Modulation of gel formation and drug-release characteristics of lidocaine-loaded poly(vinyl alcohol)-tetraborate hydrogel systems using scavenger polyol sugars

Polyol sugars, displaying a plurality Of hydroxyl groups, were shown to modulate tetra hydroxyborate (borate) cross-linking in lidocaine hydrochloride containing poly(vinyl alcohol) scini-solid hydrogels. Without polyol, demixing of borate cross-linked PVA hydrogels into two distinct phases was noticeable upon lidocaine hydrochloride addition, preventing further use as a topical System. D-Mannitol incorporation was found to be particularly suitable in cicumventing network constriction induced by ionic and pH effects upon adding the hydrochloride salt of lidocaine. A test formulation (4% w/v lidocaine HCl, 2% W/V D-mannitol, 10% w/v PVA and 2.5%, w/v THB) was shown to constitute an effective delivery system, which was characterised by an initial burst release and a drug release mechanism dependent on temperature, changing from a diffusion-controlled system to one with the properties of a reservoir system. The novel flow properties and innocuous adhesion of PVA-tetrahydroxyborate hydrogels Support their application for drug delivery to exposed epithelial surfaces, Such as lacerated wounds. Furthermore, addition of a polyol, such as mannitol, allows incorporation of soluble salt forms of active therapeutic agents by modulation of cross-linking density. (C) 2008 Elsevier B.V. All rights reserved.

Effect of lycopene supplementation on insulin-like growth factor-1 and insulin-like growth factor binding protein-3: a double-blind, placebo-controlled trial

Objective: Studies have suggested a link between lycopene and insulin-like growth factor-1 ( IGF-1). The aim of this study was to test the effect of lycopene supplementation on IGF-1 and binding protein-3 ( IGFBP-3) status in healthy male volunteers.

Effect of the incorporation of hydroxy-terminated liquid silicones on the cure characteristics, morphology, and release of a model protein from silicone elastomer-covered rods

Silicone elastomer systems have previously been shown to offer potential for the sustained release of protein therapeutics. However, the general requirement for the incorporation of large amounts of release enhancing solid excipients to achieve therapeutically effective release rates from these otherwise hydrophobic polymer systems can detrimentally affect the viscosity of the precure silicone elastomer mixture and its curing characteristics. The increase in viscosity necessitates the use of higher operating pressures in manufacture, resulting in higher shear stresses that are often detrimental to the structural integrity of the incorporated protein. The addition of liquid silicones increases the initial tan delta value and the tan delta values in the early stages of curing by increasing the liquid character (G '') of the silicone elastomer system and reducing its elastic character (G'), thereby reducing the shear stress placed on the formulation during manufacture and minimizing the potential for protein degradation. However, SEM analysis has demonstrated that if the liquid character of the silicone elastomer is too high, the formulation will be unable to fill the mold during manufacture. This study demonstrates that incorporation of liquid hydroxy-terminated polydimethylsiloxanes into addition-cure silicone elastomer-covered rod formulations can both effectively lower the viscosity of the precured silicone elastomer and enhance the release rate of the model therapeutic protein bovine serum albumin. (C) 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Characterisation of protein stability in rod-insert vaginal rings

A major goal in vaccine development is elimination of the ‘cold chain’, the transport and storage system for maintenance and distribution of the vaccine product. This is particularly pertinent to liquid formulation of vaccines. We have previously described the rod-insert vaginal ring (RiR) device, comprising an elastomeric body into which are inserted lyophilised, rod-shaped, solid drug dosage forms, and having potential for sustained mucosal delivery of biomacromolecules, such as HIV envelope protein-based vaccine candidates. Given the solid, lyophilised nature of these insert dosage forms, we hypothesised that antigen stability may be significantly increased compared with more conventional solubilised vaginal gel format. In this study, we prepared and tested vaginal ring devices fitted with lyophilised rod inserts containing the model antigen bovine serum albumin (BSA). Both the RiRs and the gels that were freeze-dried to prepare the inserts were evaluated for BSA stability using PAGE, turbidimetry, microbial load, MALDI-TOF and qualitative precipitate solubility measurements. When stored at 4 oC, but not when stored at 40 oC / 75% RH, the RiR formulation offered protection against structural and conformational changes to BSA. The insert also retained matrix integrity and release characteristics. The results demonstrate that lypophilised gels can provide relative protection against degradation at lower temperatures compared to semi-solid gels. The major mechanism of degradation at 40 oC / 75% RH was shown to be protein aggregation. Finally, in a preliminary study, we found that addition of trehalose to the formulation significantly reduces the rate of BSA degradation as compared to the original formulation when stored at 40 oC /75% RH. Establishing the mechanism of degradation, and finding that degradation is decelerated in the presence of trehalose, will help inform further development of RiRs specifically and polymer based freeze-dried systems in general.

