Wolfgang Wohlrab

Modification of drug penetration into human skin using microemulsions

Abstract The objective of this study was to investigate the penetration of the hydrophilic substance diphenhydramine hydrochloride from a W/O-microemulsion into human skin under ex vivo conditions. The focus of the study was to determine the amount of a model substance in the different skin layers, rather than measure the transdermal rate. Modifications of the vehicle components clarified the extent, to which it is possible to control the penetration of a hydrophilic drug incorporated in a microemulsion (ME) system. A standard ME showed an accumulation of penetrated drug in the dermis, indicating a potential following high absorption rate. Incorporation of cholesterol into the system leads to an even higher penetration rate and a shifting of the concentration profile further towards the epidermis. In comparison, addition of oleic acid had no effect. The results are in concordance with the assumption that diphenhydramine hydrochloride follows hydrophilic structures into the stratum corneum. Therefore, an alteration of the barrier properties can be achieved obviously only by influencing this hydrophilic pathway.

How deep do intact liposomes penetrate into human skin

Abstract Attempts were made to visualise intact liposomes in the human skin by fluoromicrography. To this end the aqueous compartment of the liposomes as well as the lipid bilayer were followed by labelling with hydrophilic and lipophilic fluorophores, respectively. Micrographs at various times after external application suggested that intact liposomes do not penetrate deeper than the horny layer.

Tumor hypoxia, p53, and prognosis in cervical cancers

BACKGROUND The p53 protein is involved in the regulation of initiation of apoptosis. In vitro, p53-deficient cells do not respond to hypoxia with apoptosis as do p53-normal cells, and this may lead to a relative growth advantage of cells without a functioning p53 under hypoxia. On the basis of this hypothesis, a selection of cells with a functionally inactive p53 may occur in hypoxic tumors. The development of uterine cervical carcinomas is closely associated with infections of human papilloma viruses, which may cause a degradation of the tumor suppressor gene p53, resulting in a restriction of apoptosis. Thus, cervical cancers have often a functionally inactive p53. The purpose of our clinical study was therefore to investigate the association between p53, hypoxia, and prognosis in cervical cancers in which the oxygenation status can be determined by clinical methods. MATERIAL AND METHODS Seventy patients with locally advanced squamous cell cervical cancer Stages IIB (n = 14), IIIB (n = 49), and IVA (n = 7) were investigated in the period from 1996 through 1999. All were treated with definitive radiotherapy with curative intent by a combination of external radiotherapy plus high-dose-rate afterloading. Before therapy, tumor oxygenation was measured with a needle probe polarographically using the Eppendorf histograph. Hypoxic tumors were defined as those with pO(2) measurements below 5 mm Hg (HF5). Pretreatment biopsies were taken and analyzed immunohistologically for p53 protein expression with the DO-7 antibody. The DNA index was measured by flow cytometry. The statistical data analysis was done with SPSS 9.0 for Windows. RESULTS The 3-year overall survival was 55% for the whole group of patients. Clinical prognostic factors in a multivariate analysis were pretreatment hemoglobin level (3-year survival 62% for patients with a pretreatment hemoglobin > or =11 g/dl vs. 27% for hemoglobin <11 g/dl, p = 0.006) and FIGO stage (Stage IIB: 65%; Stage IIIB: 60%; Stage IVA: 29%, p = 0.01). Sixty of the 70 tumors showed positive immunohistologic staining for p53 protein (transformed p53 = tp53), and 10/70 were negative (wild-type p53 = wtp53); p53 expression had no significant impact on survival (50% for tp53 vs. 79% for wtp53, p = 0.11). FIGO stage and anemia had no impact on p53 expression. Forty-nine of 70 tumors were hypoxic (HF5+), and 21 showed no hypoxia (HF5-). Hypoxic carcinomas were more frequently positive for p53 as compared to nonhypoxic tumors (27% vs. 13%, p = 0.011) and showed a trend toward a lower survival (48% vs. 70%, p = 0.07). In a further multivariate analysis, the impact of a combination of p53 expression and hypoxia on survival was examined. After adjusting for FIGO stage and pretreatment anemia, patients with wtp53 tumors had the best prognosis (3-year survival 79%) followed by tp53-HF5(-) patients (57%), and the most unfavorable prognosis was observed for tp53-HF5(+) patients (47%). The DNA index was higher in tp53 carcinomas compared to wtp53 tumors, 1.97 +/- 0.4 vs. 1.67 +/- 0.1, p = 0.05. The highest DNA index was found in hypoxic tumors with transformed p53 (2.2 +/- 3.1). CONCLUSIONS Advanced stage and pretreatment hemoglobin level are independent prognostic factors in cervical carcinomas. The immunohistologic detection of (a functionally inactive) p53 and the presence of hypoxia had no prognostic impact, if analyzed as single parameters. However, the combination of both parameters was able to discriminate different prognostic subgroups. Moreover, hypoxic cancers were more often immunohistologically positive for tp53 protein and had a higher DNA index with the highest DNA index in tumors with both hypoxia and tp53 protein expression. These findings in summary support the theory that the tumors microenvironment may influence the biologic behavior via hypoxia.

