Heat-separated Epidermis Research Articles

Human Skin Drug Metabolism: Relationships between Methyl Salicylate Metabolism and Esterase Activities in IVPT Skin Membranes.

The presence of esterase enzymes in human skin and their role in drug metabolism has been reported, but their distribution in the various skin layers and the relative contributions of those layers to metabolism is poorly defined. To gain further insight into esterase distribution, we performed in vitro skin permeation of a commercial 28.3% methyl salicylate (MeSA) cream (Metsal™) in Franz diffusion cells, using a range of human skin membranes, all from the same donor. The membranes were viable epidermis separated by a dispase II enzymatic method, heat separated epidermis, dermatomed skin, and dermis separated by a dispase II enzymatic method. Methyl salicylate and its metabolite, salicylic acid (SA), were measured by high-performance liquid chromatography. Alpha naphthyl acetate and Hematoxylin and Eosin staining provided qualitative estimations of esterase distribution in these membranes. The permeation of methyl salicylate after 24 h was similar across all membranes. Salicylic acid formation and permeation were found to be similar in dermatomed skin and dermis, suggesting dermal esterase activity. These results were supported by the staining studies, which showed strong esterase activity in the dermal-epidermal junction region of the dermis. In contrast with high staining of esterase activity in the stratum corneum and viable epidermis, minimal stained and functional esterase activity was found in heat-separated and dispase II-prepared epidermal membranes. The results are consistent with dispase II digesting hemidesmosomes, penetrating the epidermis, and affecting epidermal esterases but not those in the dermis. Accordingly, whilst the resulting dispase II-generated dermal membranes may be used for in vitro permeation tests (IVPT) involving esterase-based metabolic studies, the dispase II-generated epidermal membranes are not suitable for this purpose.

Human Skin Permeation Enhancement Using PLGA Nanoparticles Is Mediated by Local pH Changes.

The steady improvement and optimization of transdermal permeation is a constant and challenging pharmaceutical task. In this study the influence of poly(lactide-co-glycolide) (PLGA) nanoparticles on the dermal permeation of the anti-inflammatory drug flufenamic acid (FFA) was investigated. For this aim, different vehicles under non-buffered and buffered conditions and different skin models (human heat separated epidermis and reconstructed human epidermis equivalents) were tested. Permeation experiments were performed using static Franz diffusion cells under infinite dosing conditions. Already the presence of drug-free nanoparticles increased drug permeation across the skin. Drug permeation was even enhanced when applying drug-loaded nanoparticles. In contrast, buffered vehicles with different pH values (pH 5.4–7.4) revealed the influence of the pH on the permeation of FFA. The change of the surrounding pH of the biodegradable nanoparticulate system was demonstrated and visualized using pH-sensitive fluorescent probes. While a potential contribution of hair follicles could be ruled out, our data suggest that the enhanced permeation of FFA through human skin in the presence of PLGA nanoparticles is mediated by a locally decreased pH during hydrolytic degradation of this polymer. This hypothesis is supported by the observation that skin permeation of the weak base caffeine was not affected.

Quantitative MALDI mass spectrometry imaging for exploring cutaneous drug delivery of tofacitinib in human skin

In skin penetration studies, HPLC-MS/MS analysis on extracts of heat-separated epidermis and dermis provides an estimate of the amount of drug penetrated. In this study, MALDI-MSI enabled qualitative skin distribution analysis of endogenous molecules and the drug molecule, tofacitinib and quantitative analysis of the amount of tofacitinib in the epidermis. The delivery of tofacitinib to the skin was investigated in a Franz diffusion cell using three different formulations (two oil-in-water creams, C1 and C2 and an aqueous gel). Further, in vitro release testing (IVRT) was performed and resulted in the fastest release of tofacitinib from the aqueous gel and the lowest from C2. In the ex vivo skin penetration and permeation study, C1 showed the largest skin retention of tofacitinib, whereas, lower retention and higher permeation were observed for the gel and C2. The quantitative MALDI-MSI analysis showed that the content of tofacitinib in the epidermis for the C1 treated samples was comparable to HPLC-MS/MS analysis, whereas, the samples treated with C2 and the aqueous gel were below LOQ. The study demonstrates that MALDI-MSI can be used for the quantitative determination of drug penetration in epidermis, as well as, to provide valuable information on qualitative skin distribution of tofacitinib.

