Identification of Retinoic Acid Receptor Agonists as Potent Hepatitis B Virus Inhibitors via a Drug Repurposing Screen.

Currently available therapies for chronic hepatitis B virus (HBV) infection can efficiently reduce viremia but induce hepatitis B surface antigen (HBsAg) loss in very few patients; also, these therapies do not greatly affect the viral covalently closed circular DNA (cccDNA). To discover new agents with complementary anti-HBV effects, we performed a drug repurposing screen of 1,018 Food and Drug Administration (FDA)-approved compounds using HBV-infected primary human hepatocytes (PHH). Several compounds belonging to the family of retinoic acid receptor (RAR) agonists were identified that reduced HBsAg levels in a dose-dependent manner without significant cytotoxicity. Among them, tazarotene exhibited the most potent anti-HBV effect, with a half-maximal inhibitory concentration (IC50) for HBsAg of less than 30 nM in PHH. The inhibitory effect was also observed in HBV-infected differentiated HepaRG (dHepaRG) models, but not in HepG2.215 cells, and HBV genotypes A to D were similarly inhibited. Tazarotene was further demonstrated to repress HBV cccDNA transcription, as determined by the levels of HBV cccDNA and RNAs and the activation of HBV promoters. Moreover, RNA sequence analysis showed that tazarotene did not induce an interferon response but altered the expression of a number of genes associated with RAR and metabolic pathways. Inhibition of RARß, but not RARα, by a specific antagonist significantly attenuated the anti-HBV activity of tazarotene, suggesting that tazarotene inhibits HBV in part through RARß. Finally, a synergistic effect of tazarotene and entecavir on HBV DNA levels was observed. Therefore, RAR agonists as represented by tazarotene were identified as potential novel anti-HBV agents.

The essential oil from Citrus limetta Risso peels alleviates skin inflammation: In-vitro and in-vivo study.

ETHNOPHARMACOLOGICAL RELEVANCE: Citrus fruit peels are traditionally used in folk medicine for the treatment of skin disorders but it lacks proper pharmacological intervention. Citrus limetta Risso (Rutaceae) is an important commercial fruit crops used by juice processing industries in all continents. Ethnopharmacological validation of an essential oil isolated from its peels may play a key role in converting the fruit waste materials into therapeutic value added products. AIM OF THE STUDY: To evaluate the chemical and pharmacological (in-vitro and in-vivo) profile of essential oil isolated from Citrus limetta peels (Clp-EO) against skin inflammation for its ethnopharmacological validation. MATERIALS AND METHODS: Hydro-distilled essential oil extracted from Citrus limetta peels (Clp-EO) was subjected to gas chromatography (GC) analysis for identification of essential oil constituents and its anti-inflammatory evaluation through in vitro and in vivo models. RESULTS: Chemical fingerprint of Clp-EO revealed the presence of monoterpene hydrocarbon and limonene is the major component. Pre-treatment of Clp-EO to the macrophages was able to inhibit the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß) in LPS-induced inflammation as well as the production of reactive oxygen species (ROS) in H2O2-induced oxidative stress. In in-vivo study, topical application of Clp-EO was also able to reduce the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear thickness, ear weight, lipid peroxidation, pro-inflammatory cytokines production and ameliorate the histological damage in the ear tissue. In-vitro and in-vivo toxicity study indicate that it is safe for topical application on skin. CONCLUSION: These findings suggested the preventive potential of Clp-EO for the treatment of inflammation linked skin diseases.

Rescue of Hypovitaminosis A Induces Non-Amyloidogenic Amyloid Precursor Protein (APP) Processing.

