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The aim of the present study was the evaluation of lipid nanoparticles (solid lipid nanoparticles, SLN, and nanostructured lipid carriers, NLC) as potential carriers for octyl-methoxycinnamate (OMC). The release pattern of OMC from SLN and NLC was evaluated in vitro, determining its percutaneous absorption through excised human skin. Additional in vitro studies were performed in order to evaluate, after UVA radiation treatment, the spectral stability of OMC-loaded lipid nanoparticles. From the obtained results, ultrasonication method yielded both SLN and NLC in the nanometer range with a high active loading and a particle shape close to spherical. Differential scanning calorimetry data pointed out the key role of the inner oil phase of NLC in stabilizing the particle architecture and in increasing the solubility of OMC as compared with SLN. In vitro results showed that OMC, when incorporated in viscosized NLC dispersions (OMC-NLC), exhibited a lower flux with respect to viscosized SLN dispersions (OMC-SLN) and two reference formulations: a microemulsion (OMC-ME) and a hydroalcoholic gel (OMC-GEL). Photostability studies revealed that viscosized NLC dispersions were the most efficient at preserving OMC from ultraviolet-mediated photodegradation., (Copyright © 2011 Wiley-Liss, Inc.)

The aim of this study was to optimize the formulation of lipid nanoparticles (NPs), intended for brain targeting, with the aid of a computer generated experimental design. The high pressure homogenization technique, selected for this purpose, was suitable to formulate the 3 investigated lipids (i.e., Softisan(®) 142, SOFT; Compritol(®) 888 ATO, COMP; cetyl palmitate, CP) into nanometre-length particles, while the computer generated experimental design helped to individuate the best preparation conditions with a small number of experimental assay. Even though all the 3 optimized formulations were suitable for intravenous infusion, CP NPs showed the smallest particle size and the appropriate thermal behaviour to be used as carriers in brain targeting applications., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The present study concerns the in vitro and in vivo evaluation of benzocaine (BENZO) and lidocaine (LIDO) topical delivery from nanostructured lipid carriers (NLCs). Morphology and dimensional distribution of NLCs have been, respectively, characterized by differential scanning calorimetry (DSC) and photon correlation spectroscopy. The release pattern of BENZO and LIDO from NLCs was evaluated in vitro determining drug percutaneous absorption through excised human skin. Radiant heat tail-flick test was carried out in mice to determine the antinociceptive effect of BENZO and LIDO from NLC. DSC studies revealed that the inner oil phase of NLC plays a significant role in stabilizing the particle architecture and increasing the drug solubility. In vitro evidences show that BENZO and LIDO, when incorporated in viscosized NLC dispersions, exhibited a lower flux with respect to formulations containing the free drugs in the aqueous phase. In vivo study enabled to demonstrate that BENZO and LIDO can be released in a prolonged fashion when incorporated into lipid carriers. The results obtained pointed out NLC capability to act as an effective drug reservoir, thus prolonging the anesthetic effect of BENZO and LIDO., (Copyright © 2010 Wiley-Liss, Inc.)

In the present study, a complex of compounds (red orange complex, ROC), obtained from three red orange varieties (Citrus sinensis varieties: Moro, Tarocco and Sanguinello), containing cyanidin glycosides, hydroxycinnamic acids, flavanone glycosides and ascorbic acid, was screened to discover new lead compounds in the suppression of the production of key molecules released during inflammatory events in interleukin-1beta (IL-beta) stimulated human primary chondrocytes. The expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX)-2 and intercellular adhesion molecule-1 (ICAM-1), and the release of nitric oxide, prostaglandin (PG)E(2) and interleukin-8 (IL-8) were determined. Indomethacin was used as an anti-inflammatory drug reference. ROC acts as a potent inhibitor of iNOS and COX-2 gene expression while also suppressing the production of PGE(2) and nitrite in human chondrocytes. In addition, ROC induces a significant decrease in ICAM expression and IL-8 release. These findings suggest that ROC exerts anti-inflammatory effects probably through the suppression of COX-2 and iNOS expression.

The present study concerns the percutaneous absorption of naproxen (NPX), as model anti-inflammatory drug, included in liposome formulations constituted of different lipids: stratum corneum lipids (SCL) and phosphatidylcholine/cholesterol (PC/CHOL). Liposome dispersions were produced using two different methods: reverse-phase evaporation (REV) and thin layer evaporation (TLE). Morphology and dimensions of the disperse phase were characterized by cryo-transmission electron microscopy (cryo-TEM) and photon correlation spectroscopy, respectively. X-ray diffraction was employed to determine the structural organization of the vesicles. In vitro diffusion was studied by Franz cell on liposome dispersions viscosized by carbomer. Tape stripping was performed to investigate in vivo the performance of differently composed liposomes as NPX delivery system. Cryo-TEM showed spherical vesicles and bigger irregular elongated nanoparticles for TLE SCL liposomes. REV resulted in spherical and elongated multilamellar vesicles. Also X-ray diffraction evidenced L alpha or L beta multilamellar vesicles for PC/CHOL and SCL liposome respectively. The in vitro study showed a lower NPX flux for SCL with respect to PC/CHOL liposome. Tape stripping corroborate the in vitro findings regarding SCL, suggesting that liposomes create a drug reservoir mixing with SC lipids, whilst PC/CHOL liposome promoted NPX permeation through the skin. Liposome lipid composition seems to affect NPX permeation through the skin., ((c) 2009 Wiley-Liss, Inc. and the American Pharmacists Association)

Nano-emulsions are innovative colloidal systems characterized by high kinetic stability, low viscosity, and optical transparency, which make them very attractive in many dermatological applications. Furthermore their small size seems to favor the topical administration of actives which scarcely cross the skin. In the light of these interesting features, the present study was aimed to the evaluation, in vitro and in vivo, of glycyrrhetic acid (GA) release through the skin from the nanoemulsion system. GA-loaded nanoemulsion (GA(N)) was prepared by phase inversion temperature (PIT) method, and was characterized in order to determine mean droplet size and its stability during a well-defined storage period. Further Cryo-TEM studies were performed to obtain information regarding nanoemulsion structure. The GA release pattern from nanoemulsion was evaluated in vitro, to determine its percutaneous absorption through excised human skin (stratum corneum and epidermis, SCE), and in vivo evaluating GA topical anti-inflammatory activity on healthy human volunteers by the UVB-induced erythema model. Nanoemulsions prepared by PIT method showed a mean droplet diameter of 210 nm that drastically changed during a storage of 5 weeks at room temperature. In vitro and in vivo evidence showed that the nanoemulsion system significantly increased the transdermal permeability of GA in comparison to a control O/W emulsion (GA(O/W)) containing the same amount of active compound.

