Ex vivo model of human skin (hOSEC) for assessing the dermatokinetics of the anti-melanoma drug Dacarbazine.

Alternative models to replace animals in experimental studies remain a challenge in testing the effectiveness of dermatologic and cosmetic drugs. We proposed a model of human organotypic skin explant culture (hOSEC) to assess the profile of cutaneous drug skin distribution, adopting dacarbazine as a model, and respective new methodologies for dermatokinetic analysis. The viability tests were evaluated in primary keratinocytes and fibroblasts, and skin by MTT and TTC assays, respectively. Then, dacarbazine was applied to the culture medium, and the hOSEC method was applied to verify the dynamics of skin distribution of dacarbazine and determine its dermatokinetic profile. The results of cell and tissue viability showed that both were considered viable. The dermatokinetic results indicated that dacarbazine can be absorbed through the skin, reaching a concentration of 36.36 µg/mL (18,18%) of the initial dose (200 µg/mL) after 12 h in culture. Histological data showed that the skin maintained its structure throughout the tested time that the hOSEC method was applied. No apoptotic cells were observed in the epidermal and dermal layers. No visible changes in the dermo-epidermal junction and no inflammatory processes with the recruitment of defense cells were observed. Hence, these findings suggest that the hOSEC concept as an alternative ex vivo model for assessing the dynamics of skin distribution of drugs, such as dacarbazine, and determining their respective dermatokinetic profiles.

Evaluation of critical parameters for in vitro skin permeation and penetration studies using animal skin models.

In vitro skin permeation/penetration studies may be affected by many sources of variation. Herein, we aimed to investigate the major critical procedures of in vitro skin delivery studies. These experiments were performed with model drugs according to official guidelines. The influence of skin source on penetration studies was studied as well as the use of a cryopreservation agent on skin freezing evaluated by transepidermal water loss, electrical resistance, permeation/penetration profiles and histological changes of the skin. The best condition for tape stripping procedure was validated through the evaluation of the distribution of corneocytes, mass of stratum corneum (SC) removed and amount of protein removed using finger pressure, a 2kg weight and a roller. The interchangeability of the tape stripping procedures followed by the epidermis and dermis homogenate and the micrometric horizontal cryostat skin sectioning methods were also investigated, besides the effect of different formulations. Noteworthy, different skin sources were able to ensure reliable interchangeability for in vitro permeation studies. Furthermore, an increased penetration was obtained for stored frozen skin compared to fresh skin, even with the addition of a cryoprotectant agent. The best method for tape stripping was the finger pressure followed by the addition of a propylene glycol solvent leading to better SC removal. Finally, no significant difference was found in skin penetration studies performed by different methods suggesting their possible interchangeability.

Liquid Crystalline Systems Based on Glyceryl Monooleate and Penetration Enhancers for Skin Delivery of Celecoxib: Characterization, In Vitro Drug Release, and In Vivo Studies.

Celecoxib (CXB) is a widely used anti-inflammatory drug that also acts as a chemopreventive agent against several types of cancer, including skin cancer. As the long-term oral administration of CXB has been associated with severe side effects, the skin delivery of this drug represents a promising alternative for the treatment of skin inflammatory conditions and chemoprevention of skin cancer. We prepared and characterized liquid crystalline systems based on glyceryl monooleate and water containing penetration enhancers which were primarily designed to promote skin delivery of CXB. Analysis of their phase behavior revealed the formation of cubic and hexagonal phases depending on the systems' composition. The systems' structure and composition markedly affected the in vitro CXB release profile. Oleic acid reduced CXB release rate, but association oleic acid/propylene glycol increased the drug release rate. The developed systems significantly reduced inflammation in an aerosil-induced rat paw edema model. The systems' composition and liquid crystalline structure influenced their anti-inflammatory potency. Cubic phase systems containing oleic acid/propylene glycol association reduced edema in a sustained manner, indicating that they modulate CXB release and permeation. Our findings demonstrate that the developed liquid crystalline systems are potential carriers for the skin delivery of CXB.

Ketoprofen Microemulsion for Improved Skin Delivery and In Vivo Anti-inflammatory Effect.

