Using a model of pure epidermal wounds in normal human volunteers, we have studied the effects of Biafine emulsion firstly on inflammatory cell migration, vascular permeability and cytokine release during the first 24 h, and secondly on epidermal wound healing by measuring transepidermal water loss from day 1 to day 7. Under these conditions, Biafine does not improve epidermal healing, in contrast to what is observed with bleeding dermoepidermal wounds. Our results suggest that the effects of Biafine are essentially at the dermis level. The analysis of epidermal wound exudates leads to the same conclusion. As a matter of fact, we demonstrated that Biafine is chemotactic for macrophages and increases the IL-1/IL-6 ratio, chiefly by reducing the secretion of IL-6. This study permits to progressively clarify the mode of action of Biafine, that seems to be located at the level of granulation tissue formation and not at the epidermal level.
We investigated the antipruritic effect of a 15-min application of dimethindene maleate (Fenistil gel) and other local analgesics (Optiderm, EMLA, Xylocain ointment 5%) on subsequent focal histamine stimulus (20 mC) given by iontophoresis in 12 patients suffering from acute atopic eczema (AE). The results were compared to histamine after pretreatment with the respective placebo and to non-pretreated skin. Wheal and flare areas were planimetrically evaluated. Itch or pain ratings were performed over a 24-min period using a rating scale. The examination also comprised alloknesis, i.e. induction of a perifocal itch sensation by a non-itching mechanical stimulus. None of the antihistaminic and anaesthetic agents reduced the itch intensity significantly. Three of the AE patients had a total lack of alloknesis. We conclude that these substances, when applied for 15 min, are not sufficiently effective in atopic skin suppressing histamine-induced reactions under experimental conditions. The diminished elicitation of alloknesis in these patients may be a result of central nervous system alteration.
We measurd the superoxide dismutase (SOD) activity in human skin from tissue homogenates after topical application of hydrocortisone-21 -acetate and clobetasol proprionate, dissolved in propylene glycol. SOD was measured spectropho-tometrically. SOD activity was higher in the treated skin than in the control untreated skin. We separated epidermis from the dermis by curettage and measured the level of SOD in each homogenized layer; SOD activity was higher in the epidermis compared to the dermis in untreated skin. After corticoid application, SOD activity was higher in the dermis compared to the epidermis to a degree dependent on corticoid potency. These experiments demonstrate that the epidermis may have a role in the barrier function of the skin by its antioxidant capacity and that the dermis is the major location of the metabolic activity in the skin. On the other hand, our results suggest that these corticosteroids may stimulate SOD production and may release antioxidants. This could be another anti-inflammatory property of corticosteroids.
The aim of our study was to further investigate and to objectively assess the effects of tacalcitol on psoriasis by means of double-blind comparisons with placebo and betamethasone valerate, documented by instrumental evaluation. The study was conducted as intrasubject half-side right-left comparisons. Twelve subjects entered a double-blind placebo-controlled treatment (tacalcitol/placebo group), whereas 14 subjects received double-blind medication with tacalcitol/betamethasone valerate. Medications were applied once daily without occlusion to affected skin areas. Instrumental evaluations were carried out by means of a colorimeter and a 20-MHz B scanner. The colour co-ordinate a*, representing the colour range from green (-) to red (+), was used for assessing erythema. B scan images were processed according to segmentation procedures. A 0-10 interval, marking the hyporeflecting dermal areas, was used for assessing oedema and inflammatory infiltration at psoriatic plaque sites. Twenty-two patients (11 of the tacalcitol/betamethasone group and 11 of the tacalcitol/ placebo group) completed the treatment period. Mean a* values showed an increase in the tacalcitol/betamethasone group after the first week of therapy on both sides, followed by a slight decrease, the values of which did not reach statistical relevance for either treatment. The decrease observed at tacalcitol-treated sites in patients of the tacalcitol/placebo group was not significant either with respect to baseline values during the first weeks of therapy. The extension of 0-10 dermal areas decreased both in betamethasone- and tacalcitol-treated areas in both patient groups. No significant differences were noticeable between the two treatments at all assessment points, although both by clinical and echographic evaluation a more pronounced decrease in clinical scores and in echogenicity values was observable at betamethasone-valerate-treated sites, especially at weeks 6 and 8. According to our data, assessment of erythema does not always represent a valid method for monitoring the response to therapy in psoriatic patients. In fact, modifications of a* values did not allow a distinction between different treatments, whereas processing of echographic images by the 0-10 segmentation enabled tacalcitol to be classified as a topical drug, the activity of which approaches that of a potent steroid.
