Neuroimmunomodulatory compound for sensitive skin care: in vitro and clinical assessment

Background The pathophysiology of sensitive skin consists of an inflammatory reaction resulting from the abnormal penetration in the skin of potentially irritating substances, which occurs due to skin barrier dysfunction and changes in the production of local neuromediators. Aims The therapeutic potential of l ‐carnosine and Rhodiola rosea, as antioxidant and neuromodulatory, respectively, leads us to investigate the effects of the R. rosea extract/l ‐carnosine–associated compound (RCAC) on sensitive skin alterations. Methods A double‐blind comparative study was conducted on 124 volunteers with sensitive skin, who were selected by their reactivity to stinging test. Two randomized groups of 62 each received either a formulation containing 1% of RCAC or placebo, which was applied twice a day for 28 consecutive days. One perceptibility questionnaire was applied at the onset and at the end of the treatment to evaluate the subjective response to test product. Additionally, in vitro studies were performed to investigate RCAC neuroimmunomodulatory mechanisms. Results RCAC treatment produced in vivo protective effects in skin barrier function and a positive subjective response of sensitive skin volunteers. In vitro treatment promoted the release of proopiomelanocortin peptides and restored to normal the increased levels of neuropeptides and cytokines produced by keratinocytes exposed to ultraviolet radiation. Clinical effectiveness was measured by reduction of transepidermal water loss, positive perceptions of improvements in skin dryness and skin comfort sensation, and reduction of discomfort sensation after stinging test. Conclusions The protective effect of RCAC in skin barrier function and the positive response produced in human subjects with sensitive skin could be partially explained by our in vitro results showing a significant increase in opioid peptides release, an inhibitory effect on neuropeptides production, and modulation of cytokines production by keratinocytes under ultraviolet stress.     

The lactic acid stinging test predicts susceptibility to cumulative irritation caused by two lipophilic irritants

Background. Skin exposure to lipophilic irritants at workplaces is a risk factor for the development of occupational irritant hand dermatitis. Suitable lipophilic model irritants to be used in repeated irritation studies in volunteers are needed to gain a better understanding of the chemical‐dependent mechanisms of skin irritation. Objectives. To investigate acute and cumulative irritation elicited by two new lipophilic model irritants, octane (CAS No. 111‐65‐9) and cumene (CAS No. 98‐82‐8), in stingers as compared with non‐stingers. Methods. Short‐time occlusive and repetitive occlusive irritation patch tests were performed over 5 days with octane and cumene in two groups of healthy volunteers (n = 15 each) who were classified as stingers and non‐stingers according to a preceding lactic acid stinging test. Acute and cumulative irritation was quantified using clinical assessments and bioengineering methods (laser Doppler flowmetry, transepidermal water loss, capacitance measurements, and skin colour reflectance). Results. Significantly stronger cumulative irritation was observed in stingers than in non‐stingers for both irritants (visual scoring, stratum corneum hydration, and skin colour reflectance). Conclusions. The preliminary results suggest the existence of a distinct sub‐population with increased susceptibility to cumulative irritation induced by lipophilic irritants that can be identified by the lactic acid stinging test.     

Comparison of irritant potential of shampoos using cultured human epidermal keratinocytes model and patch test reaction measured by laser Doppler flowmetry

The keratinocyte culture model has previously been used as an in vitro method for testing skin irritating potential of common skin irritants. However, solubility limits its use for finished products. Shampoo is very soluble in water which should make it an ideal product category for the cell culture model. To determine the skin irritant potential of several commercial shampoos, we employed cultured human keratinocytes as an in vitro model. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test (MTT) and lactic dehydrogenase release (LDH) test were used to document cell toxicity. 7 volunteers were patch tested and their reactions evaluated using laser Doppler flowmetry and compared with the in vitro data. MTT and LDH have a good negative correlation with each other. Patch test reaction, especially at high concentrations, correlates relatively well with the in vitro test, especially with shampoos of strong and weak irritancy. However, the rank order of the shampoos of moderate toxicity was not the same as in the in vitro data. This suggests that the cell culture technique cannot directly replace in vivo methods, and that data obtained by the cell culture method should be interpreted carefully.     

