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Leukocyte adhesion in the microvasculature influences blood rheology and plays a key role in vaso-occlusive manifestations of sickle cell disease. Notably, polymorphonuclear neutrophils (PMNs) can capture circulating sickle red blood cells (sRBCs) in inflamed venules, leading to critical reduction in blood flow and vaso-occlusion. Recent studies have suggested that E-selectin expression by endothelial cells plays a key role by sending activating signals that lead to the activation of Mac-1 at the leading edge of PMNs, thereby allowing RBC capture. Thus, the inhibition of E-selectin may represent a valuable target in this disease. Here, we have tested the biologic properties of a novel synthetic pan-selectin inhibitor, GMI-1070, with in vitro assays and in a humanized model of sickle cell vaso-occlusion analyzed by intravital microscopy. We have found that GMI-1070 predominantly inhibited E-selectin–mediated adhesion and dramatically inhibited sRBC-leukocyte interactions, leading to improved microcirculatory blood flow and improved survival. These results suggest that GMI-1070 may represent a valuable novel therapeutic intervention for acute sickle cell crises that should be further evaluated in a clinical trial.

Abstract 951 INTRODUCTION: Multiple Myeloma (MM) is characterized by widespread disease at diagnosis with the presence of multiple lytic lesions and disseminated involvement of the bone marrow (BM), implying that the progression of MM involves a continuous re-circulation of the MM cells in the peripheral blood and re-entrance into the BM. Selectins are adhesion molecules expressed by activated endothelium of venules and leukocytes, and are involved in the primary interaction of lymphocytes with the endothelium of blood vessels. The binding of selectins serves as a biologic brake, making leukocyte quickly decelerate by rolling on endothelial cells, as the first step of extravasation. In this study, we have investigated the role of selectins and their ligands in the regulation of homing of MM Cells to the BM and the therapeutic implications of this role. METHODS AND RESULTS: We have used flow cytometry to characterize the expression of E, L and P-selectins and their ligands on MM cell lines, patient samples and on plasma cells from normal subjects. We found that all MM cell lines and patient samples showed high expression of L and P, but little of no E-selectin. While normal plasma cells showed low expression of all selectins and ligands.(give numbers) A pan-selectin inhibitor GMI-1070 (GlycoMimetics Inc., Gaithersburg, MD) inhibited the interaction of recombinant selectins with the selectin-ligands on the MM cells in a dose response manner. We have tested the role of the selectins and their ligands on the adhesion of MM cells to endothelial cells and found that MM cells adhered preferentially to endothelial cells expressing P-selectin compared to control endothelial cells and endothelial cells expressing E-selectin (p CONCLUSION: In summary, we showed that P-selectin ligand is highly expressed in MM cells compared to normal plasma cells, and that it plays a major role in homing of MM cells to the BM, an effect which was inhibited by the pan-selectin inhibitor GMI-1070. This provides a basis for testing the effect of selectin inhibition on tumor initiation and tumor response to therapeutic agents such as bortezomib. Moreover, it provides a basis for future clinical trials for prevention of MM metastasis and increasing efficacy of existing therapies by using selectin inhibitors for the treatment of myeloma. Disclosures: Patton: GlycoMimetics, Inc: Employment. Smith:GlycoMimetics, Inc: Employment. Sarkar:GlycoMimetics, Inc: Employment. Anderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Magnani:GlycoMimetics, Inc.: Employment. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Previous studies using intravital microscopy in a sickle cell disease (SCD) mouse model (Berkeley) suggest that adherent leukocytes (WBCs) play a key role in vaso-occlusion by capturing circulating erythrocytes (RBCs) in venules. In addition, mice deficient in both P-and E-selectins are protected from vaso-occlusion (VOC) induced by surgical trauma and TNF-α stimulation, suggesting that targeting selectins or their ligands represents a potentially useful strategy. Selectins bind to specific sialylated and fucosylated carbohydrate structures presented by glycoprotein or glycolipid ligands. Here, we tested the effect of novel small glycomimetic selectin inhibitors, GMI-1070 and GMI-1077, on leukocyte behavior and sickle cell VOC. Berkeley SCD mouse bone marrow was transplantated into lethally irradiated C57BL/6 animals to generate age- and gender-matched genetically identical cohorts of SCD mice. Fully engrafted male SCD mice were treated with TNF-α and prepared for intravital microscopy examination of the cremaster muscle 90 min later. GMI-1070, GMI-1077 (both 20 mg/kg) or vehicle (PBS) were