Retinoic acid, the bioactive metabolite of beta-carotene or vitamin A, plays a pleiotropic, multifunctional role in vertebrate development. Studies in rodents revealed that a diet deficient in vitamin A results in a complex neonatal syndrome (the VAD syndrome), manifested in many organs. In humans, the function of retinoic acid (RA) extends into adulthood, where it has important roles in fertility, vision, and suppression of neoplastic growth. In recent years, it has also been suggested that retinoic acid might potentially act as a therapeutically relevant drug in attenuating or even preventing neurodegenerative diseases such as Alzheimer's disease (AD). Here, we report that VAD leads to an increase in A-beta peptide levels while only minor effects were observed on expression levels of the amyloid precursor protein (APP) processing proteinases in wild type mice. In line with these findings, rescue of hypovitaminosis reduced A-beta amount to baseline and induced sApp-alpha secretion in combination with an increase of alpha-secretase Adam10. By comparing retinoic acid treatment starting from a full nutrition status and a "VAD" situation in human neuroblastoma cells, we show that while intensities of differential gene expression were higher in replenished cells, a large overlap in AD-related, regulated genes was observed. Our data suggest that hypovitaminosis A can contribute to onset or progression of AD by increasing synthesis of A-beta peptides and that several AD-related genes such as ADAM10 or BDNF are regulated by retinoic acid. We suggest that dietary supplementation with retinoic acid derivatives is likely to have a beneficial effect on AD-pathology in individuals with hypovitaminosis and patients with normal vitamin A status.

The effects of retinoic acid on human corneal stromal keratocytes cultured in vitro under serum-free conditions.

PURPOSE: Retinoic acid (RA) is a metabolite of vitamin A that plays a fundamental role in the development and function of the human eye. The purpose of this study was to investigate the effects of RA on the phenotype of corneal stromal keratocytes maintained in vitro for extended periods under serum-free conditions. METHODS: Keratocytes isolated from human corneas were cultured up to 21 days in serum-free media supplemented with RA or DMSO vehicle. The effects of RA and of its removal after treatment on cell proliferation and morphology were evaluated. In addition, the expression of keratocyte markers was quantified at the transcriptional and protein levels by quantitative PCR and immunoblotting or ELISA, respectively. Furthermore, the effects of RA on keratocyte migration were tested using scratch assays. RESULTS: Keratocytes cultured with RA up to 10 × 10(-6) M showed enhanced proliferation and stratification, and reduced mobility. RA also promoted the expression of keratocyte-characteristic proteoglycans, such as keratocan, lumican, and decorin, and increased the amounts of collagen type-I in culture while significantly reducing the expression of matrix metalloproteases 1, 3, and 9. RA effects were reversible, and cell phenotype reverted to that of control after removal of RA from media. CONCLUSIONS: Retinoic acid was shown to control the phenotype of human corneal keratocytes cultured in vitro by regulating cell behavior and extracellular matrix composition. These findings contribute to our understanding of corneal stromal biology in health and disease, and may prove useful in optimizing keratocyte cultures for applications in tissue engineering, cell biology, and medicine.

Effect of retinoic acid on epithelial differentiation and mucin expression in primary human corneal limbal epithelial cells.

PURPOSE: Retinoic acid (RA) is essential for epithelial differentiation and maintenance of the mucous phenotype. This study investigated the effect of RA on corneal epithelial differentiation and mucin expression in a primary human corneal limbal epithelial cell (HCLEC) culture model. METHODS: HCLECs were grown in RA-supplemented media at various concentrations (0, 10(-9) to 10(-6) M). Stratified HCLECs were examined using immunohistochemical or immunofluorescent staining for p63, ABCG2, CK3, CK19, and Western blotting for ABCG2 and CK12 to assess differentiation. Ultrastructural morphology was investigated using scanning and transmission electron microscopy. They were incubated with rose bengal dye to examine barrier function. The effects of RA on the expression of MUC1, -4, and -16 were analyzed by immunohistochemistry, quantitative real-time PCR and Western blot analysis. RESULTS: HCLEC grown without RA showed hyperkeratosis, whereas those grown with 10(-8) to 10(-7) M RA induced non-keratinized stratified epithelium with a normal appearance. Under these conditions, p63, ABCG2, CK3, CK19, MUC1, -4, and -16 staining patterns were similar to in vivo limbal epithelium. A higher concentration (10(-6) M) of RA resulted in abnormal differentiation. HCLECs grown with RA were tightly apposed and maintained intact barrier function against dye penetration. In addition, MUC1, -4, and -16 expressions were highly associated with RA concentrations. CONCLUSIONS: This study showed that cultured HCLEC could mimic physiologic and functional phenotypes by controlling RA concentrations in medium. Also, our results suggested modulating effect of RA on differentiation and mucin expression in corneal epithelium.

