Celastrol Nanoemulgel Formulation And In Vitro Penetration Test

Objective: To obtain formulations of Celastrol (Cst) nanoemulgel via transdermal route. Celastrol is classified in BCS 4 class as an anti-inflammatory drug. These routes are considered to reduce the risk of Celastrol side effects and have the same characteristics as skin morphology. Methods: Celastrol nanoemulgel was prepared by a high-pressure homogenizer (HPH) technique. To find the optimum nanoemulsion area by using the Chemix 7.00 ternary phase program. Celastrol nanoemulgel was evaluated by measuring the particle size, PDI, morphology, zeta potential, stability tests and in vitro using Franz diffusion cell Results: Results showed the ideal formula based on the ternary phase diagram using chemix 7.00 is oil: smix: water (5:45:50), with particle size 89.9±5 nm, PDI 0.1, and zeta-21 mV. The morphological shape is quite spherical ≤ 100±5 nm. The pH value of this formula is 4.5, which compatible with the pH of the skin. The highest recovery rate of Celastrol and encapsulation efficiency (EE) were formulas 3 μg/ml and 5 μg/ml, with EE 91.70% and 94.54%, respectively. In vitro test results showed that the formula 3 μg/ml and 5 μg/ml give better penetration results than the formula 2.5 μg/ml. Thus, Celastrol nanoemulgel formula has good potential to be developed as a transdermal anti-inflammatory drug. Conclusion: Transdermal nanoemulgel containing Celastrol has been successfully developed with particle size ≤ 200±2 nm.

Improving Transdermal Drug Delivery System For Medroxyprogesterone Acetate By Olive Oil And Dimethylsulfoxide (DMSO) As Penetration Enhancers: In Vitro Penetration Study

Objective: Medroxyprogesterone Acetate (MPA) using a transdermal drug delivery system for contraception by passive diffusion is limited by the skin barrier properties. Penetration enhancers such as olive oil (fatty acid permeation enhancer) and DMSO (chemical enhancer) can be used. The objective of this study was to overcome MPA penetration problem by using olive oil and DMSO. Methods: An in vitro penetration study using the Franz diffusion cells was performed. The first penetration study used MPA in olive oil (O) and MPA in coconut oil (C) with the concentration 100 μg/ml to each sample and MPA suspension as a control with the same concentration. The second study used MPA in olive oil with the concentration 200.0 μg/ml (A), MPA in olive oil with 0.5% DMSO with the concentration 200.0 μg/ml (B), and MPA in olive oil with 1% DMSO with the concentration 200 μg/ml (C). Results: MPA penetration test for olive oil+0.5% DMSO had flux value 4.24±0.074 μg/cm2. hr and it was not significantly different (t-test, P>0.05) with olive oil+1% DMSO. While the MPA penetration test in only Olive oil had flux value 0.90±0.0087 μg/cm2. hr. Conclusion: This research concluded that olive oil and 0.5% DMSO could improve the penetration of MPA into skin membrane by 4.5 times more than olive oil alone.

Actividad in vitro contra Leishmania y permeación en piel humana de liposomas ultradeformables de miltefosina / In vitro activity against Leishmania and human skin permeation of miltefosine ultradeformable liposomes

Introducción: los liposomas ultradeformables de miltefosina (LUD-MIL) constituyen una opción para el tratamiento tópico en leishmaniasis cutánea penetrando los estratos de la piel hasta la dermis, sitio donde habita el parásito. Objetivo: diseñar LUD-MIL y determinar su actividad contra L. (Viannia) panamensis y L. (V.) braziliensis y la permeación en piel humana. Métodos: los LUD-MIL, liposomas convencionales de fosfatidilcolina (LConv) y LUD-MIL-fluorescente (LUD-MIL-Fluo) fueron preparados por el método de rehidratación de película lipídica. Se caracterizaron fisicoquímicamente y se determinaron: la liberación en membrana semisintéticas, la retención en las capas de la piel y la permeación en piel humana. La citotoxicidad en THP-1 fue determinada por el ensayo colorimétrico de MTT y la actividad en promastigotes y amastigotes intracelulares por recuento microscópico. Resultados: el tamaño, índice de polidispersión, potencial Z y concentración de fosfolípidos de los LUD-MIL fue de 100,7 nm, 0,147, -12,0 mV y 53,24 mM respectivamente. El flujo de MIL a través de la membrana fue mayor con LUD-MIL que con MIL-libre. El tratamiento con LUD-MIL indujo menor acumulación de la MIL en el estrato corneo y mayor permeación que el tratamiento con MIL libre. Los LUD-MIL y los LConv-MIL mantuvieron la actividad de la MIL en los parásitos y células. Los LUD-MIL fueron más tóxicos para las células que los LConv y la MIL y más activos en amastigotes intracelulares de L. (V.) braziliensis. Conclusión: los LUD-MIL preparados conservaron la actividad anti-Leishmania de la MIL y permitieron la liberación del compuesto en membranas y piel humana. Ensayos en modelos experimentales de leishmaniasis cutánea para evaluar la actividad de estas formulaciones son urgentes de realizar(AU)