Correlation between stinging, TEWL and capacitance

Background/aims: To study the correlation between stinging and skin barrier and to make the lactic acid stinging test much safer.
Methods: The modified lactic acid stinging test with 3% and/or 5% aqueous solution of lactic acid combined with transepidermal water loss (TEWL) and capacitance (CAP) was used. The amount of 50 µL-test material was applied on test areas, the skin of both sides nasolabial fold of 50 healthy subjects. Subjects assessed stinging basing on a 4-point scale at 0, 2.5, 5.0 and 8 min TEWL and CAP were measured at baseline and 8 min.
Results: The results show that the tendency of decrease in baseline CAP and increase in baseline TEWL are proportional with the increase of the sum of clinical scores (CS_S) . In 5% lactic acid test, negative correlation between changing ratio of TEWL (P_TEWL) and clinical score at 5 min (CS_5 min), and negative correlation between changing ratio of capacitance (P_CAP) and CS_5 min are registered.
Conclusion : Our data indicate that there is certain relationship between the degree of stinging and the skin barrier. The stinging test method modified by lower concentration aqueous lactic acid and the assessment combined with P_TEWL and P_CAP is suitable for Chinese to evaluate the skin susceptibility.     

Photocontact Allergy Due to Suprofen

A 79-year-old Japanese woman developed edematous erythema on sun-exposed areas 3 months after applying Sulprotin® ointment, which contains 1% suprofen (SP), a phenylacetic acid derivative. A patch test with Sulprotin® ointment as is was negative, but a photopatch test with Sulprotin® ointment as is was positive. A photopatch test with 10^–3% SP was positive, as was that obtained with an SP analogue, 3% ketoprofen, but that with the ointment base of Sulprotin® was negative. Photopatch tests with other SP analogues such as flurbiprofen, ibuprofen, and pranoprofen were negative. She developed abnormal erythema with monochromatic irradiation in the range from 320 to 380 nm on the uninvolved abdominal skin where 1% SP had been applied 24 hours (h) before irradiation. The action spectrum for this erythema agreed with the absorption spectrum of SP in the range of UVA. The MED after applying SP was below that after applying w. pet. in the range from 250 to 290 nm and from 300 to 320 nm. Positive intracutaneous test results were obtained with an irradiated mixture of human serum albumin (HSA) and SP at 48 and 72 h after injection and negative ones with a mixture of HSA and irradiated SP.     

Contact urticaria due to the common stinging nettle (Urtica dioica)—histological, ultrastructural and pharmacological studies

A frequent cause of contact urticaria is skin exposure to the common stinging nettle (Urtica dioica). The urticaria is accompanied by a stinging sensation lasting longer than 12 h. Little is known of the cellular and molecular mechanism of stinging-nettle urticaria. After preliminary pharmacological analysis of pro-inflammatory activity in nettle stings, the cellular response of mononuclear cells, polymorphonuclear cells and mast cells was examined in six people 5 min and 12 h after nettle contact. Only mast cell numbers were significantly increased at 12 h. Ultrastructurally, some mast cells showed evidence of degranulation at 5 min and 12 h. At 12 h mast cells were closely associated with dermal dendritic cells and lymphocytes suggesting a functional unit. The mean histamine and serotonin contents of a nettle hair were found to be 6.1 ng and 33.25 pg, respectively. Nettle-sting extracts did not demonstrate histamine release from dispersed rat mast cells in vitro. These results suggest that part of the immediate reaction to nettle stings is due to histamine introduced by the nettle. However, the persistence of the stinging sensation might suggest the presence of substances in nettle fluid directly toxic to nerves or capable of secondary release of other mediators.     

Stinging and rosacea

A total of 32 rosacea patients (25 with the papulopustular type of rosacea and 7 with the erythematotelangiectatic type) and 32 healthy persons were single-blind tested with a solution of 5% lactic acid and pure water applied to their cheeks. Twenty-four patients and 6 controls reacted positively as "stingers" (p<0.001) in this objective test of sensitive skin. All 7 of the patients with erythematotelangiectatic rosacea, but only 17/25 with the papulopustular type, were stingers (n.s.). The reason why some patients react with subjective symptoms, such as itching, burning, stinging, prickling or tingling, is unclear. The findings in this study are not surprising, but do support the theory that impairment due to different stimuli, most likely because of vascular sensitivity, is a central mechanism in the aetiology of rosacea. The correlation between sensitive vessels and sensitive skin has, however, not yet been determined.     

