O/W emulsions compromise the stratum corneum barrier and improve drug penetration

BACKGROUND: W/O emulsions improve the stratum corneum barrier, while microemulsions tend to compromise it. We, therefore, were interested to explore the effects of O/W emulsions on the stratum corneum barrier. METHODS: Aqueous Cream BP 2001, Clioquinol Cream BP 1999 without clioquinol, Nonionic Hydrophilic Cream DAB 2001 without glycerol, Hydrophilic Skin Emulsion Base NRF S. 25., point of time 2001, without glycerol, and Base Cream DAC were tested versus untreated controls in 29 healthy volunteers for 7 days. Outcome measures included transepidermal water loss (TEWL), skin redness (chromametry a*-value) and erythrocyte circulation in the subpapillary vessels (laser Doppler). Barrier compromise was subsequently explored by performing the hydrocortisone blanching test using Hydrocortisone Cream 0.5% NRF 11.36. (outcome measure: a*-value) in 15 subjects and the sodium lauryl sulfate (SLS) irritation test (outcome measures: TEWL, a*-value, laser Doppler) in 14 subjects. RESULTS: Pretreatment with the test emulsions produced increases in TEWL (statistically significant for all test emulsions), a*-value (statistically significant for Aqueous Cream BP 2001 and Base Cream DAC), and laser Doppler value (statistically significant for all emulsions except Base Cream DAC). Hydrocortisone penetration was statistically significantly increased with all test emulsions versus untreated contols. SLS irritation was mostly statistically significantly increased versus untreated controls when analyzing the study endpoint-baseline difference. CONCLUSIONS: O/W emulsions may compromise the stratum corneum barrier and improve drug penetration.     

In vitro percutaneous absorption of metronidazole and glucose: Comparison of o/w,w/o/w and w/o systems

The percutaneous absorption of model hydrophilic drugs with intermediate and high polarity, metronidazole and glucose, respectively, from three emulsion types (o/w, w/o/w and w/o) has been studied. All the emulsions were prepared with exactly the same composition in order to avoid the influence of the formulation and to study the role of the emulsion type alone. The yield of the w/o/w emulsion containing glucose was high (95.8%), while that of the w/o/w emulsion containing metronidazole was lower (77.6%), although the multiple character of the emulsion was not in question. Absorption of metronidazole across hairless rat skin over 24 h ranged from 55 to 69% of the applied dose and was similar for the three emulsions studied. Absorption of glucose ranged from 1 to 4% and was found to be greater from the o/w emulsion than that observed from the w/o/w or w/o emulsions. The amount of glucose found in the dermis seems to be dependent on the emulsion type: o/w > w/o/w ≅ w/o. The differences between the emulsions cannot be attributed only to the evaporation rate of water alone. X-ray diffraction patterns obtained after treatment of the stratum corneum with the three emulsions did not display any difference, but were somewhat different from that observed with untreated stratum corneum.     

Effects of microemulsions on the stratum corneum and hydrocortisone penetration.

We tested a high-water-content hydrophilic microemulsion (ME 1) and a low-water-content lipophilic microemulsion (ME 2) for their suitability for use in dermatology, in general, and as alternative hydrocortisone (HC) vehicles, in particular. The lipophilic component of both study products was isopropyl myristate. The surfactant/cosurfactant system of ME 1 consisted of two sucrose esters and that of ME 2 was a mixture of Tagat S and Plurololeat. Both MEs showed no in vitro irritability in the hen's egg test on chorioallantoic membranes.In 14 subjects, stratum corneum water content was determined by corneometry and transepidermal water loss (TEWL) by the Tewameter before and after 3 days use of ME 1 or ME 2 as well as on two untreated control sites. ME 1 produced dehydration and increased TEWL as evidence of barrier compromise. ME 2 also produced an increase in TEWL but had no dehydrating effect. Subjects then underwent standardized washing with a surfactant solution. Under these conditions, pretreatment with ME 2 also produced dehydration, but to a lesser extent than did pretreatment with ME 1.In the same subjects, the impact of the two MEs on HC penetration (0.5%, 24h occlusion) was evaluated in terms of the chromameter-determined blanching effect compared with that on a site treated only with an occlusive film dressing. The comparator was an ambiphilic cream (Basiscreme (BC) Deutscher Arzneimittel Codex (German Formulary)). Irritative skin redness produced by ME 1 was significant and that produced by ME 2 was slight but visible, compared with BC. HC penetration was demonstrable from all the study products via the blanching effect and was significantly greater from ME 1 and slightly greater from ME 2 than from BC. However, neither ME would improve HC therapy because the irritative effects were so great that the blanching effect of HC formulated in ME 1 was significantly smaller and that of HC in ME 2 slightly smaller than that of HC formulated in BC.     

