Pathways of lateral spreading.

In the case of topically applied substances, usually both lateral spreading and competitive penetration into the skin occur in parallel. In the present study, the pathways of lateral spreading were studied quantitatively and visually. The local distribution and lateral spreading of the UV filter substance butyl methoxydibenzoylmethane applied in an o/w emulsion was studied on the forearm and the back. The tape stripping procedure was used to determine the recovery rates inside and outside the area of application. The skin characteristics of transepidermal water loss, pH value, hydration of the stratum corneum and sebum rate were determined at both anatomic sites. Photography and laser scanning microscopy were used to visually investigate the lateral spreading of topically applied dyes. On the back, a preferred direction of lateral spreading parallel to the body axis was observed. This result was caused by differences in the network of furrows. The furrows functioned as a pathway for lateral spreading, whereas the follicles formed a reservoir for the topically applied substance.

The pathogenetic mechanism of anthracycline-induced palmar-plantar erythrodysesthesia.

BACKGROUND: Anthracyclines, such as pegylated liposomal doxorubicin (PLD) and epirubicin (EP), are effective for the treatment of malignant tumors. Unfortunately, their implementation in therapy is limited due to severe side-effects such as palmar-plantar erythrodysesthesia (PPE). PATIENTS AND METHODS: As the exact pathogenesis of PPE still remains unclear, laser scanning microscopy was utilized to detect PLD, EP and their metabolites in and on the skin surface of patients. RESULTS: It was shown that PLD was significantly more frequently detectable on the skin than was EP (p<0.05), whereas both substances were most frequently seen in the palms and soles. Additionally, it has been visualized that the substances reach the skin surface via sweat, where they distribute and then penetrate back into the skin. CONCLUSION: It was concluded that a high density of sweat glands and a thick stratum corneum might represent important predestined factors for the development of PPE. These findings will help to develop efficient prevention and therapy strategies for PPE.

The tape stripping procedure--evaluation of some critical parameters.

Tape stripping is a simple and efficient method for the assessment of quality and efficacy of cosmetical and dermatological formulations. After topical application and penetration of formulations, the cell layers of the stratum corneum are successively removed from the same skin area using adhesive films. The tape strips contain the amount of corneocytes and the corresponding amount of the penetrated formulation, which can be determined by classical analytical chemical methods. Different formulations can strongly influence the amount of stratum corneum removed with every tape strip. Therefore, it is essential for the comparison of the penetration of different formulations that the amount of formulation detected on the single tape strip is not related to the tape strip number as a relative measure of the penetration depths, but to their standardized real position in the stratum corneum. Therefore, different methods are reported for the determination of the amount of stratum corneum removed with every tape strip. The tape stripping method in its standardized form is well-suited to determine the dermatopharmacokinetics of topically applied substances. Additionally, the method can be used to obtain information about the homogeneity and the distribution of formulations on the skin and in the stratum corneum. This is used, e.g., for the determination of the homogeneity of the distribution and the ex vivo determination of a universal sun protection factor (USPF) characterizing the efficacy of sunscreens.

Human percutaneous absorption of a direct hair dye comparing in vitro and in vivo results: implications for safety assessment and animal testing.

Although in vitro skin absorption studies often detect small residues of applied test material in the epidermis/dermis, it is uncertain whether the residue is within the living skin. We studied the dermal absorption of a hair dye hydroxyanthraquinone-aminopropyl methyl morpholinium methosulphate (HAM) in human skin in vivo and in vitro. In vivo, skin (back and scalp) received 0.5% HAM in a commercial formulation at 20microg/cm2 After 0.5 or 48h, skin was tape stripped, followed by cyanoacrylate biopsies (CAB). Sebum from scalp sites was collected for 48h. In vitro, skin was treated with 20mg/cm2 dye for 0.5h, penetration determined after 24h. In vivo, at 0.5h, total recovery (back) was 0.67microg/cm2 (tape strips+CAB). Fluorescence microscopy showed HAM in the hair follicle openings (HFO). At 0.5h, scalp tape strips contained 1.80microg/cm2, HFO 0.82microg/cm2. At 48h, HFO contained 0.21microg/cm2, sebum 0.80microg/cm2. In vivo, skin residues were in the non-living skin and eliminated via desquamation and sebum secretion. In vitro, the SC contained 1.50microg/cm2, epidermis/dermis 0.86microg/cm2, receptor fluid<0.04microg/cm2, a total of 0.90microg/cm2 was considered to be bioavailable. In vitro epidermis/dermis residues were nearly identical to those located in non-living skin in vivo. In conclusion, in vitro percutaneous penetration studies may produce seemingly bioavailable material , which raises the need for a Threshold of Skin Absorption (TSA) addressing a negligible dermal absorption in order to avoid unnecessary in vivo toxicity studies on substances that produce no significant human systemic exposure.

