Comparison of human skin or epidermis models with human and animal skin in in-vitro percutaneous absorption.

For the study of in-vitro skin penetration of candidate drugs, excised animal skin is frequently used as a replacement for human skin. Reconstructed human skin or epidermis equivalents have been proposed as alternatives. We compared the penetration properties of human, pig and rat skin with the Graftskin LSE (living skin equivalent) and the Skinethic HRE (human reconstructed epidermis) models using four topical dermatological drugs (salicylic acid, hydrocortisone, clotrimazole and terbinafine) with widely varying polarity. In agreement with published data, pig skin appeared as the most suitable model for human skin: the fluxes through the skin and concentrations in the skin were of the same order of magnitude for both tissues, with differences of at most two- or fourfold, respectively. Graftskin LSE provided an adequate barrier to salicylic acid, but was very permeable for the more hydrophobic compounds (e.g. about 900-fold higher flux and 50-fold higher skin concentrations of clotrimazole as compared to human skin), even more than rat skin. In the case of the Skinethic HRE, we found similar concentrations of salicylic acid as in human skin and an approximately sevenfold higher flux. In contrast, the permeation of hydrophobic compounds through the epidermal layer was vastly higher than through split-thickness human skin (up to a factor of about 800). To conclude, currently available reconstituted skin models cannot be regarded as generally useful for in-vitro penetration studies.

Expression of epidermal keratins and the cornified envelope protein involucrin is influenced by permeability barrier disruption.

In previous studies we have shown that experimental permeability barrier disruption leads to an increase in epidermal lipid and DNA synthesis. Here we investigate whether barrier disruption also influences keratins and cornified envelope proteins as major structural keratinocyte proteins. Cutaneous barrier disruption was achieved in hairless mouse skin by treatments with acetone +/- occlusion, sodium dodecyl sulfate, or tape-stripping. As a chronic model for barrier disruption, we used essential fatty acid deficient mice. Epidermal keratins were determined by one- and two-dimensional gel electrophoresis, immunoblots, and anti-keratin antibodies in biopsy samples. In addition, the expression of the cornified envelope proteins loricrin and involucrin after barrier disruption was determined by specific antibodies in human skin. Acute as well as chronic barrier disruption resulted in the induction of the expression of keratins K6, K16, and K17. Occlusion after acute disruption led to a slight reduction of keratin K6 and K16 expression. Expression of basal keratins K5 and K14 was reduced after both methods of barrier disruption. Suprabasal keratin K10 expression was increased after acute barrier disruption and K1 as well as K10 expression was increased after chronic barrier disruption. Loricrin expression in mouse and in human skin was unchanged after barrier disruption. In contrast, involucrin expression, which was restricted to the granular and upper spinous layers in normal human skin, showed an extension to the lower spinous layers 24 h after acetone treatment. In summary, our results document that acute or chronic barrier disruption leads to expression of keratins K6, K16, and K17 and to a premature expression of involucrin. We suggest that the coordinated regulation of lipid, DNA, keratin, and involucrin synthesis is critical for epidermal permeability barrier function.

Penetration of Sandimmune (cyclosporin A) in rat skin in vitro. Effects of penetration enhancers and solvents.

The effect of various fatty acids or alcohols on the penetration rates and skin concentrations of cyclosporin A (Sandimmune; CyA) was evaluated in an in vitro model using skin of hairless rats. The influence of chain length, number and position of double bonds and branching of the carbon chain of the enhancer were investigated. In addition the penetration dependency of CyA on the concentration of both enhancer and CyA was studied. CyA was quantitated by high-performance liquid chromatography. The penetration rates of CyA through rat skin decreased with increasing number of double bonds of the enhancer and decreasing CyA concentrations in the donor solution, and increased with increasing chain length of the enhancer. Enhancers increase penetration rates by a factor of up to 20-90 in alcoholic vs. maximally 5-fold in oily compositions. Enhancers increase skin concentrations of CyA by a factor of up to 10-25 in alcoholic and about 4-20 in oily compositions.

[Systemic reactions in rats following the initiation of a local inflammatory process by subcutaneous administration of spirits of turpentine]. / Systemische Reaktionen in Ratten nach Setzung lokal entzündlicher Prozesse durch subcutane Terpentinöl-Gaben.

Local inflammation was induced in rats by single (1 x 4 ml/kg) or multiple (14 X 0.2 ml/animal) infections of turpentine. The induction of inflammatory processes in both groups resulted in anemia and granulocytosis following an initial leukopenia. Thrombopenia on the second day, followed by thrombocytosis, was also observed in both groups. Studies on blood chemistry parameters revealed a decline in serum albumin; elevation of alkaline phosphatase in serum was observed only after multiple injection of turpentine. In these animals an elevation in the weights of spleen and adrenals and a reduction in the weight of thymus were also found.

Composition of fluids from diffusion chambers implanted in the soft tissue and kidneys of rabbits.

The fluids from diffusion chambers implanted in soft tissue and kidneys of rabbits were analysed for total protein, albumin, enzymes, ions, glucose, creatinine, urea, uric acid, bilirubin and cholesterol. These date were compared with the corresponding values in plasma. Our data for chamber fluid are in good agreement with data reported for interstitial fluids. The composition of the kidney chamber fluid is nearly constant from three to ten weeks after implantation. The low urea, uric acid and creatinine concentrations indicate that the chamber is not located in the urine collecting area of the kidney. Three days after subcutaneous implantation of chambers, the fluid contains less protein than plasma but has an equal concentration of ions, thus meeting the principal requirements for interstitial fluid. There are indications that the healing process lasts up to ten days after the surgical implantation. In order to examine the permeability of the diffusion chambers, the equilibration half-life times of antibiotics and substances of high and low molecular weights were determined in vitro.

Enhancement of the anticoccidial activity of polyether antibiotics in chickens by tiamulin.

The anticoccidial activities of monensin and lasalocid have been studied separately and in combination with tiamulin, a new pleuromutilin derivative. Combinations of constant tiamulin concentration (.0125%) in drinking water with various levels of polyether anticoccidials (6.3 to 125 ppm) in feed and conversely of constant levels of anticoccidials with various concentrations of tiamulin were used. The prophylactic efficacy of these combined treatments in battery raised broiler chickens infected with Eimeria tenella was evaluated. Assessment of the parameters mortality, weight gain, dropping scores, lesion scores, and oocyst output showed that simultaneous application of tiamulin significantly improved the anticoccidial activity of the polyethers. As tiamulin alone is without anticoccidial activity, this phenomenon was considered to result from an interaction between tiamulin and the polyethers leading to a slower metabolic degradation of the latter. Thus tissue levels adequate for maximum anticoccidial activity would be attained with lower polyether dose levels. Experiments using isolated perfused rat liver showed that elimination of monensin was reduced by 60% in the presence of tiamulin.