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Peat and various peat extracts have been successfully applied for a variety of clinical indications. Quite apart from the physico-thermal effects, new studies point towards the so-called "chemical effects" of peat containing substances. These effects include a stimulatory response of the spontaneous contractile activity (SCA) of smooth muscle (SM) tissue. The effects are, however, dependent on the possible permeability of pharmacologically active substances as naturally occurring ingredients of peat. Since peat is a mixture of various products it is necessary to examine the various peat types based upon their biological activity on SM tissue. In order to unequivocally prove the pharmacological activity of cutaneous peat treatment, in vitro permeation measurements of these actives across excised human skin can be used.HPLC analysis revealed that aqueous peat extracts contain up to 18 fractions of water-soluble compounds of fulvic and ulmic acids. These compounds have been found to have a stimulatory response on the contractile activity of SM tissue. In vitro diffusion studies showed that the permeability of these substances across human full thickness skin (thickness: 200 um(-1)) is highly selective and the resulting stimulatory activity is dependent on the permeated fraction. Especially, the HPLC fractions 7-11 and 14 are able to permeate human skin. Fractions 7-11 show a moderate stimulatory effect of SCA on SM for more than 90 min whereas fraction 14 shows the strongest stimulatory effect which was, however, suppressed after 87 min. These results show that the cutaneous therapy with peat treatment results in transcutaneaous permeation of biologically active fulvic and ulmic acid derivatives explaining the additional "chemical" effect of peat treatment in clinical practice.

Purpose: To study at the ultrastructural level which part of the skin is associated with percutaneous iodide transport by passive diffusion and iontophoresis., Methods: Following passive diffusion or iontophoresis of iodide, the morphology and the ion distribution of the skin was preserved by rapid freezing. The skin was kept frozen until and during examination by transmission electron microscopy (TEM) and X-ray microanalysis (XRMA). The intrinsic electron absorbing characteristics of cryopreserved skin allow direct TEM examination without additional staining. XRMA can be used to obtain in a relatively nondestructive way in situ information on ion distributions across the skin., Results: After passive diffusion, iodide was mainly found in the stratum corneum (SC), whereas there was little iodide in the viable epidermis. Iontophoresis up to 300 microA/cm2 did not significantly affect this distribution. With iontophoresis at 1,000 microA/cm2, the amount of iodide increased dramatically and was equally distributed over the SC and viable epidermis. The presence of iodide in the SC suggests that iodide is present inside corneocytes., Conclusions: Iontophoresis up to 300 microA/cm2 does not significantly perturb skin structures in contrast to iontophoresis at 1,000 microA/cm2. The presence of iodide inside corneocytes suggests the possibility of transcellular percutaneous iodide transport.