Penetration of pollen proteins into the skin.

Atopic dermatitis is a chronic remittent skin disease. In the extrinsic form of atopic dermatitis, type IgE-mediated reactions play an important pathophysiological role. The aim of the present study was to examine whether type I allergens can penetrate into the skin. Therefore, pollen proteins were labeled with fluorescein isothiocyanate (FITC), and their penetration profile was studied qualitatively. Solutions of FITC-labeled pollen proteins were applied in vitro on porcine skin and in vivo on human skin. In vitro, the FITC-labeled proteins were observed within the complete stratum corneum (SC) and inside the hair follicles even 15 min after application. They were also distributed inside the dermis around the hair follicles. In vivo, a similar pattern of distribution within the SC and the hair follicles was observed. These results indicate penetration via the SC lipid layers and a faster penetration via the hair follicles. The FITC-labeled proteins entered the dermis via the follicular pathway. Therefore, the follicular penetration should be considered in the development of skin protection strategies. To evaluate such strategies, the developed method can be used, and further studies in atopic dermatitis patients are necessary to determine whether the penetration of type I allergens is increased.

The number of stratum corneum cell layers correlates with the pseudo-absorption of the corneocytes.

The removal of the stratum corneum (SC) using adhesive tapes is a common technique in cutaneous studies. The determination of the varying amounts of the SC removed would be a helpful tool in such investigations. In the present study, the cell layers of porcine SC were counted before and after removal of several tape strips using histological techniques. In addition, the pseudo-absorption of the corneocytes reflecting the amount of these cells was determined using spectroscopy. Different amounts of SC were removed using 20 tape strips. The spectroscopically determined data correlate linearly with the number of removed cell layers. Based on these results, the pseudo-absorption of the corneocytes can be used to calculate the absolute number of cell layers removed with a standard deviation of less than 11%. In this way, the SC can be quantified using the procedure of tape stripping in combination with the spectroscopic determination of the corneocytes.

Comparison of transepidermal water loss and spectroscopic absorbance to quantify changes of the stratum corneum after tape stripping.

The objective and quantitative application of tape stripping in pharmaceutics and dermatopharmacokinetics requires the determination of the exact position of each removed tape strip inside the stratum corneum (SC) and/or the determination of the relative SC thickness. In this study, transepidermal water loss (TEWL) and the optical spectroscopic data of the corneocytes were measured simultaneously during the complete removal of the SC by tape stripping. The spectroscopic data quantitatively reflect the amount of corneocytes removed by the individual tape strips, whereas TEWL and 1/TEWL are not sensitive enough to measure the relatively small changes in the SC thickness realized by the removal of the individual strips. The relative SC thickness can be determined directly by the spectroscopic data, while the 1/TEWL values require a second independent method. The results demonstrate the importance of tape stripping characterizing the behaviour of topically applied substances.

Estimation of the relative stratum corneum amount removed by tape stripping.