We have designed a microemulsion (ME) containing Ketoprofen (KET) for anti-inflammatory effect evaluated using the rat paw edema model. The ME was prepared by adding propylene glycol (PG) loaded with 1% KET/water (3:1, w/w), to a mixture of sorbitan monooleate and polysorbate 80 (47.0%) at 3:1 (w/w) and canola oil (38.0%). The physicochemical characterization of KET-loaded ME involved particle size and zeta potential determination, entrapment efficiency, calorimetric analysis, and in vitro drug release. The in vivo anti-inflammatory study employed male Wistar rats. Measurement of the foot volume was performed using a caliper immediately before and 2, 4, and 6 h after injection of Aerosil. KET-loaded ME showed particle size around 20 nm, with zeta potential at -16 mV and entrapment efficiency at 70%. Moreover, KET was converted to the amorphous state when loaded in the formulation and it was shown that the drug was slowly released from the ME. Finally, the in vivo biological activity was similar to that of the commercial gel, but ME better controlled edema at 4 h. These results demonstrated that the ME formulation is an alternative strategy for improving KET skin permeation for anti-inflammatory effect. Furthermore, our findings are promising considering that the developed ME was loaded with only 1% KET, and the formulation was able to keep a similar release profile and in vivo effect compared to the commercial gel with 2.5% KET. Therefore, the KET-loaded developed herein ME is likely to have a decreased side effect compared with that of the commercial gel, but both presented the same efficacy.

Avaliação da citotoxicidade da microemulsão contendo o fotossensibilizador ZNPCSO4, sobre cultura de célula de linhagem de câncer de mama / Evaluación de la citotoxicidad de la microemulsión que contiene el fotosensibilizador ZNPCSO4, en la cultura celular del cáncer de mama / Cytotoxicity evaluation of microemulsion containing the photosensibilizer ZNPCSO4, on breast cancer cell culture line

Introdução: O câncer de mama tem alta incidência e mortalidade dentre todos os tipos de câncer e, apesar de todos os avanços tecnológicos recentes,mantém-se ainda como um problema mundial de saúde pública. Novas estratégias terapêuticas, como a Terapia Fotodinâmica, têm sido usadas para tratarvários tipos de tumores de tecidos moles e hiperplasia. Objetivo: Caracterizar o efeito citotóxico da microemulsão contendo zinco ftalocianina tetrasulfonada(ZnPcSO4). Material e Método: A microemulsão (ME) foi preparada por adição da fase aquosa (15%) composta de propilenoglicol e água (3:1), em umamistura de fase oleosa composta pelos surfactantes monooleato de sorbitanol e polissorbato 80 (47%) e óleo de canola (38%). Para o teste de citotoxicidadefoi realizado o ensaio de MTT com as células MCF-7 de adenocarcinoma de mama humana. Resultados: Os baixos valores de condutividade obtidos (0,63e 0,53 para ME com e sem ZnPcSO4, respectivamente) indicaram que a ME era de tipo água/óleo. O diâmetro interno das partículas foi na ordem de 60nmna presença e ausência do ZnPcSO4 sugerindo que a presença do fotossensibilizante não alterou as propriedades do sistema nanoestruturado. O índicede polidispersividade foi menor do que 0,10, enquanto que a carga superfi cial da nanopartícula foi negativa, em concordância com as característicasdos compostos utilizados para o preparo da ME. A atividade metabólica das células não sofreu interferências com as MEs não demonstrando toxicidade...

Introduction: Breast cancer has a high incidence and mortality among all types of cancer and, despite all the recent technological advances, still remains aworldwide public health problem. New therapeutic strategies, such as Photodynamic Therapy, have been used to treat various types of soft tissue tumorsand hyperplasia. Objective: To characterize the cytotoxic effect of the zinc-containing microemulsion containing tetrasulf...

Introducción: El cáncer de mama tiene alta incidencia y mortalidad entre todos los tipos de cáncer y, a pesar de todos los avances tecnológicos recientes,sigue siendo un problema mundial de salud pública. Nuevas estrategias terapéuticas, como la Terapia Fotodinámica, se han utilizado para tratar varios tiposde tumores de tejidos blandos e hiperplasia. Objetivo: Caracterizar el efecto citotóxico de la microemulsión que contiene el cinc ftalocianina tetrasulfonada(ZnPcSO4). Material y método: La microemulsión (ME) fue preparada por adición de la fase acuosa (15%) compuesta de propilenglicol y agua (3: 1), enuna mezcla de fase oleosa compuesta por los surfactantes monooleato de sorbitanol y polisorbato 80 (47%), Y el aceite de canola (38%). Para la pruebade citotoxicidad se realizó el ensayo de MTT con las células MCF-7 de adenocarcinoma de mama humana. Resultados: Los bajos valores de conductividadobtenidos (0,63 y 0,53 para ME con y sin ZnPcSO4, respectivamente)...