Red oranges are an important component of the so-called Mediterranean diet and they have been used by traditional medicine for their health protective properties, particularly to heal sore throat and cough, suggesting an interesting antiinflammatory activity. The purpose of this study was to evaluate the antiinflammatory activity of a red orange (Citrus sinensis varieties: Moro, Tarocco, Sanguinello) complex (ROC), characterized by high levels of anthocyanins, flavanones, hydroxycinnamic acids and ascorbic acid, on the human keratinocyte line NCTC 2544 exposed to interferon-gamma (IFN-gamma) and histamine. The expression of immunomodulatory membrane molecules such as inter-cellular adhesion molecule-1 (ICAM-1) by Western blot analysis, and the release of chemokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) through ELISA kits, were determined. ICAM-1 modulates the permanence and activation of T lymphocytes in the epidermis. MCP-1 is a specific chemoattractant for monocytes and dendritic cells. IL-8 is important for the recruitment of both neutrophils and T lymphocytes. Addition of ROC at different concentrations together with IFN-gamma and histamine induced a dose-dependent inhibition of ICAM-1 expression and MCP-1 and IL-8 release. ROC shows interesting antiinflammatory properties in human keratinocyte cells NCTC 2544. This natural complex could have a topical employment and mitigate the consequences of some skin pathologies., ((c) 2009 John Wiley & Sons, Ltd.)

Objectives: Diethyltoluamide and ethylhexyl p-methoxycinnamate (OMC) are two active ingredients in insect repellent and sunscreen products, respectively. The concurrent application of these two substances often increases their systemic absorption, compromising the safety and efficiency of the cosmetic product. In this study, diethyltoluamide and OMC were incorporated into solid lipid nanoparticles, a colloidal drug delivery system, to reduce percutaneous absorption and avoid toxic effects and also maintain the efficacy of the two active compounds on the skin surface for a long duration., Methods: Solid lipid nanoparticles were prepared based on an ultrasonication technique and characterized by differential scanning calorimetry (DSC) analyses. In-vitro studies determined the percutaneous absorption of diethyltoluamide and OMC., Key Findings: DSC data carried out on unloaded and diethyltoluamide- and/or OMC-loaded solid lipid nanoparticles highlighted that diethyltoluamide and OMC modified the temperature and the enthalpy change associated to the calorimetric peak of solid lipid nanoparticles. The concurrent presence of the two compounds in the solid lipid nanoparticles caused a synergic effect, indicating that the lipid matrix of nanoparticles guaranteed a high encapsulation of both diethyltoluamide and OMC. Results from the in-vitro study demonstrated that the particles were able to reduce the skin permeation of the two cosmetic ingredients in comparison with an oil-in-water emulsion., Conclusions: This study has provided supplementary evidence as to the potential of lipid nanoparticles as carriers for topical administration of cosmetic active compounds.

Dermal therapy is still a challenge due to the difficulties in controlling the active pharmaceutical ingredient (API) fate within the skin. Recently, lipid nanoparticles have shown a great potential as vehicle for topical administration of active substances, principally owing to the possible targeting effect and controlled release in different skin strata. Ketoprofen and naproxen loaded lipid nanoparticles were prepared, using hot high pressure homogenization and ultrasonication techniques, and characterized by means of photo correlation spectroscopy and differential scanning calorimetry. Nanoparticle behavior on human skin was assessed, in vitro, to determine drug percutaneous absorption (Franz cell method) and, in vivo, to establish the active localization (tape-stripping technique) and the controlled release abilities (UVB-induced erythema model). Results demonstrated that the particles were able to reduce drug penetration increasing, simultaneously, the permeation and the accumulation in the horny layer. A prolonged anti-inflammatory effect was observed in the case of drug loaded nanoparticles with respect to the drug solution. Direct as well as indirect evidences corroborate the early reports on the usefulness of lipid nanoparticles as carriers for topical administration, stimulating new and deeper investigations in the field.

The skin's uppermost thin layer, stratum corneum, plays a crucial role in protecting the body against unwanted influences from the environment. Disruption of the stratum corneum, by tape stripping or chemical injury, results in epidermal recovery of the skin barrier. Soy phytosterols are widely used in the cosmetic field as active ingredients in creams and lipsticks. Furthermore, they deserve an important place among nutracosmeceuticals; in fact, after their absorption from the diet they are transferred from the plasma to the skin, playing an important role in the constitution of skin surface lipids. The aim of the present work was to study the effect of the topical application of soybean phytosterols on skin barrier recovery in human volunteers using the extent of methyl nicotinate (MN)-induced erythema in damaged skin as a parameter to evaluate the rate of stratum corneum recovery. MN was chosen as an erythematogenous substance for its capability to cause an erythema whose intensity and duration are proportional to the quantity of the substance that has entered the living epidermis over time. MN-induced erythema was monitored using reflectance spectrophotometry as a noninvasive instrumental technique. The results show clearly that soy phytosterols exert positive results on skin repair; in fact, three days after tape stripping, the sites treated with a formulation containing phytosterols showed an appreciable recovery of barrier function compared to those treated with a vehicle control without soy phytosterols.

The aim of our present study was to evaluate the in vitro percutaneous absorption of atenolol, a well-known antihypertensive, from a series of formulations containing various penetration enhancers. Particularly the promoting effect of EPA and DHA, two polyunsaturated fatty acids (PUFAs), has been studied, and drug permeation data have been compared with those obtained with the other formulations containing "classic" penetration enhancers such as transcutol, d-limonene, and PG. Not all the penetration enhancers tested were effective in increasing atenolol percutaneous flux and the best permeation profile for atenolol was obtained with the formulation containing transcutol (B) and PUFA (E). To explain the enhancer mechanism, the atenolol diffusion and partitioning coefficients from the different formulations were calculated. The results indicated that PUFAs increased the apparent diffusion coefficient of the drugs but did not affect their apparent stratum corneum (SC)/vehicle partition coefficient (K(m)). At this same time transcutol exerted its enhancer effect increasing significantly the apparent SC/vehicle partition coefficient (K(m)) and in a minor amount the apparent diffusion coefficient of skin permeation process (D(m)). The potential application of formulations B and E in atenolol percutaneous absorption was determined from the calculation of the steady-state plasma concentrations (C(ss)). These values resulted within the drug therapeutic range and suggest that atenolol transdermal delivery could be feasible.