administered immediately prior to cytokine stimulation (t=0 min), and an additional dose was given at t=70min. Another group of mice was injected with antibodies against P-and E-selectins (PES, 1 mg/kg) as positive control. Several post-capillary and collecting venules were examined between t= 90min and t= 150min. Antibody blockade of endothelial selectins completely ablated leukocyte rolling, whereas GMI-1070 and GMI-1077 significantly increased the rolling flux fractions (PBS: 5.0±1.2 GMI-1070: 10.6±1.3%%; GMI-1077: 9.9±1.0%; p< 0.001). Furthermore GMI-1070 and GMI-1077 significantly reduced the recruitment of adherent leukocytes (914±172 and 1433±119 cells/mm2, respectively) compared to sickle mice injected with PBS control (2400±392 cells/mm2, p< 0.001). Although the reduction in leukocyte adhesion was not as marked as with anti-P and E-selectins (61±25 cells/mm2, p< 0.001), GMI-1070, in particular, dramatically inhibited the capture of sickle RBCs by adherent leukocytes (PBS: 0.9±0.4, GMI-1077: 0.6±0.2, GMI-1070: 0.07±0.05 and PES: 0.01±0.01 RBC interactions/WBC/min, p< 0.05) and markedly improved the blood flow in venules (PBS: 312±24, GMI-1077: 398±41, GMI-1070: 710±68 and PES: 683±75 nL/s, p< 0.001), to levels observed in non-sickle mice. The increased leukocyte rolling fluxes by these glycomimetics suggest that they inhibit E-selectin > P-selectin. Since the hallmark of E-selectin-mediated adhesion is the slow leukocyte rolling, we analyzed leukocyte rolling velocities in the various group and indeed found a 2-fold increase in rolling velocities in sickle mice treated with GMI-1070 compared to PBS control (PBS: 21±1 μm/s, GMI-1070: 38±1 μm/s, p

Rational design of glycomimetic inhibitors based on the bioactive conformation of functional carbohydrates provides new therapeutic opportunities for the development of small molecule drugs with improved activity, pharmacokinetics, and bioavailability relative to native ligands. In designing more active glycomimetic selectin inhibitors, the low enthalpy (ΔH0) of the reaction is compensated by pre-forming the bioactive conformation thereby improving the entropy (ΔS0), known as S/H compensation. Modifications of the molecule that also stabilize the core structure, further improve ΔS0 and binding activity. To improve ΔH0, second site interactions were explored. To address the requirements for P and L-selectins, interactions were combined for both carbohydrate and sulfate-binding domains to produce heterobifunctional molecules. By stabilizing the bioactive core and exploring second site molecular interactions, we have produced a family of pan-selectin antagonists, one of which (GMI-1070) is now in development and scale-up synthesis as a lead compound. GMI-1070 is a potent inhibitor of E, P, and L-selectins in vitro and inhibits E and P-selectin-mediated leukocyte adhesion to endothelial monolayers under flow conditions. More importantly, GMI-1070 is active in several animal models of diseases requiring leukocyte adhesion and migration such as: a delayed-type hypersensitivity (DTH) response, cardiac ischemia/reperfusion injury, and vaso-occlusive crisis in sickle cell disease. GMI-1070 significantly inhibited infarct size in an ischemia/reperfusion cardiac injury model in rats. A single dose at 10mg/kg gave maximal inhibition. The effects of GMI-1070 on T-cell migration was studied in a DTH model. Mice were sensitized with oxazolone on the abdomen and then, 7 days later challenged on the ear. Donors T-cells from a different cohort of similarly sensitized mice were fluorescently labeled and injected into the test cohorts at the time of administration of GMI-1070. Three hours following injection, migration of fluorescent T-cells to the challenged area was determined. Selectin-dependent T-cell migration in the DTH response was completely eliminated with a dose of 10mg/kg of GMI-1070, suggesting the potential clinical application in diseases involving skin homing T-cells such as graft vs. host disease (GVHD) after bone marrow transplantation, and other inflammatory skin diseases. We have also shown that acute myelogenous leukemia (AML) cells adhere to endothelial cells under flow in a selectin-dependent mechanism suggesting that graft vs. leukemia as well as graft vs. host may be affected by treatment with GMI-1070. GMI-1070 was also tested in a model of vaso-occlusive crisis in sickle cell disease using Berkeley sickle cell mice. Blood flow was restored to normal values and adhesion of sickle red blood cells to adherent leukocytes was essentially eliminated as determined intravital microscopy using a dose of 20mg/kg administered at the time of elicitation of the vaso-occlusive challenge (TNFα) and at the start of intravital microscopy (70 minutes later). Based on the encouraging results in disease models, we are advancing the clinical development of GMI-1070 into Phase 1 studies to support indications which include the treatment of sickle cell patients in vaso-occlusive crisis.