Comparação do perfil de liberação de nitrato de isoconazol em formas farmacêuticas semissólidas usando modelo matemático de diferença (f1) e semelhança (f2) / Comparación de perfiles de liberación del nitrato isoconazol en formas farmacéuticas semisólido utilizando el modelo de diferencia matemática (f1) y semejanza (f2) / Comparison of isoconazole nitrate release profile in pharmaceutical semisolid forms using f1 (differential) and f2 (similarities) mathematical models

Introdução: A comprovação da eficácia e segurança de produtos é dada por meio dos estudos de equivalência farmacêutica e bioequivalência. Abioequivalência entre os dois produtos farmacêuticos pode ser demonstrada por meio de estudos diferentes: farmacocinético, farmacodinâmico,clínicos comparativos ou ensaios in vitro. Objetivo: Comparar o perfil de liberação in vitro do nitrato de isoconazol a partir de medicamentosreferência e genéricos, aplicando dois diferentes modelos estatísticos para a análise dos resultados baseados nos fatores F1 e F2. Material eMétodo: Os perfis de liberação do nitrato de isoconazol foram determinados em condições previamente otimizadas em célula de difusão verticalcontendo solução 0,01M de fosfato de sódio (pH 7,4) como meio receptor (7 mL) e membrana sintética de acetato de celulose (com área dedifusão de 1,77 cm2). Após 1, 2, 3, 4, 6 e 8 horas de experimento, exatos 1,0 mL da solução receptora foi coletada e a quantidade de nitratode isoconazol presente na solução foi analisada por método validado de Cromatografia Líquida de Alta Eficiência. O fluxo de entrega do nitratode isoconazol foi obtido com base no perfil de liberação utilizando a porção linear da curva. Os valores de diferença e semelhança, F1 e F2,respectivamente, obtidos entre as curvas de liberação de nitrato de isoconazol, foram aplicados como uma ferramenta de análise da equivalênciafarmacêutica de formas semissólidas. Resultados: Houve equivalência farmacêutica entre os medicamentos referência e genérico, considerando osnúmeros de lotes utilizados nestes ensaios...

Introduction: Evidence of efficacy and safety of products is given through pharmaceutical equivalence and bioequivalence studies. Bioequivalencebetween the two pharmaceutical products can be demonstrated by different studies: pharmacokinetics, pharmacodynamics, comparative clinicaltrials or in vitro assay. Objective: To compare the in vitro release profile of isoconazole nitrate from reference and generic drugs using twodifferent statistical models based on factors F1 and F2. Material and Method: The release profiles of isoconazole nitrate were determined underpre-optimized conditions in a vertical diffusion cell containing 0.01M sodium phosphate solution (pH 7.4) as receptor medium (7mL) and syntheticcellulose acetate membrane (with diffusion area of 1.77 cm2). After 1, 2, 3, 4, 6 and 8 hours of permeation, exactly 1.0 mL of the receptor solutionwas collected and the amount of isoconazole nitrate was analyzed by validated High Performance Liquid Chromatography method. The isoconazolenitrate delivery flow was obtained based on the release profile using the linear portion of the curve. The values of difference and similarity, F1 andF2 respectively, obtained between the release curves of isoconazole nitrate from generic and reference products, were applied as a tool for analysisof the pharmaceutical equivalence of semisolid forms. Results: There was a pharmaceutical equivalence between reference and generic drugs,considering the batches number...