Six new 1-alkylazacycloalkan-2-one esters of ketorolac (1-6) were synthesized and evaluated as potential dermal prodrugs. In vitro experiments were carried out to evaluate their chemical and enzymatic stability and permeation through excised human skin. Furthermore, partition coefficients n-octanol-water of ketorolac and its esters were determined to obtain information about their lipophilicity. Esters 1-6 showed increased lipophilicity compared to the parent drug, good stability in phosphate buffer pH 7.4, and were readily hydrolyzed in human plasma. Results from in vitro percutaneous absorption studies showed that, among all esters synthesized, only for esters 2 and 4 did a higher cumulative amount of drug penetrate through the skin, compared with that obtained after topical application of ketorolac.

Three oily extracts, obtained by acetone extraction from the entrails of different varieties of Mediterranean fishes, such as mackerel (Scomber scombrus), sardine (Sardina pilchardus) and horse mackerel (Trachurus mediterraneus), were characterized to determine their unsaturated fatty acid content. In an in-vitro model, their inhibitory effect was then evaluated against protein aggregation and their protective efficacy against heat-induced albumin denaturation assessed. The fish oil extracts tested in this study presented a significant amount of unsaturated fatty acids; in particular the extract obtained from the entrails of horse mackerel proved to have higher concentrations of DHA (docosahexaenoic acid) and oleic acid compared with the other two oils. The in-vitro study revealed an interesting protective effect of the oil extracts (particularly the horse mackerel extract) against heat-induced denaturation of albumin.

In the present study, 2 alternative strategies to optimize ketorolac transdermal delivery, namely, prodrugs (polyoxyethylene glycol ester derivatives, I-IV) and nanostructured lipid carriers (NLC) were investigated. The synthesized prodrugs were chemically stable and easily degraded to the parent drug in human plasma. Ketorolac-loaded NLC with high drug content could be successfully prepared. The obtained products formulated into gels showed a different trend of drug permeation through human stratum corneum and epidermis. Particularly, skin permeation of ester prodrugs was significantly enhanced, apart from ester IV, compared with ketorolac, while the results of drug release from NLC outlined that these carriers were ineffective in increasing ketorolac percutaneous absorption owing to a high degree of mutual interaction between the drug and carrier lipid matrix. Polyoxyethylene glycol esterification confirmed to be a suitable approach to enhance ketorolac transdermal delivery, while NLC seemed more appropriate for sustained release owing to the possible formation of a drug reservoir into the skin.

The present work was aimed at evaluating the in vitro effects of a lyophilized extract of wine (JW-E) obtained from Jacquez grapes (Vitis aestivalis-cinereaxVitis vinifera grapes) on the production of key molecules released in inflammatory disease utilising interleukin-1beta (IL-1beta) activated chondrocytes. The extract contains large amounts of phenolic components, in particular some flavonoids (flavan-3-ols, also known as catechins) and proanthocyanidins, as hydroxycinnamic acids and anthocyanins, that possess several biological features such as antiinflammatory and antioxidant effects and a "radical scavenger" activity too. In this study, we assayed the effect of JW-E on the production of key molecules released during chronic inflammatory events as nitric oxide (NO), prostaglandins E(2) (PGE(2)) and reactive oxygen species (ROS) in human chondrocytes culture, stimulated with proinflammatory cytokine interleukin-1beta. The JW-E proved to possess good ability against the harmfull effects of IL-1beta. Our data showed the protective effects of JW-E in cartilage alteration, that appears greater than that elicited by indomethacin, a not steroidal antiinflammatory drug (NSAID), commonly employed in joint diseases.

In traditional medicine extracts of polysaccharide-containing plants are widely employed for the treatment of skin and epithelium wounds and of mucous membrane irritation. The extracts of Opuntia ficus-indica cladodes are used in folk medicine for their antiulcer and wound-healing activities. The present study describes the wound-healing potential of two lyophilized polysaccharide extracts obtained from O. ficus-indica (L.) cladodes applied on large full-thickness wounds in the rat. When topically applied for 6 days, polysaccharides with a molecular weight (MW)>10(4)Da from O. ficus-indica cladodes induce a beneficial effect on cutaneous repair in this experimental model; in particular the topical application of O. ficus-indica extracts on skin lesions accelerates the reepithelization and remodelling phases, also by affecting cell-matrix interactions and by modulating laminin deposition. Furthermore, the wound-healing effect is more marked for polysaccharides with a MW ranging 10(4)-10(6)Da than for those with MW>10(6)Da, leading us to suppose that the fine structure of these polysaccharides and thus their particular hygroscopic, rheologic and viscoelastic properties may be essential for the wound-healing promoter activity observed.

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.

Novel polyoxyethylene esters of 18 beta-glycyrrhetic acid (GA) were synthesized and evaluated as potential dermal prodrugs. The permeation of these prodrugs (1a-e) was studied in-vitro, using excised human skin membranes (SCE; stratum corneum/epidermis) mounted in Franz type cells, and in-vivo, evaluating the ability of these compounds to inhibit methyl nicotinate (MN)-induced skin erythema in healthy human subjects. All the esters synthesized showed a good water stability, while the enzymatic hydrolysis rate was significantly affected by the length of the polyoxyethylenic chain used as promoiety. In in-vitro percutaneous absorption studies, only esters 1b and 1c (respectively triethylen- and tetraethylenglycol derivatives) showed an increased flux through SCE membranes compared with GA. Furthermore, we observed an appreciable and sustained in-vivo topical anti-inflammatory activity of esters 1b and 1c compared with the parent drug.

