536 In vitro and in vivo efficacy of nitric oxide-releasing antiviral therapeutic agents KA McHale 1 , K Balogh 2 , H Wang 3 , S Hollenbach 1 , N Christensen 2 , L Chow 3 , T Broker 3 and N Stasko 1 1 Novan, Inc, Durham, NC, 2 Department of Experimental Pathology, Pennsyl- vania State University at Hershey Park, Hershey, PA and 3 Department of Biochemistry & Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL The efficacy of topical nitric oxide-releasing therapeutics to inhibit viral replication was demonstrated in vitro and in vivo. NVN1000 or NVN4000 drug substance, dissolved in phosphate buffered saline, was applied 1 hour/day for 6 days to organotypic cultures of primary human keratinocytes containing HPV-18 genomic replicons. Preliminary results indicate a dose responsive reduction of viral DNA copy number to less than 20% of the untreated cultures. At the highest concentrations applied (1.5 mg or 2 mg/ml), no cytotoxicity was detected, as judged from hematoxylin and eosin staining of normal or HPV-18 containing raft cultures. The NVN1000 drug substance was formulated into SB206 gel and the ability of a daily topical drug product (5 doses/week for 8 weeks) to inhibit papilloma growth in vivo was assessed in the Cottontail Rabbit Papillomavirus (CRPV) model. Dosing of SB206 gel was initiated two weeks after inoculation of CRPV viral DNA into the scarified skin sites. Topical SB206 treatment inhibited papilloma formation in a dose dependent fashion with the highest dose, 10% SB206 gel, achieving 82% inhibition compared to the Vehicle control group. Blinded histological analysis of biopsies from 10% SB206-treated animals exhibited mild hyperplasias, with an absence of viral infection as evidenced by a lack of intra-nuclear basophilic inclusions. Qualitative assessment of inflammation and quantitative cytokine gene expression was similar across all dose groups, suggesting immune activation did not account for the efficacy observed in the CRPV model and supports a direct anti-viral effect of Novan’s nitric oxide releasing therapeutic agents. SB206 Gel is currently being evaluated as a topical therapy for external genital warts in a Phase 2 clinical trial. 537 Topical application of chitosan-based nanoformulated green tea polyphenol EGCG ameliorates imiquimod-induced psoriasis-like skin lesion in mice J Chamcheu, I Siddiqui, V Adhami, S Dodwad, D Bharali, G Wood, S Mousa and H Mukhtar Dermatology, University of Wisconsin Madison, Madison, WI Psoriasis is a chronic and currently incurable inflammatory skin disease characterized by hyperproliferation, aberrant differentiation and inflammation, leading to disrupted skin barrier function. The use of natural agents that possess the ability to abrogate these effects could be useful for the treatment of psoriasis. Earlier studies have shown that treatment of keratinocytes and mouse skin with the green tea polyphenol, epigallocatechin-3-gallate (EGCG), increased the expression of caspase-14 that is involved in epidermal differentiation and cornification. However, dose and bioavailability restrict the therapeutic development and application of EGCG in the treatment of psoriasis. To overcome these limitations, we developed fully characterized, chitosan-based nanocarrier to encapsulate EGCG (hereafter termed as nano- EGCG) suitable for topical delivery. Here, we assessed the efficacy of nanoEGCG (48mg/ animal) using an IMQ-induced mouse psoriasis-like skin model and compared the effects to free EGCG (1mg/amimal). Treatment with nanoEGCG resulted in significant (p<0.01) amelioration of pathological markers of psoriasiform lesions including reduction in i) ear and skin thickness, erytherma and scales, ii) proliferation (Ki-67), iii) infiltratory immune cells (mast cells, neutrophils, macrophages and CD4+ T cells), iv) angiogenesis (CD31), and v) increase in the protein expression of caspase-14, early (K1 and k10) and late (filaggrin and loricrin) differentiation markers and AP-1 factors (JunB and cJun), and (v) modulates several psoriasis-related inflammatory cytokines and chemokines analyzed compared to high dose of free EGCG (p<0.05). Taken together, topical application of nanoEGCG was found to ameliorate IMQ-induced Balb/c mouse psoriasis-like lesions with greater than 25-fold dose advantage over free EGCG. Our observations warrant further in vivo efficacy studies of this unique nanoEGCG formulation in robust inducible and genetic models of psoriasis. 