Introducción: La prueba de la eficacia y seguridade del producto está dada por los estudios de equivalência farmacéutica y bioequivalência. Labioequivalencia entre las dos productos farmacêuticos puede ser demostrada en diversos estudios: farmacocinética, farmacodinámica, clínicos yencomparación in vitro. Objetivo: Comparar el perfil de liberación in vitro de nitrato de isoconazol de medicamentos genéricos y de referencia,utilizando dos diferentes modelos estadísticos para el análisis de los resultados en función de los factores F1 y F2. Material y Método: Los perfilesde liberación de isoconazol han sido determinados en condiciones de nitrato isoconazol previamente optimizados en la célula de difusión verticalque contiene solución de fosfato de sodio 0,01 M (pH 7,4) como medio receptor (7 ml) y membrana sintética (con 1,77 cm2 área de difusión).Después de 1, 2, 3, 4, 6 y 8 horas de experimento se recogieronexactamente 1,0 ml de solución de receptor y la cantidad de nitrato isoconazolen lasolución se analizó por lacromatografía líquida de alto rendimiento. Se obtuvo el flujo de entrega isoconazol nitrato basado en el perfil deliberación usando la parte lineal de la curva. Las diferencias y semejanza de los valores, F1 y F2, respectivamente, obtenidos a partir de las curvasde liberación de nitrato isoconazol, se aplicaron como una herramienta de análisis de equivalência farmacéutica de formas semisólidas. Resultados:Se demostró la equivalência farmacéutica entre lós...

Development and characterization of novel 1-(1-Naphthyl)piperazine-loaded lipid vesicles for prevention of UV-induced skin inflammation.

1-(1-Naphthyl)piperazine (1-NPZ) has shown promising effects by inhibiting UV radiation-induced immunosuppression. Ultradeformable vesicles are recent advantageous systems capable of improving the (trans)dermal drug delivery. The aim of this study was to investigate 1-NPZ-loaded transethosomes (NPZ-TE) and 1-NPZ-loaded vesicles containing dimethyl sulfoxide (NPZ-DM) as novel delivery nanosystems, and to uncover their chemopreventive effect against UV-induced acute inflammation. Their physicochemical properties were evaluated as follows: vesicles size and zeta potential by dynamic and electrophoretic light scattering, respectively; vesicle deformability by pressure driven transport; rheological behavior by measuring viscosity and I-NPZ entrapment yield by HPLC. In vitro topical delivery studies were performed in order to evaluate the permeation profile of both formulations, whereas in vivo studies sought to assess the photoprotective effect of the selected formulation on irradiated hairless mice by measuring myeloperoxidase activity and the secretion of proinflammatory cytokines. Either NPZ-TE or NPZ-DM exhibited positive results in terms of physicochemical properties. In vitro data revealed an improved permeation of 1-NPZ across pig ear skin, especially by NPZ-DM. In vivo studies demonstrated that NPZ-DM exposure was capable of preventing UVB-induced inflammation and blocking mediators of inflammation in mouse skin. The successful results here obtained encourage us to continue these studies for the management of inflammatory skin conditions that may lead to the development of skin cancers.

Quantification of lipoic acid from skin samples by HPLC using ultraviolet, electrochemical and evaporative light scattering detectors.

Lipoic acid (LA) is an endogenous organosulfur compound with potent antioxidant property. LA is often used as a drug for the treatment of skin disorders. For the accomplishment of topical applications of LA appropriate drug quantification methods are essential. Thus far, no HPLC methods have been reported for the measurement of LA extracted from skin. In this article we report on the development and validation of three sensitive and specific HPLC methods for LA and dihydrolipoic acid (DHLA) using ultraviolet (UV), electrochemical (EC) or evaporative light scattering (ELS) detection. These methods demonstrate different linearity ranges. The chromatographic separations were performed by RP-HPLC (250 × 4 mm, 5 µm) with isocratic elution using an acidic mobile phase for the three detection techniques. The lower limits of detection and quantification were 0.04 and 0.08 ng LA, respectively, for HPLC coupled to ELS, an innovative detector for LA with high sensitivity. The extraction of LA from skin samples showed recoveries greater than 71%. The recovered LA concentrations from stratum corneum and epidermis+dermis layers were: 5.41 ± 0.56 and 4.92 ± 0.33 µg/mL, respectively for HPLC/UV and 6.52 ± 0.49 and 5.01 ± 0.41 µg/mL, respectively, for HPLC/EC for the added LA concentration (6.67 µg/mL), and 8.88 ± 0.46 and 8.95 ± 0.08 µg/mL, respectively, for HPLC/ELS for the added LA concentration (10 µg/mL). These three optimized HPLC methods allowed for a simple, rapid and reliable determination of LA in human skin. They should be useful for the development of drug delivery systems for topical applications of LA.

Advances in the bioanalytical study of drug delivery across the skin.