Determination of formaldehyde in formaldehyde‐releaser patch test preparations

Background: Positive patch test reactions to formaldehyde‐releasers in patients co‐reacting to formaldehyde are often ascribed to formaldehyde allergy. However, the formaldehyde content of patch test materials has not been investigated. Objectives: To demonstrate and quantify free formaldehyde in commercial patch test materials and in prepared aqueous solutions of formaldehyde releasers. Materials: Free formaldehyde was measured by ¹³C NMR Spectroscopy in (i) all formaldehyde‐releasers in water available from Chemotechnique® and Brial®, (ii) 5 releasers in petrolatum, (iii) 12 prepared aqueous solutions of formaldehyde‐releasers and (iv) water that had been in contact with petrolatum test samples. Results: In none of the five petrolatum test substances was free formaldehyde found. In all nine commercial aqueous patch test substances and 9 of the 12 prepared solutions, free formaldehyde was demonstrated with concentrations ranging from 0.019% to 0.37% (detection limit 0.01%). Contact of the petrolatum test samples with water resulted in the release of formaldehyde. Conclusions: Most aqueous solutions of formaldehyde‐releasers contain free formaldehyde. Petrolatum‐based patch test materials with formaldehyde‐releasers do not contain free formaldehyde, but probably start releasing it upon contact with water. Therefore, in future studies, determination of free and releasable formaldehyde may be preferable.     

In vitro phototoxicity test using artificial skin with melanocytes

BACKGROUND: Exposure to ultraviolet radiation (UVR) has been shown to induce cutaneous biological changes and phototoxic reactions. OBJECTIVE: This study was designed to evaluate the usefulness of artificial skin (AS) composed of keratinocytes and melanocytes as an in vitro phototoxicity model for topical agents. MATERIALS AND METHODS: AS was manufactured with three-dimensionally cultured keratinocytes and melanocytes on a de-epidermized dermis (DED). The photobiological responses in AS with and without melanocytes were comparatively examined after ultraviolet A (UVA) exposure. Three test chemicals (8-methoxypsoralen, 6-methylcoumarin and tetracycline) were topically applied onto the AS with melanocytes and after UVA irradiation, the released inflammatory cytokines (IL-1alpha, IL-beta and IL-6) were analyzed. RESULTS: AS with melanocytes showed better epidermal structures, stronger resistance to UVA exposure and photobiological responses closer to in vivo human skin. Releases of inflammatory cytokines were well correlated with the increased phototoxicities of test chemicals. Among the measured cytokines, IL-6 could be the most reliable in vitro marker indicative of phototoxic potential, because it showed statistically significant increase only in case of concurrent exposure to chemicals and UVA. CONCLUSION: The AS with melanocytes may be a useful tool especially for examining UV-induced cutaneous changes and a promising in vitro phototoxicity test model for topical agents.     

Sensory irritation caused by two organic solvents-short-time single application and repeated occlusive test in stingers and non-stingers

Background. Neurosensory irritation is a subjective phenomenon induced by a number of chemicals. Objectives. To investigate the sensory irritation induced by two organic solvents – n‐octane and cumene – between two groups of volunteers, stingers and non‐stingers, identified as such according to the results of a lactic acid stinging test (LAST). Methods. The immediate effects of the solvents were directly compared in a single simultaneous application test. The reaction intensities over time were studied in a repetitive irritation test over 4 days. The volunteers graded the reaction intensities by the use of a labelled magnitude scale. Results. Cumene induced significantly stronger sensory irritation than octane in both the single and the repeated applications. Both substances induced less subjective irritation the more times the volunteers were exposed. The decline with time for cumene was statistically significant for the non‐stingers but not for the stingers. However, no significant differences regarding the reaction intensities were detected in the direct comparisons of stingers and non‐stingers. Conclusions. Further studies with larger sample sizes are needed to investigate a potential connection between the responsiveness to the sensory irritation caused by lipophilic irritants and lactic acid.     

Treatment of ocular rosacea with 40 mg doxycycline in a slow release form

Background: About 30–50 % of rosacea patients have ocular involvement. The symptoms range from a foreign‐body sensation to conjunctivitis or blepharitis and may even include severe corneal ulcerations. Systemic treatment is generally with tetracycline. Side effects can occur with the usual antimicrobial dose. Patients and Methods: In a retrospective study, seven patients were evaluated who had been treated for ocular rosacea with a sub‐antimicrobial dose of doxycycline 40 mg in a slow‐release form (Oraycea^®). The responses were evaluated on the basis of clinical findings. Results: Seven patients with an average age of 63 took slow release doxycycline 40 mg every day for at least two months. In five patients, other systemic drugs had already failed. All patients experienced a clear improvement in their ocular rosacea after an average of 2.29 months of treatment. One patient had complete clearance and another had almost complete clearance. None of the patients experienced side effects. Conclusions: A sub‐antimicrobial dose of slow release doxycycline 40 mg daily is an effective long‐term therapy for ocular rosacea. It is not associated with the side effects of long‐term antibiotic therapy or the risk of resistance.     