Introduction: miltefosine ultradeformable liposomes (MIL-LUD) are an option for the topical treatment of cutaneous leishmaniasis penetrating the skin layers to the dermis where the parasite inhabits. Objective: to design MIL-LUD and determine their in vitro activity against L. (Viannia) panamensis and L. (V.) braziliensis and to determine human skin permeation. Methods: MIL-LUD, phosphatidylcholine liposomes (MIL-LConv) and fluorescent MIL-LUD (MIL-LUD-Fluo) were prepared by lipid film rehydration method. They were physicochemically characterized to determine drug release in semisynthetic membrane, retention in skin layers and permeation on human skin membranes. Cytotoxicity in THP-1 was determined by the MTT colorimetric test and activity in promastigotes and intracellular amastigotes by microscopic counting. Results: the size, the polydispersion index, the Zeta potential and phospholipid content were 100.7 nm, 0.147, -12.0mV and 53.24mM, respectively for MIL-LUD. MIL flow through the semisynthetic membrane was greater with MIL-LUD than MIL-free treatment. MIL-LUD treatment induced lower MIL accumulation in the stratum corneum and increased permeation than MIL free treatment. The MIL-LUD and MIL-Conv maintained MIL activity in parasites and cells. The MIL-LUD was more toxic to cells than MIL-Conv and more active against intracellular amastigotes of L. (V.) braziliensis. Conclusion: prepared LUD -MIL retained the anti-leishmanial activity of the MIL and allowed the compound release in human skin and membranes. Testing of experimental cutaneous leishmaniasis models to evaluate the activity of these formulations are urgently needed(AU)

Permeación en piel humana de una nanoemulsión de ftalocianina de aluminio clorada para la optimización de tratamientos tópicos de leishmaniasis cutánea / Human Skin Permeation of a Chloroaluminum Phthalocyanine Nanoemulsion for Optimization of Topical Cutaneous Leishmaniasis Formulations / Permeação em pele humana de uma nanoemulsão de ftalocianina de alumínio clorada para a otimização de tratamentos tópicos de leishmaniose cutânea

Introducción: Las nanoemulsiones son excelentes sistemas de transporte y entrega de fármacos. La ftalocianina de aluminio clorada (PcAlCl) en terapia fotodinámica constituye una alternativa de tratamiento en leishmaniasis cutánea. Objetivo: Determinar la difusión y retención en piel humana de la PcAlCl contenida en una nanoemulsión (nano-PcAlCl) para su optimización en formulaciones tópicas. Materiales y métodos: Se prepararon y caracterizaron fisico-químicamente dos formulaciones (nano-PcAlCl y solución-PcAlCl) y sus vehículos sin-PcAlCl. La permeación se determinó en ensayos en celdas de difusión de Franz y la retención por el método de la cinta adhesiva. La concentración de PcAlCl fue determinada fluorométricamente (nM/cm2). Biopsias de piel fueron analizadas histotécnicamente. Resultados: El tamaño promedio, el potencial Z y el índice de polidispersión de la nano-PcAlCl en agua fue de 132,9 nm, -19,23 y 0,14 y diluida en PBS fue 125,33 nm, -13,69 y 0,139. Las concentraciones de PcAlCl se mantuvieron estables. La PcAlCl no atravesó la piel y fue retenida en sus capas, en estrato córneo y epidermis+dermis con valores de 44,17 nM y 8,48 nM postratamiento con nano-PcAlCl, y 96,90 nM y 9,80 nM postratamiento con solución-PcAlCl. Esta última promovió mayor retención en estrato córneo y ambas formulaciones promovieron similar retención en epidermis+dermis. Se observó desprendimiento del estrato córneo y fragmentación del colágeno. Conclusión: La PcAlCl no atravesó la piel, se retuvo en estrato córneo y epidermis+dermis. Se sugiere realizar ensayos de permeación utilizando piel humana desprovista de estrato córneo y ensayos de distribución en animales con leishmaniasis cutánea.