Development of liposome gel based formulations for intravaginal delivery of the recombinant HIV-1 envelope protein CN54gp140

Mucosally-administered vaccine strategies are widely investigated as a promising means of preventing HIV infection. This study describes the development of liposomal gel formulations, and novel lyophilised variants, comprising HIV-1 envelope glycoprotein, CN54gp140, encapsulated within neutral, positively charged or negatively charged liposomes. The CN54gp140 liposomes were evaluated for mean vesicle diameter, polydispersity, morphology, zeta potential and antigen encapsulation efficiency before being incorporated into hydroxyethyl cellulose (HEC) aqueous gel and subsequently lyophilised to produce a rod-shaped solid dosage form for practical vaginal application. The lyophilised liposome-HEC rods were evaluated for moisture content and redispersibility in simulated vaginal fluid. Since these rods are designed to revert to gel form following intravaginal application, mucoadhesive, mechanical (compressibility and hardness) and rheological properties of the reformed gels were evaluated. The liposomes exhibited good encapsulation efficiency and the gels demonstrated suitable mucoadhesive strength. The freeze-dried liposome-HEC formulations represent a novel formulation strategy that could offer potential as stable and practical dosage form.

Insulin-like growth factor binding protein-3 mediates vascular repair by enhancing nitric oxide generation

Insulin-like growth factor binding protein (IGFBP)-3 modulates vascular development by regulating endothelial progenitor cell (EPC) behavior, specifically stimulating EPC cell migration. This study was undertaken to investigate the mechanism of IGFBP-3 effects on EPC function and how IGFBP-3 mediates cytoprotection following vascular injury.

Release of β-galactosidase from poloxamine/α-cyclodextrin hydrogels

All mammals lose their ability to produce lactase (β-galactosidase), the enzyme that cleaves lactose into galactose and glucose, after weaning. The prevalence of lactase deficiency (LD) spans from 2 to 15% among northern Europeans, to nearly 100% among Asians. Following lactose consumption, people with LD often experience gastrointestinal symptoms such as abdominal pain, bowel distension, cramps and flatulence, or even systemic problems such as headache, loss of concentration and muscle pain. These symptoms vary depending on the amount of lactose ingested, type of food and degree of intolerance. Although those affected can avoid the uptake of dairy products, in doing so, they lose a readily available source of calcium and protein. In this work, gels obtained by complexation of Tetronic 90R4 with α-cyclodextrin loaded with β-galactosidase are proposed as a way to administer the enzyme immediately before or with the lactose-containing meal. Both molecules are biocompatible, can form gels in situ, and show sustained erosion kinetics in aqueous media. The complex was characterized by FTIR that evidenced an inclusion complex between the polyethylene oxide block and α-cyclodextrin. The release profiles of β-galactosidase from two different matrices (gels and tablets) of the in situ hydrogels have been obtained. The influence of the percentage of Tetronic in media of different pH was evaluated. No differences were observed regarding the release rate from the gel matrices at pH 6 (t50 = 105 min). However, in the case of the tablets, the kinetics were faster and they released a greater amount of 90R4 (25%, t50 = 40–50 min). Also, the amount of enzyme released was higher for mixtures with 25% Tetronic. Using suitable mathematical models, the corresponding kinetic parameters have been calculated. In all cases, the release data fit quite well to the Peppas–Sahlin model equation, indicating that the release of β-galactosidase is governed by a combination of diffusion and erosion processes. It has been observed that the diffusion mechanism prevails over erosion during the first 50 minutes, followed by continued release of the enzyme due to the disintegration of the matrix.

