The thermal behaviour of human stratum corneum (SC) with various hydration levels was studied using differential thermal analysis DSC within the temperature range of −130 to 120°C. SC containing 20% water, resembling the intact condition, shows thermal transitions at around −20°C (representing water in skin), −10, 40, 70°C (representing skin lipids), 85°C (representing protein-associated lipids) and 100°C (representing skin protein). Dehydration of SC causes the transitions at −20 and 100°C to be invisible. Lipid extraction followed by dehydration eliminates all transitions. Further hydration produces a transition of water at around 0°C with a huge change in enthalpy. The perturbation effects of penetration enhancers fatty acids (FA) and propylene glycol (PG) were studied using DTA on SC after pretreatment with PG alone and FA/PG. The application of PG alone shifted the transitions at 70 and 85°C to lower temperatures. Additionally, the application to dehydrated stratum corneum removes the transitions at −10°C. Saturated fatty acids, e.g. nonanoic and decanoic acids, exert barely noticeable effects on the thermal behaviour of SC suggesting that they easily mix with the skin lipids. Thermal analysis also revealed that the cis -9- and 13-isomers of octadecenoic acid (monounsaturated fatty acids) form a separate domain containing mostly the pure fatty acids within the SC lipids and suppress the lipid transitions at 70/80°C. Polyunsaturated fatty acids linoleic and α-linolenic acids — form separate domains but do not completely suppress the SC lipid transitions at 70/80°C as monounsaturated acids do. This study suggests different ways of perturbation by various fatty acids.

The effects of current density on the temperature dependence of the electrical properties of human stratum corneum were investigated in vitro at two different current densities: 13 and 130 μA cm−2. To obtain information on the structural basis of the current-induced effects on the electrical resistances and capacitances, stratum corneum samples were subjected to three heating-and-cooling cycles (20-45-20 °C; 20-75-20 °C and 20-95-20 °C) which included four lipid phase transitions. At both current densities three characteristic temperature intervals could be distinguished: (1) A lower interval, from 20 to about 60 °C at the lower current density and from 20 to about 50 °C at the higher current density. In this interval a constant activation energy for ion transport and a gradual decrease of the resistances were found, whereas the capacitances were almost constant; all changes within this interval were thermo-reversible; (2) A middle interval, from 60 to about 75 °C at the lower and from 50 to about 75 °C at the higher current density. Within these temperature ranges, a rapid and thermo-irreversible decrease of the resistances was observed, accompanied by an increase of the capacitances; and (3) A higher interval, from 75 to 95 °C, within which the resistance did not decrease any further, although the capacitance increase continued. At a current density of 13 μA cm−2, the activation energy of ion transport across human stratum corneum was 5.4 ± 0.7 kcal mol−1, which suggested the presence of highly conductive pathways. The middle temperature interval at 13 μA cm−2, 60–75 °C, in which irreversible changes occurred, corresponded with the temperature interval of the gel-liquid phase transition of stratum corneum lipids. The irreversible increase of the capacitances further continued from 75 to 95 °C. Hence, the capacitances are determined by free lipids as well as protein-bound lipids. At 130 μA cm−2 the temperature dependence of the electrical properties had the same features as observed at 13 μA cm−2, but with two important differences: (1) A 15-fold decrease of the activation energy to 0.37 ± 0.03 kcal mol−1; and (2) A downward shift by almost 10 °C of the temperature, at which the thermal transition of the electrical properties starts. In conclusion, the thermal analysis of electrical properties has shown that the resistances of human stratum corneum are closely associated with the intercellular lipid lamellae, whereas the capacitances are determined by both the intercellular lipid lamellae and protein-bound lipids. Furthermore, under influence of an electrical field the lipid phase transition temperature is shifted downward, indicating that the electrical field is capable of modifying the arrangements of stratum corneum lipids.

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Delivery of drugs via the buccal mucosa is an alternative for the low oral absorption and inconvenient parenteral administration of hydrophilic macromolecular drugs. Due to the low permeability of the buccal epithelium the use of absorption enhancers is a prerequisite. In this study, buccal delivery of fluorescein isothiocyanate-labelled dextran 4400 (FD4) and the peptide drug buserelin was investigated in vivo, in pigs. The delivery device consisted of an application chamber with a solution of FD4 or buserelin, and was attached to the buccal mucosa for 4 h using an adhesive patch. A randomized cross-over study including intravenous administration and buccal delivery without and with 10 mM sodium glycodeoxycholate (GDC) as absorption enhancer was performed in pigs. After buccal administration steady-state plasma levels were rapidly achieved. Go-administration of 10 mM GDC increased the absolute bioavailability from 1.8 +/- 0.5% to 12.7 +/- 2.0% for FD4, and from 1.0 +/- 0.3% to 5.3 +/- 1.1% for buserelin. From the present studies it is concluded that buccal administration is a suitable route of delivery for macromolecules and hydrophilic compounds such as peptide drugs.

