Mupirocin ointments: In vitro x In vivo bioequivalence evaluation

Abstract Bioequivalence (BE) assessment of topical drug products is a long-standing challenge. Agencies such as the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have published several drafts in recent years suggesting different approaches as alternative to evaluate the BE. A proposed Topical Classification System (TCS) has even been discussed. Given the above, the objective of this research was to use in vitro and in vivo BE approaches to evaluate Brazilian marketed mupirocin (MPC) ointments, previously classified as TCS class The in vitro permeation test (IVPT) was performed by applying formulations to pig skin by Franz cells. The in vivo methodology was dermatopharmacokinetic (DPK). These approaches (in vivo tape stripping and IVPT) demonstrated capability of distinguishing among different formulations, thus making them useful methodologies for BE evaluation.

Antilipoperoxidative and anti-inflammatory efficacy of multifunctional sunscreens containing ferulic acid

Unprotected chronic exposure to ultraviolet radiation generates many harmful effects to human skin and UV filters are essential to health, however, traditional sunscreens do not provide enough protection against cutaneous oxidative stress, a process amplified by UV radiation. Therefore, is been proposed the development of multifunctional photoprotective formulations, acting in the absorption/reflection of UV radiation and assisting in cutaneous homeostasis. In the present study, ferulic acid is used in conjunction with two sunscreens, bemotrizinol and ethylhexyl triazone, for the determination of biosafety and efficacy methods, using techniques that better elucidate the effects of ferulic acid. Skin permeation assays were performed by applying a formulation containing the three substances in the stratum corneum of volunteers, which were removed by the tape stripping method (ex vivo) with follow quantification by high performance liquid chromatography (HPLC). The test was able to evaluate the penetration depth of the substances, characterizing them. In addition, the simultaneous quantification of the three substances was performed by a single and fast method, facilitating their analysis and improving the technique. Also, TBARS (thiobarbituric acid reactive substances) assays were performed in stratum corneum removed by tape stripping (ex vivo), evaluating the potential of cutaneous lipid peroxidation, with or without ferulic acid. To date, it is the first time that TBARS method is used to characterize the stratum corneum (ex vivo) and quantified by HPLC. The protocol developed may aid in the efficacy of antioxidant agents in studies aimed at elucidating the level of lipid peroxidation caused by drugs and cosmetics, and even in carrying out baseline studies characterizing different ethnicities and genders. As last, an anti-inflammatory in vivo assay with Laser Doppler flowmetry equipment was used to compare the sunscreen formulation with or without ferulic acid. Data indicated that the antioxidant reduced the angular coefficient of the perfusion units, mitigating the inflammatory effects. Furthermore, a significant difference was found between the genders, suggesting a more pronounced inflammatory reaction in women. Ferulic acid proved to be a valuable resource, besides being safe and raise the SPF of sunscreens, it also mitigates the effects of inflammation