OBJECTIVE:In order to assess the proliferative changes induced by all-trans retinoic acid (RA) and retinol (ROL), we have carried out a study of the DNA content of basal and suprabasal keratinocytes after epicutaneous application on the rhino mouse.STUDY DESIGN:Skin sections were analyzed stereologically and cytophotometrically using the Feulgen technique. The diploid DNA value (2C) was obtained from hepatocyte nuclei of control animals. Whereas cells in phase G0-G1 will show a 2C content, cells during phase S and in phase G2-M will show DNA values ranging from 2C to 4C and 4C, respectively.RESULTS:Although epidermal thickness (ET) increased significantly in all treated animals, surface density only increased in animals treated with all-trans RA. Quantification of DNA content of basal keratinocytes showed reduction of 2C and 2C-4C populations with a commensurate increase in proportions of cells with 4C and > 4C in the animals treated with 0.025% all-trans RA and ROL. Suprabasal keratinocytes of mice treated with 0.025% all-trans showed a decrease of the 2C population and an increased proportion of cells with 4C. Whereas 0.025% all-trans RA induced an increase of both basal and suprabasal DNA indices, ROL enhanced only the basal DNA index significantly.CONCLUSION:Animals treated with 0.025% ROL showed a significant increase in the basal proliferative index (PI) while the suprabasal PI remained constant; treatment with 0.025% all-trans RA produced a significant increase of both basal and suprabasal PIs and parakeratotic hyperkeratosis probably due to incomplete differentiation.
Minoxidil is the most used drug with proved effects in the treatment of androgenetic alopecia (AGA), but little is known about its pharmacological activity and target cells in hair follicles. As AGA is characterized by follicle atrophy, accelerated hair cycles and hair fiber thinning, we postulated that keratinocyte proliferation/differentiation is affected and we tested Minoxidil's effects on those parameters. Normal human keratinocytes (NHK) of follicular or epidermal origin were cultured in the presence of Minoxidil (0, 0.1, 1, 10, 100, 1,000 microM) during 5-8 days in various media (high-/low-calcium content, with or without serum). Proliferation was assessed by mitochondrial dehydrogenase activity (XTT), BrdU incorporation, lysosome numeration (neutral red incorporation) and total protein dosage. Drug-induced cytotoxicity was measured by lactate dehydrogenase release in culture supernatant, and pro-differentiating effects were evaluated by relative involucrin expression (ELISA dosage). On this basis, we showed that Minoxidil had biphasic effects on the proliferation and differentiation of NHK: Minoxidil stimulated NHK proliferation at micromolar doses, while antiproliferative, pro-differentiative and partially cytotoxic effects were observed with millimolar concentrations. We can hypothesize that Minoxidil hypertrichotic activity in vivo is possibly mediated by the maintenance of proliferative potential in follicular keratinocytes precociously committed to differentiation.
Using a new computerized methodological procedure a separate analysis and a quantitative determination of the viscous and elastic parameters of the scalp hair shaft were performed in 37 neonates of both sexes with a gestational age of 28-29 weeks (n = 16) and 39-40 weeks (n = 21), respectively. A statistically significant (p < 0.001) decrease in the values of modulus of elasticity (E alpha) was found in the hair shaft of premature neonates, as compared to the full-term ones, whereas the values of post yield slope (E beta) and of SDIS/SSTOR (viscous parameter) did not significantly differ in the two groups. The decrease in modulus of elasticity in the hair shaft of premature neonates may be interpreted in terms of an insufficient number of disulphide bonds between the alpha-helical keratin units of the hair cortex or of a disordered arrangement of microfibrils within the matrix. Further studies are now warranted to determine the pattern of mechanical parameters of the scalp hair shaft in large numbers of newborn infants of different gestational ages and to answer the question as to whether this pattern might be useful in the accurate postnatal assessment of fetal maturation.