Novel stain-free lecithinized coal tar formulation for psoriasis.

BACKGROUND: Coal tar has been a very popular traditional treatment for various types of psoriasis for over a century. It is the first-line treatment for scalp, hand, and foot psoriasis. However, the application of coal tar on hair invariably causes staining, which results in a high degree of patient non-compliance, especially in patients with non-black hair. Thus, the treatment of scalp psoriasis with a topical coal tar formulation requires that special concern be paid to product esthetics. OBJECTIVE: This study aimed to evaluate the hair-staining characteristics of a novel lecithinized coal tar (LCT) formulation on different types of mammalian hair. METHODS: Samples of hair from different mammals, including human, sheep, rabbit, and goat, were repeatedly exposed to the LCT formulation over 14 days. The color of hair samples treated with LCT was compared with that of untreated control hair samples. RESULTS: The study revealed the distinct non-staining potential of the LCT formulation. CONCLUSIONS: This LCT formulation lacks the propensity to stain hair and thus has excellent potential to be exploited in the treatment of scalp psoriasis.

Acitretin revisited in the era of biologics.

Acitretin is a time-honored treatment for psoriasis. During the last decade biologics have revolutionized the treatment of psoriasis. This raises the question: What is the position of acitretin as a classical systemic treatment for psoriasis in the era of biologics? Based on the mode of action of acitretin, it is evident that at least one antipsoriatic treatment has to be available in the armamentarium of antipsoriatic treatments that is not immunosuppressive, intended for those patients who are contraindicated for immunosuppressive treatment. It is attractive to speculate that at least an additive or possibly a synergistic effect can be expected in case of combination of acitretin with a biologic. The efficacy of acitretin in chronic plaque psoriasis as a monotherapy is below methotrexate and cyclosporine. However, acitretin in combination with phototherapy has an efficacy at least comparable with the other classical systemic treatments. Based on several clinical studies it can be concluded that acitretin is an important treatment option in case of contraindications for immunosuppression, such as patients with infections or cancer-prone patients. Furthermore, some evidence is available for high efficacy of the combination of acitretin and biologics.

A review of acitretin for the treatment of psoriasis.

BACKGROUND: Acitretin is an oral retinoid that is approved for the treatment of psoriasis. It is unique compared to other systemic therapies for psoriasis such as methotrexate and cyclosporine in that it is not immunosuppressive. It is, therefore, safe for use in psoriasis patients with a history of chronic infection such as HIV, hepatitis B, hepatitis C or malignancy who have a contraindication to systemic immunosuppressive therapy and require systemic therapy because topical therapy is inadequate and they are unable to commit to phototherapy. Acitretin is one of the treatments of choice for pustular psoriasis. Even though acitretin is less effective as a monotherapy for chronic plaque psoriasis, combination therapy with other agents, especially UVB or psoralen plus UVA phototherapy, can enhance efficacy. OBJECTIVE: To provide an updated review of the safety and efficacy of acitretin in the treatment for psoriasis. METHODS: Literature review of journal articles from 2008 to 2009 since the last review of acitretin evaluated medical literature from 2005 to 2008. RESULTS/CONCLUSION: Acitretin is an effective systemic therapy for psoriasis and is generally well tolerated at low doses for long-term use. If monotherapy with acitretin is inadequate, it can be used in combination with other treatments, particularly UVB phototherapy, to increase efficacy.

Randomised, placebo-controlled, double-blind, split-face study on the clinical efficacy of Tricutan on skin firmness.

