Background Heterogeneity of clinical manifestations and underlying biology has confounded treatment development and optimal care of systemic lupus erythematosus (SLE) patients. About half of adult SLE patients tend to have stable, high expression levels of genes in the interferon (IFN) signaling pathways. We and others have previously reported differences in expression levels between IFN groups of genes in pathways other than IFN signaling pathways1,2,3,4,5. Objectives To determine how gene expression changes with disease flare in SLE patients with high versus low expression of IFN pathway genes. Methods The BOLD study enrolls patients with active disease who are withdrawn from potentially confounding background immunosuppressive therapy and given brief intramuscular steroids (depomedrol) to induce improvement (Improving Visit), and followed until flare (Flare Visit). An interim analysis was conducted by TaqMan® RT-PCR on 23 patients who completed the study. Expression levels of 272 genes (selected based on reported associations with lupus and/or inflammation) were compared between Improving Visit and Flare Visit. Results Striking expression differences between Improving Visit and Flare Visit were observed for some genes, predominantly associated with the IFN High Group. Expression of most (76%) genes tested did not change with flare (p>0.1). IFN groups did not differ in demographic features. There was a trend towards higher disease activity (CLASI score, p=0.06, BILAG scores p=0.1) at baseline in the IFN high group. Conclusions This interim report suggests biomarkers in several pathways might be tested as sensitive indicators to guide patient selection and dosing in the development of treatments which affect the TLR/IFN pathway, and to optimize the eventual use of these agents in clinic. Given the heterogeneity of patients with SLE it could be that some of these downstream indicators could help differentiate between patients who are or are not intrinsically good candidates for targeting of IFN signaling pathways. References O9Toole et al., ACR 2011, Abstract 1407. Baechler et al., PNAS 2003. Bennett et al., J. Exp. Med 2003. Kirou et al., Arthritis and Rheumatism 2005. Petri et al, Lupus 2009. Disclosure of Interest A. Seyhan Employee of: Pfizer, M. O9Toole Employee of: Pfizer, Y. Zhang Employee of: Pfizer, F. Immermann Employee of: Pfizer, A. Hill Employee of: Pfizer, P. Reddy Employee of: Pfizer, J. Masferrer Employee of: Pfizer, T. Zhou Employee of: Pfizer, W. Mounts Employee of: Pfizer, M. Whitley Employee of: Pfizer, T. Walker Employee of: Pfizer, S. Kamp: None Declared, J. James: None Declared, S. Sridharan Employee of: Pfizer, J. Merrill: None Declared, M. Honczarenko Employee of: Pfizer

Background Elevated expression of IFN pathway genes, known as the IFN signature (IS) is found in roughly half of SLE patients1. Various studies have used different IFN genes to define the IS, and different methods to assign patients to “IFN high” or “low” expression groups. Importantly a recent report from the Genentech ROSE study found higher responsiveness to an interferon antagonist in patients with lower IFN expression using a 7 gene set (G-7), suggesting that IS might inform optimal selection of treatments in the future2. Objectives As a step toward understanding the clinical relevance and molecular drivers of the IS, we used data from the BOLD study to test the difference between IS-derived patient groupings derived from different sets of IFN genes. Methods The BOLD study enrolled SLE patients with active disease (minimum SLEDAI of 6 or two BILAG B scores). 41 patients had background immune suppressives withdrawn, were given brief steroids until improvement, then followed with serial samples until disease flare. 62 additional active SLE patients were evaluated only once. RNA expression levels for 329 genes were assayed using TaqMan® Low Density Arrays. 238 SLE patient-visits were partitioned into two groups by unsupervised hierarchical clustering of the expression levels of IFN-related genes. Results Clustering was done separately for 5 gene sets, denoted B-30, P-11, M-20, G-7, and O-3. Two of the gene sets (M-20 and G-7) were 95% and 100% matched to sets used in clinical trials of IFN-inhibiting treatments. Consistent with previous reports, all of the gene sets assigned about 40-50% of visits (96 to 118 patient-visits) to an “IFN high” group characterized by IFN pathway expression that was higher than healthy volunteers. In a cross-sectional analysis using the P-11 gene set, membership in the IFN high group was associated with higher SLEDAI score than IFN low assignment (9.73 vs. 7.70, p<0.0001) and higher TNFSF13B (BLYS/BAFF) gene expression (2.4 fold increase, p=0.0005). Most of the patients followed longitudinally remained in the same group over the course of the study, despite changes of gene expression with disease improvement and flare. IFN group assignments were similar for the B-30 and P-11 gene sets, and also similar among the M-20, G-7, and O-3 gene sets, but differed more across these groups. For instance, 21 patient-visits (9%) were assigned to the IFN high group by the G-7 gene set, but placed in the IFN low group by the P-11 gene set. Conclusions The comparison of IFN group assignments for the same samples, derived using different IFN gene sets, highlights the need for more investigation of the molecular and cellular drivers of IFN gene expression in SLE blood. An optimal IFN gene set, once determined, might improve how patients are selected for targeted therapies in the future. References Baechler et al., PNAS 100(5) 2610-2615 (2003); Kalunian et al., ACR 2012 Washington D.C, Abstract 2622. Disclosure of Interest None Declared