538 The synergistic effect of cinnamon cassia oil and erythromycin on overcoming antibiotic resistant Staphylococcus aureus GR Delost 1,2 , C Haydanek 2 , N Carty 2 and C Keller 2 1 Dermatology, UH Regional Hospitals, Richmond Heights, OH and 2 Department of Human Pathogens, Lake Erie College of Osteopathic Medicine, Erie, PA Increasing antibiotic resistance in Staphylococcus aureus has prompted the need for alter- native treatments. One potential strategy involves synergism between antibiotics and essential plant oils. The antimicrobial activity of essential oils has long been recognized, but used only recently synergistically with antibiotics. The goal of this study was to assess the inhibitory effect of varying combinations of cinnamon oil and erythromycin on the growth of erythro- mycin-resistant S. aureus. Using the disk diffusion method, antibiotic susceptibility profiles were established for S. aureus clinical isolates (n¼110). Erythromycin-resistant isolates (n¼35) were selected for further study due to a high rate of resistance (31.8%) and their clinical significance. The inhibitory effect of 32 essential plant oils was assessed against a S. aureus laboratory control strain. Cinnamon cassia oil exhibited the most prominent inhibitory effect and was therefore selected for further testing. Checkerboard microdilution assays were used to test combinations of cinnamon cassia oil and erythromycin against erythromycin-resistant S. aureus isolates. Fractional inhibitory concentrations of erythromycin and cinnamon oil were calculated for each checkerboard assay, and results were plotted using an isobologram. A synergistic effect between cinnamon cassia oil and erythromycin was demonstrated against 68.6% of the erythromycin-resistant isolates. Of the remaining isolates, 14.3% showed additive effects and 17.1% showed indifferent effects; no combina- tion displayed an antagonistic effect. These results show that combinations of sub-inhibitory concentrations of cinnamon oil and erythromycin increased the susceptibility of erythro- mycin-resistant S. aureus to erythromycin. This study carries the broader implication that synergism with essential oils may renew the effectiveness of antibiotics, and perhaps lead to novel topical therapeutic regimes for common skin infections. 539 Design of experiments approach using an in vitro skin model to evaluate irritancy of topical formulations M Patel 1 , M Doherty 2 , V Desai 1 , N Gogda 1 and V Nalamothu 1 1 Tergus Pharma, Durham, NC and 2 Lq3 Pharma, Morrisville, NC Purpose: The purpose of this study was to test formulations with varying levels of excipients in the Mattek Epiderm model and determine which excipient could potentially lead to irritation. Methods: Fusion software was used to create a design of experiments (DOE) matrix of for- mulations with varying levels of penetration enhancers, co-solvents, and gelling agents. The formulations were dosed on a 3D human skin model from Mattek for up to 4 hours and an MTT assay was performed to determine cell viability. Tissue viability less than 50% was considered irritant. Results: The DOE matrix consisted of 41 test formulations and 1 control formulation. The test formulations had varying levels of a solvent, penetration enhancer, alcohol, gelling agent, and water. The control formulation was a clear to slightly hazy gel in appearance and resulted in 7.66 Æ 1.56% cell tissue viability. All formulations without alcohol and addition of a co-solvent resulted in average cell tissue viability of 58.98 Æ 6.29%. In contrast, formulations containing alcohol resulted in only 8.19 Æ 1.41% average cell viability. Cell viability with formulations made with gelling agent 1 or gelling agent 2 was 58.43 Æ 10.98% and 57.86 Æ 3.21%, respectively. Conclusions: DOE was used as a screening tool to evaluate multiple parameters simultaneously and investigate the irritation potential of several formulations. In vitro studies performed on a human skin model suggest that alcohol content may be contributing to dermal irritation. Data from