Analytical methods for measuring urea in pharmaceutical formulations

Two new methods are described for the routine determination of urea that utilize HPTLC-densitometry and colorimetry. The methods involve derivatization of urea with p-dimethylaminobenzaldehyde to a yellow-coloured compound. Validation of the methods was accomplished with respect to linearity, accuracy, reproducibility and limit of detection/quantification. Both methods were compared with an enzymatic method previously described in the literature and were found to be in close agreement. The proposed methods have the advantages of being simple, rapid and involve a single step sample preparation. Under experimental conditions HPTLC was the most sensitive method.

Betulinic acid induces apoptosis in skin cancer cells and differentiation in normal human keratinocytes.

Abstract:  Betulinic acid (BA), a pentacyclic triterpene of plant origin, induces cell death in melanoma cells and other malignant cells of neuroectodermal origin. Little is known about additional biological effects in normal target cells. We show, in this study, that BA induces differentiation as well as cell death in normal human keratinocytes (NHK). Cytotoxicity profiles of BA are compared among normal human keratinocytes, HaCaT cells, IGR1 melanoma cells and normal melanocytes. As expected, BA is toxic to all cell types, normal and malignant, but varies in its cytotoxic potency and in the extent of induction of apoptotic vs. necrotic cell death in the four different skin cell types. Apoptosis is proved by annexin V and Apo2.7 binding and by DNA fragmentation. Induction of differentiation‐associated antigens in keratinocytes – filaggrin and involucrin – is demonstrated, together with specific morphological changes in treated cell cultures. BA, apart from its cytotoxic activities in cellular systems, is capable of inducing differentiation of NHK into corneocytes without immediately provoking apoptotic cell death.

Quantitative evaluation of urea in stratum corneum of human skin

Urea, as a final product of protein metabolism, is a physiological substance in the human organism. The elimination of urea in humans amounts to 30 g per day; the amount in urine is of the order of 2% and in the blood 250 mg per ml. The urea content of human skin is about 1%. Urea, as one of the natural moisturizing factors, influences the capacity of the horny layer to bind water and it also has keratoplastic, penetration-promoting, antimicrobial and antipruritic properties. Therefore, the observation that pathological skin alternations are accompanied by alterations in the content of urea is not unexpected. This has been studied and confirmed previously by several investigators using various analytical methods. Schwarz determined urea in aqueous extracts of scrapings from the horny layer of healthy volunteers and of patients with atopic dermatitis or psoriasis using thin layer chromatography followed by detection with Ehrlichs reagent [6] and, in a separate study, using an amino acid analyser [7]. For the investigation of urea content in aged skin Kfigelgen and Schwarz [5] also used an amino acid analyser. The amount of urea in callus was determined by Jacobi [3] using urease [1]. The procedure applying the amino acid analyser permits a determination of urea values only related to the part of amino acids. A disadvantage of all the methods previously described is that, in using scrapings from the horny layer, only the very superficial parts of the stratum corneum are considered. The analytical method described here allows the quantitative evaluation of urea in absolute values over the whole thickness of the horny layer in a defined area of 2.5 cm 2. The method was selected using the following die critera: detection limit, sensitivity, specifity, cost and

A multilayer membrane system for modelling drug penetration into skin

Abstract A new model system for modelling drug penetration profiles in human skin is presented with a multilayer membrane system as receptor. For this purpose, membranes were used with dodecanol (DD) as lipid and collodion as matrix. The variability of the model system was demonstrated by the addition of propylene glycol and by changing the DD content of the membrane. Dithranol (DT) was used as a model drug. The penetration of DT was found to take place rapidly into two-, three- and six-layer membrane systems. The penetration profiles of DT in the six-layer membrane systems could be controlled by varying the DD content of the first membrane and by the use of intermediate propylene glycol membranes. Consequently, it was possible to adapt the penetration profiles of DT in the multilayer membrane system to those in human skin.