One of the possibilities for increasing the penetration rate of drugs through the skin is the use of vesicular systems. Currently, special attention is paid to the elastic properties of liquid-state vesicles, which are supposed to have superior properties compared to gel-state vesicles with respect to skin interactions. In this study, the effects of vesicles on hairless mouse skin, both in vivo and in vitro, were studied in relation to the composition of vesicles. The interactions of elastic vesicles containing the single chain surfactant octaoxyethylene laurate-ester (PEG-8-L) and sucrose laurate-ester (L-595) with hairless mouse skin were studied, in vivo, after non-occlusive application for 1, 3 and 6 h. The skin ultrastructure was examined by ruthenium tetroxide electron microscopy (TEM) and histology. The extent, to which vesicle constituents penetrated into the stratum corneum, was quantified by thin layer chromatography (TLC). The interactions of the elastic vesicles containing PEG-8-L and L-595 surfactants were compared with those observed after treatment with rigid vesicles containing the surfactant sucrose stearate-ester (Wasag-7). Furthermore, skin permeability experiments were carried out to investigate the effect of treatment with PEG-8-L micelles, elastic vesicles (containing PEG-8-L and L-595 surfactants) or rigid Wasag-7 vesicles on the 3H(2)O transport through hairless mouse skin, in vitro, after non-occlusive application. Treatment of hairless mouse skin with the elastic vesicles affected the ultrastructure of the stratum corneum: distinct regions with lamellar stacks derived from the vesicles were observed in intercellular spaces of the stratum corneum. These stacks disrupted the organization of skin bilayers leading to an increased skin permeability, whereas no changes in the ultrastructure of the underlying viable epidermis were observed. Treatment with rigid Wasag-7 vesicles did not affect the skin ultrastructure or skin permeability. TLC measurements showed that after 1 h of non-occlusive application of elastic or rigid vesicles, a six-fold increased amount of elastic vesicle material was present within the stratum corneum compared to rigid vesicle material. After 3 and 6 h of application the amount of PEG-8-L vesicle material in SC decreased to approximately three- and two-fold, respectively, compared to Wasag-7 vesicle material. Pretreatment of the hairless mouse skin with the elastic vesicles containing 70 mol% PEG-8-L increased the diffusion of 3H(2)O with an optimum application dose of 2.5 mg lipids/cm(2) compared to PBS pretreatment. No significant difference in the enhancement of the 3H(2)O-diffusion was observed between PEG-8-L micelles or elastic vesicles containing 30 or 70 mol% PEG-8-L. Pretreatment with the rigid Wasag-7 vesicles decreased the diffusion rate of 3H(2)O, most probably by the formation of a lipid layer on the skin surface. The effect of the elastic vesicles on the skin permeability is supported by the ultrastructural changes observed by TEM in the intercellular lipid domains. The elastic vesicles containing 70 mol% PEG-8-L disorganize the lipid bilayers thereby creating or modifying pathways for possible drug penetration.

Interactions between vesicle formulations and human skin were studied, in vitro, in relation to their composition and elasticity. The skin ultrastructure was investigated using transmission electron microscopy (TEM), freeze-fracture electron microscopy (FFEM) and two-photon fluorescence microscopy (TPE). The main difference between the vesicle formulations was their elasticity. Elastic vesicle formulations contained bilayer forming surfactants/lipids and single-chain surfactant octaoxyethylenelaurate-ester (PEG-8-L), whereas rigid vesicles contained bilayer surfactants in combination with cholesterol. TEM results showed three types of interactions after non-occlusive application of elastic PEG-8-L containing vesicle formulations on human skin: (1) the presence of spherical lipid structures containing or surrounded by electron dense spots; (2) oligolamellar vesicles were observed between the corneocytes in the upper part of the stratum corneum; and (3) large areas containing lipids, surfactants and electron dense spots were observed deeper down into the stratum corneum. Furthermore, after treatment with vesicles containing PEG-8-L and a saturated C12-chain surfactant, small stacks of bilayers were found in intercellular spaces of the stratum corneum. Rigid vesicles affected only the most apical corneocytes to some extent. FFEM observations supported the TEM findings. Major morphological changes in the intercellular lipid bilayer structure were only observed after treatment with PEG-8-L containing elastic vesicles. TPE showed a distinct difference in penetration pathways after non-occlusive application of elastic or rigid vesicles. After treatment with elastic vesicles, thread-like channels were formed within the entire stratum corneum and the polygonal cell shape of corneocytes could not be distinguished. Fluorescent label incorporated in rigid vesicles was confined to the intercellular spaces of the upper 2-5 micrometer of the stratum corneum and the cell contours could still be distinguished.

One major problem in (trans)dermal drug delivery is the low penetration rate of drugs through the barrier of the skin. Encapsulation of a drug in lipid vesicles is one strategy to increase the penetration rate of a drug across the skin. In this study, the interactions between fluorescent-labelled liposomes and skin are visualized by confocal laser scanning microscopy (CLSM). Bilayer labelled gel-state and liquid-state liposomes (conventional or with flexible bilayers) were non-occlusively applied on human skin in vitro. The penetration pathway and penetration depth of the lipophilic fluorescent label into the skin were visualized. From the CLSM images, it was clear that the label applied in micelles and gel-state liposomes did not penetrate as deep into the skin as the label applied in liquid-state vesicles. Among the liquid-state vesicles, the suspensions with the flexible bilayers showed the highest fluorescence intensity in the dermis. Thus, the thermodynamic state of the bilayer and, to a smaller extent, the flexibility of the bilayer influence, strongly the penetration depth of the label into the skin. The label applied non-occlusively in flexible liposomes penetrated deeper into the skin than after occlusive application., (Copyright 1998 Elsevier Science B.V.)