this study will aid in the development of an effective and tolerable topical formulation for treatment of pain. 540 Targeting pruritus in vitro by two non-histaminergic pathways F Lestienne, M Leveque, H Delga, M Aries, S Bessou-Touya and N Castex-Rizzi Pierre FABRE Dermo Cosme´tique, TOULOUSE, France Pruritus is one of the most common skin complaints, not always with a skin disease associ- ated. Pruritus could hit every age group but its prevalence is high in elderly patients. Itch constitutive of pruritus can trigger sensitisation by histaminergic and non-histaminergic pathways. H1/H4 Histamine receptors stimulation, the histaminergic classical pathway will not be addressed in this study. Indeed, epidermal PAR-2 receptors (Protease-activated re- ceptor-2) activation mediates cutaneous itch directly, not suppressed by antihistamine com- pound with a specific neuronal stimulation pathway (Davidson et al., 2010). TRPV1 channel (Transient receptor potential vanilloid 1) can be activated at the skin level by heat, pH and osmolarity modulation, physicochemical effects present during itch. Furthermore, its expression is increased in elderly patients epidermis (Lee YM et al., 2008). The aim of this work is to evaluate active ingredients to target PAR-2 and TRPV1 activation pathways to inhibit pruritus induction. The use of keratinocytes cell line HaCat allowed us to assess PAR-2 activity after stimulation by trypsin or SLIGKV-NH2 peptide agonist in monitoring Ca2+ flux signals. In this model, Polidocanol showed a significant dose response inhibitory activity after trypsin stimulation and a competitive activity against peptide agonist at the highest con- centration. Prucidine-4 Ò efficacy was tested on TRPV1 recombinant CHO cell based assay. Ca2+ flux signals modulation was measured after capsaı ¨cin agonist stimulation. Prucidine-4 Ò showed a significant dose response inhibition with a full reverse effect. These data suggest that a topical Dermo-Cosmetic product combining Polydocanol and Prucidine-4 Ò com- pounds for their PAR-2 and TRPV1 inhibitory effects respectively could be useful to protect against pruritus induction. 541 EGR1 as a molecular target for the treatment of skin inflammatory disease O Sarig 1 , L Fried 1 , L Megal 1 , A Keren 2 , D Vodo 1,3 , D Hershkovitz 2,4 , J Uitto 5 , A Gilhar 2 and E Sprecher 1,3 1 Department of Dermatology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel, 2 Technion, Haifa, Israel, 3 Department of Human Molecular Genetics & Biochemistry, Tel Aviv University, Tel Aviv, Israel, 4 Department of Pathology, Rambam Medical center, Haifa, Israel and 5 Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA Mutations in SAMD9 underlie normophosphatemic familial tumoral calcinosis, a recessive condition resulting from an aberrant inflammatory response heralding calcinosis cutis. We recently demonstrated that SAMD9 functions in the skin by down-regulating the expression of EGR1, a critical regulator of cutaneous inflammatory responses. We therefore sought to identify small inducers of SAMD9 transcriptional activity to be used as novel anti-inflam- matory therapeutics. We generated cell lines stably expressing a luciferase gene under the regulation of a functional SAMD9 promoter. Using this system, we screened over 1400 small molecules and identified two compounds, 6H-Pyrido[4,3-b]carbazole-1-carboxamide, 5,11-dimethyl-, monohydrochloride (CaCx) and 10-n-Propyl-1,3-dichloro-7-amino-pheno- thiazine-5,5-dioxide (ApDx), which reproducibly induced SAMD9 promoter activity as well as endogenous SAMD9 expression in HeLa, TERC-transformed fibroblasts and primary fibroblasts cells in a time- and dose-dependent manner. Accordingly, the two molecules were found to concomitantly down-regulate the expression of EGR1. We then administered the two compounds systemically (i.p) to chimeric mice carrying human psoriatic skin. Both com- pounds resulted in significant clinical and histological attenuation of the psoriasiform phenotype (ApDx p<0.0001, CaCx p<0.05), suggesting that EGR1-targeting therapies may serve as an alternative or as an adjunct to current therapies for hyperproliferative skin inflammatory diseases. Pharmacology & Drug Development | ABSTRACTS www.jidonline.org S95