A exposição crônica desprotegida à radiação ultravioleta (UV) contribui para o desenvolvimento de câncer de pele e os filtros solares são relevantes para evitar tais efeitos prejudiciais, porém, os protetores solares tradicionais não geram proteção suficiente contra o estresse oxidativo cutâneo. Logo, espera-se o desenvolvimento de formulações fotoprotetoras multifuncionais, atuando não somente na absorção e/ou reflexão da radiação UV, mas, também, auxiliando na homeostase cutânea, com presença de agentes antioxidantes. No presente estudo foi utilizado o ácido ferúlico conjuntamente com dois filtros solares, o bemotrizinol e a triazona de octila, para determinação de métodos de segurança e eficácia, utilizando técnicas que melhor elucidem e comprovem os efeitos do ácido ferúlico. Foram realizados ensaios de permeação cutânea pela aplicação tópica de formulação contendo as três substâncias em voluntários, sendo o estrato córneo retirado pelo método de tape stripping (ex vivo) com subsequente quantificação por cromatografia líquida de alta eficiência (CLAE). O ensaio pôde avaliar a profundidade de penetração das substâncias, caracterizando-as. Ademais, a quantificação simultânea das três substâncias foi efetuada por método único e rápido, facilitando análise com aprimoramento da técnica. Em adição, foi realizado ensaios de TBARS (substâncias reativas ao ácido tiobarbitúrico) em estrato córneo removido por tape stripping (ex vivo), para avaliar o potencial de peroxidação lipídica cutânea, contendo ou não o ácido ferúlico. Até o presente momento, é a primeira vez que o método TBARS é utilizado para caracterização do estrato córneo (ex vivo) e quantificada por CLAE. O protocolo desenvolvido pode auxiliar na eficácia de agentes antioxidantes, em estudos que visam elucidar o nível de peroxidação lipídica causada por medicamentos e cosméticos e, até mesmo, na realização de estudos de base, caracterizando etnias e gêneros. Ademais, um ensaio anti-inflamatório in vivo com equipamento de fluxometria Laser Doppler foi utilizado para comparar a formulação fotoprotetora com ou sem ácido ferúlico. Os dados indicaram que o antioxidante reduziu o coeficiente angular das unidades de perfusão, mitigando os efeitos inflamatórios. Ainda, foi identificada diferença entre os gêneros, sugerindo reação inflamatória mais pronunciada em mulheres. O ácido ferúlico provou ser um recurso valioso, além de ser seguro e elevar o FPS dos fotoprotetores, também atenuando os efeitos da inflamação

Enhancing The Sunscreen Efficacy Of Bemotrizinol Micropigment By Using O/W Nanoemulsion Topical Preparations

Objective: Bemotrizinol (BEMT) is the most efficient broad-spectrum UV-absorber having a dual mechanism of action in absorbing and reflecting photons. The main objective of this work was to develop successful oil in water (o/w) nanoemulsion for improving the solubility of BEMT and its protective characteristics. Methods: Pseudo-ternary phase diagrams were constructed using labrafac PG and isopropyl myristate as oil phase, tween 80 as surfactant (S) and cremophor EL as cosurfactant (CoS) the ratio of S/CoS was determined according to highest percent of water incorporation to the system. Full factorial study design (24) using Design-Expert® software was adopted to study the effect of four independent variables namely: oil type, oil concentration, S/CoSmix (3:1) concentrations and BEMT concentration on the particle size and the in vitro release at 2 h (Q2h) of the prepared nanoemulsion formulae. Two systems each of eight formulae were developed and evaluated through droplet size analysis, zeta potential measurement, refractive index, in vitro drug release and according to the desirability value two formulae (F6 and F14) were used for further evaluations including in vitro sun protection factor (SPF), ex-vivo deposition by tape stripping technique, permeation test and photostability study. Results: Formula (F14) was chosen as the optimum formula having an in vitro SPF of 16.08±0.39, lowest permeation of 140±0.06 μg/cm2after six h and highest photostability (t90% = 168.02) after 120 min. Conclusion: Despite the poor solubility of bemotrizinol, it could be enhanced by novel drug delivery systems with good SPF value while maintaining its photostability.

Comparison of three paeonol gels in their percutaneous penetration performance / 中成药

AIM To compare the percutaneous penetration performance of three paeonol gels.METHODS Franz diffusion cell method was applied to investigating the penetration and retention behaviors of eutectic mixturebased nanoemulsion,ordinary nanoemulsion and saturated solution gels onto mouse skins in vitro.The retention and permeation amounts in stratum corneum and hair follicles of volunteers smeared with gels were compared by tape stripping method.RESULTS The accumulative permeation and retention amounts of various gels onto mouse skins in vitro were in sequence of eutectic mixture-based nanoemulsion gel ＞ ordinary nanoemulsion gel ＞ saturated solution gel.The main retention of all the three gels was observed on the volunteers' skin surface,and the permeation amounts of eutectic mixture-based nanoemulsion,ordinary nanoemulsion gels,and their accumulative permeation amounts in stratum corneum and hair follicles were significantly higher than those of saturated solution gel (P ＜0.05).CONCLUSION Nanoemulsion technology can significantly promote the percutaneous penetration performance of paeonol.