Purpose: The present study concerns the production and characterization of monooleine (MO) dispersions as drug delivery systems for indomethacin, taken as model anti-inflammatory drug., Methods: Dispersions were produced by emulsification and homogenization of MO and poloxamer in water. Morphology and dimensional distribution of the disperse phase have been characterized by cryo-transmission electron microscopy and photon correlation spectroscopy, respectively. X-ray diffraction has been performed to determine the structural organization of the disperse phase. Sedimentation field flow fractionation (SdFFF) has been performed to investigate drug distribution in the dispersion. An in vitro diffusion study was conducted by Franz cell associated to stratum corneum epidermis membrane on cubosome dispersions viscosized by carbomer. In vivo studies based on skin reflectance spectrophotometry and tape stripping were performed to better investigate the performance of cubosome as indomethacin delivery system., Results: Microscopy studies showed the coexistence of vesicles and cubosomes. X-ray diffraction revealed the presence of a bicontinuous cubic phase of spatial symmetry Im3m (Q229). SdFFF demonstrated that no free drug was present in the dispersion. Indomethacin incorporated in viscosized MO dispersions exhibited a lower flux with respect to the analogous formulation containing the free drug in the aqueous phase and to the control formulation based on carbomer gel. Reflectance spectroscopy demonstrated that indomethacin incorporated into MO dispersions can be released in a prolonged fashion. Tape-stripping experiments corroborated this finding., Conclusions: MO dispersions can be proposed as nanoparticulate systems able to control the percutaneous absorption of indomethacin.

The preparation and detailed composition of an oil newly extracted from pure soy germ (not less than 96 % hypocotyle) are presented. Experiments in vivo showed that soybean-germ oil (SGO) possesses a remarkable protective activity against UVB-induced skin inflammation, exceeding that of tocopherol acetate by a factor of 2. These results suggest that SGO might have interesting therapeutic and cosmetic applications in the management of skin diseases initiated, sustained, or exacerbated by an over production of free radicals.

Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) are interesting nanoparticulate delivery systems produced from solid lipids. Both carrier types are submicron size particles but they can be distinguished by their inner structure. In the present paper, indomethacin (IND)-loaded SLN and NLC were prepared and the organization and distribution of the different ingredients originating each type of nanoparticle system were studied by differential scanning calorimetry (DSC) technique. Furthermore, mean particle size and percentage of drug encapsulation were also determined. From the results obtained, NLC lipid organization guaranteed an increased indomethacin encapsulation in comparison with SLN. DSC static and dynamic measurements performed on SLN and NLC showed that oil nanocompartments incorporated into NLC solid matrix drastically influenced drug distribution inside the nanoparticle system. Controlled release from NLC system could be explained considering both drug partition between oil nanocompartments and solid lipid and a successive partition between solid lipid and water.

We have investigated the influence of charge and lipid concentration on the in-vivo percutaneous absorption of a model compound, methyl nicotinate (MN), from liposomal vesicles. MN-loaded liposomes were produced by the reverse-phase evaporation method (REV) using different concentrations of phosphatidyl choline (PC), in association with surfactants such as dioctadecyl dimethyl ammonium bromide (DDAB18) and dicetyl phosphate (DCP), which impart a positive or negative charge to the systems, respectively. The liposomal suspensions were then processed to hydrogels and used to study in-vivo the MN permeation profile. MN was chosen as the model compound since it was capable of causing cutaneous erythema, the intensity and duration of which was proportional to the amount entering the living epidermis over time. The extent of the erythema was monitored by reflectance spectrophotometry, a non-invasive technique. In-vivo findings showed an interesting MN delayed release, which was proportional to the amount of phospholipids in each liposomal formulation. Furthermore, it could be noted that the erythematous effect was more prolonged when MN was delivered from neutral or negatively-charged liposomal forms.

The aim of the present study was to evaluate the in vitro percutaneous absorption and the in vivo anti-inflammatory activity of EPA and DHA fatty acids from three oily extracts, obtained by acetonic extractions from the entrails of different varieties of Mediterranean fishes such as mackerel (Scomber scombrus), sardine (Sardina pilchardus) and horse mackerel (Trachurus mediterraneus). In the first part of our research, we focused our attention on the characterization of the oily extracts to determine their omega-3 polyunsaturated fatty acid content, then, we evaluated the in vitro percutaneous absorption through excised human skin (stratum corneum/epidermis membranes; SCE) of EPA and DHA contained in the extracts. In the second part, the fish oil which guaranteed the best in vitro permeation profile of these omega-3 fatty acids was studied in order to evaluate its inhibiting ability towards the in vivo UVB-induced skin erythema. From the results obtained, all the fish oils tested in this study presented significant amounts of omega-3 fatty acids EPA and DHA, and particularly sardine oil extract showed higher concentrations of these substances compared to the other two fish oils. The in vitro experiments revealed interesting fluxes of these compounds from sardine extract through the stratum corneum/epidermis membranes and an appreciable anti-inflammatory activity against UVB-induced erythema in human volunteers was also observed.

The aim of this study was the evaluation, in vitro and in vivo, of indomethacin (IND) release through the skin from nanostructured lipid carriers (NLC). NLC were prepared by ultrasonication, and were characterized in order to determine drug content, and particle size; finally the NLC were processed to hydrogels (A and B). The IND release pattern from NLC hydrogels was evaluated in vitro, to determine its percutaneous absorption through excised human skin (stratum corneum and epidermis, SCE), and in vivo. To evaluate the in vivo IND release, two methods were employed: (1) the IND topical anti-inflammatory activity was determined at different time-points after its cutaneous application; in this case, the UVB-induced erythema on healthy human volunteers, chosen as inflammatory model, was monitored by reflectance visible spectrophotometry; (2) the extent of IND absorption into human skin was performed by the tape-stripping technique. The in vitro percutaneous absorption studies showed lower fluxes of IND through SCE membranes from NLC hydrogels (A and B) in comparison to an aqueous dispersion (C) and a hydro-alcoholic gel (D) both containing free IND. The findings from the former in vivo method showed that the anti-inflammatory effect, following IND topical application, was more prolonged with IND-loaded NLC gel formulation (A) if compared to formulation C and D. The results from tape stripping technique confirmed the trend obtained by the former in vivo method and indicated that IND topical bioavailability in the stratum corneum varied substantially depending upon the formulations (A-D)., (Copyright 2005 Wiley-Liss, Inc)

