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The aim of this study was to obtain and to characterize microemulsions containing 5-aminolevulinic acid (5-ALA) and to investigate the influence of these systems in drug skin permeation for further topical photodynamic therapy (PDT). 5-ALA was incorporated in water-in-oil (W/O), bicontinuous (Bc), and oil-in-water (O/W) microemulsions obtained by the titration of ethyl oleate and PEG-8 caprylic/capric glycerides:polyglyceryl-6 dioleate (3:1) mixtures with water. Selected systems were characterized by conductivity, viscosity, size of the droplets, and drug release. The stability of the drug in the microemulsions was also assessed. Moreover, the in vitro and in vivo skin permeation of 5-ALA was investigated using diffusion cells and confocal scanning laser microscopy (CSLM), respectively. Despite the fact that the O/W microemulsion decreased the 5-ALA diffusion coefficient and retarded the drug release, it also significantly increased the in vitro drug skin permeation when compared to other 5-ALA carriers. It was observed by CSLM that the red fluorescence of the skin increased homogeneously in the deeper skin layers when the 5-ALA microemulsion was applied in vivo, probably due to the formation of the photoactive protoporphyrin IX. The microemulsion developed carried 5-ALA to the deeper skin layers, increasing the red fluorescence of the skin and indicating the potentiality of the system for topical 5-ALA-PDT.

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Topical chemotherapy with the antineoplastic doxorubicin (DXR) could be an alternative to treat skin cancer, however its poor skin penetration often limits the efficacy of topical formulations. The aim of this work was to study the effect of monoolein (MO), a penetration enhancer, on the in vitro skin permeation and retention of DXR. DXR was incorporated in a propylene glycol preparation containing 0-20% of MO. DXR release rate and topical delivery were evaluated in vitro using acetate cellulose membrane and porcine skin, respectively, mounted in a Franz diffusion cell. At 5%, MO did not significantly change DXR release rate, but MO concentrations larger than 10% decreased almost twice its release. In vitro skin penetration studies showed that the presence of MO in the propylene glycol formulations markedly increased DXR presence in the stratum corneum (SC). At 5%, MO significantly increased the amount of DXR in the SC already in the first hours, attained a maximum in 6h. Comparing propylene glycol formulations containing more than 10% MO with that containing 5%, the former took the double of the time (12h) to reach the same amount of DXR in the skin, result that is in agreement with in vitro release studies. Interesting, despite the fact that MO significantly increased the amount of DXR in the SC, drug transdermal delivery did not change. These findings suggest a cutaneous delivery of DXR that is an important condition for topical treatment of skin tumors. Further in vivo experiments can show DXR delivery to deeper skin layers.

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Avaliamos os efeitos do ultra-som pulsado de baixa intensidade sobre a cicatrização de lesão cutânea produzida na região dorsal de ratos. Utilizamos 60 ratos machos (Wistar, peso médio de 300g) divididos em dois grupos, sendo: 1) irradiação simulada; 2) irradiação efetiva (freqüência fundamental de 1,5MHz, freqüência de repetição de pulsos de 1KHz, largura de pulso de 200 æs, intensidade de 30mW/cm² -SATA, 10 minutos de aplicação em dias alternados). Estes foram subdivididos em subgrupos, de acordo com o tempo de avaliação da lesão, de 3, 7 e 14 dias, e a cicatrização foi avaliada através de análises planimétrica e histo-morfométrica. Aumento significante (p<0,05) da área cicatrizada foi observado no Grupo 2 (141,88±18,50 mm²) em relação ao Grupo 1 (117,38±15,14 mm²), no 14° dia. Houve diminuição significante do número de células inflamatórias (p<0,05), associada a um incremento da angiogênese, no Grupo 2 (2196,56 cel/mm²±234,93) em relação ao Grupo 1 (2611,68 cel/mm²±423,82), no 3° dia. Não observamos diferenças significantes na formação de colágeno, área de derme e epiderme entre os grupos. Concluiu-se que o ultra-som pulsado de baixa intensidade não apresenta efeitos deletérios e estimula moderadamente a cicatrização cutânea por segunda intenção em condições experimentais, com potencial para aplicação clínica em humanos.

We evaluated the effects of low-power pulsed ultrasound on skin injury healing at dorsal region of rats. Sixty male rats were used (Wistar, mean weight: 300 g) divided into two groups, namely: 1) simulated irradiation; 2) effective irradiation (basic rate of 1.5MHz, pulse cycle rate of 1KHz, pulse width of 200 æs, power of 30mW/cm2 -SATA, 10 minutes of application in alternate days). These were further divided into subgroups, according to the time of injury assessment, as 3, 7, and 14 days, and healing was assessed by planimetrical and histomorphometrical analysis. A significant increase (p<0.05) of the healing area was seen for Group 2 (141.88±18.50 mm²) compared to Group 1 (117.38±15.14 mm²), at the 14th day. There was a significant reduction of the number of inflammatory cells (p<0.05), associated to an increment of angiogenesis for Group 2 (2196.56 cel/mm²±234.93) in comparison to Group 1 (2611.68 cel/mm²±423.82), at the 3rd day. No significant differences were seen in collagen formation, or on dermis and epidermis area between groups. It was concluded that low-power pulsed ultrasound does not cause any deleterious effects and can moderately stimulate skin second-intention healing in experimental environments, showing a potential to clinical use in human beings.

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PURPOSE: In topical photodynamic therapy, 5-ALA and its esters are enzymatically converted in the endogenous photosensitizing compounds such as, for example, protoporphyrin IX (PpIX). In order to elucidate in more detail their enzymatic fate, we have determined in vitro the enzymatic degradation of methyl, butyl, hexyl, and octyl-5-ALA ester derivatives in skin homogenate. Furthermore, in vivo porphyrin accumulation was measured in healthy hairless mice skins. METHODS: Hairless mouse skins were homogenized in isotonic phosphate buffer pH 7.4. 5-ALA esters were added, and aliquots were colleted for HPLC-fluorimetric determinations of remaining content of 5-ALA esters. Furthermore, oil-in-water emulsions containing esters were topically applied to mice skin for 6 h, and the amount of accumulated PpIX in the treated areas was determined by quantitative extraction and confocal fluorescence microscopy. RESULTS: The enzymatic degradation of esters follows pseudo first-order kinetics. The octyl ester had the largest rate constant for enzymatic degradation, followed by hexyl-, butyl-, and methyl-ALA. The long-chained 5-ALA esters, butyl-, hexyl-, and octyl ester, induced significantly more porphyrins than 5-ALA and 5-ALA methyl ester as shown by confocal microscopy and quantitative extraction studies. CONCLUSIONS: 5-ALA derivatives differ widely with respect to their enzymatic degradation. The presence of alkyl chains in 5-ALA esters significantly influences the in vitro enzymatic metabolism and the in vivo PpIX formation in healthy hairless mice skins.

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