Ácido ferúlico em protetores solares: desenvolvimento e eficácia multifuncional in vitro, ex vivo e in vivo / Ferulic acid in sunscreens: development and multifunctional effectiveness in vitro, ex vivo and in vivo

A conscientização da população com relação aos malefícios da radiação ultravioleta (UV) levaram à adoção de hábitos preventivos, com destaque para a utilização diária de formulações fotoprotetoras. Ainda que os filtros solares tradicionais sejam efetivos na absorção ou reflexão da radiação UV, sabe-se que a sua atuação contra os radicais livres é limitada, fator que exige a incorporação de compostos antioxidantes às preparações, visando proteção cutânea de excelência. O presente trabalho objetivou o desenvolvimento e avaliação de formulações multifuncionais contendo os filtros solares bemotrizinol e octil triazona, acrescidos do composto bioativo ácido ferúlico - reconhecido pelas suas propriedades antioxidante e fotoprotetora. As formulações foram desenvolvidas de acordo com um delineamento experimental do tipo fatorial (DoE 23) e avaliadas por meio de metodologias in vitro, in vivo e ex vivo para determinação de sua eficácia fotoprotetora e potencial antioxidante. As emulsões foram consideradas estáveis, seguras, com valor de pH biocompatível com a pele e viscosidade aparente adequada ao uso proposto. O delineamento experimental comprovou o potencial antioxidante e fotoprotetor do ácido ferúlico, visto que a presença deste promoveu aumento de até 90% na atividade antioxidante in vitro das formulações, bem como acréscimo de 32% no Fator de Proteção Solar (FPS) in vivo e 24% no Fator de Proteção UVA (UVA-PF). Por meio do ensaio ex vivo, a formulação contendo o ácido ferúlico apresentou tendência em aumentar a atividade antioxidante natural da pele, comparativamente à formulação contendo apenas os filtros solares e à pele não tratada; ademais, houve queda no potencial antioxidante da pele após a exposição à radiação UV, enfatizando a necessidade de proteção cutânea frente aos danos oxidativos. Os resultados comprovaram os efeitos antioxidante e fotoprotetor combinados do ácido ferúlico, em especial quando em sinergismo com filtros solares, tornando o composto bioativo um candidato promissor no desenvolvimento de formulações multifuncionais inovadoras, seguras e eficazes contra os efeitos nocivos decorrentes da exposição solar

The public awareness regarding the harmful effects of ultraviolet (UV) radiation led to preventive habits, especially the daily use of sunscreens. Although traditional solar filters are effective in UV radiation absorption or reflection, it is known that their activity against free radicals is restricted, an aspect that requires the incorporation of antioxidants to preparations, aiming superior skin protection. Here, we developed and evaluated multifunctional formulations containing the sunscreens bemotrizinol and ethylhexyl triazone plus the bioactive compound ferulic acid - known for its antioxidant and photoprotective properties. Formulations were developed according to a factorial experimental design (DoE 23) and evaluated using in vitro, in vivo and ex vivo methods to determine its photoprotective effectiveness and antioxidant potential. The emulsions were considered stable, safe, with pH value biocompatible with the skin and apparent viscosity suitable for the proposed use. The experimental design evidenced the photoprotective and antioxidant potential of ferulic acid, once its presence increased to 90% the in vitro antioxidant activity of the formulations, as well as 32% increase in the in vivo Sun Protection Factor (SPF) and 24% increase in the UVA Protection Factor (UVA-PF). Through the ex vivo assay, the formulation containing ferulic acid tended to increase the natural antioxidant activity of the skin, compared to the formulation containing only UV filters or the untreated skin; moreover, there was a decrease in the antioxidant potential of the skin after UV radiation exposure, emphasizing the need for skin protection against oxidative damage. The results confirmed the antioxidant and photoprotective effects of ferulic acid, especially when in synergy with UV filters, making the bioactive compound a promising candidate in the development of innovative, safe and effective multifunctional formulations against the harmful effects of sun exposure