A study was made of the validity of freeze-drying to visualize the distribution of 3H-estradiol in human stratum corneum after topical application of a dry dose, a patch or a buffer solution. Each of these donor formulations was applied to human dermatomed skin for 24 h using Franz permeation cells. Subsequently, small pieces of skin were subjected to cryofixation, freeze-drying, osmium tetroxide vapor fixation, Spurr resin embedding and electron microscopic autoradiography. Stratum corneum from dry dose and patch application experiments was well preserved by freeze-drying, allowing an accurate localization of 3H-estradiol. In contrast, stratum corneum from buffer solution experiments suffered from cryofixation artifacts due to excessive hydration of the skin. The corresponding autoradiographs showed strong redistribution of 3H-estradiol. Thus, the visualization method under investigation has its limitations regarding the hydration level of the skin.

The intercellular lipid bilayers of the stratum corneum provide the permeability barrier of the skin. To perform an electron microscopical examination of the ultrastructure of these bilayers, ruthenium tetroxide fixation is required. In this study an optimal fixation protocol was developed and selected upon comparing seven different fixation procedures, using glutaraldehyde (GA) and the postfixatives ruthenium red, osmium tetroxide (OsO4) and ruthenium tetroxide (RuO4) in combination with potassium ferrocyanide (K4Fe(CN)6) and potassium ferricyanide (K3Fe(CN)6). Instead of fixing skin with either OsO4 or RuO4, these two fixatives were combined in one protocol. In addition, the use of RuRed was introduced and the influence of both K4Fe(CN)6 and K3Fe(CN)6 in combination with RuO4 were examined. Furthermore, we compared two dehydration solvents, methanol and acetone. The most satisfying results were obtained when the skin was prefixed in GA and postfixed in OsO4 and RuO4 with K3Fe(CN)6, i.e. with Fe in its highest oxidation state (Fe3+). No differences were observed between dehydration in methanol and acetone.

In transdermal iontophoresis, drugs can be driven across the skin by electrorepulsion, but their transport can also be enhanced by electrical perturbation of the skin barrier. Our objective was to study perturbing effects of electrical current on human stratum corneum lipid fine structure combining techniques including freeze-fracture electron microscopy. Human stratum corneum was subjected to pulsed constant currents, varying from 0.013-13 mA.cm-2. The voltage across the stratum corneum was high-frequency-sampled and s.c. impedence values derived from it. Upon termination of the current, skin samples were rapidly frozen and processed for freeze-fracture electron microscopy or subjected to X-ray diffraction analysis. Initially a rapid decrease of the resistance and, overall, a rapid increase of the capacitances was observed; generally, these effects became more pronounced with increasing current density. Wide- and small-angle X-ray diffractograms of human stratum corneum exposed for 1 h to the highest current indicated a disordering of both the lateral packaging arrangement and long-range lamellar stacking of the intercellular lipids of stratum corneum. Furthermore, an increase in the stratum corneum hydration level as a result of electrical current application was observed. On electron micrographs of freeze-fracture replicas of human stratum corneum, exposed for 1 h to current densities between 0.013 and 13 mA.cm-2, perturbations of the intercellular lipid structure were observed in accordance with the results of X-ray diffraction; these perturbations aggravated with increasing current density. Together, the data suggest that both the lateral and the longitudinal disordering of the intercellular lipids observed with X-ray diffraction may be responsible for the appearance of perturbed structures observed with freeze-fracture electron microscopy. The lipid disordering may be due to polarization of the lipid head groups induced by the electrical field, followed by mutual repulsion.