Tricutan is a combination product of herbal extracts traditionally used for treatment of skin conditions, together with dimethylaminoethanol. The effectiveness of Tricutan in improving skin firmness and elasticity in photoaged facial skin was examined in a randomised, placebo-controlled, double-blind, split-face study in 28 women, 34-67 years old. Treatment with Tricutan and placebo was given for 4 weeks. Skin firmness and elasticity was evaluated using the speed of propagation of ultrasound shear waves in the skin as end point (Reviscometer RVM 600). The study was completed by 25 women. The Tricutan treatment resulted in a significantly reduced propagation speed indicating increased firmness. There was no immediate effect by Tricutan application on propagation speed. At self evaluation the women evaluated the treatment effect of Tricutan to be significantly better than the treatment effect of placebo. The clinical evaluation also showed Tricutan to give a significantly better treatment result than placebo. Tolerance to Tricutan was generally good. However, three women did not complete the study because of mild irritative contact dermatitis. The results show that Tricutan can increase skin firmness both objectively and subjectively. Further studies are warranted, especially to investigate if Tricutan can delay the need for surgical face-lift procedures.

Development of a pluripotent ES-like cell line from Asian sea bass (Lates calcarifer)--an oviparous stem cell line mimicking viviparous ES cells.

We report a pluripotent embryonic stem cell-like cell line designated as SBES from blastula stage embryos of Asian sea bass (Lates calcarifer), which is an economically important cultivable and edible marine fish species in India. The SBES cells were cultured at 28 degrees C in Leibovitz L-15 medium supplemented with 20% fetal bovine serum without a feeder layer. The ES-like cells were round or polygonal and grew exponentially in culture. The SBES cells exhibited an intense alkaline phosphatase activity and expression of transcription factor Oct 4. The undifferentiated state of these cells was maintained at low seeding densities and the cells formed embryoid bodies when seeded in bacteriological plates. On treatment with all-trans retinoic acid, these cells differentiated into neuron-like cells, muscle cells, and beating cardiomyocytes, indicating their pluripotency. This embryonic ES-like cell line derived from an oviparous fish blastula conserved several peculiar features of viviparous mammalian embryonic stem cell lines. The present study highlights the importance and potential of piscine ES-like cell line for stem cell research without evoking ethical issues and invasive interventions sparing mammalian embryos.

Vehicle and enhancer effects on human skin penetration of aminophylline from cream formulations: evaluation in vivo.

The effects of four essential oils (rosemary, ylang, lilacin, and peppermint oils), and three plant oils (jojoba oil, corn germ oil, and olive oil) on the permeation of aminophylline were studied using human skin. The permeation effects of these oils were compared with those of three chemical penetration enhancers. Although all oils enhanced the permeation of aminophylline, their effects were less than that of ethanol. Jojoba oil was found to be the most active, causing about a 32% peak height decrease of N-H bending absorbances in comparison with the control, while peppermint, lilacin, rosemary, and ylang oils caused 28%, 24%, 18%, and 12% peak height decreases, respectively. Microemulsions containing 10% jojoba oil and 30% corn germ oil were found to be superior vehicles for the percutaneous absorption of aminophylline. Comparision with results obtained from high-performance liquid chromatography shows good agreement.

The effects of various irradiation doses on the growth and differentiation of marrow-derived human mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) are progenitors of mesenchymal tissues including bones. Irradiation can damage the osteogenic activity of human marrow by suppressing osteoblasts leading to post-irradiation osteoporosis. However, the effect of therapeutic irradiation on MSC remains unexplored. We investigated the effects of various doses of X-ray irradiation on human MSC (hMSC) by measuring its post-irradiation proliferation and differentiation activities. Standard immunophenotypes and differentiating functions of the MSC were determined. Irradiation inhibited proliferation of hMSC up to two wk post-irradiation but thereafter, those residual surviving cells regained their normal proliferation rate. Bone forming activity as reflected by alkaline phosphatase (ALP) and calcium deposition were both reduced in a dose-dependent fashion. Maximum suppressive effect on osteogenic activity was noted in MSC treated with high-dose irradiation (12 Gy). Adipocyte percentage was also reduced by 50% in cultures that received >4 Gy. Attempts to protect the irradiated cells with 1 microM all-trans retinoic acid did not show any beneficial effect on MSC proliferation and differentiation. The direct impairment of proliferation and osteogenic differentiation potential of MSC by irradiation may contribute partly to the post-transplant osteoporosis.