Basic considerations in the dermatokinetics of topical formulations

Assessing the bioavailability of drug molecules at the site of action provides better insight into the efficiency of a dosage form. However, determining drug concentration in the skin layers following topical application of dermatological formulations is a great challenge. The protocols followed in oral formulations could not be applied for topical dosage forms. The regulatory agencies are considering several possible approaches such as tape stripping, microdialysis etc. On the other hand, the skin bioavailability assessment of xenobiotics is equally important for topical formulations in order to evaluate the toxicity. It is always possible that drug molecules applied on the skin surface may transport thorough the skin and reaches systemic circulation. Thus the real time measurement of molecules in the skin layer has become obligatory. In the last two decades, quite a few investigations have been carried out to assess the skin bioavailability and toxicity of topical/dermatological products. This review provides current understanding on the basics of dermatokinetics, drug depot formation, skin metabolism and clearance of drug molecules from the skin layers following application of topical formulations.

A avaliação da biodisponibilidade de moléculas de fármacos no sítio de ação oferece melhor compreensão sobre a eficiência da forma de dosagem. Entretanto, a determinação da concentração de fármaco nas camadas da pele em seguida à aplicação tópica de formulações dermatológicas é um grande desafio. Os protocolos seguidos para as formulações orais não podem ser aplicados para as formulações tópicas. As agências regulatórias consideram várias abordagens possíveis, tape stripping, microdiálise etc. Por outro lado, a avaliação da biodisponibilidade de xenobióticos na pele é igualmente importante para as formulações tópicas para se avaliar a toxicidade. É sempre possível que as moléculas de fármaco aplicadas na superfície da pele sejam transportadas através da pele e alcancem a circulação sistêmica. Assim, a medida em tempo real de moléculas na camada da pele tem se tornado obrigatória. Nas últimas duas décadas, realizaram-se poucas pesquisas para avaliar a biodisponibilidade da pele e a toxicidade de produtos tópicos/dermatológicos. Esta revisão fornece a compreensão atual com base na dermatocinética, formação de fármaco de depósito, metabolismo da pele e o clearance das moléculas de fármaco das camadas da pele em seguida à aplicação de formulações tópicas.

Dynamic observation of repigmentation in vitiligo lesions after narrow-band ultraviolet B phototherapy by using an adhesive tape stripping technique / 中华皮肤科杂志

Objective To observe the changes of melanin granules in superficial corneocytes in vitiligo lesions after irradiation with narrow-band ultraviolet B (NB-UVB) by using an adhesive tape stripping technique.Methods Vitiligo lesions were selected from 6 patients and irradiated with NB-UVB every other day for 31 sessions.Superficial corneocytes were obtained by an adhesive tape stripping technique from the vitiligo lesions and perilesional normal skin before every treatment.The morphology,distribution and color of melanin granules were observed after Masson-Fontana silver staining.The percentage of area occupied by melanin granules in superficial corneocytes were calculated by using the Image-Pro Plus 6.0 software.Statistical analysis was conducted by the SPSS11.5 software.Results There were still a few superficial corneocytes containing melanin granules remaining in the vitiligo lesions before treatment.The percentage of area occupied by melanin granules was (5.31 ± 4.12)％ before treatment,(6.24 ± 2.65)％ after 10 treatment sessions,(10.14 ± 5.73)％ after 20 sessions,and (13.05 ± 6.17)％ after 30 sessions,with significant differences between these time points (F =4.334,P ＜ 0.05).Multiple comparisons revealed a significant increase in the percentage of area occupied by melanin granules after 30 treatment sessions compared with those before treatment and after 10 treatment sessions (both P ＜ 0.01 ).The morphology and color of melanin granules in repigmented lesions after treatment differed from those in perilesional normal skin before treatment.Conclusion The adhesive tape stripping technique may serve as a useful tool for the evaluation of repigmentation in vitiligo lesions after phototherapy.