Arachidonic acid is an important polyunsaturated fatty acid involved in cell signaling. It is derived primarily from dietary linoleic acid, and the rate-limiting step in its biosynthesis is the initial desaturation of linoleic acid via Delta-6 desaturase. Evidence suggests that downstream metabolic products of arachidonic acid, e.g. prostaglandins, are involved in colorectal cancer, but involvement of the biosynthetic pathway of arachidonic acid has not been previously investigated. In the present study, we report the effects of a novel selective Delta-6 desaturase inhibitor, SC-26196, on tumorigenesis in two in vivo models of intestinal cancer. SC-26196 treatment resulted in 36-37% fewer tumors in Apc(Min/+) mice and 35% decrease in primary tumor size in nude mice bearing HT-29 human colon cancer cell xenografts (P<0.05). As expected, SC-26196 treatment resulted in significantly higher linoleic acid levels in tissue phospholipids and lower levels of arachidonic acid. The effects on both tissue fatty acid composition and tumorigenesis in Apc(Min/+) mice were abrogated by concomitant treatment with dietary arachidonic acid, indicating that the observed effects were due to interference with the biosynthetic pathway of arachidonic acid.

Two cyclooxygenase (COX) isoforms have been identified: COX-1 and COX-2. COX-1 is the constitutively expressed form of the enzyme and is ubiquitous in its distribution. COX-2 is inducible and is present in inflammatory foci, tumors, and neovasculature. Expression of COX-2 appears to be important in tumor promotion, growth, and metastasis. It is up-regulated in a variety of premalignant disorders and malignancies. COX inhibitors have a major role in the treatment of inflammation and pain. Epidemiologic evidence in patients who take nonsteroidal anti-inflammatory drugs links COX inhibition with decreases in malignant esophageal, stomach, colon, lung, and breast tumors. Nonselective COX inhibitors have demonstrated efficacy in control of familial adenomatous polyposis, a disorder associated with the development of thousands of benign intestinal polyps. The selective COX-2 inhibitor celecoxib (Celebrex (R), Pharmacia) has been shown to reduce the number of adenomatous colorectal polyps in familial adenomatous polyposis as an adjunct to usual care. Celecoxib has recently been approved for this indication and offers the potential for equivalent or greater efficacy than that seen with nonselective COX inhibitors but without the gastrointestinal mucosal toxicity and the inhibition of platelet function associated with those agents. Angiogenesis is a feature of both benign and malignant disease. Because COX-2 is up-regulated in the neovasculature of the rheumatoid pannus and in malignant tumors and their surrounding stroma, selective COX-2 inhibitors may be able to modify the progression of these disorders through the control of angiogenesis.