The efficiency of the epicutaneous microcirculation is important to the elicitation of pharmacological effects and percutaneous absorption of drugs. Laser Doppler flowmeter, chromameter and measurement of transepidermal water loss (TEWL) were used to assess the extent of skin blood flow, water loss and light absorption induced after topical administration of 8-methoxypsoralen (8-MOP). TEWL monitors barrier function directly, while the chromameter measures the light reflected by the skin. Irrespective of the concentration of 8-MOP applied, there is a good correlation between chromameter and evaporimeter readings. However, different drug concentrations elicited significant differences between laser Doppler flowmetry (LDF) and TEWL readings. The response measured with LDF was faster and greater when compared with the other measurements, suggesting that 8-MOP caused a rapid and intense response in skin blood flow. Superficial responses, such as those produced by light reflectance (chromameter) and transepidermal perturbations, were weaker following topical application of 8-MOP combined with UV irradiation (PUVA). This information may be important in delineating guidelines for the clinical practice and instrumental monitoring of topical 8-MOP. These observations suggest that LDF provides a useful, noninvasive physical technique to monitor skin after PUVA. However, caution should be exercised in the interpretation of physical measurements for evaluating the degree of the clinical vasoresponse produced by PUVA treatment.
Topical metronidazole is effective in the treatment of papulo-pustular rosacea, a common inflammatory disease of the face. In this study, a comparison of the bio-availability of two topical formulations (A and B) of 0.75% metronidazole has been made. Twenty-four subjects took part in a double-blind study where each was randomly allocated to four groups corresponding to 0.5, 1, 2 and 4 h of treatment. A single application was made to each forearm and after increasing times of contact, samples of stratum corneum were removed by the skin surface biopsy (SSB) technique. After SBB removal, a full-thickness skin biopsy was taken. The tissues were extracted and analysed by an HPLC method. Both formulations showed rapid percutaneous penetration. Formulation A gave the highest concentrations at the lowest levels of the stratum corneum at 4 h. Thus in SSB5, the mean drug concentrations at 4 h were as follows: A, 6.20 ± 1.16 μg cm-2; B, 1.93 ± 0.98 μg cm-2. A similar difference was observed when the levels in the punch biopsies were determined. At 4 h, the mean drug levels were: A, 6.07 ± 2.17 μgmg-1; B, 0.95 ± 1.16 μgmg-1. Formulation A clearly gives a higher rate of percutaneous penetration leading to higher tissue levels of drug. Whether this increase leads to a clinically more effective treatment has yet to be determined.
The European Community directive, imposing that effects claimed for cosmetic actives must be validated using non-animal procedures, has stimulated the use of in vitro models for pharmacotoxicological trials. In this paper, an efficacy study of a new biopeptide, a hydrolysate obtained by fermentation of milk proteins, was performed using an in vitro skin equivalent (SE). This SE is obtained by seeding normal human keratinocytes onto a dermal equivalent comprising a collagen-glycosaminoglycan(GAG)-chitosan porous matrix populated by normal human fibroblasts, which neosynthesize their own extracellular matrix (ECM). A gel containing 2% milk biopeptide was applied topically (10 microliters) every 2 days during 15 days. Subsequent investigations of the biopeptide effects were based on morphological criteria after histological analysis and on synthesis of ECM components. Collagen and GAG synthesis were measured by tritiated proline, glucosamine and Na2(35)SO4 incorporation. Qualitatively, the histological features of the biopeptide-treated SEs showed a thicker epidermis than the untreated control SEs, where only a few layers of stratum corneum were observed. The dermal porous matrix seems to be more filled by neosynthesized ECM than the control. Quantitatively, milk biopeptide treatment induced a significant activation of hyaluronic acid (+46%) and sulfated GAG (+53%) synthesis, whereas only non-significant increases of total protein and collagen synthesis were observed (Student's test, p < 0.001).
Lipid-soluble psychotropics are often used to treat skin diseases with psychosomatic indications. Although these drugs are known to exert their effects through the central nervous system, relatively little is known about their mechanism of action in skin. In this communication, several lipid-soluble psychotropic drugs have been examined for their ability to inhibit protein kinase C (PKC)-catalyzed phosphorylation of exogenous substrates and endogenous skin proteins. Phosphorylation of three discrete skin protein substrates at 64, 42 and 28 kDa and a group crowded together at 15-18 kDa was prevented by the antidepressants/antipsychotics. Inhibition was more pronounced in a phospholipid (PL) dependent system, but both drug-PL and drug-PKC interactions seem to be important in the mechanism of action of these drugs. In addition to the tricyclic nucleus, the propanamine side chain or its N-methyl form may influence the interaction of these drugs with PKC and its substrate(s). Chlorpromazine, imipramine, fluoxetine, doxepin, amitriptyline and hydroxyzine used in the practice of dermatology may exert their therapeutic effects by modulating skin PKC activity.
