Cíntia Delai da Silva Horinouchi

Simvastatin ointment, a new treatment for skin inflammatory conditions

BACKGROUND Statins represent a class of drugs that effectively lowers cholesterol, however they also possess pleiotropic effects, like promotion of angiogenesis, prevention of bone loss, immunomodulatory and anti-inflammatory effects. OBJECTIVES Thus, the aim of this study was to investigate the activity of simvastatin topically applied in mice in acute and chronic skin inflammation models. METHODS Skin inflammation was induced in mice ears by topical application of 12-O-tetradecanoylphorbol acetate (TPA). In the acute model, ear oedema was measured by the increase of ear thickness 6h after TPA (2.5μg/ear). The chronic inflammatory process was induced by multiple applications of TPA (2.0μg/ear) for nine alternate days, and the oedema was measured daily as the increase in ear thickness. RESULTS Topical treatment was applied immediately after TPA in acute model or started at 5th day of chronic experiment. For acute model treatment was simvastatin (0.24, 0.71 and 2.40μM), dexamethasone (0.13μM), both in acetone or vehicle alone (acetone). In chronic model simvastatin (1% and 3%) and dexamethasone (0.5%) were incorporated in ointment preparations, and a group received ointment alone (vehicle). Samples of ear tissue (6mm) were taken from acute and chronic models, weighted and prepared for histological analysis and myeloperoxidase (MPO) enzymatic activity evaluation. Application of simvastatin in acetone reduced the ear oedema after a single TPA application in a dose dependent manner [ID(50) of 0.47 (0.22-1.13) μM], and the MPO enzymatic activity up to 61±10%. Also, both simvastatin ointment preparations 1% and 3% reduced acute TPA-induced ear oedema in 55±7% and 65±8%, respectively. In the chronic model, simvastatin ointment 1% was able to reduce ear oedema (25±3%) and ear weight (10±1%), though 3% formulation augmented both parameters. Histological analysis revealed a reduction of swelling and leukocyte migration in the acute model for both the formulations of simvastatin (1% and 3%), while in chronic model simvastatin 1% decreased ear swelling and epidermal thickness, but simvastatin 3% increased both parameters. CONCLUSION The results confirm the anti-inflammatory activity of simvastatin when applied topically in both acute and chronic models of skin inflammation. Besides, the formulation of simvastatin ointment 1% shows to be a very effective formulation for a chronic usage.

Effectiveness of Vernonia scorpioides ethanolic extract against skin inflammatory processes.

ETHNOPHARMACOLOGICAL RELEVANCE Vernonia scorpioides (Asteraceae) is a native Brazilian medicinal plant that is commonly used to treat skin disorders. Considering the traditional use of Vernonia scorpioides and the lack of information about its pharmacological properties, we investigated the topical anti-inflammatory effect of the ethanolic extract of Vernonia scorpioides (EEVS) on acute and chronic cutaneous inflammation models in mouse. MATERIALS AND METHODS The topical anti-inflammatory effect of EEVS was evaluated against acute models (12-O-tetradecanoylphorbol acetate (TPA)- and arachidonic acid (AA)-induced mouse ear oedema) and chronic models (multiple applications of croton oil). RESULTS The EEVS caused a dose-related inhibition of oedema in both the TPA- and AA-induced acute models (DI(50)=0.24 and 0.68 mg/ear with an inhibition of 80 ± 5% and 65 ± 5%, respectively, for 1mg/ear). In addition, the TPA-induced increase in myeloperoxidase activity (MPO) in the ear was reduced (77 ± 8%) by the topical application of EEVS. In the chronic model, the EEVS reduced all parameters evaluated: oedema formation (31 ± 2%), epidermal hyperproliferation (histology) and MPO (25 ± 10%). However, the topical treatment of EEVS had no effect on N-acetyl-β-d-glucosaminidase activity. The EEVS effectively interfered in the ear oedema on the delayed-type hypersensitivity reaction induced by oxazolone. The topical treatment with EEVS performed on both phases or only on the elicitation phase caused the inhibition of the ear oedema-induced by oxazolone in 42.9% and 63.4%, respectively, when compared to control animals (sensitized and challenged). CONCLUSIONS The results suggest that EEVS is effective as a topical anti-inflammatory agent in acute and chronic inflammatory processes and that its action is markedly influenced by the inhibition of neutrophil migration into inflamed tissue as well as by epidermal hyperproliferation.