BACKGROUND. The cyclooxygenase (COX) enzyme catalyzes the formation of prostaglandins, which can affect cell proliferation and alter the response of the immune system to malignant cells. The inducible form of COX, COX-2, has been shown to be important in carcinogenesis.METHODS, The authors studied COX-1 and -2 expression in 20 tumors of the lung, colon, and breast (60 total) by using commercially available monoclonal and polyclonal antibodies on formalin fixed, paraffin embedded tissue. Our evaluation also included seven carcinoma-associated colonic adenomas and 10 mammary ductal carcinomas in situ (DCIS). Quantitation of immunoreactivity was accomplished using an immunohistochemical scoring system that approximates the use of image analysis-based systems.RESULTS. Ninety percent of lung tumors (squamous cell carcinomas and adenocarcinomas), 71% of colon adenocarcinomas and 56% of breast tumors (DCIS and infiltrating ductal and lobular carcinomas) expressed COX-2 at a moderate to strong level, which was significantly different from the negligible expression in distant nonneoplastic epithelium (controls; P < 0.0001). Poorly differentiated histologic features were correlated with low COX-2 expression overall, especially in colon carcinomas. Among breast carcinomas, DCIS was more likely to: express COX-2 than invasive carcinomas. Adenomatous colonic epithelium showed moderate COX-2 expression, as did adjacent nonneoplastic epithelium. COX-1 immunoreactivity was essentially weak to moderate in all tissues evaluated.CONCLUSIONS. COX-2 expression is upregulated in well and moderately differentiated carcinomas of the lung, colon, and breast whereas COX-1 appears to be constitutively expressed at low levels. A possible COX-2 paracrine effect is suggested by moderate immunoreactivity in adjacent nonneoplastic epithelium. Cancer 2000;89:2637-45. (C) 2000 American Cancer Society.

A series of sulfonamide-substituted 4,5-diarylthiazoles was prepared via three synthetic routes as selective COX-2 inhibitors. Recently in the synthesis of selective COX-2 inhibitors we have discovered that the sulfonamide moiety is a suitable replacement for the methylsulfonyl moiety yielding compounds with activity both in vitro and in vivo.

Extensive documentation has validated the role of UV irradiation as a tumor initiator and promoter, inducing both squamous and basal cell carcinomas. Human epidermis is a tissue which undergoes active metabolism of arachidonic acid to prostaglandins which is regulated by the action of prostaglandin H synthase (also known as cyclooxygenase). One mechanism for the promotional activity of UV light may involve its ability to induce prostaglandin formation. Work in our laboratory has demonstrated that acute exposure of human keratinocytes to UVB irradiation results in increased production of prostaglandin E2 (PGE2). When cultured human keratinocytes were examined after irradiation with 30 mJ/cm2 UVB in vitro, Western blot analysis showed a 6-fold increase in COX-2 protein which was evident at 6 h and peaked 24 h after irradiation. Furthermore, when human subjects were irradiated on sun-protected skin with up to four times their minimal erythema dosage (MED) and biopsied 24 h later, upregulation of COX-2 protein expression was observed via immunofluorescence microscopy. RNAase protection assays supported this observation, showing induction of COX-2 message which peaked at approximately 12 h following irradiation in vitro. Furthermore, human squamous cell carcinoma biopsies exhibited strongly enhanced staining for COX-2 protein via immunohistochemistry and Western analysis when compared to normal non-sun-exposed control skin. Together, these data demonstrate acute upregulation of COX-2 via UVB irradiation and suggest the need for further studies of COX-2 expression as a potential pharmacological target mediating human skin tumor development.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of a number of inflammatory diseases and are believed to act via inhibition of the enzyme cyclooxygenase (COX). This enzyme catalyzes the conversion of arachidonic acid to the prostaglandins (PGs). Although commercially available NSAIDs are efficacious anti-inflammatory agents, significant side effects limit their use. Recently two forms of COX were identified — a constitutively expressed COX-1 and a cytokine-inducible COX-2. Potent anti-inflammatory agents like the glucocorticoids are known to inhibit specifically the expression of COX-2 while commercially available NSAIDs like indomethacin inhibit both COX-1 and COX-2. These findings have led to the hypothesis that toxicities associated with NSAID therapy are due to inhibition of the non-regulated or constitutive form of COX (COX-1), whereas therapeutic benefit derives from inhibition of the inducible enzyme, COX-2. We have examined the relative distribution of COX-1 and COX-2 in both normal and inflamed tissues and report that COX-1 expression dominates normal tissues while COX-2 mRNA is induced at the inflammatory site. Furthermore, compounds that selectively inhibit COX-2 are anti-inflammatory without gastric toxicity.