Application of quality by design principles in the development and evaluation of semisolid drug carrier systems for the transdermal delivery of lidocaine

The aim of this work was the formulation and comparison of different drug carrier systems for the transdermal delivery of lidocaine. Quality target product profile and quality attributes were identified, and an initial risk assessment was made according to the Quality by Design methodology. The critical quality attributes influencing the quality and efficacy of the final drug formulation were then defined in order to select the control points. Four formulation types, each containing 5% lidocaine were compared: conventional hydrogel, oleogel, lyotropic liquid crystal and nanostructured lipid carrier. Microscopic analysis, particle size and zeta potential measurements, Fourier transform infrared spectroscopy and Raman spectroscopy were performed to characterize the critical parameters in case of the different vehicles. Membrane diffusion and penetration studies were completed as well for each formulation in vitro and ex vivo, followed by measurements on skin hydration and transepidermal water loss in vivo. Our results lead us to the conclusion that the nanostructured lipid carrier is the most promising vehicle for the topical delivery of lidocaine. It showed the best penetration properties through heat separated epidermis and the highest moisturizing effect which are the most critical parameters based on the Quality by Design initial risk assessment and evaluation.

In Vitro Drug Transfer Due to Drug Retention in Human Epidermis Pretreated with Application of Marketed Estradiol Transdermal Systems

Study objective was to assess skin-to-skin drug transfer potential that may occur due to drug retention in human epidermis (DRE) pretreated with application of estradiol transdermal drug delivery systems (TDDS) and other estradiol transdermal dosage forms (gels and sprays). TDDS (products-A, B, and C) with varying formulation design and composition, and other estradiol transdermal products (gel and spray) were applied to heat separated human epidermis (HSE) and subjected to in vitro drug permeation study. Amounts of DRE were quantified after 24h. The DRE with product-B was significantly (P < 0.001) higher than that with product-C, product-A, gel, and spray. However, products-A and C, gel, and spray showed almost the same (P > 0.05) amounts of DRE. A separate in vitro permeation study was carried out to determine amounts of drug transferred from drug-retaining epidermis to untreated HSE. The amounts of drug transferred, due to DRE after 8h, with product-C were significantly (P < 0.001) higher than those with products-A and B, gel, and spray. The in vitro study results indicate a high potential of skin-to-skin drug transfer due to the DRE after labeled period of using estradiol TDDS, though the clinical relevance of these findings is yet to be determined.

Porcine Ear Skin as a Biological Substrate for in vitro Testing of Sunscreen Performance

Purpose: The purpose of the study was to examine the use of skin from porcine ears as a biological substrate for in vitro testing of sunscreens in order to overcome the shortcomings of the presently used polymethylmethacrylate (PMMA) plates that generally fail to yield a satisfactory correlation between sun protection factors (SPF) in vitro and in vivo. Procedures: Trypsin-separated stratum corneum and heat-separated epidermis provided UV-transparent substrates that were laid on quartz or on PMMA plates. These were used to determine surface roughness by chromatic confocal imaging and to measure SPF in vitro of 2 sunscreens by diffuse transmission spectroscopy. Results: The recovered skin layers showed a lower roughness than full-thickness skin but yielded SPF in vitro values that more accurately reflected the SPF determined in vivo by a validated procedure than PMMA plates, although the latter had in part roughness values identical to those of intact skin. Combination of skin tissue with a high roughness PMMA plate also provided accurate SPF in vitro. Conclusions: Besides roughness, the improved affinity of the sunscreen to the skin substrate compared to PMMA plates may explain the better in vitro prediction of SPF achieved with the use of a biological substrate. i 2014 S. Karger AG, Basel