Combretum leprosum Mart. (Combretaceae): Potential as an antiproliferative and anti-inflammatory agent

ETHNOPHARMACOLOGICAL RELEVANCE Combretum leprosum is a species that is popularly used in Brazil as a healing agent to treat skin problems and lesions. In this study we investigated the possible potential of this extract to treat inflammatory and hyperproliferative skin conditions. MATERIALS AND METHODS Classical models of skin inflammation such as TPA- and croton oil-induced mouse ear oedema were applied in order to verify the potential topical anti-inflammatory activity of the ethanolic extract from flowers of Combretum leprosum. RESULTS Topical application of ethanolic extract promoted a dose-dependent inhibition of phorbol ester-induced ear oedema, reduced myeloperoxidase activity and IL-6 tissue levels with inhibition comparable to dexamethasone (positive control). Histological and immunohistochemical analysis revealed that ethanolic extract also suppressed cell infiltration. Ethanolic extract altered inflammatory parameters on a chronic skin inflammation model induced by repeated applications of croton oil, decreasing ear oedema, epidermal hyperproliferation and cell infiltration. In addition, immunohistochemical analysis showed that the extract decreased PCNA expression on the epidermis. CONCLUSION Taken together, these results suggest that the extract from flowers of Combretum leprosum could be considered as a new potential tool for the treatment of several skin inflammatory diseases since it reversed the skin inflammatory and hyperproliferative process in a very significant manner. Further investigations are needed in order to verify the cellular mechanism and safety of Combretum leprosum extract.

Hyperpigmentant activity of leaves and flowers extracts of Pyrostegia venusta on murine B16F10 melanoma.

ETHNOPHARMACOLOGICAL RELEVANCE Pyrostegia venusta is a native Brazilian plant which has a variety of uses in traditional folk medicine including the treatment of vitiligo. However, its effectiveness on melanogenesis is not yet elucidated. AIM OF THE STUDY This study aimed to investigate the melanogenic activity of hydroalcoholic extracts from the leaves and flowers of P. venusta on murine B16F10 melanoma cells. MATERIALS AND METHODS Different concentrations of the hydroalcoholic extracts of flowers and leaves of P. venusta were evaluated in trials of spontaneous melanin content (4 days), and cell viability by the MTT assay in murine B16F10 cells, and in the mushroom tyrosinase activity in vitro. RESULTS Both extracts, leaves (0.1; 0.3; 1 and 3 μg/mL) and flowers (0.03 and 0.1 μg/mL) increased the melanin content in a concentration dependent manner after 4 days of incubation on melanoma cells. Leaves extract promoted enhancement of melanogenesis with maximum effect of 33.3±3% (3 μg/mL), and the flower extract increased in 23.4±3% (0.1 μg/mL). The cell viability test using MTT showed that in the same tested concentrations of both extracts no cell death was detected. Actually, either extract was not able to cause any change in the tyrosinase activity. HPLC analysis of P. venusta extracts found 0.09% and 1.08% of allantoin on leaves and flowers extracts, respectively. CONCLUSIONS The leaves and flowers extracts of P. venusta stimulates B16F10 melanogenesis at very low concentrations. These findings support the folk medicinal use of P. venusta on the treatment of hypopigmentation diseases, such as vitiligo.