Introduction: The nanoemulsions constitute excellent drug delivery systems for carrying and delivering active drugs. Chloroaluminum phthalocyanine (ClAlPc) in photodynamic therapy constitutes an interesting alternative in cutaneous leishmaniasis treatment. Objective: To determine the diffusion and retention of ClAlPc contained in a nanoemulsion (nano-ClAlPc) in human skin membranes for optimization of topical formulations. Materials and methods: Two formulations (ClAlPc-nano- and ClAlPc-solution) and vehicles without ClAlPc were prepared and physicochemical characterized. The permeation was tested in Franz-diffusion cells and the retention by the tape stripping method. ClAlPc concentration was determined fluorometrically (nM/cm2). Skin biopsies were analyzed by histologic technics. Results: The ClAlPc-nano average size, zeta potential and polydispersity index diluted in water was 132.9 nm, -19.23 and 0.14 and diluted in phosphate-buffer-saline was 25.33 nm, -13.69 and 0.139. ClAlPc maintains its stability in each formulation. ClAlPc was unable to pass completely through the skin; it was retained in the different skin layers. A ClAlPc retention in stratum corneum and epidermis+dermis was observed with values of 44.17 nM and 8.48 nM after ClAlPc-nano treatment and 96.90 nM and 9.80 nM after ClAlPc-solution treatment. The ClAlPc-solution promoted greater retention in stratum corneum and both formulations showed similar ClAlPc-retention in epidermis+dermis. Histological changes as stratum corneum detachment and collagen-fragmentation were observed. Conclusion: ClAlPc was not able to cross completely the skin, it was retained in stratum corneum and epidermis+dermis Human permeation test using skin membranes without stratum corneum, and distribution assays in cutaneous leishmaniasis-infected animals, are suggested.

Introdução: as nanoemulsões são excelentes sistemas de transporte e entrega de fármacos. A ftalocianina de alumínio clorada (PcAlCl) em terapia fotodinâmica constitui uma alternativa de tratamento em leishmaniose cutânea. Objetivo: determinar a difusão e retenção em pele humana da PcAlCl contida em uma nanoemulsão (nano-PcAlCl) para sua otimização em formulações tópicas. Materiais e métodos: se prepararam e caracterizaram fisico-quimicamente duas formulações (nano-PcAlCl e solução-PcAlCl) e seus veículos sem-PcAlCl. A permeação determinou-se em ensaios em celas de difusão de Franz e a retenção pelo método da fita adesiva. A concentração de PcAlCl foi determinada fluorometricamente (nM/cm²). Biopsias de pele foram analisadas histotecnicamente. Resultados: o tamanho médio, o potencial Z e o índice de polidispersão da nano-PcAlCl em água foi de 132,9 nm, -19,23 e 0,14 e diluída em PBS foi 125,33 nm, -13,69 e 0,139. As concentrações de PcAlCl mantiveram-se estáveis. A PcAlCl não atravessou a pele, foi retido em suas capas. A PcAlCl foi retida em estrato córneo e epiderme+derme com valores de 44,17 nM e 8,48 nM pós-tratamento com nano-PcAlCl e 96,90 nM e 9,80 nM pós-tratamento com solução-PcAlCl. A solução-PcAlCl promoveu maior retenção em estrato córneo e ambas as formulações promoveram similar retenção em epiderme+derme. Observou-se desprendimento do estrato córneo e fragmentação do colágeno. Conclusão: a PcAlCl não atravessou a pele; reteve-se em estrato córneo e epiderme+derme. Sugere-se realizar ensaios de permeação utilizando pele humana desprovida de estrato córneo e ensaios de distribuição em animais com leishmaniose cutânea.

Permeación en piel y biodistribución de una nanoemulsión de ftalocianina de aluminio clorada (PcAlCl) aplicada tópicamente en ratas Wistar / Skin permeation and biodistribution of chloroaluminum phthalocyanine (ClAlPc) nanoemulsion applied topically in Wistar rats