Comparison of three different sampling methods for canine skin lipids

Epidermal lipids are of major interest in dermatological research, especially in canine atopic dermatitis. Owing to the existence of several sampling methods, the interpretation of study results is often complicated. This study aimed to compare three different sampling methods and to establish a minimally invasive method for collecting canine epidermal lipids. Skin samples from five dogs with no obvious skin abnormalities were taken from the caudal back and the inguinal region postmortem. Samples consisted of heat-separated epidermis of three skin biopsies, three scrapes and three skin scrubs. Lipids were analysed by high-performance thin-layer chromatography; the resulting bands were identified by using corresponding standards, retardation factors and mass spectrometry. The influences of the sampling method, the body site and the ceramide standards were investigated. Between body sites, significant differences were found for cholesterol sulphate, cholesteryl esters and triglycerides. Significant differences between sampling methods were detected for all lipid fractions except for cholesterol sulphate and glucosylceramides within the lipid profile, and for at least four ceramide classes within the ceramide profile. The most obvious discrepancies were found between heat-separated epidermis and skin scrub. The reproducibility was high for scraping and skin scrub, but was lowest for heat-separated epidermis. Furthermore, this study revealed a marked influence of ceramide standards on the results regarding the ceramide profile. Scraping and skin scrub are comparably suitable methods for skin lipid sampling, whereas the analysis of heat-separated epidermis may not be the method of first choice.

Human skin penetration of a copper tripeptide in vitro as a function of skin layer.

Objective and designSkin retention and penetration by copper applied as glycyl-l-histidyl-l-lysine cuprate diacetate was evaluated in vitro in order to assess its potential for its transdermal delivery as an anti-inflammatory agent.Materials and methodsFlow-through diffusion cells with 1 cm2 exposure area were used under infinite dose conditions. 0.68% aq. copper tripeptide as permeant was applied on isolated stratum corneum, heat-separated epidermis and dermatomed skin and receptor fluid collected over 48 h in 4 h intervals using inductively coupled plasma mass spectrometry to analyze for copper in tissues and receptor fluid.ResultsThe permeability coefficient of the compound through dermatomed skin was 2.43 ± 0.51 × 10−4 cm/h; 136.2 ± 17.5 μg/cm2 copper permeated 1 cm2 of that tissue over 48 h, while 97 ± 6.6 μg/cm2 were retained as depot.ConclusionsCopper as tripeptide was delivered in potentially therapeutically effective amounts for inflammatory disease.

Human skin retention and penetration of a copper tripeptide in vitro as function of skin layer towards anti-inflammatory therapy.

Objective and designThe skin retention and penetration characteristics of copper applied as glycyl-l-histidyl-l-lysine cuprate diacetate were evaluated in vitro in order to assess the potential for its transdermal delivery as anti-inflammatory agent.Materials and methodsFlow-through diffusion cells with 1 cm2 exposure area were used under infinite dose conditions. 0.68% aq. Copper as a tripeptide was applied on isolated stratum corneum, on heat-separated epidermis and on dermatomed skin. Receptor fluid collected over 48 h in 4 h intervals was analyzed by inductively coupled plasma mass spectrometry for copper in tissues and receptor fluid.ResultsThe permeability coefficient of the compound through dermatomed skin was 2.43 ± 0.51 × 10−4 cm/h; 136.2 ± 17.5 μg/cm2 copper permeated 1 cm2 of that tissue over 48 h, while 82 ± 8.1 μg/cm2 of copper were retained there as depot.ConclusionsApplied tansdermally as the tripeptide on human skin ex vivo, copper permeated the skin and was also retained in skin tissue in amounts potentially effective for the treatment of inflammatory diseases.

Variation of in vitro Human Skin Permeation of Rose Oil between Different Application Sites

Background: Rose oil is the essential oil extracted from the petals of the damask rose. It is an ingredient of cosmetic as well as health care products and it is used in folk medicine and in aromatherapy. It is well known that essential oils are able to permeate the skin whereas the lipophilic properties of the compounds as well as the application site have an influence on their permeation behaviour. In this paper, we investigated the influence of the application site on the permeation of rose oil. Method: We tested the permeation of rose oil and its major ingredients through human heat-separated epidermis of abdominal skin, breast skin, and skin from the upper arm. Results: We found that no application site is suited best for all substances tested to permeate the skin. It depends on the structure of the essential oil ingredients which application site should be chosen for topical application, which is already known for pharmaceuticals. Furthermore, when investigating the permeation of essential oils, we found that not only the P_app value but also the lag-time should be considered. Conclusion: The application site of substances to permeate the skin has a great influence on the permeation behaviour. As essential oils are volatile mixtures and their retention on the surface of the skin amounts only to a few hours, the lag-time should be considered in addition to the P_app-value.