Purpose: This study aims to elucidate the skin permeation enhancement and the skin perturbation effects of a number of fatty acids, i.e. straight-chain saturated (SFA), monounsaturated (MUFA) and polyunsaturated acids (PUFA)., Methods: The skin permeation enhancement effects were studied using human stratum comeum (SC) and p-aminobenzoic acid (PABA) as a model permeant. The fatty acids in propylene glycol (FA/PG) were applied according to a pre-treatment/co-treatment protocol. The perturbation effects were studied using differential thermal analysis (DTA) on SC after pretreatment with FA/PG., Results: SFA with 6 to 12 carbons exhibit a parabolic correlation between enhancement effect and chain-length, with a maximum at nonanoic-decanoic acids (with 9 and 10 carbons). Nonanoic and decanoic acids exert barely noticeable effects on the thermal behaviour of SC, suggesting that they easily mix with the skin lipids. All cis-6-, 9-, 11- or 13-octadecenoic acids (MUFA) enhance the permeation of PABA to the same extent. DTA revealed that the cis-9- and 13-isomers form a separate domain containing mostly the pure fatty acids within the SC lipids and suppress the lipid transitions at 70 degrees/80 degrees C. PUFA--linoleic (LA), alpha-linolenic (ALA) and arachidonic acids--enhance PABA permeation stronger than MUFA but additional double bonds do not further increase the degree of enhancement. LA and ALA form separate domains but do not completely suppress the SC lipid transitions at 70 degrees/80 degrees C. Increase in the enthalpy changes of 70 degrees/80 degrees transitions linearly correlates to the decrease in the permeability coefficients, suggesting that an increased perturbation of the skin lipids not necessarily has to yield an increased PABA permeation., Conclusions: The enhancement effects of fatty acids on the PABA penetration through SC are structure-dependent, associated with the existence of a balance between the permeability of pure fatty acids across SC and the interaction of the acids to skin lipids.

The structure of fully hydrated human stratum corneum was investigated by means of freeze-fracture electron microscopy. Mammary and abdominal stratum corneum were incubated for 48 h with phosphate-buffered saline, pH 7.4, occlusively or phosphate buffer, pH 7.4, occlusively and non-occlusively. The micrographs showed the corneocytes aligned parallel to the surface of the stratum corneum embedded in intercellular lipids. The corneocytes were swollen by the uptake of water. New features located in the intercellular lamellar regions were rough structures, water pools, and occasionally vesicle-like structures. The nature of the vesicle-like structures was not completely clear. The presence of water pools, mostly in close contact with the rough structures, suggests that a lipid-water phase separation occurred. The localization of water in the intercellular region and the corneocytes offers new insights into the penetration enhancement property of water (and into the pathways of drug penetration).

Purpose: 1. The assessment of the role of hair follicles and sweat glands in skin resistance and percutaneous iontophoretic flux of 9-desglycinamide, 8-arginine vasopressin (DGAVP) by comparing two skin species: human stratum corneum which contained hair follicles, sweat and sebaceous glands, and shed snake skin which lacked all appendages. 2. The effect of 1-dodecylazacycloheptan-2-one (dodecyl-Azone, a lipid perturbing agent) on the iontophoretic DGAVP flux., Methods: Iontophoresis in vitro was performed in a transport cell (0.79 cm2 area available for percutaneous transport) by 8-hours application of a pulsed constant current of 100 Hz, 50% duty cycle and 0.26 mA.cm-2 current density delivered by a pair of Ag/AgCl electrodes, of which the anode was facing the anatomical surface of the skin samples., Results: The initial resistances of human stratum corneum and shed snake skin samples were of the same order of magnitude (20-24 k omega.cm2) and both skin species showed a comparable resistance-decrease profile during 8-hours iontophoresis, indicating that the resistances were mainly determined by the stratum corneum and not greatly influenced by the appendageal structures. The initial resistances of the skin samples pretreated with dodecyl-azone were less than 50% of the values of untreated samples. Because dodecylazone is known to perturb the ordering of the intercellular lipids, the effect of azone on the resistance confirms that the resistance mainly resides within the intercellular lipids of the stratum corneum. No correlation was found between the iontophoretic DGAVP-flux and the conductance of human skin. For shed snake skin, however, a good correlation was found, indicating that the iontophoretic permeability of human skin in vitro for a peptide such as DGAVP is, unlike shed snake skin, not related to its overall permeability to ions. While the initial resistances of both human and snake skin were in the same order of magnitude and showed the same declining profile during iontophoresis, the steady state iontophoretic DGAVP flux across human stratum corneum was approximately 140 times larger than through shed snake skin. These findings suggest that small ions follow pathways common to both skin types, presumably the intercellular route, while the peptide on the other hand is transported differently: across snake skin presumably along intercellular pathways only, but across human stratum corneum along additional pathways (most likely of appendageal origin) as well. This interpretation is supported by the observations made of the effects of dodecyl-azone on DGAVP-iontophoresis. Pretreatment with dodecyl-azone did not significantly change steady state fluxes and lag times of DGAVP-iontophoresis across human stratum corneum, but resulted in a significant 3-fold lag time decrease and a 3-fold flux increase of DGAVP-iontophoresis across snake skin., Conclusions: The results of these in vitro studies emphasize the importance of the appendageal pathway for iontophoretic peptide transport across human stratum corneum.