Quercetin in w/o microemulsion: In vitro and in vivo skin penetration and efficacy against UVB-induced skin damages evaluated in vivo.

The present study evaluated the potential of a w/o microemulsion as a topical carrier system for delivery of the antioxidant quercetin. Topical and transdermal delivery of quercetin were evaluated in vitro using porcine ear skin mounted on a Franz diffusion cell and in vivo on hairless-skin mice. Skin irritation by topical application of the microemulsion containing quercetin, and the protective effect of the formulation on UVB-induced decrease of endogenous reduced glutathione levels and increase of cutaneous proteinase secretion/activity were also investigated. The w/o microemulsion increased the penetration of quercetin into the stratum corneum and epidermis plus dermis at 3, 6, 9 and 12h post-application in vitro and in vivo at 6h post-application. No transdermal delivery of quercetin occurred. By evaluating established endpoints of skin irritation (erythema formation, epidermis thickening and infiltration of inflammatory cells), the study demonstrated that the daily application of the w/o microemulsion for up to 2 days did not cause skin irritation. W/o microemulsion containing quercetin significantly prevented the UVB irradiation-induced GSH depletion and secretion/activity of metalloproteinases.     

Percutaneous penetration of topically applied melatonin in a cream and an alcoholic solution

In a clinical study, the skin penetration properties of melatonin 0.01% in a cream and 0.01 and 0.03% in a solution were investigated by evaluation of the serum melatonin levels over a 24-hour time course in 15 healthy volunteers. Blood samples for melatonin measurements were taken at 9.00 a.m. before applying the test preparations and 1, 4, 8 and 24 h after application. The measurements were carried out by radioimmunoassay for melatonin. In 15 volunteers, the serum levels of melatonin before application of the topical preparations were between 0.6 and 15.9 pg/ml. After application of the 0.01% melatonin cream, there was a steady increase starting from 9.00 a.m. up to a mean serum value of 9.0 pg/ml at 9.00 a.m. the next day. The solution of 0.01% melatonin also showed an increase, starting from 5.00 p.m., up to a mean melatonin level of 12.7 pg/ml 24 h after application. The solution containing 0.03% melatonin resulted in elevated melatonin levels 1 and 8 h after application. The values were 18.1 and 19.0 pg/ml. The cumulative melatonin values for each preparation were 7.1, 8.6 and 15.7 pg/ml, respectively. This study shows that the strongly lipophilic substance melatonin is able to penetrate through the skin and leads to dose- and galenic-dependent melatonin levels in the blood. No increase of melatonin above the physiological range was observed.     

Interaction of lipophilic moisturizers on stratum corneum lipid domains in vitro and in vivo

Dry skin symptoms such as scaling and itching are often treated with lipophilic moisturizers. The aim of this study was to investigate the effect of lipophilic moisturizers on the stratum corneum (SC) ultra-structure and lipid organization. Lipophilic moisturizers were applied on the forearms of 4 healthy volunteers for 3 h. Subsequently, the application sites were tape stripped, and selected tape strips prepared for Freeze Fracture Electron Microscopy (FFEM), a method to visualize the SC intercellular lipid parallel to the skin surface. To investigate the effect of lipid moisturizers on the lipid lamellae, isolated SC was pretreated with the lipophilic moisturizers for 24 h prior to performing small angle X-ray diffraction (SAXD) measurements. Additionally, the lipid organization of mixtures prepared with ceramides, cholesterol, free fatty acids and lipophilic moisturizer in a 2:1:1:1 molar ratio were studied using SAXD. The FFEM data (in vivo) as well as the SAXD data (in vitro) show that the lipophilic moisturizers do not change the lipid lamellar organization in the SC. Addition of 20% m/m lipophilic moisturizer to the ceramide:cholesterol:free fatty acids mixture did not inhibit the formation of the long periodicity phase, the characteristic lamellar phase in the SC, even though there was clear evidence that two of the three moisturizers were at least partially incorporated in the long periodicity phase. Concluding, all findings suggest that the lipophilic moisturizers investigated in this study do not drastically change the lamellar organization of the SC intracellular lipid matrix, but that the moisturizers form separate domains in the SC, as was visualized by FFEM.     