The study of a drug's dermal penetration profile provides important pharmaceutical data for the rational development of topical and transdermal delivery systems because the skin is a broadly used delivery route for local and systemic drugs and a potential route for gene therapy and vaccines. Monitoring drug penetration across the skin and quantifying its levels in different skin layers have been constant challenges due to the detection limitations of the available techniques, as well as the inherent interference in this tissue. This review explores and discusses several bionalytical methods that are indispensable tools to study drugs across the skin. In addressing the main topic, we structure the review highlighting the skin as an important route of drug administration and its structure, skin membrane models most used and its properties, in vitro and in vivo assays most used in the study of drug delivery to the skin, the techniques for processing the skin for subsequent analysis by bioanalytical methods that have a theoretical and practical approach showing its applicability, limitations and also including examples of its use. This review has a comprehensive approach in order to help researchers design their experiments and update the applicability and advances in this area of expertise.

Liquid crystalline systems for transdermal delivery of celecoxib: in vitro drug release and skin permeation studies.

Liquid crystalline systems of monoolein/water could be a promising approach for the delivery of celecoxib (CXB) to the skin because these systems can sustain drug release, improve drug penetration into the skin layers and minimize side effects. This study evaluated the potential of these systems for the delivery of CXB into the skin based on in vitro drug release and skin permeation studies. The amount of CXB that permeated into and/or was retained in the skin was assayed using an HPLC method. Polarizing light microscopy studies showed that liquid crystalline systems of monoolein/water were formed in the presence of CXB, without any changes in the mesophases. The liquid crystalline systems decreased drug release when compared to control solution. Drug release was independent of the initial water content of the systems and CXB was released from cubic phase systems, irrespective of the initial water content. The systems released the CXB following zero-order release kinetics. In vitro drug permeation studies showed that cubic phase systems allowed drug permeation and retention in the skin layers. Cubic phase systems of monoolein/water may be promising vehicles for the delivery of CXB in/through the skin because it improved CXB skin permeation compared with the control solution.

A Validated Reverse Phase HPLC Analytical Method for Quantitation of Glycoalkaloids in Solanum lycocarpum and Its Extracts.

Solanum lycocarpum (Solanaceae) is native to the Brazilian Cerrado. Fruits of this species contain the glycoalkaloids solasonine (SN) and solamargine (SM), which display antiparasitic and anticancer properties. A method has been developed for the extraction and HPLC-UV analysis of the SN and SM in different parts of S. lycocarpum, mainly comprising ripe and unripe fruits, leaf, and stem. This analytical method was validated and gave good detection response with linearity over a dynamic range of 0.77-1000.00 µg mL(-1) and recovery in the range of 80.92-91.71%, allowing a reliable quantitation of the target compounds. Unripe fruits displayed higher concentrations of glycoalkaloids (1.04% ± 0.01 of SN and 0.69% ± 0.00 of SM) than the ripe fruits (0.83% ± 0.02 of SN and 0.60% ± 0.01 of SM). Quantitation of glycoalkaloids in the alkaloidic extract gave 45.09% ± 1.14 of SN and 44.37% ± 0.60 of SM, respectively.

Celecoxib determination in different layers of skin by a newly developed and validated HPLC-UV method.

A simple, rapid and sensitive analytical procedure for the measurement of celecoxib (CXB) levels in skin samples after in vitro penetration studies was developed and validated. In vitro permeability studies in porcine skin were performed for quantification of CXB at different layers of skin, the stratum corneum (SC) and epidermis plus dermis (EP + D) as well as in the acceptor solution (AS) to assess CXB permeation through skin. CXB was quantified by HPLC using a C18 column and UV detection at 251 nm. The mobile phase was methanol-water 72:28 (v/v) and the flow-rate was 0.8 mL/min. The CXB retention time was 5 min. The assay was linear for CBX in the concentration range of 0.1-3.0 µg/mL in the AS (drug permeated through skin) and 5.0-50.0 µg/mL for drug retained in SC and [EP + D] in vitro. The linear correlation coefficients for the different calibration curves were equal or greater than 0.99. Intra- and inter-assay variabilities were below 8.0%. Extraction of CXB from skin samples showed recoveries higher than 95.0% after 15 min of ultrasonic sound and centrifugation at 2500 rpm for 3 min. The method was considered appropriate for the assay of CXB in skin samples, after in vitro cutaneous penetration studies.