Wistar rat skin as surrogate for human skin in nortriptyline hydrochloride patch studies

Six different matrices were prepared containing nortriptyline hydrochloride (NTH) with hydroxypropyl-methyl-cellulose as polymer. A mixture of transdermal enhancers was included as part of the vehicle. Diffusion studies were carried out through Wistar rat full thickness skin using Franz cells. They were compared with previously determined human heat separated epidermis in order to test if this animal can be used as model for in vivo assays. A linear correlation was obtained between NTH diffusion coefficients through both skin types ( r 2 = 0.996).

Nortriptyline for smoking cessation: Release and human skin diffusion from patches

The objective of this work was to develop a simple and inexpensive transdermal formulation containing Nortriptyline Hydrochloride (NTH) for smoking cessation support therapy. Hydroxypropyl-methyl-cellulose was chosen as polymer and a mixture of transdermal enhancers (selected from previous research) was incorporated. The formulations were characterised in terms of appearance, thickness, uniformity of NTH content, release and skin permeation. Release studies demonstrated controlled release for four formulations. Diffusion studies were performed through human heat separated epidermis (HHSE) using Franz Diffusion Cells (FDC). Patches provided different fluxes varying from 20.39 ± 7.09 μg/(cm 2 h) to 256.19 ± 94.62 μg/(cm 2 h). The penetration profiles of NTH within the stratum corneum (SC) and deeper skin layers (DSL) were established after three administration periods (3 h, 6 h, and 24 h). Skin changes induced by the application of the patches were observed by confocal laser scanning microscopy (CLSM). The highest flux obtained would provide the recommended doses for smoke cessation support therapy (25–75 mg per day) with a 2 cm × 2 cm patch or a 3.5 cm × 3.5 cm patch, respectively, without skin damage evidence.

Stability of triptorelin in the presence of dermis and epidermis

An important issue with respect to the transdermal delivery of peptides is their stability during transit through the epidermis and dermis before entry into the systemic circulation. The objective of the present study was to evaluate the effect of epidermal and dermal tissue on the stability of the luteinizing hormone releasing hormone superagonist, triptorelin. The decapeptide was dissolved in PBS (pH 7.4) and placed in contact with (i) heat separated epidermis (HSE), (ii) dermatomed skin (0.75 mm; DS) and (iii) full thickness skin (FTS) and the extent of peptide biotransformation monitored as a function of time by HPLC. The results showed that triptorelin was metabolized when in contact with each of the skin tissues. However, there were marked differences with respect to the extent of peptide degradation. Triptorelin was least stable in the presence of FTS. After 3 h exposure to HSE, DS and FTS, the extent of triptorelin degradation was 15.0 ± 6.0%, 64.8 ± 9.9% and 100%, respectively. After 24 h, further triptorelin degradation had occurred in the samples in contact with HSE and DS—with 51.3 ± 6.0% and 87.8 ± 4.4%, respectively, of the peptide being degraded. The chromatograms revealed the presence of a degradation peak at a higher retention time than the parent molecule—most probably the free acid.

Transdermal delivery of car vedilol in rats: probing the percutaneous permeation enhancement mechanism of soybean extract–chitosan mixture

Background: This study was designed for investigating the effect of soybean (SS) extract and chitosan (CTN) in facilitating the permeation of carvedilol (CDL) across rat epidermis. Method: Transdermal flux of carvedilol through heat-separated rat epidermis was investigated in vitro using vertical Keshary–Chien diffusion cells. Biophysical and microscopic manifestations of epidermis treated with SS-extract, CTN, and SS extract–CTN mixture were investigated by using DSC, TEWL, SEM, and TEM. Biochemical estimations of cholesterol, sphingosine, and triglycerides were carried out for treated excised as well as viable rat epidermis. The antihypertensive activity of the patches in comparison to that after oral administration of carvedilol was studied in deoxycorticosterone acetate-induced hypertensive rats. Results: The solubility of CDL was found to be maximum in the presence of 1% (w/v) SS extract. The KIPM/PB of CDL decreased with increase in concentration of SS extract. The in vitro permeation of CDL across rat epidermis increased and was maximum with combination of SS extract and chitosan (CTN). Biochemical and microscopic studies revealed the initiation of reversal of barrier integrity after 12 hours. Furthermore, the application of patches containing SS extract–CTN mixture resulted in sustained release of carvedilol, which was able to control the hypertension in deoxycorticosterone acetate (DOCA) induced hypertensive rats through 24 hours. CTN was found to potentiate the permeation enhancing activity of SS extract. Conclusion: The developed transdermal patches of CDL containing SS extract–CTN mixture exhibited better performance as compared to oral administration in controlling hypertension in rats.