Reservoir function of the stratum corneum: development of an in vivo method to quantitatively determine the stratum corneum reservoir for topically applied substances

Investigations on the stratum corneum (SC) reservoir for topically applied substances are of importance in dermatologic science in order to assess the pharmacokinetics of these substances. In the present study, an in vivo method was developed to determine the SC reservoir quantitatively and to investigate the temporal behavior of this reservoir. Therefore, increasing amounts of an oil-in-water emulsion (o/w emulsion) containing 4% of a chemical UV filter were topically applied onto the flexor forearms of 5 healthy volunteers. The saturation of the SC reservoir was determined utilizing the tape stripping technique 1 and 6 h after application. The capacity of the SC reservoir for the o/w emulsion was found to be approximately 2.7 mg/cm(2). Furthermore, a correlation of the capacity of the SC with transepidermal water loss was observed. Extending the time between the topical application and SC removal did not affect the distribution or the recovery rate of the UV filter in the SC. The results indicate that the reservoir of the SC is limited. This is reflected by the saturation level, which depends on the individual volunteer and, presumably, the topically applied substances and formulations used. The results show that the method developed is suited to quantitatively determine in vivo the SC reservoir for topically applied substances.     

Controlled release of methotrexate from w/o microemulsion and its in vitro antitumor activity

The objective of this study was to prepare the microemulsion of methotrexate (M-MTX) for oral use and to investigate the suppressive effect of MTX-loaded microemulsion on MCF-7 human breast cancer cells. At the same time this effect of M-MTX was compared with those of a solution of the drug (Sol-MTX). Microemulsion was composed of soybean oil as oil phase, a mixture of Cremophore EL and Span 80 as surfactants, and isopropyl alcohol as co-surfactant, and 0.2 N NaOH as the aqueous phase. MTX was added into microemulsion at the last stage. We clearly demonstrated that M-MTX had a significant cytotoxic effect on breast cancer cell lines and the cytotoxic effect of M-MTX was significantly more than that of solutions (p < 0.05) and IC(50) value for M-MTX was 40 ng/mL. We also examined M-MTX and Sol-MTX on a model biological environmental model. For this purpose a gastrointestinal cell culture model, the Caco-2 cell line, was used to investigate the cytotoxic effects of the polymeric carrier and its effect on the cell monolayer integrity. The differences between the viability of cells for M-MTX and Sol-MTX were significantly different when applied to ANOVA according to 2 x 8 factorial randomized design (p:0.016; for alpha: 0.05, power : 0.695). According to the in vitro cytotoxicity studies, we concluded that when MTX was incorporated into the microemulsion (M-MTX), which is a new drug carrier system, it suppresses tumour cell growth on multiple tumor lines. These results indicate that M-MTX may exert a low cytotoxic effect on normal cells and may be effective as an antitumor agent that induces apoptosis.     

A lamellar matrix model for stratum corneum intercellular lipids. II. Effect of geometry of the stratum corneum on permeation of model drugs 5-fluorouracil and oestradiol

The principal barrier to transdermal delivery of most drugs is the lamellar intercellular lipid domain of the stratum corneum (SC). The low permeability of SC in comparison to other lipid barriers is in part due to its geometry. Here, effect of geometry of the SC on permeation of 5-fluorouracil (5-FU) - a model hydrophilic drug - and oestradiol (OE) - a model lipophilic drug - was investigated using a lamellar model matrix for SC intercellular lipids. Release studies at 32°C showed that the diffusion coefficients of 5-FU and OE in the matrix are approximately 6000 —7000 and 60–260-times greater, respectively, than apparent human epidermis values, in good agreement with theoretical considerations. Release studies from 13 to 44°C revealed that 5-FU has a maximum diffusion coefficient around the main transition temperature of the matrix (35°C) in agreement with other lamellar systems reported in the literature. The diffusional activation energy of 5-FU in the matrix was 27.8 kcal mol⁻¹ which correlates well with human epidermal data. Release experiments were then combined with permeation studies and the permeability of model drugs through the SC at 32°C was predicted from matrix data. The predicted permeability coefficients of 5-FU (5.5–18 × 10⁻⁵ cm h⁻¹) and OE (0.07-0.24 × 10⁻³ cm h⁻¹) were in agreement with human epidermis data. Our results show the effect of the SC morphology on the percutaneous absorption of drugs and illustrate that 5-FU and OE permeate the SC through intercellular lipids.     