The antiallergic properties of two lyophilized extracts obtained from Capparis spinosa L. flowering buds (capers) by methanol extraction, carried out at room temperature (CAP-C) or with heating at 60 degrees C (CAP-H), were investigated. The protective effects of CAP-H and CAP-C, orally administered (14.28 mg[sol ]kg), were evaluated against Oleaceae antigen challenge-induced and histamine-induced bronchospasm in anaesthetized guinea-pigs. Furthermore, the histamine skin prick test was performed on humans, applying a gel formulation containing 2% CAP-C (the only extract able to protect against histamine-induced bronchospasm) on the skin for 1 h before histamine application and monitoring the erythema by reflectance spectrophotometry. The CAP-H showed a good protective effect against the bronchospasm induced by antigen challenge in sensitized guinea-pigs; conversely, a significant decrease in the responsiveness to histamine was seen only in CAP-C pretreated animals. Finally, the CAP-C gel formulation possessed a marked inhibitory effect (46.07%) against histamine-induced skin erythema. These two caper extracts displayed marked antiallergic effectiveness; however, the protective effect of CAP-H was very likely due to an indirect mechanism (for example, inhibition of mediator release from mast cells or production of arachidonic acid metabolites); conversely, CAP-C is endowed with direct antihistaminic properties. The different mechanisms of action of CAP-H and CAP-C may be related to a difference in the extraction procedure and, thus, in their qualitative[sol ]quantitative chemical profile., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

The aim of this study was to evaluate the in-vivo drug release profile of indometacin-loaded liposomes into the skin. Large unilamellar vesicles (LUVs), composed of dipalmitoyl-L-alpha-phosphatidylcholine and cholesterol (9:1), were obtained using the extrusion method and then incorporated in hydrogels (LUV-A and LUV-B). The delivery of indometacin from the liposomal system was evaluated by determining its in-vivo local anti-inflammatory activity after cutaneous application of liposomal gel formulations; the anti-inflammatory activity is directly proportional to the amount of drug that actually crosses the skin. UVB-induced erythema on healthy human volunteers was chosen as the inflammatory model and the extent of erythema was monitored by the non-invasive technique of reflectance spectrophotometry. The results showed that LUV dispersions containing indometacin provided a high percentage of entrapped drug (approximately 84%). Furthermore, in-vivo findings revealed that the anti-inflammatory effect was more prolonged when indometacin was delivered from a liposomal gel formulation rather than from a gel formulation without liposomes. In particular, the indometacin-loaded gel formulation LUV-A showed a sustained effect, probably related to an interaction between LUV lipids and stratum corneum lipid structure. This interaction produces a depot in the stratum corneum that ensures sustained release of the drug to deeper skin layers.

Six new 1-alkylazacycloalkan-2-one esters of ketoprofen (1-6) were synthesized and evaluated as potential dermal prodrugs of ketoprofen. Their lipophilicity by both experimental lipophilicity indices (log k') and calculated ClogP was also determined. In vitro experiments were carried out to evaluate the chemical and enzymatic stability and permeation through excised human skin of these new ketoprofen derivatives. Furthermore, we investigated the in vivo topical anti-inflammatory activity of ester 5, which showed the best in vitro profile, evaluating the ability of this compound to inhibit methyl nicotinate-induced skin erythema on healthy human volunteers. Esters 1-6 showed increased lipophilicity compared with the parent drug (ketoprofen), good stability in phosphate buffer pH 7.4, and were readily hydrolyzed by porcine esterase. Results from in vitro percutaneous absorption studies showed that, among all esters synthesized, only for esters 1 and 5 did a higher cumulative amount of drug penetrate through the skin, compared with that obtained after topical application of ketoprofen. In vivo results showed an interesting delayed and sustained activity of ester 5, compared with the parent drugs.

Novel glycosyl derivatives of dopamine and L-dopa (I-IV) are synthesized in order to overcome the problem of blood-brain barrier low permeability of dopamine and of low bioavailability of its precursor L-dopa. Esters synthesized link dopamine and L-dopa, by a succinyl linker, to C-3 position of glucose (I and II) and to C-6 of galactose (II and IV). The chemical and enzymatic stabilities of esters synthesized were evaluated in order to determine both their stability in aqueous medium and their feasibility in undergoing enzymatic cleavage by rat plasma to regenerate the original drug. Furthermore, we have shown the central effects of esters I-IV on classic dopaminergic models, such as morphine induced locomotion and reserpine-induced hypolocomotion. From the result obtained compounds I-IV appeared moderately stable in a pH 7.4 buffered solution and in rat plasma. Furthermore, pharmacological studies showed that both dopamine derivatives (I and II) were equiactive in reversing reserpine-induced hypolocomotion in rats, and both were more active than L-dopa or ester III and IV, while II and III were more potent in reducing morphine-induced locomotion than I and IV. The minimal vascular effects of these derivatives allow us to underline the possibility to use them in pathologies, such as Parkinson disease, characterised by an evident decreasing of dopamine concentration in the brain.

Thiocolchicoside, a semi-synthetic derivative of colchicoside, is used in topical formulations for its anti-inflammatory and muscle-relaxant properties. The objective of this study was to evaluate the effect of a (propylene glycol diperlagonate) DPPG and (propylene glycol) PG mixture present in an innovative foam formulation (Miotens) on the flux of thiocolchicoside through excised human skin. Furthermore, the in vitro permeation behaviour of this new formulation (Miotens foam) was compared to another commercial product (Muscoril ointment) and to a control gel formulation (thiogel), both enhancer free. The best permeation profile was obtained from the foam formulation (Miotens) which was able to increase the thiocolchicoside flux about three fold compared to control formulation (thiogel) and about two fold compared to the commercial formulation Muscoril ointment.