Patient-reported outcomes in psoriasis research and practice

MADAM, Several studies have shown that psoriasis vulgaris and psoriatic arthritis affect patients not only physically, but also socially, emotionally and psychologically, as much as cancer or heart disease. Additionally, traditional clinical outcomes measures, such as Psoriasis Area and Severity Index, show only weak associations with patient-reported outcomes (PRO). Considering that and the fact that psoriasis is still not curable, the most important treatment aims are to control the disease progress and to reduce its harmful effects on the patient’s quality of life (QoL), including parameters such as well being, anxiety, depression and work limitations, among others. Consequently, various QoL measures have been proposed and used to evaluate the impact of psoriasis and its treatment on patients’ health-related QoL (HRQoL). The aim of this correspondence is to provide information about the tools most used to evaluate the impact of treatments on patients’ QoL or other PRO between 2000 and 2010. The data were obtained from the PubMed (National Library of Medicine) database by systematic review using the keywords ‘psoriasis’ and ‘quality of life’ and a sensitive, broad clinical query using research methodology filters to therapy category (http://www.ncbi.nlm.nih.gov/entrez/query/static/ clinicaltable.html). Abstracts retrieved were analysed to determine which clinical outcomes and ⁄or PRO measures had been used in the studies. We analysed a total of 499 publications, of which 238 were considered to report PRO in psoriasis. The countries which contributed the most articles were the U.S.A. (33Æ1%), Germany (9Æ6%) and the U.K. (8Æ4%). The greatest proportion of papers was published in the British Journal of Dermatology (11Æ2%), followed by the Journal of the American Academy of Dermatology (6Æ2%) and the Journal of the European Academy of Dermatology and Venereology (4Æ4%). The most frequently used tools are shown in Table 1. The Dermatology Life Quality Index was the most used measure (40Æ8%). There were seven different generic QoL instruments used most frequently: Short-Form-36 Health Survey (SF-36), Health Assessment Questionnaire, EuroQoL, Beck Depression Inventory, Hospital Anxiety and Depression Scale, Brief Symptom Inventory and Nottingham Health Profile. The SF36 was the generic HRQoL measure used the most (14Æ7%). Regarding psoriasis-specific instruments used to evaluate the impact of specific aspects of the disease on patients’ HRQoL, nine different ones were most used in the last 10 years, the most frequently applied being the Psoriasis Disability Index (8Æ4%). Pavlovsky et al. showed that between 1993 and 2007 there was a significant increase in the number of all types of psoriasis publications. The authors suggest that this increase is supported by basic research discoveries, where the better understanding of psoriasis immunopathology has led to an increase in clinical studies, and by the discovery of biological treatments. Similarly, our results show that concern about PRO in psoriasis care has increased continuously in the last 10 years, being more evident from 2003, coinciding with the emergence of biological agents for psoriasis treatment (Fig. 1). In summary, the use of instruments for measuring PRO and QoL has grown over the years in studies involving patients with psoriasis. This demonstrates the growing importance of issues that go beyond the strictly clinical aspects and severity of the disease. The high variability in the use of humanistic outcome assessment tools to evaluate the impact of psoriasis and its treatments is a consequence of the lack of standardized Table 1 Most commonly used patient-reported outcomes measures in psoriasis articles published between 2000 and 2010

In vivo participation of nitric oxide in hyperproliferative epidermal phenomena in mice.

A significant involvement of nitric oxide (NO) in the process of keratinocyte proliferation is reported with many divergences. To determine the involvement of NO in the hyperproliferative process of epidermis in vivo, non-selective inhibitor (N(G)-nitro-L-arginine-methyl ester.HCl: L-NAME) and selective inhibitors for inducible NO synthase (iNOS) and neuronal NO synthase (nNOS) (Aminoguanidine: AG and 7-Nitroindazole: 7-NI, respectively) and a NO-donor (Sodium nitroprusside: SNP) were topically applied twice a day in mice ear treated with multiple applications of croton oil. L-NAME and 7-NI treatments decreased and SNP increased ear edema formation. However, ear weight was reduced in groups that received L-NAME and 7-NI, while the AG and SNP groups presented an increment. The histological evaluation of epidermis thickness showed that all NOS inhibitors were able to prevent the increase in epidermis width caused by croton oil, while SNP contributed to enlargement. The same results were observed in the PCNA staining, where treatments with NOS inhibitors caused a reduction in the number of cells in the epidermis, while SNP caused an enhancement. 7-NI treatment reduced polymorphonuclear and mononuclear leukocytes migration when compared to the control group. The AG application increased the migration of polymorphonuclear and mononuclear cells, while the SNP enhanced only the polymorphonuclear cells. Therefore, in the skin NO produced by nNOS is involved in the control of keratinocyte hyperproliferation, with the contribution of iNOS. In the animal model of cutaneous chronic inflammation by croton oil, NO is involved in the exudation and leukocyte migration, with participation of all three enzymes.

Inhibitory effect of GB-2a (I3-naringenin-II8-eriodictyol) on melanogenesis.

