Dextran-based hydrogel microspheres obtained in w/o emulsion: preparation, characterisation and in vivo studies.

The cross-linking reaction in w/o emulsions of dextran (DEX) functionalised with methacrylic groups, having or not acid residues in side chain, can be used to easily prepare polysaccharide hydrogel microspheres with properties suitable for drug delivery applications. The formation of a chemical network within the obtained particles was evaluated with FT-IR spectroscopy, whereas morphology and dimensions of the microspheres were investigated with optical and scanning electron microscopy. At the same time, swelling measurements were carried out on freeze-dried particles in different aqueous media simulating biological fluids. Preliminary release experiments performed with ibuprofen, betamethasone and vitamin B12 chosen as model drugs, showed that these microspheres could be suitable as modified drug delivery systems in oral formulations. Finally, in vivo writhing experiments were carried out in mice in order to verify the antinociceptive activity of betamethasone loaded into the new polysaccharide hydrogel microspheres.

Novel pH-sensitive physical hydrogels of carboxymethyl scleroglucan.

A carboxymethyl derivative of scleroglucan (Scl-CM) with derivatization degree 300 ± 10 was synthesized and characterized by Fourier transform infrared spectroscopy, potentiometer titration, mucus adhesion studies, and rheological measurements. Rheological measurements showed the ability of the polymer to undergo sol-gel transitions even in the absence of salts. Swelling experiments, performed on freeze-dried samples in different media, showed good affinity of these hydrogels toward the aqueous media and a pH-sensitive behavior. Four nonsteroidal anti-inflammatory drugs (NSAIDs) were loaded into the physical hydrogels obtained from 2.0% (w/v) solutions of the polymer. The results of the release studies carried out in conditions simulating the gastrointestinal tract showed that the new hydrogels could be suggested for the modified oral delivery of NSAIDs, particularly damaging for the gastric mucosa. In vivo studies proved the biocompatibility of the matrix and the absence of any gastric damage for administration of ulcerogenic doses of diclofenac loaded into the hydrogel (DIC/Scl-CM-300). Moreover, DIC/Scl-CM-300 was found to be effective in peripheral analgesia.

Antiproliferative and cytotoxic activity of extracts from Cistus incanus L. and Cistus monspeliensis L. on human prostate cell lines.

Benign prostatic hypertrophy (BPH) is a common condition in elderly men that impairs quality of life and leads to a number of medical complications. The use of phytotherapeutic compounds in patients with relatively moderate BPH symptoms has been growing steadily. In the present study, acute toxicity of lyophilised aqueous extracts of Cistus incanus L. and Cistus monspeliensis L., collected in Sicily, was evaluated on the shrimp (Artemia salina L.) lethality assay, an alternative test to determine the toxicity of natural products. The cytotoxic and growth inhibitory effects were studied on normal human prostate cells (PZ-HPV-7 and PNT1A) and on a lung fibroblast cell line (V79-4). Cell proliferation was evaluated by MTT and SRB assays. Cytotoxicity was measured using the Trypan blue exclusion assay. Cistus extract treatment on prostate cell lines resulted in an almost identical growth inhibitory response and in a significant decrease in an cell viability. These findings indicate the biologically relevant effect of polyphenolic compounds present in Cistus extracts, and suggest that these substances may prove beneficial in BPH treatment.

Physical carboxymethylscleroglucan/calcium ion hydrogels as modified drug delivery systems in topical formulations.

A carboxymethyl derivative of scleroglucan (Scl-CM) with a 65+/-5% carboxylic group degree of derivatization (DD) was recently synthesized and characterized. Aqueous solutions of the polymer underwent to a sharp transition toward a gel like behaviour in the presence of divalent ions such as Ca(+2). Physical hydrogels with different Scl-CM/Ca(+2) ratios were prepared and characterized for their rheological behaviour. Their potential as drug delivery systems was also evaluated. To this end three non steroidal anti-inflammatory drugs (NSAIDs) were loaded into the hydrogels obtained with 2% w/v solution of Scl-CM and 0.05 and 0.1 M CaCl(2). The release rate of the drugs was critically related to the salt concentration. By an appropriate combination of the hydrogels prepared using different amounts of salt, it was possible to obtain a system able to release diclofenac with zero-order kinetics. Primary skin irritation tests showed a good biocompatibility of the new polymer, as well as of its hydrogels. These results suggest a potential of the new hydrogels for the development of modified delivery systems in topical formulations.

pH-sensitive hydrogels of dextran: synthesis, characterization and in vivo studies.