BACKGROUND/PURPOSE: The tape stripping procedure is a suitable minimal invasive tool to study, e.g. the penetration and dermatopharmacokinetics of topically applied substances. In the present study, this procedure was used to remove the stratum corneum (SC) completely and to study the penetration of the UVA filter substance butyl methoxydibenzoylmethane after application in two different vehicles. METHODS: The amount of corneocytes removed by each tape strip from the flexor forearm of human volunteers was determined via their pseudo-absorption. In a second part, the penetration profiles of a UVA filter substance applied in two different vehicles were determined following the developed standard protocol using the tape stripping procedure in combination with UV/VIS spectroscopy. RESULTS: The amount of corneocytes removed by each tape strip was related to the number of tape strips used for removal. Mean values with a deviation of less than 20% concerning the relative amount of SC removed by a constant number of tape strips were obtained. For instance, a relative amount of 66 +/- 12% was removed with the first 20 tape strips, while nearly the complete SC (95 +/- 3%) was removed using 50 tape strips. In addition, these results were used to estimate the relative SC amounts removed, studying the penetration of the UVA filter substance after application in two different vehicles. No significant differences between the distributions of the UV filter substance applied in both emulsions were obtained (P > 0.05). CONCLUSIONS: The reported procedure for the estimation of the removed SC amount provides the possibility to avoid the complete removal of the SC and to compare the penetration characteristics obtained for different volunteers and different products in relation to the relative horny layer profile.

Synergy effects between organic and inorganic UV filters in sunscreens.

The influence of the synergy effects between organic and inorganic UV filter substances on the sun protection factor (SPF) of topically applied sunscreen formulations is investigated. The medium is considered to have reflection, absorption, and scattering properties. The distribution of photons in this medium is investigated by Monte Carlo calculation. Typical optical parameters of the skin and substances are used to characterize the synergy effect. The results of the model calculation are checked by in vitro and in vivo measurements investigating the influence of different types of scattering microparticles on the absorption efficacy of topically applied formulations. It is found that the inorganic filter substances act as scattering microparticles in the upper skin layers. They increase the optical pathway of the photons in the topically applied absorbing formulation also localized there. In this way, more photons are absorbed, increasing the SPF. The results obtained are important for the optimization of the SPF of sunscreen formulation containing organic and inorganic UV-filter components.

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.

Comparison of four different in vitro systems to study the reservoir capacity of the stratum corneum.

Four in vitro test systems were used to study the reservoir capacity of porcine stratum corneum (SC) for flufenamic acid and its drainage via penetration into the deeper skin layers: Franz diffusion cell using full thickness skin and split skin of 300 mum; Saarbruecken penetration model (SB) and intact porcine tissue (IP). Each skin sample was segmented 1, 4 and 21 h after application of an 'infinite dose' of flufenamic acid. The lipophilic drug was extracted from the SC and the deeper skin layers (viable epidermis and dermis) and determined using high-performance liquid chromatography (HPLC). For each test system, an increase in the drug amount in the deeper skin layers and the acceptor fluid, respectively, was observed in combination with a decreased amount in the SC with increasing time after application. The drainage of the SC reservoir was only reflected by a linear correlation of the drug amount in the SC with the amount in the deeper skin layers in the case of IP. The absolute drug concentrations previously detected in human skin in vivo and in vitro were compared with the present data, affording the best accordance in the case of IP.

The porcine snout--an in vitro model for human lips?

The morphology and histology of test sites commonly used to study the penetration of lip products differ significantly from those of the human lip itself. The aim of this study was to investigate whether the porcine snout could serve as an equivalent in vitro model for human lips. The lips of human test subjects and biopsies of porcine snout tissue were compared using histological and microscopic techniques. Using a dermatological laser scanning microscope, the penetration of topically applied fluorescent sodium fluorescein was investigated in vivo on human lips and in vitro on the porcine snout. Biopsies from the in vitro experiments were studied using fluorescence microscopy. Some parts of the porcine snout show a similar morphology and histology as human lips. The stratum corneum (SC) and the epidermis of the porcine snout are thicker than those of human tissue. Both in vivo and in vitro, the topically applied fluorescent dye was detected only on the skin surface and within the uppermost SC layer. These results indicate that porcine snout can be used as an in vitro model for human lips in penetration studies. Both human and porcine tissues exhibit an efficient barrier against the penetration of topically applied substances.

Penetration profile of microspheres in follicular targeting of terminal hair follicles.