Iontophoresis is believed to have great added value for the delivery of drugs to the systemic circulation via the transdermal route. Whether iontophoretic transdermal systems can be applied for a wide variety of drugs, will at the same time depend on the intrinsic safety of current application to the skin. We investigated the effect of electric current in vivo on human skin by measuring two different aspects of the irritant response: (a) the effect on the stratum corneum barrier function by measuring transepidermal water loss (TEWL); (b) effects at the level of the dermal vascular bed by measuring the erythematous response with Laser Doppler Flowmetry (LDF). Nine volunteers entered the study. DC (direct constant current) profiles were applied to the volar forearms of the subjects for 30 min. Following the application of DC and pulsed DC current profiles, TEWL and LDF measurements were carried out on these sites, on so-called passive control sites (occluded by the application chamber containing the buffer solution, but without applying current) and on untreated sites. TEWL values measured directly after patch removal were 1.5-2.0 times higher than the baseline values for TEWL regardless the presence or absence of electric current. The effect of the electric current application on the TEWL followed the same relaxation profile as the effect of buffer application without current. The erythematous response however did differ significantly from the controls. The response on the anodal sites was 1.5–2.7 times higher than at the cathodal sites. The erythema lasted from 25 to 69 min. The time span for relaxation of the erythema was independent of the current profile (DC vs. pulsed DC) but was longer at the anodal sites than at the cathodal sites. It was concluded from this study that after a single current application the effect on the barrier to water loss is negligible because the effect on TEWL cannot be distinguished from the occlusive effect resulting from the application of the aqueous buffer alone, without current. The erythematous response to current application was relatively moderate and dependent on current direction.

The effects of non-ionic surfactant vesicles (NSVs) on human skin in vitro were studied in relation to the physico-chemical properties of the vesicles. The interactions between NSVs and skin were visualized using both freeze fracture electron microscopy and confocal laser scanning microscopy. The physico-chemical properties of the NSVs were varied in a systematic way, using a broad series of polyoxyethylene monoalkyl ether type surfactants (CnEOm). The number of oxyethylene units (m) was varied between 3, 7 and 10, and the number of carbon atoms (n) was either 12 or 18. Both the effects of liquid state vesicles composed of C12Eo3,7 and C9=9EO10 surfactants and gel state vesicles (C18EO37) were investigated. After the application of the NS V suspension on the stratum corneum surface two essentially different types of vesicle-skin interactions were visualized. Firstly, an interfacial interaction, involving the adsorption of vesicles, and the deposition of bilayer sheets on the outermost layers of the stratum corneum observed for all NSV formulations tested in this study. Secondly, effects on the ultrastructure of the stratum corneum were observed: the appearance of water pools observed for the liquid state vesicles only, and ultrastructural changes of the intercellular lipid domains are induced only observed after treatment with C12EO3 NSVs. Neither changes in the ultrastructure of the viable epidermis, nor changes in deeper skin layers were observed.

The influence of temperature on the resistive and capacitative properties of human stratum corneum in vitro was studied to determine where within substructures of stratum corneum, the electrical resistance R and capacitance C components reside. Heating-cooling cycles were designed in accordance with earlier calorimetric and spectroscopic studies of thermal transitions of human stratum corneum lipids and/or proteins. Two different protocols were used. (A) Heat treatment and electrical analysis were carried out simultaneously in pH 7.4 phosphate buffered saline, starting with prehydrated stratum corneum (70% w/w) of pH 7.4. (B) Heat treatment was performed before electrical analysis, using dried stratum corneum (< 10% w/w), followed by prehydration and measurement of the electrical properties in phosphate buffered saline at 20-degrees-C. Square-wave alternating current pulses of 13 muA cm-2 were applied every 60 s. Analysis of the resulting voltage waveform across stratum corneum yielded an equivalent electrical model of stratum corneum composed of a series connection of two RC circuits (R1 parallel-to C1 and R2 parallel-to C2). Below 60-degrees-C a constant activation energy of 5.4 +/- 0.7 kcal mol-1 was measured, which was close to the activation energy of K+ diffusion in a fluid aqueous medium. The total resistance of stratum corneum was less than 100 kOMEGA cm2, which is very low compared to the resistance of black lipid membranes (1-10 MOMEGA cm2). Both the low activation energy and resistance of human stratum corneum suggest the presence of highly conductive pathways through the membrane. Between 60 and 75-degrees-C an abrupt decline of the resistances R1 and R2 and a rapid rise of the capacitances C1 and C2 was observed. This temperature interval corresponded to the temperature interval of the second thermal transition observed in human stratum corneum, which is a lipid phase transition. Beyond 75-degrees-C, the resistances were fairly constant, while the capacitances continued to increase. The changes in the resistances and capacitances brought about by heating to 75 and 95-degrees-C were completely irreversible. This is in agreement with X-ray diffraction studies, which