Una formulación tópica en leishmaniasis cutánea debe garantizar la permeación y acumulación del compuesto en la dermis, sitio donde se encuentran los macrófagos infectados con Leishmania. Se determinó la difusión y retención de PcAlCl contenida en una nanoemulsión (nano-PcAlCl) y en solución-PcAlCl y su distribución en ratas Wistar, con piel sana o lesionada quirúrgicamente. La nano-PcAlCl se preparó por el método de emulsificación espontánea y la solución-PcAlCl en mezcla DMSO: Tween 80: agua tipoI; la estabilidad se determinó espectrofotométricamente. La difusión se determinó utilizando celdas de Franz y la retención en el estrato córneo (EC) y la epidermis-dermis (E+D) por "tape stripping". Las formulaciones mantuvieron las características espectroscópicas del compuesto. La PcAlCl no difundió al medio receptor. Después de 12 y 24 horas la retención del compuesto en EC con nano-PcAlCl fue 99,73 y 79,40nM y con solución-PcAlCl 66,73 y 57,01nM; en E+D la retención con nano-PcAlCl fue 40,94 y 62,49nM y con solución-PcAlCl 81,92 y 50,28nM. En EC la nano-PcAlCl registró mayor retención y en E+D la solución-PcAlCl, registró mayor retención a las 12 horas y la nano-PcAlCl a las 24 horas. Se observó desprendimiento y disminución en las capas del epitelio. El tratamiento con nano-PcAlCl retuvo el compuesto en EC y E+D de los animales con valores de 6,43nM y 33,18nM con piel sana y 0,99nM y 17,08 con piel lesionada; valores menores de 1,92nM fueron detectados sólo en pulmón. El tratamiento con solución-PcAlCl retuvo el compuesto en EC y E+D de los animales con valores de 31,86 y 202,40nM con piel sana y 5,93 y 63,83nM con piel lesionada; se encontraron valores de 2,36nM y 20,33nM en los pulmones y valores entre 1,58 y 5,80nM en otros órganos. La piel lesionada presentó regeneración del epitelio, desprendimiento de queratina, infiltrado celular y neovascularización. La PcAlCl retenida en EC y E+D indicó la eficacia de las formulaciones para penetrar el EC y retener el compuesto en las capas viables de la piel. El tratamiento con nano-PcAlCl permeó el EC, se retuvo en E+D y no presentó distribución hacia órganos.

A successful formulation contained aluminum phthalocyanine chloride (ClAlPc) able for cutaneous leishmaniasis (CL) ensures the skin permeation and retention of the compound in dermis, a place where macrophages infected with Leishmania are located. The aim of this study was to determine the ClAlPcex vivo permeation and retention in Wistar rat skin and its distribution in animals with healthy skin and surgically damaged skin. Two formulations were prepared: nanoemulsion (ClAlPc-nano) and solution (ClAlPc-solution). The diffusion was determined using Franz diffusion cells with Wistar rat skin as membrane after 12 and 24 hours of testing. Retention in stratum corneum (SC) and epidermis plus dermis (E+D) was determined by the tape stripping method and organic solvent extraction. The distribution was performed after the topical application of formulations analyzing the CLAlPc concentration in the skin layers and organs by fluorometry. The results were expressed as nM/mg of tissue of ClAlPc. Simultaneously, skin biopsies were taken in order to determine their histological characteristics. ClAlPc formulations were effective in maintaining compound solubility and spectroscopic characteristics. ClAlPc was unable to pass completely through the skin; its skin retention was related with the used formulation, time of assays and type of skin layer. After 12 and 24 hours, the SC retention induced by ClAlPc-nano treatment was 99.73 and 79.40nM and by ClAlPc-solution was 66.73 and 57.01nM. In E+D the retention induced by ClAlPc-nano was 40.94 and 62.49nM and by ClAlPc-solution was 81.92 and 50.28nM. In SC, ClAlPc-nano showed greater retention after 12 and 24 hours, while in E+D, ClAlPc-solution was greater retained at 12 hours and ClAlPc-nano at 24 hours. After the permeation assays, detachment and reduction in epithelial layers were observed in skin membranes. In Wistar rats, ClAlPc-nano placed on healthy skin retained the compound in E+D and SC with values of 33.18nM and 6.43nM and placed on damaged skin retained in E+D in SC with values of 17.08nM and 0.99nM respectively; no organs distribution occur, except in lung where values of 0.82nM in animals with healthy skin and 1.92nM in animals with damaged skin were observed. ClAlPc-solution induced retention of 31.86 in SC and 202,40nM in E+D placed on healthy skin and 5.93 in SC and 63.83nM E+D placed on damaged skin; concentrations in lung with values of 2,36nM after application in animals with healthy skin and 20,33nM in animals with damaged skin were observed. In addition, ClAlPc-solution promoted, after application on damaged skin, distribution of ClAlPc to other organs with values between 1.58 and 5.80nM. In animals without skin injury, normal histologic skin anatomy were observed after treatment. In contrast, epithelial regeneration, detachment of keratin, loss of cellular differentiation, cellular infiltration and neovascularization were observed in animals with skin injury. The efficacy of the formulation to penetrate the SC and be retained in the viable skin layers was demonstrated. The in vivo ClAlPc-biodistribution was related on its concentration and skin state. A nanoemulsion contained ClAlPc, represents a good system of drug delivery because induce both the SC penetration and E+D retention, without systemic organ distribution even when presented tissue repair and regeneration. A ClAlPc-nano prepared in this work constitutes a good formulation for the topical application of ClAlPc in LC treatment.