Daily treatments of skin in hairless mice with concentrates of rice wine, Japanese traditional alcohol, lowered transepidermal water loss levels compared to the controls on the 3rd day after ultraviolet B (UVB) irradiation. These findings indicate that the concentrates of rice wine suppress the murine skin barrier disruption caused by UVB. Ethyl alpha-D-glucoside (alpha-ethylglucoside), one of the peculiar components in rice wine, showed the same effect, whereas beta-ethylglucoside had no effect. In order to clarify the functions of alpha-ethylglucoside on murine skin, we examined the effects of this compound on the expression of some phenotypes in human keratinocytes in vitro. As a result, alpha-ethylglucoside as well as beta-ethylglucoside enhanced cell proliferation weakly, and the formation of cornified envelopes and differentiated type keratin (K1) in keratinocytes was accelerated by alpha-ethylglucoside but not by beta-ethylglucoside. From the results, we conclude that alpha-ethylglucoside enhanced the differentiation of keratinocytes, which might be related to reduced barrier disruption by UVB.
Cyclosporin A (CsA) is efficacious in many dermatoses as an oral but not as a topical form, while tacrolimus (FK506) has been shown to be effective in both forms. As inadequate skin absorption has been proposed as the reason for inefficacy of topical CsA, factors contributing to percutaneous absorption of FK506 and CsA were studied. Partitioning of FK506 and CsA between octanol and water, stratum corneum and water, and stratum corneum and isopropyl myristate was determined. Absorption of FK506 and CsA through dermatomed human cadaver skin was determined with in vitro flow-through cells. In partitioning experiments, CsA was more lipophilic than FK506. Both drugs were seen in comparable amounts in skin layers, but FK506 permeated the skin to a greater extent than CsA.
The hormone melatonin is produced in the pineal gland and secreted into the blood in a circadian rhythm. Due to its antioxidative and immunomodulatory effects it might play a role as a topical drug in dermatology. In this study we investigated the penetration kinetics of melatonin applied to the skin of 6 healthy volunteers aged 26-34 years (M/F = 2/4). First the individual physiologic daytime melatonin serum levels were determined by radioimmunoassay. Then 3 of the participants were treated once topically on their scalp with 20 mg melatonin dissolved in 70% ethanol, while the other 3 individuals received 100 mg. The application was performed at 9.00 a.m., and blood samples were collected repeatedly for a total of 8 h. Physiologic daytime melatonin levels in the 6 individuals were 16.8 +/- 10.0 pg/ml. The 3 individuals treated with 20 mg melatonin displayed peak serum levels of 762, 918 and 3,440 pg/ml, respectively, those treated with 100 mg melatonin showed peak levels of 1,136, 3,360 and 4,230 pg/ml lasting throughout the entire observation period of 8 h. These findings indicate that melatonin might accumulate in the stratum corneum with prolonged release into the blood system from this depot.
The 5-lipoxygenase (5-LO) product of arachidonic acid, leukotriene (LT-)B4, is considered to play a significant role in the pathogenesis of psoriasis. In vitro LTB4 is a potent chemoattractant for leukocytes, and it increases DNA synthesis in human cultured keratinocytes. Intradermal injection of LTB4 into human skin in vivo results in a wheal and flare reaction, and topical application produces intraepidermal microabscesses and induces hyperproliferation. Furthermore, LTB4 has been determined in biologically active amounts in psoriatic skin lesions. Despite the importance of LTB4 in psoriasis, the capacity of the human epidermis to synthesize LTB4 has remained controversial. Recently, a very limited 5-LO activity was reported in human epidermis. Thus, it was shown that human epidermis can contribute significantly to LT formation by transcellular LT synthesis. By this mechanism, LTA4 released from activated leukocytes is further transformed into LTB4 in the keratinocytes by the LTA4 hydrolase. Transcellular metabolism may be of importance in psoriasis where neutrophils migrate into the epidermis, because in human neutrophils the LTA4 hydrolase has been shown as the rate-limiting step in LTB4 formation. The LTA4 hydrolase was localized in the epidermis by activity determination, by inhibition of enzyme activity with known LTA4 hydrolase inhibitors, by Western blotting and by immunohistochemical staining. Moreover the enzyme was purified and further characterized from human cultured keratinocytes and human epidermis. Because of these recent results it is concluded that LTB4 is of significance in the pathogenesis of psoriasis, and it is suggested that future work should focus on developing potent LTA4 hydrolase inhibitors for treatment of psoriasis.