The transfollicular administration of pharmacologically active molecules is of current therapeutic interest, mainly with regard to delivery to specific sites of the hair follicle (HF) and the reduction of hepatic metabolism and systemic toxicity. HF are privileged pathways for specific molecules depending on formulations, which enter faster into these shunts than through the stratum corneum. The aim was to optimize the delivery of fluorescent microspheres into the HF, thereby, developing a standardized protocol for follicular targeting with microspheres. The number of HF showing penetration, as well as the depth of penetration, was determined. Freshly excised skin samples with terminal HF were divided into groups, with or without prior treatment with cyanoacrylate skin surface stripping-technique (CSSS). Thereafter microspheres at a size of 0.75-6.0 microm were applied according to the developed standardized protocol. Skin biopsies were obtained, shock-frozen, and sectioned in 5 microm slices. We demonstrated a selective penetration route of the microspheres into the HF. Optimal microsphere size proved to be approximately 1.5 microm, with a 55% rate of all HF, and with a maximum penetration depth of >2300 microm. Without previous CSSS treatment of the skin, the transfollicular microsphere penetration was below 27% with a maximum penetration depth of 1000 microm. Thus, the basis for follicular targeting of essential structures containing stem cells for keratinocytes, melanocytes, and mast cells has been laid.

Lateral spreading of topically applied UV filter substances investigated by tape stripping.

The lateral spreading of topically applied substances is a competitive process to the penetration into the stratum corneum (SC). The penetration of topically applied UV filter substances into the human SC and the lateral spreading were investigated in vivo. Tape stripping in combination with spectroscopic measurements was used to study both processes of two UV filter substances. The concentration of both UV filters was determined inside and outside the application area by varying the application and tape stripping protocol. A spreading of the topically applied substances from the treated to the untreated areas was observed, which caused a concentration gradient. This lateral spreading depends on the time between application and tape stripping and the size of the treated skin area. Significant amounts of topically applied substances were found adjoining the application area, due to the lateral spreading which takes place on the skin surface. In general, the lateral spreading must be considered to be a competitive process when studying penetration processes of topically applied substances. It has to be considered during drug treatment of small limited skin areas and for the interpretation of recovery rates obtained in penetration studies.

[Follicular penetration. An important pathway for topically applied substances]. / Follikuläre PenetrationEin entscheidender Penetrationsweg von topisch applizierten Substanzen.

The knowledge of penetration pathways in and through the skin is a prerequisite for the development and optimization of topically applied drugs and cosmetics. Skin penetration has been assumed to occur via diffusion though the lipid domains of the stratum corneum. Recent studies show that the skin appendages, especially the hair follicles, play an important role in skin penetration processes. Topically applied substances cannot enter all follicles. We discuss the reasons for the phenomenon of open and closed hair follicles.

Identification of the leukocyte adhesion molecules CD11 and CD18 as receptors for type 1-fimbriated (mannose-specific) Escherichia coli.

Attachment of bacteria to phagocytic cells may be mediated by lectin-carbohydrate interactions, resulting in lectinophagocytosis. The best-studied system is the interaction of type 1-fimbriated (mannose-specific) Escherichia coli with human phagocytic cells. Here we demonstrate that the leukocyte integrins CD11 and CD18 (CD11/CD18) constitute the major receptors for type 1-fimbriated E. coli. Bacteria were bound in a dose-dependent and saturable manner to CD11/CD18, which was immobilized to microwells, whereas nonfimbriated E. coli cells failed to bind. The binding was efficiently inhibited (82 to 85%) by methyl-alpha-mannoside but not by galactose, and it was reduced by treatment of the immobilized CD11/CD18 with sodium metaperiodate, endoglycosidase H, or a mixture of endoglycosidase F and N-glycosidase. The fimbriated bacteria also bound to CD11a,b,c and CD18 separated by polyacrylamide gel electrophoresis with sodium dodecyl sulfate and blotted onto nitrocellulose paper. This binding was inhibited specifically by methyl-alpha-mannoside and was significantly diminished by treatment of the blots with sodium metaperiodate. Only minimal binding to the blotted CD11/CD18 that had been deglycosylated enzymatically prior to electrophoresis was observed. On blots of granulocyte lysates, specific binding to two glycoproteins (Mrs, 90,000 to 100,000 and 165,000) with mobilities similar to that of CD11/CD18 was observed. Monoclonal antibodies to CD11a, CD11b, or CD18 inhibited the binding of the bacteria to intact human granulocytes by 55 to 80%, whereas antibodies against other leukocyte surface antigens were not inhibitory. We conclude that type 1-fimbriated E. coli binds to human granulocytes via the oligomannose and hybrid N-linked units of CD11/CD18. Since CD11b/CD18 and CD11c/CD18 are known to serve as receptors for complement fragment iC3b, this study provides a link between opsonophagocytosis and lectinophagocytosis.