Galanin contributes to ultraviolet irradiation‐induced inflammation in human skin

Exposure of the skin to ultraviolet (UV) irradiation causes various consequences such as inflammation and photoageing. Galanin is an active neuropeptide expressed widely in the central nervous system and peripheral tissues including the skin. Galanin promotes or inhibits inflammation in a context‐dependent manner, but its role in UV irradiation‐induced responses in human skin was still unknown. UV irradiation induced a substantial expression of galanin in primary epidermal keratinocytes in vitro and in human epidermis in vivo. Galanin knock‐down by siRNA transfection markedly inhibited UV irradiation‐induced expression of matrix metalloproteinase (MMP)‐1, interleukin (IL)‐1β, IL‐6 and cyclooxygenase (COX)‐2. Moreover, siRNA‐mediated knock‐down of GAL₂, a principal galanin receptor in the skin, led to a considerable decrease in these mediators in keratinocytes. Collectively, our findings suggest that galanin is an important messenger between the neuroendocrine system and UV irradiation‐damaged skin.     

Clinical and instrumental assessment of the effects of a new product based on hydroxypropyl chitosan and potassium azeloyl diglycinate in the management of rosacea

Background Rosacea is a chronic inflammatory skin disease affecting mostly facial skin. Its origin is multifactorial. Important steps in its treatment are avoidance of any triggering factor and control of skin inflammation. Aim To assess the benefit of topical applications of a new product (P‐3075). Patients/Methods A randomized, multicenter, double‐blind, placebo‐controlled, parallel‐group, pilot study was carried out to evaluate the efficacy and tolerability of a cream (P‐3075) based on 5% potassium azeloyl diglycinate (PAD, Azeloglicina^®) and 1% hydroxypropyl chitosan (HPCH). Forty‐two patients (rosacea stages I and II) were enrolled and randomized, 28 in the P‐3075 group and 14 in the placebo group. They were asked to apply the cream twice daily for 4 weeks. The main assessments were the objective quantification of erythema and skin hydration using the Mexameter^® and Corneometer^® devices, respectively. Clinical signs and symptoms were evaluated on a four‐point scale. Results The P‐3075 cream applied for 28 days was effective in skin protection by reducing erythema, evaluated both instrumentally and clinically. In addition, the clinical assessments of other symptoms such as flushing, stinging, and burning supported the beneficial effect of the P‐3075 cream. Conclusions The anti‐inflammatory and moisturizing effects of potassium azeloyl diglycinate combined with the protective properties of HPCH allow the new product to be a good candidate for controlling signs and symptoms of rosacea.     

Astaxanthin,a xanthophyll carotenoid,inhibits ultraviolet‐induced apoptosis in keratinocytes

Intra‐cellular reactive nitrogen/oxygen species and apoptosis play important roles in ultraviolet (UV)‐induced inflammatory responses in the skin. Astaxanthin (AST), a xanthophyll carotenoid, exhibits diverse clinical benefits. The protective effects of AST against UV‐induced apoptosis were investigated in the present study. Astaxanthin (5 μm ) caused a significant decrease in the protein content and the mRNA levels of inducible nitric oxide (iNOS) and cyclooxygenase (COX)‐2, and decreased the release of prostaglandin E2 from HaCaT keratinocytes after UVB (20 mJ/cm²) or UVC (5 mJ/cm²) irradiation. No significant protective effects against UV‐induced reactive oxygen species (ROS) were observed in AST‐pretreated cells. Astaxanthin caused a significant inhibition of UV‐irradiation‐induced apoptosis, as evidence by a DNA fragmentation assay. Furthermore, we found that the treatment with AST caused a reduction in the UVB‐ or UVC‐induced protein and mRNA expression of macrophage migration inhibitory factor (MIF), IL‐1β and TNF‐α in HaCaT keratinocytes. These results suggest that AST effectively protects against UV‐induced inflammation by decreasing iNOS and COX‐2, and thereby inhibiting the apoptosis of keratinocytes.     