New glycosyl derivatives of 3'-azido-3'-deoxythymidine (AZT) (1 and 2) were synthesized in order to improve AZT retention in the blood and to guarantee its sustained release, overcoming the necessity of multiple drug administrations. The esters synthesized (1 and 2) link AZT, by a succinyl linker, to the C-3 position of glucose and to C-6 of galactose. Furthermore, the chemical and enzymatic stabilities of esters 1 and 2 were evaluated in order to determine both their stability in aqueous medium and their feasibility to undergo enzymatic cleavage by esterase to regenerate the original drug. The pharmacokinetic profiles of esters 1 and 2, obtained after systemic administration, showed an interesting controlled release, in particular for ester 2, compared to the pharmacokinetic profile of AZT.

Diabetes mellitus is associated with a high oxidative stress level, resulting from an imbalance between free radicals or reactive oxygen species production and the antioxidant systems. Inhibition of these oxidative processes by co-adjuvant therapy could therefore prevent, or at least delay, the onset and/or the development of long-term diabetic complications. Dietary supplementation with plant biophenols may be a successful strategy to decrease this risk of pathological complications. The Red Orange Complex (ROC) is a standardized red orange extract containing, as its main active principles, phenolic compounds (anthocyanins, flavanones and hydroxycinnamic acids) as well as ascorbic acid. The aim of the present preliminary study was to evaluate the effects of short-term (2 mo) dietary supplementation with ROC (50 mg/d, orally) on some serum non-invasive biomarkers of oxidative stress (total antioxidant status, or TAS, levels of thiol groups and levels of free radicals) in a group of 33 patients with Type 2 diabetes, in comparison with a group of 28 healthy volunteers. The results obtained demonstrate that in diabetic patients supplementation with ROC can improve blood levels of thiol groups on proteins (an indirect measurement of glutathione activity in serum); furthermore, it can elicit a marked decrease in serum free radical levels, in patients with high blood oxidative stress status. However, ROC supplementation appeared unable to modify serum TAS. Finally, the glycemic profile remained stable during the study period in all subjects, and no unpleasant side effects were reported. In conclusion, the treatment of diabetic patients with ROC might be of therapeutic benefit in order to protect against diabetes complications that are partially due to uncontrolled lipid oxidation. D

Sedum telephium L. is a medicinal plant used in antiquity to cure many types of inflammatory skin diseases. The leaves (without the external cuticle), are used to promote healing and reduce skin inflammation and pain, and contain various components. We found two major components: flavonol glycosides and polysaccharides, with molecular weight between 13,000 and 13,500 Da. We evaluated the in-vitro antioxidant and in-vivo skin photoprotective effects of three lyophilized extracts obtained from the juice of S. telephium L. leaves: a total lyophilized juice, a lyophilized flavonolic fraction, and a lyophilized polysaccharidic fraction. Two in-vitro models were used: the bleaching of the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH*) radical, and the protective effect against UV-induced peroxidation on phosphatidylcholine multilamellar vesicles, as model membranes. The antioxidant/radical scavenging activity of each lyophilized extract was also assessed in-vivo by determining their ability to reduce UVB-induced skin erythema (monitored by reflectance spectrophotometry) in healthy human volunteers. The findings of the in-vitro experiments clearly demonstrated that, unlike the lyophilized polysaccharidic fraction, the lyophilized flavonolic fraction and total lyophilized juice possess strong antioxidant/free radical scavenging properties, which are likely due to phenolic compounds. Consistent with these findings, gel formulations of both the total lyophilized juice and, to a greater degree, the lyophilized flavonolic fraction appeared to possess a strong protective effect against UV-induced skin erythema in-vivo, whereas the lyophilized polysaccharidic fraction was completely ineffective. The in-vitro and in-vivo results suggest that, both the total lyophilized juice and, in particular, the lyophilized flavonolic fraction, but not the lyophilized polysaccharidic fraction of S. telephium L. leaves, have photoprotective effects against UVB-induced skin damage.

Although photodamage and photoprotection have already been extensively studied in cultured cells, few data have been reported in the literature regarding the in vitro behaviour of skin cells toward a chemical stress, such as iron-induced lipid peroxidation. We investigated the susceptibilities of two human skin-derived cell lines (NCTC 2544 keratinocytes and HFFF2 fibroblasts) to lipid peroxidation induced by FeSO4/histidine, FeSO4/ascorbate and Fe2(SO4)3/ADP. NCTC 2544 cells were more susceptible than HFFF2 cells to lipid peroxidation (assessed by measuring the content of malondialdehyde [MDA]) with iron/ascorbate and iron/ADP as pro-oxidants whereas, with iron/histidine, the same level of MDA production was achieved (about 10nmol/mg protein) in the two cell populations. On the basis of these results, one experimental model ( (iron/histidine) was selected to assess the protective effect of a mixture of two classical antioxidants, Trolox C™ (50μM) and Vitamin C (1mM), added to the cell cultures according to various protocols. The maximal decrease of MDA production in both cell lines was obtained when the antioxidant mixture and the pro-oxidant were added simultaneously to the cultures. By using the same experimental design, NCTC 2544 and HFFF2 cells were exposed to a standardised extract of red oranges (ROE; 0.025-0.5mg/ml), the main active principles of which (anthocyanins 3.1%, hydroxycinnamic acid 2.07%, and flavanone glycosides 8.1%) possess antioxidant activity. Cells treated with ROE, that were still over 90% viable, as evaluated by means of neutral red uptake and tetrazolium salt reduction tests, showed a significant and dose-dependent inhibition of MDA production. This study provides new information about the behaviour of cultured skin cells exposed to chemically induced oxidative stress, and provides further support to the possibility of using skin-derived human cell lines in the evaluation of the effectiveness of antioxidant ingredients for new drugs and/or cosmetics., (2000 FRAME.)

Several fresh orange juices, obtained from five different Citrus sinensis (L.) Osbeck varieties (three pigmented varieties, Moro, Sanguinello, and Tarocco, and two blond varieties, Valencia late and Washington navel), were subjected to antioxidant profile determination (including total polyphenols, flavanones, anthocyanins, hydroxycinnamic acids, and ascorbic acid). The antioxidant activity of these juices was then assessed by means of different "in vitro" tests (bleaching of the stable 1,1-diphenyl-2-picrylhydrazyl radical; peroxidation, induced by the water-soluble radical initiator 2,2'-azobis(2-amidinopropane) hydrochloride, on mixed dipalmitoylphosphatidylcholine/linoleic acid unilamellar vesicles; scavenging activity against nitric oxide; total antioxidant status). All orange juices tested showed an evident antioxidant effect. Our findings indicate the following: (1) the antioxidant efficiency of orange juices may be attributed, in a significant part at least, to their content of total phenols, (2) while ascorbic acid seems to play a minor role; (3) the antioxidant activity of orange juices is related not only to structural features of phytochemicals contained in them, but also to their capability to interact with biomembranes; (4) finally, as to pigmented juices, their antioxidant efficiency appears to be widely influenced by the anthocyanin level. One could speculate that the supply of natural antioxidant phenols through daily consumption of orange juice might provide additional protection against in vivo oxidation of cellular biomolecules.