Evaluation of drug penetration into human skin ex vivo using branched fatty acids and propylene glycol

The influence of two middle chain methylbranched fatty acids and propylene glycol on the penetration of the highly lipophilic model substance pyrene butyric acid into human skin ex vivo has been investigated. The results obtained were compared with experimental data using oleic acid as standard enhancer. Analogously, liberation studies of pyrene butyric acid into artificial lipid acceptor membranes were performed to evaluate the contribution of vehicle effects to the skin penetration results. It was shown that the fatty acids initially improve the liberation of the model substance which corresponds to an increase in skin penetration. When the penetration process was assessed, the dermal concentration profiles of pyrene butyric acid and propylene glycol were almost the same, strongly indicating a cotransport for the lipophilic model substance. Levels of both pyrene butyric acid and propylene glycol did increase when the vehicles contained fatty acid. Furthermore, there is some indication of a more specific action of oleic acid within the stratum corneum. However, the major effect on the penetration of pyrene butyric acid arises from propylene glycol. It is caused by solvent properties and solvent drag or favoured partition, respectively, into the stratum corneum and the hydrophilic epidermis and dermis which are supposed to be the main diffusion barrier for the model penetrant.

Über den DNS-Gehalt von Epidermiszellen unbefallener Psoriatikerhaut

The epidermal DNA has been determinated cytophotometrically in clinically not-involved psoriatic skin, separate in stratum basale, the lowest three layers and the upper part of stratum spinosum. 1. The DNA patterns differ in all layers from those in normal epidermis. 2. The number of nuclei with hyperdiploid values is in all the stratum spinosum higher than in normal skin. 3. The importance, possible due to these findings, for the run down of the psoriatic reaction is discussed.

Histochemische Befunde an der klinisch gesunden Haut von Psoriatikern

SummaryThe question of latent psoriasis has been examined with histochemical methods on the symptom-free skin in an great distance from clinical focuses and above all in intervals free from eruption. The result was:1.In the keratinizing part of the epidermis of clinically sound skin of patients suffering from psoriasis it has been possible to point out regarding the histotopy of enzymes, lipoids, protein-bound SH-and SS-groups, α-aminoacids among others certain modifications in the sense of the manifest psoriatic reaction, and the were pointed out everywhere, but in different intensity, it is true.2.In contrast to this statement the conditions in the nonkeratinized part of the epidermis and in the cutis did not present any certain differences in comparison with the normal skin.3.Likewise with regard to the number of mitoses no significant difference could be established between the eruption-free skin of psoriatic persons and the sound skin of normal persons.4.The significance of these conditions for the question of morphogenesis of the psoriatic reaction has been discussed concisely.ZusammenfassungMit histochemischen Methoden wurde die Frage der latenten Psoriasis an der erscheinungsfreien Haut weit ab von klinischen Herden und vor allem in eruptionsfreien Intervallen geprüft. Es ergab sich:1.Im verhornenden Teil der Epidermis klinisch gesunder Haut von Psoriatikern konnten hinsichtlich der Histotopie von Enzymen, Lipoiden, proteingebundenen SH-und SS-Gruppen, α-Aminosäuren unter anderem gewisse Veränderungen im Sinne der manifesten psoriatischen Reaktion festgestellt werden, und zwar durchgängig, wenn auch in unterschiedlicher Intensität.2.Im Gegensatz dazu zeigten die Befunde im unverhornten Teil der Epidermis und in der Cutis keine sicheren Unterschiede gegenüber normaler Haut.3.Auch hinsichtlich der Zahl der Mitosen konnte zwischen der eruptionsfreien Psoriatikerhaut und der gesunden Haut von Normalpersonen kein signifikanter Unterschied festgestellt werden.4.Die Bedeutung dieser Befunde für die Frage der Morphogenese der psoriatischen Reaktion wurde kurz diskutiert.