Change of Skin Barrier Function According to Stratum Corneum Removal by Tape Stripping / 대한피부과학회지

BACKGROUND: The major function of the skin is to prevent loss of water and electrolytes and transepidermal penetration of harmful materials. The stratum corneum is known to play a major role in the skin barrier function. Tape stripping (TS) with adhesive cellophane tape is one of the best method of removing the stratum corneum on the skin surface, and has been used in studies of the skin barrier function when investigating material distribution in the stratum corneum and transepidermal absorption of drugs. OBJECTIVE: This study was performed to evaluate the skin barrier function according to removal of the stratum corneum by TS. METHOD: Six vitiligo patients who had undergone autologous suction blister grafting were tested. Transepidermal water loss (TEWL) was measured on the normal skin of the buttocks in each patient after every fifth TS, plus on the skin where the entire epidermis had been removed by autologous suction blister grafting. We compared these two values and also examined morphological change of the stratum corneum after TS, by both light and electron microscope. RESULTS: The TEWL value slightly increased when TS was carried out up to 30 times, but greatly increased to 20g/m2h of TEWL when TS was done 30-40 times. Thereafter, no increase in TEWL was observed when TS was carried out up to 100 times. The pattern of TEWL according to numbers of TS approximately plotted a sigmoid curve. On average, there was about 88% impairment to the skin permeability barrier function after TS. When the TEWL increased to more than 70g/m2h over baseline TEWL, the stratum corneum was almost removed, as revealed by light and transmission electron microscope. CONCLUSION: The stratum corneum acts as a major skin barrier, and the mid to lower portion of the entire stratum corneum appears to play a significant role in the skin permeability barrier function.

Comparison of Results of Lactic Acid Sting Tests between Sensitive Skin and Nonsensitive Skin after Tape Stripping / 대한피부과학회지

BACKGROUND: Sensitive skin has been described as a skin type showing higher reactivity than normal skin and developing exaggerated reactions when exposed to internal stimulants and external irritants. The Lactic acid sting test is a widely accepted method for evaluating sensitive skin and is employed for the selection of subjects experiencing invisible sensory irritation. Tape stripping of skin is a useful method for removing stratum corneum which plays a most important part in the barrier function of the skin. Measurement of the transepidermal water loss (TEWL) is the standard method to determine stratum corneum barrier status. A disturbed skin barrier is characterized by high TEWL. OBJECTIVE: The author performed this study to compare lactic acid sting test between sensitive skin and nonsensitive skin after tape stripping, not on the face but on the forearm. METHODS: A total of 40 individuals (20 individuals of the sensitive skin group and 20 individuals of the nonsensitive skin group) were selected by the method of self-assessment questionnaires related to sensitive skin. The individuals were tested a total of 6times, at internals of 2weeks, on the volar aspects of both forearms with 10% lactic acid after tape stripping, with or without a Hilltop chamber. The frequency of tape stripping was divided according to the TEWL into 3 groups as follows: mild(increase of TEWL> or = 5g/m2h compared to baseline TEWL), moderate (> or = 10g/m2h), and severe (> or = 20g/m2h). RESULTS: When 10% lactic acid sting tests were performed after mild and moderate tape stripping of skin, irrespective of the Hilltop chamber, there were more positive responders in the sensitive skin group (p<0.05). Also statistically, there was a significant difference between the sensitive skin group and the nonsensitive skin group in the comparison of the mean values of lactic acid sting scores (p<0.05). However, when 10% lactic acid sting tests were conducted after severe tape stripping of skin, irrespective of the Hilltop chamber, there were no statistically significant difference between two groups when comparison of the positive rates and the mean values were made. CONCLUSION: The Lactic acid sting test with the Hilltop chamber, after mild and moderate tape stripping is a useful method to discover sensitive skin.