Comparison of anti-atopic dermatitis activities between DHMEQ and tacrolimus ointments in mouse model without stratum corneum

This study is aimed to further investigate the anti-atopic dermatitis (AD) activities of dehydroxymethylepoxyquinomicin (DHMEQ) ointment and compare its effect with that of tacrolimus ointment based on the previous study that DHMEQ improves AD-like lesions. AD were induced by 2,4-dinitroclilorobenzene/oxazolone (DNCB/OX) repeatedly on the ears of BABL/C mice while medical tape was additionally used to disrupt stratum corneum in order to exacerbate the lesions. The mice were randomly divided into groups, which are normal, vehicle, DHMEQ (0.1%) and tacrolimus (0.1%). Those in the last two groups were externally applied with DHMEQ ointment and tacrolimus ointment, respectively. The results showed that both of them significantly improved dermatitis symptoms of DNCB/OX-induced AD-like lesions, such as redness, itching, weeping, scaling and thickening of the skin, while reducing epidermis thickness, dermis thickness and the number of mast cells as well, which were examined histopathologically. In contrast with DHMEQ, tacrolimus led to a significant decrease in body weight after long-term application. Both DHMEQ and tacrolimus suppress DNCB-induced increase of serum total IgE and attenuate expression of inflammatory factors IL-4, IL-6, IL-13, IL-1β and interferon (IFN)-γ in the disrupted ear tissues. On the other hand, the mice applied with tacrolimus became obviously irritable, jumping up and down, and inflammatory exudation on the lesioned-skin surface of the mice was remarkably observed. Contrary to the side effects made by tacrolimus, DHMEQ didn't cause any adverse stimulus response. As a conclusion, DHMEQ is safer, milder and more suitable for long-term use than tacrolimus for the treatment of AD-like lesions.     

Enhancement of skin radical scavenging activity and stratum corneum lipids after the application of a hyperforin-rich cream

Hyperforin is well-known for its anti-inflammatory, anti-tumor, anti-bacterial, and antioxidant properties. The application of a hyperforin-rich verum cream could strengthen the skin barrier function by reducing radical formation and stabilizing stratum corneum lipids. Here, it was investigated whether topical treatment with a hyperforin-rich cream increases the radical protection of the skin during VIS/NIR irradiation. Skin lipid profile was investigated applying HPTLC on skin lipid extracts. Furthermore, the absorption- and scattering coefficients, which influence radical formation, were determined. 11 volunteers were included in this study. After a single cream application, VIS/NIR-induced radical formation could be completely inhibited by both verum and placebo showing an immediate protection. After an application period of 4 weeks, radical formation could be significantly reduced by 45% following placebo application and 78% after verum application showing a long-term protection. Furthermore, the skin lipids in both verum and placebo groups increased directly after a single cream application but only significantly for ceramide [AP], [NP1], and squalene. After long-term cream application, concentration of cholesterol and the ceramides increased, but no significance was observed. These results indicate that regular application of the hyperforin-rich cream can reduce radical formation and can stabilize skin lipids, which are responsible for the barrier function.     

Supersaturation: enhancement of skin penetration and permeation of a lipophilic drug

Purpose. To increase the dermal delivery of a lipophilic model compound (LAP), and to deduce the underlying mechanism of enhanced absorption. Methods. Penetration of LAP from mixtures of up to four degrees of saturation into the stratum corneum was evaluated using a tape-stripping method; epidermal permeation of the drug was measured in Franz diffusion cells. The relative diffusion and stratum corneum-vehicle partition coefficients of LAP were determined by fitting the results to the appropriate solutions to Fick's second law of diffusion. Results. Both the skin permeation rate and the amount of LAP in the stratum corneum increased linearly with increasing degree of saturation. The apparent diffusivity and its partition coefficient deduced from the penetration experiments were independent of the degree of saturation of the drug in the applied formulation, and consistent with corresponding parameters derived from the permeation experiments. Conclusions. Supersaturation can increase the skin penetration and permeation of lipophilic drugs. The diffusion and partition parameters deduced for LAP indicate that supersaturation acts exclusively via increased thermodynamic activity without apparent effect on the barrier function of the skin per se.     