Nipecotic acid (1), one of the most potent in vitro inhibitors of neuronal and glial gamma-amino butyric acid (GABA) uptake, is inactive as an anticonvulsant when administered systemically. To obtain in vivo active prodrugs of (1), we synthesized four new nipecotic acid esters (3-6), which were obtained by chemical conjugation with glucose, galactose, and tyrosine. These compounds were assayed to evaluate their in vitro chemical and enzymatic hydrolysis. In addition, their anticonvulsant activity was evaluated in vivo in Diluted Brown Agouti (DBA)/2 mice, an excellent animal model for the study of new anticonvulsant drugs. Esters (3-6) appeared stable, at various temperatures, in a pH 7.4 buffered solution and showed susceptibility to undergoing in vitro enzymatic hydrolysis. Intraperitoneally injected nipecotic acid (1) and esters (3-5) did not protect mice against audiogenic seizures; conversely, nipecotic tyrosine ester (6) showed a significant dose-dependent anticonvulsant activity. The in vivo protective activity of the ester (6) and the inefficiency of nipecotic acid (1) in the same experimental conditions suggest that this ester prodrug could be actively transported intact across the blood-brain barrier, beyond which it could be hydrolyzed.

4-(3,4-Dihydro-2,4-dioxo-2H-1,3-benzoxazin-3-yl)-butyric acid (7) and its ester 6, two potential gamma-aminobutyric acid (GABA) prodrugs, were synthesized and studied to determine their stability in aqueous buffer and their susceptibility to undergo enzymatic hydrolysis in vitro (mouse plasma). Both compounds were fairly stable in aqueous media, (t1/2 = 68.2 h and 25.7 h, respectively). The 3,4-dihydro-2,4-dioxo-2H-1,3-benzoxazine ring underwent enzymatic hydrolysis (t1/2 = 5.8 h) in compound 7, whereas in compound 6 it seemed not to be opened by mouse plasma esterases within the observation time (3h). Both compounds were tested for their anticonvulsant activity in pentetrazole (PTZ) treated mice, and showed significant activity. Compound 7, administered as sodium salt 8, was active at relatively low doses and can be considered a very interesting GABA prodrug.

Large unilamellar vesicles (LUVs) are generally accepted to be a suitable model for peroxidation studies. In the present report, dipalmitoylphosphatidylcholine (DPPC)/linoleic acid-mixed LUVs were employed as model membranes to verify the inhibitory effect of tocopherol (an efficient representative antioxidant) against 2,2'-azobis(2-amidinopropane)hydrochloride-induced peroxidation (evaluated by monitoring conjugated diene accumulation). In this model, the appropriate experimental conditions (particularly, liposome composition and peroxidation temperature) were selected following characterization of bilayer physical state, and not only by evaluation of peroxidation rate. Thus, the experiments described provide a routine screening procedure that would be appropriate for assessing the activity profile of novel free-radical scavengers.

Purpose: To investigate the transport of alpha-tocopherol (T), tocopherol succinate (TS) and tocopherol succinate-3-glucose (a newly synthetized, less hydrophobic T ester; TSG) through bovine erythrocyte membranes., Methods: Our experiments were carried out on erythrocytes (obtained from heparinized fresh bovine blood), because they represent a suitable model for investigations of membrane transport., Results: T was shown to reside almost completely in the suspension medium, while the greater part of TS disappeared from the suspension medium and was mainly incorporated into erythrocyte membranes. In comparison with T, a larger amount of TSG was incorporated into erythrocyte membranes and taken up by cells; however the TSG intracellular accumulation was significantly lower than that observed with TS. Furthermore, the transport of TS and TSG was partially inhibited by p-chloromercuribenzenesulfonate (which inhibits monocarboxylate uptake; PCMBS) and by maltose (a competitive inhibitor of glucose transport) respectively, with a concomitant increase in drug membrane incorporation. No significant change in drug transport was observed in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonate, a selective and irreversible blocker of band 3 protein (DIDS)., Conclusions: Our results show 1) the existence of large differences in membrane incorporation of T, TS and TSG (very likely caused by differing abilities to fill spaces in the lipid bilayer) and 2) a specific contribution of the monocarboxylate transport protein and of the glucose transport protein in the cellular uptake of TS and TSG, respectively. A tempting suggestion is that the unique cytoprotective properties of TS may be related to the differences in the transmembrane mobility observed between T and its succinate ester. Furthermore, T conjugation to a monocarboxylate or glycoside moiety could provide suitable substrates for active membrane transport, thus appearing as a promising pharmaceutical strategy for the improved delivery of tocopherol derivatives.

Flavonoids, a group of phenolic compounds widely occurring in the plant kingdom, have been reported to possess strong antioxidant activity. In the present study, four flavonoids (quercetin, hesperetin, naringenin, rutin), chosen according to their structural characteristics, were tested in two different in vitro experimental models: (1) Fe(2+)-induced linoleate peroxidation (Fe(2+)-ILP), by detection of conjugated dienes; and (2) autooxidation of rat cerebral membranes (ARCM), by using thiobarbituric acid for assay of free malondialdehyde production. The results obtained were also interpreted in the light of flavonoid interactions, studied by differential scanning calorimetry, with dipalmitoylphosphatidylcholine (DPPC) vesicles as a biological membrane model. The antilipoperoxidant activity of the flavonoids tested can be classified as follows: rutin > hesperetin > quercetin > naringenin in the Fe(2+)-ILP test: quercetin > rutin > hesperetin > naringenin in the ARCM test. Quercetin, hesperetin, and naringenin interacted with DPPC liposomes causing different shifts, toward lower values, of the main transition peak temperature (Tm) typical for DPPC liposomes; however, no change in Tm of DPPC dispersion was observed in the presence of rutin. The hypothesis will be discussed that flavonoid capacity to modify membrane-dependent processes, such as free-radical-induced membrane lipoperoxidation, is related not only to their structural characteristics but also to their ability to interact with and penetrate the lipid bilayers.