Laser treatment of rosacea: a pathoetiological study

OBJECTIVE: To study the effect of laser treatment on rosacea, a common facial skin disease with symptoms of blushing, redness, telangiectasis, papules, pustules, and diffuse swelling of the skin, we focused on the stinging sensation and performed immunohistochemical evaluation of nerve density and neuropeptide expression. DESIGN: Clinical investigation as well as the lactic acid (stinger) test was performed before and 3 months after the treatment with flashlamp pulsed dye laser, when skin biopsy specimens were also taken. SETTING: University hospital. PATIENTS: Thirty-two patients with rosacea, all with positive results from the lactic acid "stinger" test, were treated by flashlamp pulsed dye laser. MAIN OUTCOME MEASURES: The biopsy specimens were taken from the stinger-positive areas in the nasolabial folds, fixed in Lanas fixative (10% formalin and 0.4% picric acid), and analyzed for the expression of protein gene product 9.5 (general nerve marker), substance P, calcitonin gene-related peptide, and vasoactive intestinal polypeptide, using a biotinylated streptavidin technique. RESULTS: Thirty-one patients who were stinger positive before treatment showed decreased scores after treatment, and 1 patient had the same stinger test score before and after treatment. The number of protein gene product 9.5-positive fibers in the epidermis (P< .05) as well as the papillary dermis (P< .01) was decreased. This was also the case for substance P in the papillary dermis (P< .001), whereas no evident difference was noted for vasoactive intestinal polypeptide and calcitonin gene-related peptide. No difference was found for contact between nerves and vessels (factor VIII positive). CONCLUSIONS: Laser treatment of rosacea that destroys small vessels has a good medical relevance because it reduces the unpleasant symptoms of the sensitive skin. A neurogenic etiology of stinging may be possible.     

Effects of sangre de drago in an in vitro model of cutaneous neurogenic inflammation

Please cite this paper as: Effects of sangre de drago in an in vitro model of cutaneous neurogenic inflammation. Experimental Dermatology 2010; 19 : 796–799. Abstract: Sangre de drago (SD) is a viscous bright red resin collected from Croton lechleri trees that grow in the South American jungle. This sap is used extensively in the native pharmacopoeia to treat skin disorders. Its effectiveness as an inhibitor of neurogenic inflammation has been recently demonstrated. To understand the underlying mechanisms of these effects, we examined the ability of SD to reduce substance P (SP) release in an in vitro model of cutaneous neurogenic inflammation (CNI). This model is based on an enzyme immunoassay of SP (an inducer of CNI) in a porcine co‐culture of dorsal root ganglion neurons and keratinocytes. After incubation with different concentrations of SD, we noted an immediate and significant dose‐dependent decrease in basal SP release, with average values of 32% at 1% SD (v/v) and 26% at 0.1% (v/v). On the other hand, pretreatment (72 or 1 h) of the co‐culture with 1% SD (v/v) was sufficient to induce a 111% (72 h) or 65% (1 h) inhibition of capsaicin‐induced SP release, while 0.1% SD (v/v) triggered a 109% (72 h) or 30% (1 h) inhibition. We conclude that sangre de drago is a potent inhibitor of CNI through direct inhibition of neuropeptide release by sensory afferent nerves.     

Comparison and correlation between stinging responses to lactic acid and bioengineering parameters

In evaluating the safety of a novel cosmetic product or a new chemical, it is important to assess susceptible population. One group of subjects is known to stingers who are more likely to experience sensory effects such as stinging and burning after contacting with cosmetics. The purpose of the study is to measure skin biophysical parameters noninvasively in stingers and non-stingers and to see their correlations with stinging responses. 298 women were evaluated by modified lactic acid stinging test with 5% lactic acid solution rather than classic 10% solution because of strong reaction in Asian populations. Transepidermal water loss (TEWL), skin hydration, sebum content, and pH were measured using the bioengineering instruments in an environment-controlled room. Correlations between stinging responses and skin biophysical parameters were statistically analysed. There was a positive correlation between stinging responses and TEWL evaluation. However, no correlations was observed between stinging responses and other parameters such as skin hydration, sebum content, and pH. Our data indicate that there is a relationship between the degree of stinging and the skin barrier function. However, we believe that various additional studies are necessary to characterize skin of stingers and the pathogenesis.     

Development of a novel method for quantitative evaluation of sensory skin irritation inhibitors

Background/aims: Sensory skin irritation is regarded as one of the most serious side effects of cosmetic use. Thus, it is desirable to develop good inhibitors of sensory skin irritation. However, it is difficult to quantify the effect of sensory skin irritation inhibitors.
Methods: We investigated the possibility of using an electrical current perception threshold (CPT) measurement for the quantitative evaluation of these inhibitors. We divided study populations into stinger and non-stinger groups based on their response to 5% lactic acid and assessed CPT values at 2000, 250, and 5 Hz on the cheek. Stingers showed significantly lower CPT values than non-stingers did at 250 and 5 Hz. We also measured CPT values before and after the application of nine materials with inhibitory effects on sensory skin irritation. To investigate the relationship between the change in CPT values and the effect of each material in the clinical stinging test, we conducted the stinging test with the test materials in a 5% lactic acid solution and with a 5% lactic acid solution (positive control).
Results: There was a positive correlation between the change in CPT values and the inhibitory effect that each material had on the stinging test.
Conclusion: The change in CPT values can be used for the quantitative evaluation of sensory skin irritation inhibitors .