pH-Sensitive hydrogels of dextran were synthesized by photochemical cross-linking reaction of methacrylate dextran (DEX-MA) at different derivatization degree, functionalized with acidic residues through reaction with phthalic anhydride. The hydrogels were characterized by FT-IR spectra, swelling measurements, experiments of chemical and enzymatic hydrolyses. The swelling data agreed with the formation of networks having pH-sensitive behaviour. This property was confirmed by the morphological examination performed by scanning electron microscopy on samples maintained in media at different pH. (S)-4-Isobutyl-2-phenylpropionic acid (ibuprofen) was loaded into the polymeric matrices. The analysis of the release profiles of the drug from the three networks showed that all the matrices were able to retain ibuprofen during the transit through the stomach, releasing it in a sustained way in the intestinal tract at a rate strictly dependent on the derivatization degree in methacrylic groups. In vivo studies verified the biocompatibility of the materials. Moreover, when the matrix loaded with ibuprofen was administered to rats, it was able to protect them from the ulcerogenic effects of the drug.

Inhibition of intestinal motility and secretion by extracts of Epilobium spp. in mice.

Ethanol extracts of the fresh aerial parts of various Epilobium species were tested to elucidate the mechanism of their gastrointestinal activity in animals. The methods of charcoal meal, castor oil-induced diarrhoea, and enteropooling assay were used to evaluate their effect on mouse gut at various dose levels. The extracts were found to have a significant activity in all models. Moreover, the extracts resulted to possess very little toxicity. Thus, it can be concluded that Epilobium possesses anti-diarrhoeal, anti-motility, and anti-secretory activities and can prove beneficial in the treatment of gastrointestinal disorders.

Effect of a standardized extract of red orange juice on proliferation of human prostate cells in vitro.

A standardized extract of red orange juice (ROE) was shown to inhibit proliferation of fibroblast and epithelial prostate cells. These data suggest that the antiproliferative properties of ROE cannot be ascribed to cytotoxic effect and highlight its potential usefulness in the management of benign prostatic hyperplasia.

Studies on antidiarrhoeal activity of an extract of wine from Jacquez grapes in mice.

The present study was designed to verify the antidiarrhoeal effects of a lyophilized extract of wine from Jacquez grapes (Ord. Rhamnales; Fam. Vitaceae; Sp. Vitis aestivalis M.-cinerea E. x Vitis vinifera L.), studying its influence on castor oil-induced diarrhoea and enteropooling, and on gastrointestinal transit (measured by a charcoal marker) in mice. The pre-treatment of the animals with the JWE (Jacquez wine extract) produced a significant inhibition against castor oil induced-diarrhoea and intestinal fluid accumulation; furthermore the extract significantly decreased the propulsive movement of the charcoal meal. These findings suggest a potential beneficial use of the JWE in the treatment of diarrhoeal diseases.

Biological evaluation of a polyvinyl siloxane impression material.

OBJECTIVES: The aim of this study was to determine the irritant properties of a new polyvinyl siloxane impression material (Ghenesil, Lascod-Italy) after single application to intact skin of the rabbit. METHODS: The material was evaluated for primary skin irritation according to the UNI EN ISO 10993-10:1996 using three healthy male New Zealand White rabbits. The back of the animals was clipped free of fur and divided into four sites with the same area 24 h before application of the sample. The material was applied to only two sites; the other two were used as controls. All the sites were covered by gauze and the back of the rabbit was wrapped with a non-occlusive bandage. After 4 h, the bandage and the test material were removed; 1h later the sites were examined for skin irritation and the observation was repeated after 24, 48 and 72 h. The Score of Primary Irritation (SPI) was calculated for each animal and the Primary Irritation Index (PII) was calculated as the arithmetical mean of SPI values. RESULTS: The PII of the test material resulted 0.06. SIGNIFICANCE: Based on present results, it can be concluded that the primary skin irritation of the polyvinyl siloxane impression material tested can be considered negligible.

Ascorbic and 6-Br-ascorbic acid conjugates as a tool to increase the therapeutic effects of potentially central active drugs.

Ascorbic acid (AA) or 6-Br-ascorbate (BrAA) conjugation has been investigated as a tool to improve brain drug delivery by the Vitamin C transporter SVCT2. To this aim, the effects of AA- or BrAA-conjugation on drug affinity and uptake have been assessed in vitro, by using human retinal pigment epithelium (HRPE) cells, and compared in vivo on mice. Nipecotic, kynurenic and diclofenamic acids were chosen as model drugs. Kinetic and inhibition experiments referred to [(14)C]AA uptake into HRPE cells showed that nipecotic and kynurenic acids became able to interact with SVCT2, as competitive inhibitors, only when conjugated to AA or BrAA. Surprisingly, diclofenamic acid itself appeared able to interact with SVCT2, with an affinity that was significantly increased or decreased by AA or BrAA conjugation, respectively. HPLC analysis, performed on HRPE cells, confirmed the SVCT2 mediated transport for the BrAA-conjugate of nipecotic acid, whereas kynurenic acids conjugates although interacting with the transporter did not enter the cells. In accordance, only the nipecotic acid conjugates showed anticonvulsant activity after systemic injection in mice.