A rapid and precise method for the isolation of vitamins A and E and their acetate or palmitate esters from table matrices was developed using supercritical fluid extraction (SFE). The vitamins were analyzed by nonaqueous reversed-phase high-performance liquid chromatography after a 15-min extraction of the dosage form with supercritical carbon dioxide at 40 degrees C and at a pressure of 250 atm. A quantitative comparison of SFE with an established liquid extraction procedure was performed on commercial tablet formulations. Vitamin recoveries of over 95.6% compared with conventional liquid extraction were achieved by the SFE technique with a much shorter sample preparation time (15 min vs 2h). Moreover, the automated SFE process minimized the number of sample handling operations, drastically reduced the consumption of harmful solvents, and provided mild extraction conditions for the analysis of the labile vitamins. The described SFE method is suitable for quality control analyses of pharmaceutical tablets.

Five indomethacin oligoethylene ester derivatives (3-7) were synthesized and evaluated for their anti-inflammatory, analgesic, and ulcerogenic activity after oral administration. The molecular weight of the oligoethylene glycols used for synthesizing esters 3-7 ranged from 106 to 282. The chemical and enzymatic stabilities of esters 3-7 were evaluated in pH 7.4 and 2.0 buffers and in human plasma, respectively. All the prodrugs showed a good stability both in pH 7.4 phosphate buffer and in pH 2.0 buffer, and they were readily hydrolyzed by human plasma. Esters 3-7 showed an anti-inflammatory activity, determined as the percent inhibition of carrageenan-induced edema, similar to that of indomethacin, although at higher doses. From writhing test results, we observed that all the prodrugs exhibited better or similar analgesic activity compared to indomethacin. Esters 3-7 were significantly less irritating to the gastric mucosa than indomethacin, after oral administration, and esters 3-5 did not show any ulcerogenic activity, although they were administered at higher doses than indomethacin.

Bioavailability studies have been performed with ten healthy volunteers on different dosage forms of acetylsalicylic acid (ASA) in order to assess the bioavailability of two different ASA gums compared with commercial ASA tablets. The results of this study show that ASA is more readily absorbed and eliminated after administration of gum formulations than after administration of tablets, but the bioavailability obtained from the gums was lower than that observed from the tablets.

Seven 2,5-diarylalkyloxysubstituted 1,3,4-oxadiazoles were synthesized. They showed pronounced antiphlogistic action together with central activity (sedative, analgesic) that principally seems to be connected with the presence in the molecule of the 3,4-dioxymethylenephenyl radical.

Following previous research which showed the antibacterial and antimycotic activity of triazolin-3-thiones (II), a series of compounds has been prepared and tested to correlate and clarify the structure-activity relationships. The compounds were of the type represented in (II) where (R = H, CH3 or C6H5) and were in detail: 3-methylthiotriazoles (III-VIII), 3-(amino-substituted)ethylthiotriazolines (IX-XXVI), 3-carboxymethylthiotriazolines (XXVII-XXX), 3,3'dithiobistriazolines (XXXI-XXXVI) and 2-amino-methyl-N-substituted triazolin-3-thiones (XLVIII-LXVI). The 2-aminooxadiazoles (XXXVII-XLII), obtained in place of the expected 1-aroyl-S-methylisothiosemicarbazides (II a) from the reaction between thiosemicarbazides (I) (R = H; CH3, C6H5) and methyl iodide, are also described.

In continuation of previous work, the Authors describe the synthesis of 3-aminoethyl derivatives of some 5-aryl-1,3,4-oxadiazol-2-ones whose structure has been confirmed by I.R., N.M.R. and mass spectral methods. The results of pharmacological testing are also reported and these further clarify some aspects of the biological activity of the oxadiazole structure.

In continuation of previous work designed to obtain derivatives of 2-mercapto-1,3,4-oxadiazole of possible biological interest, the Mannich bases (III - XXX) were prepared. The compounds prepared bear at position 5, with respect to the oxadiazole nucleus, 3,4,5-trimethoxy and 3,4-dihydroxymethylenephenyl radicals which have proved of interest in other series of derivatives. The antimycotic activity of the compounds is reported.

In continuation of previous research designed to obtain compounds of pharmacological interest, the following substances were prepared, from the potassium salts (I-III) and from the methyl esters (IV-VI) of some aroylhydrazinocarbodithioic acids, derivatives of 1,3,4-oxadiazole (VII-XV), of 3-aminorhodanine (XVI-XVIII), of 1,2,4-triazole (XIX-XXI), of 7H-(1,2,4)-triazol[3,4-b] [1,3,4] thiadiazine (XXII-XXIV) and of 1,3,4-thiadiazole (XXV-XXX). The compounds have present in the molecule a nucleus and radical which have produced marked biological activity in other series of compounds. To date some of the above compounds have shown antibacterial, antimycotic and antimitotic activity.

The behaviour of alkaline salts of some aroyl-hydrazine-carbodithioic acids [comp. Ia,b,c,] and derivatives of 1,3,4-oxadiazole [comp. IIa,b,c, e IIIa,b,c] , has been studied, on the capability of lymphoblasts in vitro, in consequence of stimulation by some mitogen agents (PHA, PWM). It has been pointed out that the considered compounds inhibit the cellular mitosis in variable percentage in relation to their structure and to the used mitogen agent, frequently causing some mitogen-like morphological modification, also revealable under a optical microscope. The higher inhibition has been obtained by the Ic, IIc, and IIIc compounds, in which Ar is the 3,4-dihydroxymethylenephenyl radical, proving selective, in some cases, for the T lymphocytes.

The preparation of a series of aroylhydrazones of halogenobenzaldehydes (I) - (XI) is described together with that of the corresponding reduction products (XII) - (XXV) and of 2,5-diaryl-1,3,4-oxadiazoles with halogen substitution (XXVI) - (XLIII). The results of pharmacological and microbiological examination of the compounds is reported.