Photodamage of the skin can be a detriment to physical and psychological health with a consequent negative impact on both personal and professional life. Medical treatments for photodamaged skin include topical all-trans-retinoic acid (tretinoin) as well as moisturizers, chemical peels, dermabrasion, Α hydroxy acids and cosmetic surgery. Of these treatment options, only tretinoin has been subjected to large-scale, controlled studies to substantiate its clinical efficacy and long-term benefits. This review summarizes the use of various therapies and presents available clinical and histologic results of systematic trials, including the few studies of retinoid compounds other than tretinoin. Among the extensive data on topical tretinoin are long-term treatment results that provide evidence of ultrastructural changes. These histologic findings suggest that fundamental cellular effects may be responsible for the clinical benefits, which are sustained even when treatment frequency is reduced. It thus appears that tretinoin therapy at least partially restores photodamaged skin to the predamaged state.
Chronic exposure to a weak irritant leads to inflammatory changes which may be followed by pigmentary changes and accommodation. The inflammatory responses to acute exposure to an irritant have been extensively studied. This study investigated quantitatively the inflammatory reactions produced in photodamaged skin with chronic application of a weak chemical irritant (tretinoin cream 0.025%) over a period of 9 months (36 weeks). Forty-eight subjects with moderately to severely photodamaged skin were enrolled in a 36-week, double-blind placebo-controlled study. Tretinoin cream was applied nightly on the distal two thirds of one dorsal forearm and placebo on the other. The proximal third of each dorsal forearm received no treatment and served as control. Clinical assessments and diffuse reflectance measurements were made at 7 time points during treatment. Apparent concentrations of oxyhemoglobin (HbO2), deoxyhemoglo-bin (Hb) and melanin were estimated by analysis of the diffuse reflectance spectra. No changes were observed in the apparent H bO2or the Hb concentration of the placebo-treated or control sites, thus establishing a reliable baseline. The apparent HbO2 concentration of the tretinoin-treated sites increased significantly from baseline to a maximum at 12-18 weeks of treatment, then returned to baseline with continued applications. The changes in HbO2 concentration agreed closely with clinical assessments of erythema. The apparent melanin concentration, corresponding to diffuse hyperpigmentation, showed a large seasonal decrease in both the control and the treated sites, with an additional decrease in the treated sites between 12 and 18 weeks. Erythema appeared after repeated applications and eventually resolved under continuous treatment. The maximum decrease in hyperpigmentation occurred simultaneously with the maximum increase in erythema.
The fuzzy rat, a genetic mutant between hairless and hairy albino rats, expresses androgen-dependent hypersecretion of sebum and hyperplastic sebaceous glands. Using this model for human acne, we examined the effects of inhibitors of human steroid 5 alpha-reductase isozymes, type I (MK 386) and type II (finasteride), and an androgen receptor blocker (RU58841) on regression of glandular and ductal hyperplasia. The above three agents, 1% weight volume, were dissolved into the vehicle (propylene glycol, alcohol and water) and applied on the backs of peripubertal male rats for 2 months. Control and castrate groups received vehicle alone. At 8 weeks, we examined the size the sebaceous glandular lobules and ducts in split epidermal preparations as well as in frozen sections of skin stained with osmium-potassium dichromate solution. The number of bromodeoxyuridine (BrdU)-positive cells was counted in the glandular lobes in split-skin tissues stained with BrdU immunochemistry. The results revealed that the sizes of both lobes and ducts in castrates were 40-60% smaller than in controls. RU58841 induced glandular and ductal regression equivalent to that in castrates. Finasteride induced a moderate degree of lobular and ductal reduction, whereas MK386 caused only ductal regression. Reduction of BrdU-positive cells in the sebaceous lobes was found in the skin treated with finasteride and RU58841. Serum concentrations of testosterone and dihydrotestosterone showed no significant changes in all drug-treated rats. The weight of the prostatic lobes was reduced significantly in rats treated with finasteride but not by the other two agents. RU58841 effectively counteracted endogenous androgens resulting in a suppression of growth of the sebaceous glands but not the prostate. This rodent model for androgen-dependent hyperplasia of the sebaceous glands is useful for the study of many pharmacological aspects comprising the rate of percutaneous absorption, stability and affinity to target organs of the testing compounds, and selection of adequate vehicle for topical application.