HPV16 Down-Regulates the Insulin-Like Growth Factor Binding Protein 2 to Promote Epithelial Invasion in Organotypic Cultures

Cervical cancer is a multi-stage disease caused by human papillomaviruses (HPV) infection of cervical epithelial cells, but the mechanisms regulating disease progression are not clearly defined. Using 3-dimensional organotypic cultures, we demonstrate that HPV16 E6 and E7 proteins alter the secretome of primary human keratinocytes resulting in local epithelial invasion. Mechanistically, absence of the IGF-binding protein 2 (IGFBP2) caused increases in IGFI/II signalling and through crosstalk with KGF/FGFR2b/AKT, cell invasion. Repression of IGFBP2 is mediated by histone deacetylation at the IGFBP2 promoter and was reversed by treatment with histone deacetylase (HDAC) inhibitors. Our in vitro findings were confirmed in 50 invasive cancers and 79 cervical intra-epithelial neoplastic lesions caused by HPV16 infection, where IGFBP2 levels were reduced with increasing disease severity. In summary, the loss of IGFBP2 is associated with progression of premalignant disease, and sensitises cells to pro-invasive IGF signalling, and together with stromal derived factors promotes epithelial invasion.

Preproendothelin-1 expression in human mesangial cells: evidence for a p38 mitogen-activated protein kinase/protein kinases- C-dependent mechanism

Endothelin-1 (ET-1) has been implicated in the pathogenesis of renal inflammation. This study investigated the mechanisms underlying the synergistic upregulation of preproET-1 gene expression in human mesangial cells after co-stimulation with thrombin and tumor necrosis factor alpha (TNFalpha). Whereas thrombin induced a moderate upregulation of preproET-1 mRNA, co-stimulation with TNFalpha resulted in a strong and protracted upregulation of this mRNA species. Thrombin+TNFalpha-induced upregulation of preproET-1 expression was found to require p38 mitogen-activated protein kinase and protein kinases C, whereas activation of extracellular signal-regulated kinase, c-Jun-N-terminal kinase, or intracellular Ca(2+) release were not required. Actinomycin D chase experiments suggested that enhanced stability of preproET-1 mRNA did not account for the increase in transcript levels. PreproET-1 promoter analysis demonstrated that the 5'-flanking region of preproET-1 encompassed positive regulatory elements engaged by thrombin. Negative modulation of thrombin-induced activation exerted by the distal 5' portion of preproET-1 promoter (-4.4 kbp to 204 bp) was overcome by co-stimulation with TNFalpha, providing a possible mechanism underlying the synergistic upregulation of preproET-1 expression by these two agonists. In conclusion, human mesangial cell expression of preproET-1 may be increased potently in the presence of two common proinflammatory mediators, thereby providing a potential mechanism for ET-1 production in inflammatory renal disease.

Diabetes-induced activation of protein kinase C inhibits store-operated Ca2+ uptake in rat retinal microvascular smooth muscle.

AIMS/HYPOTHESIS: To assess the effects of diabetes-induced activation of protein kinase C (PKC) on voltage-dependent and voltage-independent Ca2+ influx pathways in retinal microvascular smooth muscle cells. METHODS: Cytosolic Ca2+ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic and non-diabetic rats using fura-2 microfluorimetry. Voltage-dependent Ca2+ influx was tested by measuring rises in [Ca2+]i with KCl (100 mmol/l) and store-operated Ca2+ influx was assessed by depleting [Ca2+]i stores with Ca2+ free medium containing 5 micromol/l cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca2+ on adding 2 mmol/l or 10 mmol/l Ca2+ solution. RESULTS: Ca2+ entry through voltage-dependent L-type Ca2+ channels was unaffected by diabetes. In contrast, store-operated Ca2+ influx was attenuated. In microvessels from non-diabetic rats 20 mmol/l D-mannitol had no effect on store-operated Ca2+ influx. Diabetic rats injected daily with insulin had store-operated Ca2+ influx rates similar to non-diabetic control rats. The reduced Ca2+ entry in diabetic microvessels was reversed by 2-h exposure to 100 nmol/l staurosporine, a non-specific PKC antagonist and was mimicked in microvessels from non-diabetic rats by 10-min exposure to the PKC activator phorbol myristate acetate (100 nmol/l). The specific PKCbeta antagonist LY379196 (100 nmol/l) also reversed the poor Ca2+ influx although its action was less efficacious than staurosporine. CONCLUSION/INTERPRETATION: These results show that store-operated Ca2+ influx is inhibited in retinal arterioles from rats having sustained increased blood glucose and that PKCbeta seems to play a role in mediating this effect.