Confocal laser scanning microscopy is a technique that permits the direct visualization in unfixed material of diffusion pathways and the cellular distribution of fluorescent markers after topical applications. This approach, in which the tissue specimen is optically sectioned, allows the study of changes in distribution pattern of applied compounds depending on the vehicle, time and depth without the interference of chemical alterations induced by most of the current techniques used for such studies. Using this technique the permeability properties of in-vitro-reconstructed epidermis were compared with those of the native counterpart. The epidermis was reconstructed by culturing human adult keratinocytes at the air-liquid interface either on fibroblast-populated collagen or on de-epidermized dermis. A fluorescent probe — Nile red (NR) — was applied in three different vehicles — polyethylene glycol (PEG) with a molecule mass of 400 (Da), propylene glycol (PG) and dimethyl sulphoxide (DMSO) — which perturb the SC barrier function to different extents. When NR was applied in PEG and PG on native epidermis, the amount of NR penetrating into and through the SC was very low, but was markedly increased when NR was applied in DMSO. Unlike native epidermis, the reconstructed epidermis allowed rapid NR penetration after the application in any of the solvents used. Furthermore, NR applied on reconstructed epidermis, was distributed quite homogeneously between the cellular and the intercellular spaces throughout the SC, suggesting that not only intercellular lipid structures but also the properties of the cornified envelopes differed markedly from those found in native epidermis. The differences in transport pathways between reconstructed and native epidermis may be partially ascribed to the culture conditions used, since incubation of freshly isolated epidermis under the same culture conditions as used for the reconstruction of the epidermis also leads to profound changes in the NR diffusion pathways.

Metanocortins have various physiological actions on the brain.The recent cloning of neural melanecortin 0//(2) receptors Opened new avenues to study the effect of these ncuropeptides on the nervous system.We investigated the structure activity relations (SARs) of peptides derived from adrenecorticotropo hormone (ACrH) on cloned MC3, MC4 and MC5 receptors in vitro.Analysis of the effects of various melanocortin peptides on cAMP accumulation in and on binding to cells that expressed either the rat MC3 receptor, the human MC4 receptor or the ovine MC5 receptor demonstrated that different ACTH fragments and analogs could selectively activate or inhibit the MC receptor subtype activities.The SAP.. of the MC4 receptor resembled that of the induction of excessive grooming behavior by melanocortin poptides.Antagonists that blocked the MC4 receptor were also tested to block a behavioral response induced by cc-MSH, a-MSH-induced excessive grooming behavi~ in rats was inhibited by [Pbe-IT]ACTH-(4-10), [D-ArgS]ACTH-(4-10) and [ProSa~ but not by [Ala6]ACTH-(4-10).From these 4 antagonists, only the latter compound did not antagonize the MC4 receptor in vitro.Therefore, we suggest that this behavioral response is mediated by MC4 receptors.ORG2766, an ACTH 4-9 analog that is very potent in an active avoidance task, did not activate, antagonize or bind to the MC receptors.This suggests the presence of still other MC receptors than the MC3, MC4 and MC5 receptors in the brain.In order to develop more selective MC (an0agonists, the interaction of melanoeortins and receptors at molecular level were investigated.Based upon a 3D-medel for MC receptors and the differences in primary ,structure of the MC receptors, receptormutagenesis was preformed in order to identify the amino acids in the receptors that underly the selectivity that these receptors display for the different melanecortins.Therefore, the MC4 receptor, which is not activated by low doses of yMSH, was genetically modified with sequences that occur in the MC3 receptor, which is activated by yMSH.Furthermore, amino acids that according to our model were important for peptide binding were mutated.Both the binding characteristics as well as the activation of the mutated MC4 receptors by melanecorims were altered.These studies may be employed for development of novel MC-receptor-spectfie ligands.