Characterisation of epidermal lipid composition and skin morphology of animal skin ex vivo

Epidermal lipids and skin morphology are assumed to substantially influence skin permeability. Although these parameters have been studied extensively, available data are hard to interpret as data have been gathered at different experimental conditions. Therefore, the aim of this study was to provide detailed information on these parameters for four different mammalian skin types. Lipids were extracted from heat separated epidermis, the total epidermal lipid content was measured and the epidermal lipid composition was quantitatively determined by high-performance thin-layer chromatography. Furthermore, vertical and horizontal cryostate skin slices were analysed by light microscopy for thickness of the horny layer, epidermal thickness, density, depth of anchorage and diameter of the hair follicles. The highest total epidermal lipid content was detected in rat epidermis, followed by bovine udder, dog and pig epidermis. Considering the amount of single lipid fractions, cholesterol, cholesteryl ester and free fatty acids were found to be the major constituents of epidermal lipids in all the examined species. However, as confirmed by hierarchical cluster analysis the epidermal lipid profile and morphology showed marked differences between all the examined species.

Influence of Human Skin Specimens Consisting of Different Skin Layers on the Result of in vitro Permeation Experiments

The literature exhibits high variation in results from drug permeation experiments across human skin. Our purpose was to investigate the influence of human skin specimens, consisting of different skin layers and resulting from different skin preparation techniques, on the in vitro permeation of a model drug, i.e. flufenamic acid (FFA). FFA permeation across human (1) trypsin-isolated stratum corneum, (2) heat-separated epidermis and (3) dermis, (4) dermatomized skin and (5) full-thickness skin (FTS) from either a hydrophilic or lipophilic donor was investigated in Franz-type diffusion cells. Cumulative permeated drug amounts were plotted versus time, and a fit to Fick’s 2nd law of diffusion was performed. Since performing skin diffusion experiments in the laboratory is time consuming and expensive, especially when using FTS, we also investigated the possibility of calculating the resistances of composite skin layers from the diffusion resistances of the individual skin layers. Due to short lag time, practical handling and economic preparation, heat-separated epidermis appears to be superior in human skin in vitro permeation experiments compared to separated stratumcorneum sheets, dermatomized skin and FTS. Furthermore, we found a good correlation between calculated and experimental resistances which underlines that calculation of the total diffusion resistance of composed skin preparations from resistances of individual skin layers is legitimate and useful. Considering our findings, improved interpretation of literature data and more consistent results for future permeation experiments are possible.

Aerosol OT Microemulsions as Carriers for Transdermal Delivery of Hydrophobic and Hydrophilic Local Anesthetics

The skin permeation enhancement of many kinds of drugs and cosmetic substances by microemulsions has been widely known; however, the correlations between microemulsion microstructures and the efficiency of skin permeation are not fully elucidated. Therefore, the aim of our study was to investigate the influence of microemulsion types on in vitro skin permeation of model hydrophobic drugs and their hydrophilic salts. The microemulsion systems were composed of isopropyl palmitate (IPP), water, a 2:1 w/w mixture of Aerosol OT (AOT) and 1-butanol, and a model drug. The concentrations of surfactant mixture and model drug were maintained at 45% and 1% w/w, respectively. The concentrations of IPP and water were 15% and 39% w/w, respectively, for oil-in-water (o/w) type and vice versa for water-in-oil (w/o) type. The samples were prepared by simple mixing and 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. Transdermal flux of lidocaine, tetracaine, dibucaine, and their respective hydrochloride salts from the drug-loaded AOT-based microemulsions through heat-separated human epidermis was investigated in vitro using modified Franz diffusion cells. The o/w microemulsions resulted in the highest fluxes of the model drugs in base form as compared with the other formulations within the same group of drugs. Moreover, the skin permeation of drug from microemulsions depended on drug molecular structure and interaction between drug and surfactant.