Differential Modulation of Transcriptional Activity of Estrogen Receptors by Direct Protein-Protein Interactions with the T Cell Factor Family of Transcription Factors

Two major signaling pathways, those triggered by estrogen (E(2)) and by the Wnt family, interact in the breast to cause growth and differentiation. The estrogen receptors ER(alpha) and ER(beta) are activated by binding E(2) and act as ligand-dependent transcription factors. The effector for the Wnt family is the Tcf family of transcription factors. Both sets of transcription factors recognize discrete but different nucleotide sequences in the promoters of their target genes. By using transient transfections of reporter constructs for the osteopontin and thymidine kinase promoters in rat mammary cells, we show that Tcf-4 antagonizes and Tcf-1 stimulates the effects of activated ER/E(2). For mutants of the former promoter, the stimulatory effects of ER(alpha)/E(2) can be made to be dependent on Tcf-1, and for the latter promoter the effects of the T cell factors (TCFs) are dependent on ER/E(2). Direct interaction between ERs and Tcfs either at the Tcf/ER(alpha)-binding site on the DNA or in the absence of DNA is established by gel retardation assays or by coimmunoprecipitation/biosensor methods, respectively. These results show that the two sets of transcription factors can interact directly, the interaction between ERs and Tcf-4 being antagonistic and that between ERs and Tcf-1 being synergistic on the activity of the promoters employed. Since Tcf-4 is the major Tcf family member in the breast, it is suggested that the antagonistic interaction is normally dominant in vivo in this tissue.

Overexpression of small glutamine-rich TPR-containing protein promotes apoptosis in 7721 cells.

It is known that small glutamine-rich TPR-containing protein (SGT) is the member of TPR motif family. However, the biological functions of SGT remain unclear. In this paper, we report that SGT plays a role in apoptotic signaling. Ectopic expression of SGT enhances DNA fragment and nucleus breakage after the induction of apoptosis. Increasing mRNA level of SGT is also observed in 7721 cells undergoing apoptosis, knockdown the expression of endogenous SGT contributes to the decrease of apoptosis of 7721 cells. Deletion analysis reveals that TPR domain is critical to pro-apoptotic function of SGT. Furthermore, we demonstrated that the PARP cleavage and cytochrome c release are enhanced when SGT is overexpressed in 7721 cells during apoptosis. Collectively, our results indicate that SGT is a new pro-apoptotic factor.

First report of a novel plant lysozyme with both antifungal and antibacterial activities.

A novel lysozyme exhibiting antifungal activity and with a molecular mass of 14.4 kDa in SDS–polyacrylamide gel electrophoresis was isolated from mung bean (Phaseolus mungo) seeds using a procedure that involved aqueous extraction, ammonium sulfate precipitation, ion exchange chromatography on CM-Sephadex, and high-performance liquid chromatography on POROS HS-20. Its N-terminal sequence was very different from that of hen egg white lysozyme. Its pI was estimated to be above 9.7. The specific activity of the lysozyme was 355 U/mg at pH 5.5 and 30 °C. The lysozyme exhibited a pH optimum at pH 5.5 and a temperature optimum at 55 °C. It is reported herein, for the first time, that a novel plant lysozyme exerted an antifungal action toward Fusarium oxysporum, Fusarium solani, Pythium aphanidermatum, Sclerotium rolfsii, and Botrytis cinerea, in addition to an antibacterial action against Staphylococcus aureus.