was only observed in mutant huntingtin cells transfected with Val-BDNF but not Met-BDNF. Moreover, mutant huntingtin severely affectedthe KCl-evoked release of Val-BDNF, although it had little effect on Met-BDNF regulated release. The constitutive release of Val-BDNF or Met-BDNF in mutant cells was only slightly reduced. Interestingly, mutant huntingtin only perturbed post-Golgi trafficking of proteins that follow the regulated secretory pathway (epidermal growth factor receptor or atrial natriuretic factor), whereas it did not change those that follow the constitutive pathway (p75 NTR ). We conclude that mutant huntingtin differently affects intracellular transport and release of Val-BDNF and Met-BDNF. In addition, our findings reveal a new role for huntingtin in the regulation of the post-Golgi trafficking of the regulated secretory pathway.

Phospholipid bilayer perturbing-properties underlying lysis induced by pH-sensitive cationic lysine-based surfactants in biomembranes

Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Here, we report novel cationic lysine-based surfactants (hydrochloride salts of Nε- and Nα-acyl lysine methyl ester) that differ in the position of the positive charge and the length of the alkyl chain. Amino acid-based surfactants could be promising novel biomaterials in drug delivery systems, given their biocompatible properties and low cytotoxic potential. We examined their ability to disrupt the cell membrane in a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model of endosomal membranes. Furthermore, we addressed the mechanism of surfactant-mediated membrane destabilization, including the effects of each surfactant on erythrocyte morphology as a function of pH. We found that only surfactants with the positive charge on the α-amino group of lysine showed pH-sensitive hemolytic activity and improved kinetics within the endosomal pH range, indicating that the positive charge position is critical for pH-responsive behavior. Moreover, our results showed that an increase in the alkyl chain length from 14 to 16 carbon atoms was associated with a lower ability to disrupt cell membranes. Knowledge on modulating surfactant-lipid bilayer interactions may help us to develop more efficient biocompatible amino acid-based drug delivery devices.

New cationic nanovesicular systems containing lysine-based surfactants for topical administration: Toxicity assessment using representative skin cell lines

Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Cationic nanovesicles have attracted considerable interest as effective carriers to improve the delivery of biologically active molecules into and through the skin. In this study, lipid-based nanovesicles containing three different cationic lysine-based surfactants were designed for topical administration. We used representative skin cell lines and in vitro assays to assess whether the cationic compounds modulate the toxic responses of these nanocarriers. The nanovesicles were characterized in both water and cell culture medium. In general, significant agglomeration occurred after 24 h incubation under cell culture conditions. We found different cytotoxic responses among the formulations, which depended on the surfactant,cell line (3T3, HaCaT, and THP-1) and endpoint assayed (MTT, NRU, and LDH). Moreover, no potential phototoxicity was detected in fibroblast or keratinocyte cells, whereas only a slight inflammatory response was induced, as detected by IL-1a and IL-8 production in HaCaT and THP-1 cell lines, respectively. A key finding of our research was that the cationic charge position and the alkyl chain length of the surfactants determine the nanovesicles resulting toxicity. The charge on the a-amino group of lysine increased the depletion of cell metabolic activity, as determined by the MTT assay, while a higher hydrophobicity tends to enhance the toxic responses of the nanovesicles. The insights provided here using different cell lines and assays offer a comprehensive toxicological evaluation of this group of new nanomaterials.

The L-type voltage-gated calcium channel modulates microglial pro-inflammatory activity

Under pathological conditions, microglia, the resident CNS immune cells, become reactive and release pro-inflammatory cytokines and neurotoxic factors. We investigated whether this phenotypic switch includes changes in the expression of the L-type voltage-gated calcium channel (VGCC) in a rat model of N-methyl-d-aspartate-induced hippocampal neurodegeneration. Double immunohistochemistry and confocal microscopy evidenced that activated microglia express the L-type VGCC. We then analyzed whether BV2 microglia express functional L-type VGCC, and investigated the latter's role in microglial cytokine release and phagocytic capacity. Activated BV2 microglia express the CaV1.2 and CaV1.3 subunits of the L-type VGCC determined by reverse transcription-polymerase chain reaction, Western blot and immunocytochemistry. Depolarization with KCl induced a Ca2+ entry facilitated by Bay k8644 and partially blocked with nifedipine, which also reduced TNF-α and NO release by 40%. However, no nifedipine effect on BV2 microglia viability or phagocytic capacity was observed. Our results suggest that in CNS inflammatory processes, the L-type VGCC plays a specific role in the control of microglial secretory activity.