Perturbation and Recovery of the Skin Barrier Function After Tape Stripping and Sodium Lauryl Sulfate Irritation / 대한피부과학회지

BACKGROUND: The main function of the stratum corneum is to serve as a barrier preventing the penetration of irritants and transepidermal water loss (TEWL). Previous studies have shown that the cutaneous barrier disruption by application of acetone or sodium lauryl sulfate (SLS) and tape stripping leads to various metabolic changes. Recent studies showed that there was some difference in the recovery rate between tape-stripped groups and acetone-treated groups, and an individual irritant not only exerted rather specific morphologic effects on the cellular components of the keratinocyte but also induced distinct morphological alterations of the different components of the stratum corneum. In other words, this data suggested that individual irritants could show different cutaneous barrier recoveries. OBJECTIVE: This study was done to compare the recovery rate after cutaneous barrier disruption between a tape-stripped group and a SLS irritated group, and to test the relationship among baseline TEWL, TEWL after the SLS irritation, recovery of TEWL, and number of strippings needed to increase TEWL up to 20g/m2/h. METHODS: The permeability barrier of normal human skin was disrupted by tape stripping and patch of SLS and the recovery rate was assessed by measure of TEWL and erythema (E-) index. RESULTS: The number of tape strippings needed to increase TEWL up to 20g/m2/h was 32.7+-9.6 (20-50). The recovery rate of TEWL was 56.85+-17.97% twenty-four hours after tape stripping and 51.55+/-24.73% after patch removal, and 82.70+/-8.70% and 76.61+/-11.14% four days after tape stripping and patch removal. There was no significant difference in the recovery rate of TEWL after the two methods of epidermal barrier perturbation. All the correlations among baseline TEWL, TEWL after the SLS irritation, recovery of TEWL, and number of strippings were not significant statistically. E-index after the tape stripping was 12.07+/-2.95 and higher than that after removal of SLS, 11.20+/-2.51. There was irregular recovery of E-index in the SLS irritated group, but not irregular in the tape-stripped group. There was no significant difference in the recovery rate between the two groups. CONCLUSION:There was no significant difference in the recovery rates of TEWL and E-index between the tape-stripped group and the SLS irritated group. We suggest that TEWL measurements may be more desirable than the measurement of E-index in the study of perturbation and recovery of the skin barrier function.

Impact of rat skin stratum corneum on transdermal absorption characteristic of Sinomenine Liposome Patch / 中草药

Objective To study the effect of skin stratum corneum on transdermal absorption characteristic of Sinomenine Liposome Patch. Methods The normal and stripped stratum corneum skin of back and abdomen was used to study the transdermal absorption characteristic by Franz method. The transdermal absorption sample was taken at the preset sampling time point. The sample content was determined by HPLC. The cumulative transdermal amount of per unit area was fitted with sampling time point, the transdermal rate constant (J value) was selected as comparative standard. Results The transdermal rate constant (J) of skin with stratum corneum stripped increased obviously than that of the normal skin, so was the released rate constant. Conclusion The skin stratum corneum has an notable impact on transdermal absorption characteristic of Sinomenine Liposome Patch. When the skin of experimental animal being prepared, the integrality should be paid more attention.

The effects of cell proliferation by tape stripping upon sunburn cell formation by UVB / 대한피부과학회지

index (%) in unirradiated mouse skin was 11.0+/-4.3. LI was significantly increased by tape stripping to 22.1+/-4.6. 2. The number of SBC in 1cm epidermis after 50mJ/cm UVB exposure was 28.2+/-4.1. The number of SBC was increased by tape stripping to 57.4+Cell proliferation, by evaluating sunburn cell (SBC) formation, was studied in mouse skin following tape stripping and ultraviolet light B (UVB) exposun.. 1-radiation was achieved using high pressure mercury are UVB. The results are summarized as follows. 1. Labeling 19.2. These results suggest that proliferating cells are more sensitive to UVB exposure.