Development and validation of HPLC method for imiquimod determination in skin penetration studies.

A simple, rapid and sensitive analytical procedure for the measurement of imiquimod in skin samples after in vitro penetration studies has been developed and validated. In vitro penetration studies were carried out in Franz diffusion cells with porcine skin. Tape stripping technique was used to separate the stratum corneum (SC) from the viable epidermis and dermis. Imiquimod was extracted from skin samples using a 7:3 (v/v) methanol:acetate buffer (100 mM, pH 4.0) solution and ultrasonication. Imiquimod was analyzed by HPLC using C(8) column and UV detection at 242 nm. The mobile phase used was acetonitrile:acetate buffer (pH 4.0, 100 mM):diethylamine (30:69.85:0.15, v/v) with flow rate 1 mL/min. Imiquimod eluted at 4.1 min and the running time was limited to 6.0 min. The procedure was linear across the following concentration ranges: 100-2500 ng/mL for both SC and tape-stripped skin and 20-800 ng/mL for receptor solution. Intra-day and inter-day accuracy and precision values were lower than 20% at the limit of quantitation. The recovery values ranged from 80 to 100%. The method is adequate to assay imiquimod from skin samples, enabling the determination of the cutaneous penetration profile of imiquimod by in vitro studies.

Effect of iNOS and NF-kappaB gene silencing on beta-cell survival and function.

PURPOSE: Type I diabetes results from beta-cell death and dysfunction, induced by infiltration of immune cells and local production of inflammatory cytokines. Therefore, we investigated the effect of iNOS and NF-kappaB gene silencing on beta-cell survival and function. METHODS: Rat insulinoma INS-1E cells were transfected with chemically synthesized siRNA after complex formation with Lipofectamine 2000. Cells were then treated with a cocktail of inflammatory cytokines (IL-1beta+ TNF-alpha+ IFN-alpha), and glucose stimulated-insulin response and viability were determined. iNOS and NF-kappaB gene expression was assessed at mRNA level by real time RT-PCR. The effect of gene silencing was also correlated with cytokine-induced NO production and apoptosis. RESULTS: Transfection of INS-1E cells with siRNAs silenced iNOS and NF-kappaB gene expression and reduced NO production in a sequence-specific manner without causing significant loss of cell viability and function. However, the abrogation of NO production did not prevent INS-1E cells from cytokine-induced apoptosis, suggesting that this event may not be totally dependent on NO production. CONCLUSION: The gene silencing approach presented here is capable of attenuating the effects of inflammatory cytokines, such as iNOS expression and NO production and it will help to identify new target genes to improve islet transplantation.

Enhancing effect of modified beta-cyclodextrins on in vitro skin permeation of estradiol

Estradiol, the most important hormone in females, was complexed with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and randomly methylated beta-cyclodextrin (RM-b-CD). After obtaining the inclusion complexes, they were characterized by DSC, ¹H-NMR and solubility studies. The enhancing effect of CDs on estradiol skin permeation/retention was investigated in vitro using Franz diffusion cells. Both CDs increased estradiol aqueous solubility, but in different proportions. DSC and NMR-H¹ analyses showed that estradiol was complexed with both CDs and RM-beta-CD has stronger interactions with the hormone than HP-beta-CD. Moreover, complexes formation increased estradiol flux through the skin (P<0.01), but the hormone retention in the stratum corneum (SC) only increased after complexation with HP-beta-CD. On the other hand, only RM-b-CD was able to modify estradiol retention in the SC after skin pretreatment with CDs. The results suggest that the enhancing effect of CDs on estradiol flux through the skin may be mainly described as an increase in drug availability on the skin surface due to inclusion complexation. Furthermore, the formation of a significant reservoir of estradiol in the SC due to HP-beta-CD complexation makes it an interesting approach for estradiol transdermal delivery.

Estradiol, o mais importante hormônio feminino, foi complexado com hidroxipropil-beta-ciclodextrina (HP-beta-CD) e metil-beta-ciclodextrina randômica (RM-beta-CD). Após a obtenção dos complexos, os mesmos foram caracterizados por estudos de solubilidade, CDV e RMN-H¹. O efeito das CDs sobre a absorção/retenção do estradiol na pele foi investigado in vitro em células de difusão de Franz. As CDs aumentaram a solubilidade aquosa do estradiol em diferentes proporções. As análises de CVD e RMN-H¹ comprovaram a complexação do estradiol com ambas CDs, sendo que RM-beta-CD apresentou interação mais forte com estradiol do que HP-beta-CD. Mais adiante, a formação de complexos aumentou o fluxo de estradiol através da pele (P<0,01), mas o aumento de fármaco no estrato córneo (EC) foi observado somente após complexação com HP-beta-CD. Por outro lado, somente a RM-beta-CD foi capaz de aumentar a retenção do fármaco no EC após o pré-tratamento da pele com CDs. Os resultados sugerem que o efeito promotor das CDs no fluxo de estradiol pode ser descrito principalmente como aumento da disponibilidade de fármaco na superfície da pele devido à complexação. Além disso, a formação de um significativo reservatório do hormônio no EC torna a complexação com HP-beta-CD uma estratégia interessante para liberação transdérmica do estradiol.

Reverse hexagonal phase nanodispersion of monoolein and oleic acid for topical delivery of peptides: in vitro and in vivo skin penetration of cyclosporin A.

PURPOSE: To obtain and characterize reverse hexagonal phase nanodispersions of monoolein and oleic acid, and to evaluate the ability of such system to improve the skin penetration of a model peptide (cyclosporin A, CysA) without causing skin irritation. METHODS: The nanodispersion was prepared by mixing monoolein, oleic acid, poloxamer, and water. CysA was added to the lipid mixture to obtain a final concentration of 0.6% (w/w). The nanodispersion was characterized; the skin penetration of CysA was assessed in vitro (using porcine ear skin mounted in a Franz diffusion cell) and in vivo (using hairless mice). RESULTS: The obtainment of the hexagonal phase nanodispersion was demonstrated by polarized light microscopy, cryo-TEM and small angle X-ray diffraction. Particle diameter was 181.77 +/- 1.08 nm. At 0.6%, CysA did not change the liquid crystalline structure of the particles. The nanodispersion promoted the skin penetration of CysA both in vitro and in vivo. In vitro, the maximal concentrations (after 12 h) of CysA obtained in the stratum corneum (SC) and in the epidermis without stratum corneum (E) + dermis (D) were approximately 2 fold higher when CysA was incorporated in the nanodispersion than when it was incorporated in the control formulation (olive oil). In vivo, 1.5- and 2.8-times higher concentrations were achieved in the SC and [E+D], respectively, when the nanodispersion was employed. No histopathological alterations were observed in the skin of animals treated with the nanodispersion. CONCLUSION: These results demonstrate that the hexagonal phase nanodispersion is effective in improving the topical delivery of peptides without causing skin irritation.