ETHNOPHARMACOLOGY RELEVANCE GB-2a is a I3-naringenin-II8-eriodictyol compound isolated from Garcinia gardneriana (Planchon & Triana) Zappi, a plant used in folk medicine for the treatment of skin disorders. AIM OF STUDY In the search for new depigmenting agents, this study was carried out to investigate the in vitro effects of GB-2a isolated from G. gardneriana (Planchon & Triana) Zappi in B16F10 melanoma cells. MATERIALS AND METHODS The effects of GB-2a were evaluated through determination of melanin biosynthesis in B16F10 melanoma cells in comparison with the reference drug kojic acid (500µM). In parallel, the GB-2a effect was assessed in a cell viability assay. Mushroom tyrosinase activity assays were conducted to verify the effect of this enzyme. In order to ascertain the nature of enzyme inhibition on tyrosinase, kinetics analysis of the GB-2a was performed with L-tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) substrates. RESULTS The results showed that GB-2a biflavonoid significantly inhibited the melanin content, without reducing cell viability. GB-2a also showed a strong antityrosinase activity in the mushroom tyrosinase assay. GB-2a inhibited the tyrosinase activity, exerting a mixed inhibition. For the L-tyrosine substrate the inhibition was in non-competitive mode and for L-DOPA it was in uncompetitive mode. CONCLUSION GB-2a biflavonoid promoted inhibition on tyrosinase activity and reduced melanin biosynthesis in B16F10 cells, which suggests great potential for medical and cosmetic uses as a depigmenting agent.

Aliskiren: Preclinical evidence for the treatment of hyperproliferative skin disorders

Psoriasis is a complex inflammatory and hyperproliferative skin disease. The pathogenesis and mechanisms involved are not completely understood, which makes treatment a difficult issue. Angiotensin II, the most active peptide of the renin-angiotensin system, seems to be involved in processes related to psoriasis pathogenesis, such as inflammation and cell proliferation. The aim of this study was to investigate the influence of renin inhibition on inflammation parameters and keratinocyte proliferation in a mouse model of chronic skin inflammation induced by croton oil. Aliskiren had anti-inflammatory effects by reducing levels of tumor necrosis factor-α and interleukin -6, and by inhibiting myeloperoxidase activity. Aliskiren also showed antiproliferative activity by reducing epidermal hyperplasia and proliferating cell nuclear antigen levels. Aliskiren treatment did not induce alterations in the cardiovascular system, normal skin thickness, and organ weight. These results suggest that aliskiren could be a valuable tool to be incorporated in the treatment of hyperproliferative and inflammatory skin disorders such as psoriasis.

Anti-proliferative and anti-inflammatory effects of 3β,6β,16β-Trihydroxylup-20(29)-ene on cutaneous inflammation

ETHNOPHARMACOLOGICAL RELEVANCE 3β,6β,16β-Trihydroxylup-20(29)-ene (TTHL) is a triterpene isolated from the flowers of Combretum leprosum, a plant used in folk medicine in the north of Brazil for the treatment of skin disorders. AIM OF THE STUDY In the present study, TTHL was evaluated as a potential topical anti-inflammatory and anti-proliferative agent through in vivo and in vitro models. MATERIAL AND METHODS Anti-inflammmatory and anti-proliferative effects of TTHL were assessed using Swiss mice in acute and chronic models of skin inflammation induced by 12-O-tetradecanoylphorbol-acetate (TPA) application. Anti-proliferative activity was proved through in vitro experiments with the HaCaT human keratinocyte cell line. RESULTS Treatment with TTHL inhibited inflammatory parameters such as oedema formation and cellular infiltration in acute and chronic models. In the chronic model, TTHL also inhibited epidermal hyperproliferation, as evidenced by reduction of epidermis thickness and proliferating cell nuclear antigen expression. The anti-proliferative effect was confirmed by the capability of TTHL in reducing the proliferation and inducing cell apoptosis of HaCaT cells. Suggesting a mechanism of action, TTHL showed activation of corticosteroid receptors, but without the induction of corticosteroid-related cutaneous side effects. CONCLUSION Our results demonstrate consistent anti-inflammatory and anti-proliferative activity and assign TTHL as a valuable tool in the development of a new treatment for skin inflammatory and proliferative diseases, such as psoriasis.