Development and characterization of biodegradable nanospheres as delivery systems of anti-ischemic adenosine derivatives.

We report a preliminary study concerning the encapsulation modalities in nanoparticles of the anti-ischemic drug N6-cyclopentyladenosine (CPA) and its pro-drug 5'-octanoyl-CPA (Oct-CPA). The release of these compounds and the related pro-drug stability effects in human whole blood have been tested. Moreover, the influence of the delivery systems on CPA interaction toward human adenosine A1 receptor has been analysed. The nanospheres were prepared by nanoprecipitation or double emulsion solvent evaporation method using poly(lactic acid) and recovered by gel filtration or ultracentrifugation or dialysis. Free and encapsulated Oct-CPA was incubated in fresh blood and its stability was analysed with HPLC. Quite spherical nanoparticles with mean diameters ranging between 210+/-50 and 390+/-90 nm were obtained. No encapsulation occurred when CPA was used. Satisfactory results concerning drug content (0.1-1.1% w/w) and encapsulation efficiency (6-56%) were achieved when Oct-CPA was employed. The controlled release of the pro-drug was achieved, being released within a range of 1-4 h, or very slowly, depending on nanoparticle preparations. The hydrolysis rate of Oct-CPA in human whole blood appeared stabilized in human whole blood with modalities related to the release patterns. The presence of all nanoparticle preparations did not interfere with CPA interaction at its action site.

Design, synthesis and in vitro evaluation on HRPE cells of ascorbic and 6-bromoascorbic acid conjugates with neuroactive molecules.

Preliminary investigations allowed us to anticipate that conjugation of nipecotic acid with L-ascorbate (AA) gave a prodrug endowed with anticonvulsant activity in mice. In view of these results, and in order to get deepen insight into the molecular aspects at the base of the transport mechanism, a second generation of compounds, based on 6-bromo-6-deoxy-L-ascorbic acid (BrAA) as the carrier molecule was designed and synthesized. Effects of the chirality of the transported drug was also investigated on R- and S-nipecotic acid. Interaction and uptake modalities were evaluated in our in vitro model based on human retinal pigment epithelium cells (HRPE), which expresses the membrane L-ascorbic acid (AA) SVCT2 transporters. A remarkable increase on SVCT2 affinity was found going from AA to BrAA conjugates, that is, 11 (Ki=1187+/-78 microM) versus 19 (Ki=193+/-14 microM) and 12 (Ki=39.8+/-3.2 microM) versus 20, (Ki=7.4+/-0.8 microM). Taken together, these data are in agreement with our initial hypothesis on the possibility to achieve better affinities by conjugation with AA analogs, and also consent to hypothesize the presence of accessory interactions that may improve transporters recognition.

Transporter-mediated effects of diclofenamic acid and its ascorbyl pro-drug in the in vivo neurotropic activity of ascorbyl nipecotic acid conjugate.

Continuing our studies on SVCT2 ascorbic acid (AA) transporter-mediated drug delivery of neurotropic agents, we have now investigated the in vitro intracellular uptake of Diclofenac (Diclo) and its conjugate (AA-Diclo), both characterized by high affinity for the SVCT2 transporter. We have also investigated the in vivo uptake mechanism of AA-conjugate of Nipecotic acid (AA-Nipec) and the implication of the transporter-mediated effects of Diclo and AA-Diclo. Diclo resulted as a noncompetitive inhibitor of AA transport, but also showed a sodium-dependent and ascorbate-independent uptake, thus implying the possible involvement of specific transporters in the delivery to the brain of Diclo. This result opens a perspective in the discovery of new strategies in the targeting of this drug to the brain. Inhibitory effects of Diclo and AA-Diclo on the SVCT2 transporter were used to study anticonvulsant effects of AA-Nipec, confirming our hypothesis of an SVCT2-mediated transport in its neurotropic activity. AA-Diclo stability has been also investigated: it is hydrolyzed following a first-order kinetics in buffer, plasma (t(1/2) at about 10 h) and whole blood (t(1/2) at about 3 h), suggesting AA-Diclo as a potential candidate to enhance the short half-life of Diclo in vivo.

Conformational aspects of human formyl-peptide receptor agonists.

The conformation of several Met-Ile-Phe-Leu analogues was analyzed using circular dichroism and infra-red absorption. Their effect on human neutrophils was verified by receptor binding assays and measurements of lysozyme release. The results demonstrate that in amphipatic environments the compounds examined can be highly and weakly ordered in beta-turn structures, in dependence on their N-terminal substituents. The ability of the compounds to evoke neutrophil functions appears strongly and weakly influenced by N- and C-terminal modification, respectively.