Effects of acitretin on spermatogenesis of rats.

In this experimental study the possible effects of the acitretin on the spermatogenesis of the rats were investigated histopathologically. Thirty-nine male adult Wistar albino rats were divided into 3 groups as two experimental groups and one control group. The first group consisting 14 rats were applied orally standard dose (0.75 mg/kg/day) acitretin and the second group consisting 16 rats were applied high dose (1.5 mg/kg/day) acitretin. Acitretin was given within dimetil sulphoxide (DMSO), which was diluted with saline solution as a ratio of 1/10, in order to increase its solubility. The control group consisting 9 rats were given only saline solution including DMSO for 8 weeks. After 8 weeks of the administration, half of the rats in the first and second groups and the entire control group were sacrificed under deep ether anaesthesia and bilateral orchiectomy was made. The remaining rats were compared with the control group using a similar method at the end of 8 weeks of wash-off period. The orchiectomy materials were histopathologically evaluated under the light microscope for spermatogenesis according to parameters including spermatogenetic activity, spermatogenetic organization, seminiferous tubular diameter, interstitial Leydig cells and fibroblasts. The groups, which were evaluated at the end of the 8(th) and 16(th) weeks, were compared with the control group regarding the mentioned parameters and no statistical significance was observed among the groups. In our study it was concluded that the standard and high doses of acitretin do not have any effect on the spermatogenesis of the rats.

Skin penetration of terpenes from essential oils and topical vehicles.

The purpose of this study was to evaluate the in vitro cutaneous penetration of five terpenes--linalool, linalyl acetate, terpinen-4-ol, citronellol and alpha-pinene--applied in pure essential oils or in dermatological formulations (o/w emulsion, oily solution or hydrogel) containing 0.75 % w/w of the essential oils. Different skin absorption was observed depending on the type of the vehicle and terpenes' log P values. Cutaneous accumulation of terpenes is several times higher when they are applied in pure essential oils than in topical vehicles. Penetration of terpinen-4-ol to the skin was better from an oily solution (approximately 90 microg/cm (2)) than from an emulsion (60 microg/cm (2)). No penetration of linalyl acetate from topical vehicles into viable skin was observed, but also for this terpene penetration to the upper layers of the stratum corneum was 2-times higher when an oily solution was used. In contrast, the cutaneous absorption of linalool was the same from both vehicles (50-60 microg/cm (2)). The skin penetration of alpha-pinene was not traceable when it was applied in an oily solution. Only a small amount (approximately 5 microg/cm (2)) of this terpene was determined in viable skin after application as a hydrogel. Citronellol applied in a hydrogel penetrated into all skin layers in a total amount of 25 microg/cm (2), while no penetration into viable skin layers after application of an oily solution was noted. Only citronellol permeated into the acceptor medium.

Asiaticoside induces human collagen I synthesis through TGFbeta receptor I kinase (TbetaRI kinase)-independent Smad signaling.

Skin aging appears to be principally related to a decrease in the levels of type I collagen, the primary component of the skin dermis. Asiaticoside, a saponin component isolated from Centella asiatica, has been shown to induce type I collagen synthesis in human dermal fibroblast cells. However, the mechanism underlying asiaticoside-induced type I collagen synthesis, especially at a molecular level, remains only partially understood. In this study, we have attempted to characterize the action mechanism of asiaticoside in type I collagen synthesis. Asiaticoside was determined to induce the phosphorylation of both Smad 2 and Smad 3. In addition, we detected the asiaticoside-induced binding of Smad 3 and Smad 4. In a consistent result, the nuclear translocation of the Smad 3 and Smad 4 complex was induced via treatment with asiaticoside, pointing to the involvement of asiaticoside in Smad signaling. In addition, SB431542, an inhibitor of the TGFbeta receptor I (TbetaRI) kinase, which is known to be an activator of the Smad pathway, was not found to inhibit both Smad 2 phosphorylation and Type 1 collagen synthesis induced by asiaticoside. Therefore, our results show that asiaticoside can induce type I collagen synthesis via the activation of the TbetaRI kinase-independent Smad pathway.

