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Background Janus kinase (JAK) inhibitors targeting multiple JAK isoforms, JAK1 and 2, or selective against JAK1 are currently utilized in clinical practice or being developed for the treatment of various inflammatory and oncological diseases. No truly JAK3-selective inhibitor has reached the clinic. JAK3 signal in pairs with JAK1 to transduce signal elicited from six known cytokines (IL-2, IL-4, IL-7, IL-9, IL-15 & IL-21) binding to the gamma-common (g-c) chain cytokine receptors. JAK1 in addition to be required for g-c chain cytokine receptors signaling can also signal in pair with JAK2 or TYK2 and is required for other cytokine receptors signaling such as type I and II interferons and IL-6 and IL-10 families of cytokines. Other cytokines such as IL-12 and IL-23 are signaling via JAK2 and TYK2. Additionally, hematopoietic cytokines such as EPO and TPO as well as cytokines such as IL-3 and IL-5 signal via homodimers of JAK2. Objectives Develop a JAK3 selective inhibitor that will inhibit signaling from the g-c chain cytokine receptors at therapeutic dosing without inhibiting signaling by other cytokines such as hematopoietic cytokines, type I and II interferons and IL-6, IL-12 and IL-10 families of cytokines. Methods A JAK3-specific covalent ATP competitive inhibitor was assessed functionally in vitro and in vivo . Functional differentiation of JAK3 selective inhibition as compare to pan-JAK inhibition (tofacitinib) or JAK1 selective inhibition was also assessed. Results The inhibitor showed JAK3 selective inhibition in biochemical and cellular assays. It inhibits Th1 and Th17 cell differentiation and function. Importantly, sparing JAK1 inhibition, through the selectivity of this inhibitor, preserved anti-inflammatory functions such as the differentiation of alternatively activated M2a macrophages. Unlike pan-JAK or JAK1-selective inhibitors, JAK3 selective inhibition also preserved IL-10-dependent suppression of the production of pro-inflammatory cytokines following LPS treatment in macrophages while maintaining the suppression of TNF and IL-1 responses in IL-27-primed macrophages. Further characterization of this compound in the rat adjuvant-induced arthritis (AIA) demonstrated efficacy at reducing disease pathology. Conclusions We have identified the first trully selective JAK3 inhibitor with potency and properties that make it suitable for further clinical development. Disclosure of Interest J.-B. Telliez Employee of: Pfizer, L. Wang Employee of: Pfizer, J. Jussif Employee of: Pfizer, T. Lin Employee of: Pfizer, L. Li Employee of: Pfizer, E. Moy Employee of: Pfizer, W. Li Employee of: Pfizer, Y. Zhao Employee of: Pfizer, K. Crouse Employee of: Pfizer, P. Symanowicz Employee of: Pfizer, M. Hegen Employee of: Pfizer, M. E. Banker Employee of: Pfizer, F. Vincent Employee of: Pfizer, J. Clark Employee of: Pfizer, A. Thorarensen Employee of: Pfizer

Background A number of Janus kinase (JAK) inhibitors are being actively investigated for treatment of rheumatoid arthritis (RA), including tofacitinib, baricitinib, filgotinib (GLPG0634), and decernotinib (VX-509). However, it is unclear how these drugs may differentiate from each other in the clinic based on their profiles of JAK dependent cytokine inhibition. Objectives The aim of this work was to provide an integrated modelling approach using knowledge of both whole cell JAK inhibition potencies and plasma pharmacokinetics to better understand profiles of cytokine inhibition for clinical JAK inhibitors in the context of clinically meaningful doses. Methods IC 50 values for IFNα, IFNγ, IL-6, IL-15, IL-21, IL-10, IL-27, IL-12, IL-23 and erythropoietin (EPO) signalling of tofacitinib, baricitinib, filgotinib, and decernotinib were measured in total lymphocytes, CD34+ cells (EPO) and CD3+ cells (IL-6) in human whole blood by a flow cytometry-based assay, quantifying the phosphorylation state of various STAT proteins. Human daily average plasma concentrations (C av ) were used as reported or predicted for tofacitinib (5 mg BID; 68 nM), baricitinib (4 mg QD; 32 nM), and filgotinib (200 mg QD; 527 nM). Confidence in the prediction of decernotinib pharmacokinetics was considered low because of high in-vitro metabolic instability and was not assessed further. Percent levels of cytokine inhibition (IC xx =100*C av /(IC 50 + C av )) were determined at clinically meaningful doses. Results Each JAK inhibitor showed a relatively similar profile of cytokine inhibition versus type I and II interferons (IFNα, IFNγ), the common γ chain cytokines (IL-15, IL-21), and IL-6 and IL-27 (Fig. 1). Each also showed some decrease in potency for IL-10, IL-12 and 23, and EPO. Comparing between JAK inhibitors, tofacitinib and baricitinib were overall more potent inhibitors than decernotinib and filgotinib. Clinical pharmacokinetics of tofacitinib, baricitinib, and filgotinib were available and used to further compare predicted cytokine profiles of inhibition in patients with RA. The profile of cytokine inhibition for each JAK inhibitor was strikingly similar at clinically meaningful doses (Fig. 1). While the pharmacokinetics were unavailable for decernotinib, the clinical dose ranges being explored are consistent with filgotinib which showed similar in-vitro inhibitory potencies. Conclusions These analyses illustrate the importance of studying a broad range of JAK pairing potencies and clinical concentrations when comparing JAK inhibitor compounds. Clinical profiles of cytokine inhibition for a number of JAK inhibitors in RA are strikingly similar when efficacious doses are considered, suggesting limited potential for differentiation of these JAK inhibitors based on JAK pharmacology. Disclosure of Interest M. Dowty Employee of: Pfizer Inc, T. Lin Employee of: Pfizer Inc, L. Wang Employee of: Pfizer Inc, J. Jussif Employee of: Pfizer Inc, B. Juba Employee of: Pfizer Inc, L. Li Employee of: Pfizer Inc, E. Moy Employee of: Pfizer Inc, J.-B. Telliez Employee of: Pfizer Inc DOI 10.1136/annrheumdis-2014-eular.2490

Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis.

Heparan sulfate proteoglycans (HSPGs) play important roles in tumorigenesis by mediating tumor-stromal interactions through the presentation of growth factors, cytokines, and chemokines critical for tumor progression, survival and metastasis. M402 is a rationally engineered, non-cytotoxic HSPG mimetic, designed to disrupt tumor-host interactions. M402 binds and inhibits multiple factors including VEGF, FGF2, SDF-1α, and P-selectin. A single 20 mg/kg subcutaneous (s.c.) dose of M402 effectively reduced seeding of B16F10 murine melanoma cells to the lung in a syngeneic experimental metastasis model. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis of orthotopically implanted 4T1 murine mammary carcinoma in this model. M402 treatment also normalized circulating levels of GR1+ immature myeloid cells and platelet counts in 4T1 metastatic tumor bearing mice. Fluorescently-labeled M402 exhibited selective accumulation in the primary tumor. Immunohistological analyses of primary tumor presented a decrease in microvessel density in M402-treated animals, suggesting anti-angiogenesis may be one of the mechanisms involved in vivo. Importantly, M402, as monotherapy or in combination with chemotherapeutics, also revealed significant survival benefits in this aggressive tumor model. These data demonstrate that targeting HSPG biology may provide a useful approach to attenuate multiple pathways involved in tumor progression and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2269.