THIS INVENTION REFERS TO THE APPLICATION OF INHIBITORS OF CYCLOGENASE-2 OR ITS DERIVATIVES IN THE TREATMENT AND PREVENTION OF THE NEOPLASSIC PROCESS. DESCRIBE, IN PARTICULAR, THE TECHNIQUE OF PREVENTION AND TREATMENT OF THE NEOPLASSIC PROCESS OF THE EPITHELIAL CELL IN A SUBJECT. THIS TECHNIQUE IS TO TREAT THE SUBJECT WITH AN EFFICIENT AMOUNT, FROM THE THERAPEUTIC VIEW POINT, OF A FORMULA COMPOSITE (I) IN WHICH A, R 2 AND R 3 ARE AS DESCRIBED IN THE SPECIFICATION OF THIS INVENTION.

Ultraviolet light (UV) B-induced inflammation is characterized by dramatic increases in prostaglandin E2 (PGE2) synthesis due to enhanced arachidonate deacylation from the membrane. Therefore, the effect of UV on sythesis, mass, and distribution of the high-molecular-weight phospholipase A2 (cPLA2) in cultured human keratinocytes and human skin was studied. The 105-kDa cPLA2 was demonstrated to be the critical enzyme in UV-induced PGE2 synthesis and erythema in the first 6 h postirradiation. Immunoprecipitation of 35S-labeled protein showed cPLA2 synthesis increased three- to fourfold 6 h after irradiation. Immunoprecipitated 32P-labeled cPLA2 demonstrated phosphorylation of cPLA2 was concurrently induced, suggesting that UV also activates cPLA2. This increase in cPLA2 synthesis and activation also closely correlated with increased PGE2 synthesis and [3H]arachidonic acid release and was effectively blocked by both an S-oligonucleotide antisense to cPLA2 and methyl arachidonate fluorophosphate, a specific inhibitor of cPLA2. Biopsy and histochemical examination of erythematous sites expressed increased amounts of cPLA2 whereas nonerythematous irradiated sites did not. In contrast, cyclooxygenase-1 and -2 in cultures and skin explants were unaffected 6 h post-UV, and no change in cyclooxygenase activity was observed at this time. These results suggest that increased cPLA2 synthesis occurs only when skin is exposed to UV doses that are sufficient to cause erythema and indicate expression of cPLA2 participates in acute UV inflammation.

Non-steroidal antiinflammatory drugs (NSAIDs) are commonly used for the treatment of inflammation, pain, and fever. Mechanistically, these compounds are believed to act via inhibition of the enzyme cyclooxygenase (COX), which catalyzes the conversion of arachidonic acid to the prostaglandins (PGs). Although commercially available NSAIDS are efficacious antiinflammatory agents, significant side effects limit their use. Recently two forms of COX were identified- a constitutively expressed COX-1 and a cytokine-inducible COX-2. Commercially available NSAIDs like indomethacin inhibit both COX-1 and COX-2 suggesting the hypothesis that toxicities associated with NSAID therapy are due to inhibition of the non-regulated or constitutive form of COX (COX-1) in normal tissues, whereas therapeutic benefit derives from inhibition of the inducible enzyme, COX-2, at the site of inflammation. Therefore, a selective inhibitor of COX-2 may be anti-inflammatory without GI toxicity-providing a significant improvement over currently available NSAIDs.