Transdermal drug delivery by electroporation applied on the stratum corneum of rat using stamp-type electrode and frog-type electrode in vitro

Transdermal enhancement effects of electroporation applied only on the stratum corneum by two electrode types, the stamp-type electrode and the frog-type electrode, were investigated in vitro using excised rat skin. Carboxyfluorescein (CF) was selected as a model compound. The excised skin was set in a Franz type diffusion cell and a square wave electric pulse was applied to the stratum corneum under various electric pulse conditions. We determined the permeability of CF to the receptor compartment under these conditions. Voltage, electric pulse length, and number of electric pulses, were varied from 10 to 1000 V, 50 micros to 15 ms and 5 to 30 pulses, respectively. Flux rate was enhanced as the electric pulse condition strengthened. However, the maximum value was attained in the flux rate, above which no increase was observed despite strengthening of the electric pulse. Although at low electric pulses, the enhancement effect of the frog-type electrode was superior to that of the stamp-type electrode, the maximum flux rates were the same. These results indicate that electroporation on the stratum corneum using the stamp-type electrode or frog-type electrode, is useful for transdermal drug delivery.     

Transdermal delivery of hydrophobic and hydrophilic local anesthetics from o/w and w/o Brij 97-based microemulsions.

PURPOSE: To characterize the physicochemical properties of drug-loaded oil-in-water (o/w) and water-in-oil (w/o) Brij 97-based microemulsions in comparison to their blank counterparts and to investigate the influence of microemulsion type on in vitro skin permeation of model hydrophobic drugs and their hydrophilic salts. METHODS: The microemulsion systems were composed of isopropyl palmitate (IPP), water and a 2:1 w/w mixture of Brij 97 and 1-butanol. The samples were characterized by visual appearance, pH, refractive index, electrical conductivity, viscosity and determination of the state of water and IPP in the formulations using differential scanning calorimetry (DSC). Transdermal flux of lidocaine, tetracaine, dibucaine and their respective hydrochloride salts through heat-separated human epidermis was investigated in vitro using modified Franz diffusion cells. RESULTS: The physicochemical properties of drug-loaded microemulsions and their blank counterparts were generally similar; however, slight changes in some physicochemical properties (apparent pH and conductivity) were observed due to the intrinsic properties of the drugs. The o/w microemulsions resulted in the highest flux of lidocaine, tetracaine and dibucaine as compared to the other formulations with in the same group of drugs. CONCLUSIONS: The characterization results showed that incorporation of the model drugs into the microemulsions did not change the microemulsion type. The permeation data exhibited that the nature of the microemulsions was a crucial parameter for transdermal drug delivery. The o/w microemulsions containing hydrophobic drugs provided the highest skin permeation enhancement. In addition, skin permeation was depended on the molecular weight of the model drugs.     

Comparison of protocols measuring diffusion and partition coefficients in the stratum corneum

Partition (K) and diffusion (D) coefficients are important to measure for the modelling of skin penetration of chemicals through the stratum corneum (SC). We compared the feasibility of three protocols for the testing of 50 chemicals in our main studies, using three cosmetics‐relevant model chemicals with a wide range of logP values. Protocol 1: SC concentration‐depth profile using tape‐stripping (measures K_SC/v and D_SC/H_SC², where H_SC is the SC thickness); Protocol 2A: incubation of isolated SC with chemical (direct measurement of K_SC/v only) and Protocol 2B: diffusion through isolated SC mounted on a Franz cell (measures K_SC/v and D_SC/H_SC², and is based on Fick's laws). K_SC/v values for caffeine and resorcinol using Protocol 1 and 2B were within 30% of each other, values using Protocol 2A were ~two‐fold higher, and all values were within 10‐fold of each other. Only indirect determination of K_SC/v by Protocol 2B was different from the direct measurement of K_SC/v by Protocol 2A and Protocol 1 for 7‐EC. The variability of K_SC/v for all three chemicals using Protocol 2B was higher compared to Protocol 1 and 2A. D_SC/H_SC² values for the three chemicals were of the same order of magnitude using all three protocols. Additionally, using Protocol 1, there was very little difference between parameters measured in pig and human SC. In conclusion, K_SC/v, and D_SC values were comparable using different methods. Pig skin might be a good surrogate for human skin for the three chemicals tested. Copyright © 2017 The Authors Journal of Applied Toxicology published by John Wiley & Sons Ltd.