This study deals with effects of electrical (current density, frequency and duty cycle) and chemical (buffer pH and ionic strength) conditions on the flux of the octapeptide, 9-desglycinamide, 8-arginine-vasopressin (DGAVP), through dermatomed human skin. A pulsed constant current was applied during iontophoresis. The anode faced the anatomical surface of the skin samples inside the diffusion cells. The resistive and capacitative components of the equivalent electrical circuit of human skin could be calculated by fitting the voltage response to a bi-exponential equation. The skin resistance prior to iontophoresis varied between 20 and 60 k Ω.cm2. During iontophoresis a decrease of skin resistance and an increase of the series capacitances was observed, which were most pronounced during the first hour of iontophoresis; thereafter both quantities gradually levelled off to an apparent steady state value. The reduction of the resistance during iontophoresis increased non-linearly with increasing current density between 0.013–0.64 mA.cm−2. The steady state resistance and capacitances did not vary significantly with frequency and duty cycle of the current pulse. There was no pH dependence of skin resistance at steady state. Between pH 4 and 10, the steady state peptide flux had a bell-shaped pH-dependence with a maximum of 0.17 nmol.cm−2.h−1 at pH 7.4, which is close to the I.E.P. of the peptide. Lowering the ionic strength from 0.15 to 0.015 M NaCl increased the steady state flux at pH 5 and pH 8 by a factor 5 to 0.28 ± 0.21 and 0.48 ± 0.37 nmol.cm−2.h−1, respectively. Together these observations suggested that DGAVP is transported predominately by volume flow. At pH 6, at which 65% of the peptide carried a net single positive charge, the steady state flux increased with increasing current density (0.013–0.64 mA.cm−2) from 0.11 ± 0.03 to 0.19 ± 0.04 nmol.cm−2.h−1. Skin permeability during passive diffusion preceding iontophoresis at pH 6.0 was 2.9 ± 0.6 * 10−7 cm.h−7. In accordance with theoretical predictions based on the Nernst-Planck equation, to which a volume flow term was added, the flux was proportional to the mean voltage across the skin between 0.013 and 0.32 mA.cm−2.h−1. Variation of frequency or duty cycle did not result in significantly different peptide transport rates. From these studies it is concluded that DGAVP can be transported iontophoretically through human skin. The pH- and ionic strength-dependence of the iontophoretic peptide flux suggests that transport of DGAVP mainly occurs by volume flow. Furthermore, the flux of DGAVP appears to be controlled by the applied voltage rather than by the current density, as predicted by the Nernst-Planck equation.

In vivo skin hydration effects of liposomal suspensions in deuterium oxide were compared with those of pure deuterium oxide, under occlusive conditions, using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy.

SummaryThe aim of the present study was to visualize the routes of penetration of mercuric chloride through human skin in vitro at the ultrastructural level, and to find out to what extent intra- and extracellular space contribute to the percutaneous transport of the model compound. Dermatomed human skin was subjected to in vitro mercuric chloride diffusion experiments in a bicompartmental polycarbonate diffusion cell. Upon interrupting a diffusion experiment samples were treated with ammonium sulfide vapor to precipitate the mercury as mercuric sulfide, and then processed for transmission electron microscopy (TEM). The presence of mercury in the precipitates was verified using X-ray microcanalysis.An additional series of experiments involved the immersion of freshly excised human plantar callus in mercuric chloride solutions, followed by the same tissue-processing protocol as used for the dermatomed skin samples; the mercury-treated callus samples were likewise subjected to TEM.The results indicate that the intercellular route of transport the stratum corneum predominates, but that after longer transport times, apical corneocytes tend to take up material, leading to a bimodal distribution of mercury: in the apical region of the stratum corneum there is mercury both in- and outside the cells; in the medial and proximal region intercellular transport prevails. There were no signs of a discontinuity in the in-depth distribution of mercury in that an almost impermeable barrier would exist in the lower region of the stratum corneum, as suggested by, e.g., Sharata and Burnette [19, 20]; however, there was evidence of the presence of two types of cells: apical corneocytes, which tend to take up mercuric ions relatively easily, and medial and proximal corneocytes, which are less capable of doing so. Interestingly, the results further indicated that intracellular mercury uptake in the apical, squamous region of the stratum corneum occurred preferentially via the desmosomes. This was also the case in the callus immersion studies. These results suggest that the desmosomes may serve to channel material into corneocytes, especially in the squamous region where the desmosomes are beginning to disintegrate and, hence, the cellular lipid envelopes are leaky. A reservoir function for the apical zone of the stratum corneum is suggested.