The effects of ginsenoside Re and its metabolite, ginsenoside Rh1, on 12-O-tetradecanoylphorbol 13-acetate- and oxazolone-induced mouse dermatitis models.

The effects of the main constituent ginsenoside Re in ginseng and its metabolite, ginsenoside Rh1, were investigated in 12-O-tetradecanoylphorbol 13-acetate (TPA)- and oxazolone-induced mouse ear dermatitis models. Ginsenoside Rh1 potently suppressed the TPA- and oxazolone-induced swellings as well as mRNA expression levels of cyclooxygenase-2, IL-1beta and TNF-alpha, although these were only weakly inhibited by ginsenoside Re.

Cosmetic applications of selected traditional Chinese herbal medicines.

Because tyrosinase catalyzes melanin synthesis, tyrosinase inhibitors are important in cosmetic skin-whitening. Oxidative stress contributes to skin aging and can adversely affect skin health, which means antioxidants active in skin cells may support skin health. We examined 25 traditional Chinese herbal medicines that might be useful for skin-whitening and skin health. Extracts (100microg/mL) were tested for cytotoxicity on human epidermal melanocytes (HEMn); 12 exhibited low cytotoxicity. Their effects on tyrosinase and melanin inhibitory activities and free radical scavenging activities were further assessed. Phenolic contents were evaluated using Folin-Ciocalteu reagent. Four herbs, Pharbitis nil, Sophora japonica, Spatholobus suberectus, and Morus alba, exhibited potent inhibitory effects on tyrosinase (IC(50) values 24.9, 95.6, 83.9, and 78.3microg/mL, respectively). Melanin inhibition was not dose-dependent. Sophora japonica (IC(50): 14.46microg/mL, 1,1-diphenyl-2-picrylhydrazyl (DPPH); 1.95microg/mL, hydroxyl radical) and Spatholobus suberectus (IC(50): 10.51microg/mL, DPPH; 4.36microg/mL, hydroxyl radical) showed good antioxidative activities and high phenolic contents (255 and 189mg of gallic acid/g extract, respectively). Among active anti-tyrosinase extracts, Sophora japonica and Spatholobus suberectus were especially potent in HEMn cells in terms of free radical scavenging effects and high phenolic contents, making them the strongest candidates for cosmetic application found in the current study.

Topical tazarotene: a review of its use in the treatment of plaque psoriasis.

Tazarotene (Tazorac) is a topical retinoid indicated for the treatment of plaque psoriasis. When used as monotherapy, topical tazarotene was effective at controlling signs and symptoms of plaque psoriasis, and had significantly lower post-treatment relapse rates than fluocinonide cream. The most common adverse events associated with tazarotene therapy are skin-associated events, such as pruritus, burning, and erythema. Combination therapy with tazarotene and mid-to-high potency topical corticosteroids generally resulted in a greater therapeutic effect than that with tazarotene alone, reduced the irritancy of tazarotene, and decreased the risk of post-treatment disease flare seen with corticosteroids; it also has the potential to reduce the degree of skin atrophy associated with topical corticosteroids. The combination of tazarotene and phototherapy also appears promising. Thus, tazarotene, as monotherapy or in combination with topical corticosteroids or UV light therapy, represents a useful treatment option in patients with plaque psoriasis.

Matrix metalloproteinase-1 expression inhibitory compound from the whole plants of Viola ibukiana Makino.

Bioassay-guided fractionation has led to the isolation of two triterpenoid saponins 3-O-[O-beta-D-glucopyranosyl-(1 --> 3)-O-beta-D-glucopyranosyl] oleanolic acid (1), and 3-O-[O-beta-D-glucopyranosyl-(1 --> 2)-O-beta-D-glucopyranosyl] oleanolic acid (2) from the whole plants of Viola ibukiana Makino. Compound 2 showed matrix metalloproteinase-1 expression inhibition activities in a dose-dependent manner.