Vitamin E acetate is often used rather than vitamin E as an ingredient of skin care products and dermatological preparations, because it lacks the free phenolic OH group. However, because of this the acetate as such is biologically inactive. In spite of this intrinsic inactivity, the skin is protected against the harmful effects of sunlight after topical application of vitamin E acetate. Therefore it is supposed that hydrolysis takes place in the skin and that the reaction product, the radical scavenger vitamin E, is responsible for the protection observed. In this in vivo study with the rat, we have investigated the hydrolysis of RRR-alpha-tocopheryl acetate (vitamin E acetate) in the epidermis in relation to UV radiation protection. (As a measure of protection, we used the UV-induced binding of 8-methoxypsoralen to epidermal biomacromolecules.) After a period of 5 h from a single application of vitamin E acetate, hydrolysis into free vitamin E was not observed. No protection was found at this time point, corresponding with the absence of vitamin E. After treatment for 5 days, consisting of one topical application daily, the percentage of acetate present in the stratum corneum which was hydrolysed into free vitamin E was less than 1%, whereas the corresponding value for the viable layer of the epidermis was about 5%. The hydrolysis of vitamin E acetate in the epidermis proceeded very slowly. As a result, the absolute amount of free vitamin E, found in the total epidermis after treatment for 5 days with the acetate, was only a few times higher than the normal level. Yet, this very small amount of free vitamin E proved to be sufficient for maximal protection in this animal model. The results show that vitamin E acetate acts as a prodrug, which very slowly releases minute amounts of active vitamin E.

The interactions between three liposomal formulations and human stratum corneum were visualized using freeze fracture electron microscopy. A new replica cleaning method was introduced. Human stratum corneum was submerged for 48 h in liposome suspensions prepared from commercially available phospholipid mixtures. The size, lamellarity and lipid moieties of the liposomes were similar. The main difference between the three phospholipid formulations was the hydrophilicity of the headgroups. The composition dependence of the interactions between these vesicles and human stratum corneum was investigated. In essence, two types of interaction were observed: adsorption of the liposomes on to the outer surface of the stratum corneum, and ultrastructural changes in deeper layers of the stratum corneum caused by mixing of the liposomal constituents and the stratum corneum lipids. The electron microscopic observations were verified with small-angle X-ray scattering. It was found that liposomes composed of phospholipids containing relatively small hydrophilic headgroups showed a marked interaction with the skin lipids of human stratum corneum in vitro. The complexity of the phospholipid mixtures, however, made it very difficult to determine the exact effect each of these headgroups has on the interactions between these vesicles and human stratum corneum.

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 omega.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 predominantly by volume flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogel patches based on water swellable polyacrylates have been developed for long-term transdermal drug delivery. Two properties, relevant to the performance of hydrogel patches in-vivo have been investigated in humans over five days. These were: (i) the kinetics of water exchange between the skin and the patches; (ii) the skin compatibility of the patches. It was found that initially there was a gradually increasing uptake of water from the skin by the patches, but after about 20 h the water exchange followed a regular fluctuating pattern, peaking once a day and once a night. The skin compatibility of the patches was satisfactory, in that no redness or pustulation was noticed throughout the five days. This was most likely due to the capability of the patches to exchange water with the skin.