Qualitative and quantitative comparison of heat separated epidermis and dermatomed skin in percutaneous absorption studies

Transepidermal water loss (TEWL), mainly regulated by the stratum corneum, was quantitatively correlated to percutaneous absorption of compounds in human and suggested for the ex vivo assessment of skin integrity. The present study investigated qualitatively and quantitatively the relevance of 100-microm heat separated epidermis (HSE) in percutaneous absorption studies as compared to 500-microm dermatomed skin by dual complementary approaches. Percutaneous absorption of caffeine delivered from aqueous solution through dermatomed skin or HSE specimens (n = 9) was measured using vertical static diffusion cells coupled with an unventilated evaporimeter enabling the assessment of TEWL and skin integrity for 21 h. Permeation of caffeine exhibited different finite dose-like profiles ranged according to the thickness of skin specimens (cumulative dose absorbed up to 21 h: 11.5 +/- 11.5 microg/cm(2) and 29.4 +/- 36.2 microg/cm(2) through dermatomed skin and HSE, respectively). Normalized TEWL and caffeine fluxes were similar through dermatomed skin and HSE suggesting that the intrinsic permeability properties of both models were undifferentiated over time. Interestingly, a significant relationship was shown between TEWL and caffeine fluxes, suggesting the usefulness of TEWL measurement as an element in the estimation of percutaneous drug absorption. In conclusion, the present showed that percutaneous absorption through HSE was qualitatively and quantitatively similar to dermatomed skin when TEWL as endogenous standard and skin thickness were considered in permeability data comparisons.

Permeability of the reconstructed human epidermis model Episkin ® in comparison to various human skin preparations

The objective of this work was to compare the barrier function of the small diameter reconstructed human epidermis model Episkin ® ( d = 12 mm) to human skin in vitro. For that purpose a modification for the Franz diffusion cell ( d = 15 mm) had to be developed so as to allow direct comparison with the following human skin preparations: Full thickness skin (FTS), split thickness skin (STS), heat-separated epidermis (HSE), and trypsin isolated stratum corneum (TISC). Among the tested preparations, HSE appeared to be the most preferable due to its clear morphological structure and ease of preparation. The lipid profile of HSE and Episkin ® was analyzed and showed significant differences in terms of cholesterol, ceramides and triglycerides contents, whereas cholesterol esters and fatty acids were not different. Permeation data with HSE and Episkin ® were then gathered using caffeine and testosterone. Both test compounds permeated much faster through Episkin ® than through HSE. Moreover, opposed to Episkin ®, HSE differentiated between the two test compounds. In spite of the remarkable progress in developing RHEs in the past years at this time Episkin ® can obviously not yet fully replace human skin for in vitro permeability experiments.

TEWL measurements as a routine method for evaluating the integrity of epidermis sheets in static Franz type diffusion cells in vitro. Limitations shown by transport data testing

The suitability of transepidermal water loss (TEWL) measurements in vitro as a barrier integrity test for human heat separated epidermis (HSE) was investigated. A model system consisting of a Teflon membrane mounted in Franz diffusion cells (FDC) filled with phosphate buffer saline (PBS) was set up. The membrane was used intact and punctured with a needle (up to five holes). After each puncturing the TEWL was measured. Only the TEWL of intact and punctured membrane differed significantly regardless of the number of holes. From three donators intact human HSE and punctured HSE were compared and no significant difference of the TEWL was found. Permeation experiments with flufenamic acid (FFA) showed a significantly higher diffusion rate through punctured HSE. TEWL and drug permeation were compared for skin stripped three, seven and 15 times prior to heat separation to an intact control group. Only the TEWL values of intact HSE and HSE stripped 15 times differed significantly. However, seven and 15 times stripping resulted in significantly higher diffusion rate. In conclusion, TEWL measurements can detect severe damage of the stratum corneum (SC) but not small changes, which nevertheless may already influence drug diffusion. Therefore, TEWL measurements appears to be of limited use as a barrier integrity test for human HSE in in vitro test systems.