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Celebrating Diversity Within the Sickle Cell Community: Commitment, Innovation, Practice
Friday, October 12 • 5:15pm - 5:30pm
The Clinical-Stage SGC Stimulator Olinciguat Prevents Increase Of Plasma Biomarkers Of Intravascular Inflammation And Suppresses Leukocyte-Endothelial Interactions In Tnfa-Treated Mice

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Abstract

Authors:Dr. Regina Graul- IronWood Pharmaceuticals, Dr. Boris Tchernychev- IronWood Pharmaceuticals, Ms. Susan Feil- University of Tubingen, Prof. Robert Feil- University of Tubingen, Ms. Elisabeth Lonie –IronWood Pharmaceuticals, Dr. Peter Germano- IronWood Pharmaceuticals, Dr. G. Todd Milne- IronWood Pharmaceuticals, Mr. John Hadcock- IronWood Pharmaceuticals, Dr. Yueh-Tyng Chen- IronWood Pharmaceuticals, Dr. Mark Currie- IronWood Pharmaceuticals    

 Objective: Olinciguat is a soluble guanylate (sGC) stimulator in clinical development for the treatment of patients with sickle cell disease (SCD). sGC is a signaling enzyme that increases the production of cyclic guanosine 3’,5’-monophosphate (cGMP) from guanosine triphosphate in response to nitric oxide (NO) binding. The sGC stimulator olinciguat binds to sGC and enhances its NO-dependent activity. Conditions of low NO bioavailability, as seen in hemoglobinopathies such as sickle cell disease, are proposed to lead to increased vascular inflammation, cellular adhesion, and reduced blood flow due to vasoconstriction. By enhancing NO signaling through sGC, olinciguat is hypothesized to reduce vascular inflammation and restore blood flow and tissue oxygenation. Preclinically, modulation of the NO-sGC-cGMP pathway via treatment with NO or with inhibitors of the cGMP-selective phosphodiesterase 9 has been demonstrated to significantly attenuate TNFa-induced intravascular inflammation in C57BL/6 and sickle cell mice. We evaluated the effect of olinciguat alone and with hydroxyurea (HU, current SCD standard of care) on biomarkers of intravascular inflammation, leukocyte-endothelial cell interactions, and neutrophil trafficking in C57BL/6 mice.
Methods and Results: Treatment of C57BL/6 mice with TNFa (50 ng/mouse, ip) increased the plasma levels of biomarkers of endothelial cell activation (sP-selectin, sE-selectin, sICAM-1) and leukocyte activation (MIP-2, sL-selectin). Pretreatment of mice with olinciguat (10 mg/kg, po) attenuated the increase in plasma concentrations of MIP-2 and sL-selectin following TNFa challenge by 81% and 58%, respectively. The TNFa-induced increase in plasma levels of sP-selectin, sE-selectin, and sICAM-1 were attenuated by 31%, 37%, and 34%, respectively, in olinciguat-treated mice. Co-treatment with HU augmented the effect of olinciguat on plasma concentrations of sE-selectin and sICAM-1 in TNFa-treated mice.
The effects of olinciguat, HU, and the combination on leukocyte endothelial cell interactions were assessed using intravital microscopy of post-capillary venules of the mouse cremaster muscle. Compared to controls, TNFa-treated C57BL/6 mice had slower leukocyte velocity (5.5±0.7 vs 26.6±3.1 µm/sec in controls) and leukocyte rolling flux (16.1±2.7 vs 43.3±7.1 cells/min in controls). Pretreatment with olinciguat (10 mg/kg) alone resulted in faster leukocyte velocity and rolling flux (10.3±1.1 µm/sec and 24.1±2.3 cells/min, respectively) compared to TNFa-treated controls. Similarly, pretreatment with HU (100 mg/kg, po) alone led to faster leukocyte velocity and leukocyte rolling flux (5.5±1.7 µm/sec and 50.2±5.9 cells/min, respectively). Co-administration of olinciguat and HU led to even faster leukocyte velocity and leukocyte rolling flux (19.7±1.9 µm/sec and 64.5±5.5 cells/min, respectively). Treatment with either olinciguat, HU, or the combination did not change leukocyte adherence following TNFa stimulation. To further evaluate the effect of olinciguat on neutrophil extravasation and trafficking we used a peritoneal recruitment model. Intraperitoneal injection of TNFa induced accumulation of Gr.1 polymorphonuclear leukocytes (PMN) in the mouse peritoneal cavity (4±0.4×105 PMNs/peritoneal lavage). Trafficking of PMNs into mouse peritoneum was attenuated by pretreatment of mice with HU (2.7±0.5×105 PMNs/peritoneal lavage). Co-administration of olinciguat and HU further attenuated the number of neutrophils extravasated into the peritoneum of TNFa-challenged mice (1.4±0.3×105 PMNs/peritoneal lavage) relative to TNFa-treated controls.
 Conclusion: In summary, prophylactic treatment with olinciguat inhibited the TNFa-induced increase in plasma levels of biomarkers of intravascular inflammation and attenuated the effect of TNFa on transient adhesive interactions between leukocytes and endothelial cells in mice. Co-administration with HU led to significantly reduced neutrophil trafficking and augmented the effect of olinciguat on biomarkers of endothelial cell activation and transient adhesive interactions in the mouse cremaster muscle. This study supports evaluation of the sGC stimulator olinciguat in patients with SCD.

Speakers
BT

Boris Tchernychev

Principal Investigator, IRONWOOD PHARMACEUTICALS


Friday October 12, 2018 5:15pm - 5:30pm
Constellation C

Attendees (10)