[Nitrogen metabolism in the large intestine of ruminants. 3. Microbial utilization of intracecally administered 14C- and 15N-marked urea in the large intestine of sheep in simultaneous intracecal administration of partially hydrolyzed straw meal]. / Untersuchungen zum Stickstoffumsatz im Dickdarm des Wiederkäuers. 3. Mitteilung. Mikrobielle Verwertung von intrazäkal verabreichtem 14C- und 15N-markiertem Harnstoff im Dickdarm von Schafen bei gleichzeitiger intrazäkaler Gabe von teilhydrolysiertem Strohmehl.

Two experiments were performed on sheep, receiving on maintenance level a pelleted straw ration high in crude fibre (straw, 70.5%; dried sugar beet pulp, 12%; cereals, 10%; urea, 2%; ammonium hydrogen carbonate, 3%; minerals 2,5%). The animals were fitted with ileo-caecal re-entrant cannulas. The effects of the introduction of HC1-partly hydrolysed straw meal into the digesta of the large intestine on the digestion processes in that segment were studied. Under these conditions the metabolism of 14C and 15N labelled urea, which was given into the caecum, was estimated. In experiment 1 (E 1; 2 animals) unlabelled, precollected digesta were hourly reintroduced together with 14C and 15N labelled urea via the caecal cannula. In experiment 2 (E 2; 3 animals) the digesta were supplemented with partly hydrolysed straw meal (10% of the mean daily DM-intake with the ration). The supplement of partly hydrolysed straw meal caused an increase of the 15N excretion with faeces from 13.4% (E 1) to 19.8% (E 2) of the dose. The 15N was mainly incorporated in the bacterial fraction (98% E 1; 96% E 2). As a reason for the increased 15N incorporation into the bacterial fraction of 106.4 mg15N' in E 2 vs. 67.3 mg15N' in the experiment without straw meal supplement the higher supply of energy as fermentable carbohydrates was assumed.

[The effect of zeolite (clinoptilolite) on the post-feeding dynamics of N metabolism in the portal vein, jugular vein and the rumen fluid of bulls]. / Stúdium vplyvu zeolitu (klinoptilolitu) na dynamiku niektorých ukazovatel'ov N-metabolizmu po nakrmení vo v. port ae, v. jugularis a v bachorovej tekutine u býkov.

If easily digestible saccharides are deficient in the feed ration of bulls with the live weight of 300 kg and at simultaneous single application of urea at a rate of 0.2 g per 1 kg live weight, zeolite (with 50.6% clinoptilolite content) administered at a rate of 2.5% per 1 kg dry matter influenced significantly (P less than 0.05) the ammonia concentration in rumen, v. portae and v. jugularis. The rumen contents and blood were sampled at the intervals of 0, 15, 30, 60, 90, 120, 180 and 360 minutes after feeding. Basal feed ration consisted of 1 kg feed mixture and 3 kg meadow hay. After urea administration, zeolite reduced the ammonia concentration in rumen by 20-40% in comparison with the control group and in v. portae by 60-70%. In v. jugularis in the 90th minute after feeding significant hyperammonemia was observed in bulls with no zeolite supplement. Zeolite administration did not influence urea concentration in plasma.