A Novel and Potent Inhibitor of E-Selectin, GMI-1687, Attenuates Thrombus Formation and Augments Chemotherapeutic Intervention of AML in Preclinical Models Following Subcutaneous Administration

Uproleselan (GMI-1271), an E-selectin antagonist, has been shown in preclinical models to disrupt activation of cell survival pathways in acute myeloid leukemia (AML), enhance chemotherapy efficacy, and improve survival. Uproleselan received FDA breakthrough therapy designation for adult relapsed/refractory AML in 2017 and Phase III studies are ongoing. In the present studies we report on the in vitro and in vivo comparative activities of an innovative high potency E-selectin antagonist, GMI-1687, a potential subcutaneously administered follow-on drug candidate to Uproleselan.

The binding constant, association and dissociation rates of GMI-1687 to immobilized recombinant human (rh) E-selectin were determined by surface plasmon resonance (SPR) at 25^oC. The K_D of GMI-1687 was 2.4 nM, with K_on = 3 x 10⁶ M^-1s^-1 and K_off = 1 x10^-2 s^-1. Under similar experimental conditions the K_D of Uproleselan was 520 nM with K_on = 0.02 x 10⁶ M^-1s^-1 and K_off = 1 x10^-2 s^-1. GMI-1687 was evaluated for its ability to inhibit binding of sialyl Le^a to immobilized rh E-selectin. The median IC₅₀ (n=6 independent assays) of GMI-1687 and Uproleselan in this assay was 15 and 550 nM, respectively. The in vitro activity of GMI-1687 to release adherent KG1a AML cells from E-selectin coated wells was also determined. GMI-1687 at 100 nM detached approximately 55% of adherent AML cells and was significantly different from Uproleselan at an identical concentration (38% detachment, P=0.0216). The percent bioavailability (%F) of GMI-1687 was evaluated in male Sprague-Dawley rats following intravenous (IV) and subcutaneous (SC) routes of administration at 5 mg/kg. The mean (+/- SD) SC %F for GMI-1687 was 126 +/- 3.8%. GMI-1687 also showed high bioavailability in CD-1 mice after SC administration of 0.58 mg/kg with %F = 132 +/-38.

The in vivo therapeutic activity of GMI-1687 following SC administration was assessed in an acute model of inferior vena cava (IVC) thrombosis and a tumor model of AML.Immediately following the induction of a non-occlusive thrombosis via electrical stimulation (250 mAmp) of the IVC, cohorts of male C57BL/6J mice (n=5/group) were given a single SC injection of saline (0.1 mL); Uproleselan (40 mg/kg); or GMI-1687 (4 mg/kg, 0.4 mg/kg or 0.04 mg/kg), and twenty-four hrs post thrombus induction the IVC was harvested from all mice and thrombus weights were determined. Treatment with GMI-1687 decreased thrombus formation with significant inhibition at 0.04 mg/kg (92%, P<0.001 compared to saline control). The inhibition of thrombus formation with GMI-1687 dosed at 0.04 mg/kg was statistically indistinguishable from Uproleselan administered SC at 40 mg/kg (97% inhibition). The therapeutic activity of SC GMI-1687 was also observed in combination with chemotherapy in a U937 tumor model. Three days post IV injection of U937 tumor cells, bone marrow ablated, female NOD/SCID mice (n=10/group) were treated with saline (0.1 mL SC QDx14); GMI-1687 (0.04 mg/kg SC QDx14) alone; cytarabine (AraC 300 mg/kg IP QDx3) + daunorubicin (DNR 3 mg/kg IV QDx1), or the combination of GMI-1687 and AraC + DNR. All treatments were well tolerated. The median survival time (MST) of mice treated with AraC + DNR was 36 days and statistically different (P<0.001) to groups treated with saline (MST=22 days) or GMI-1687 alone (MST=23 days). Importantly, the therapeutic activity of AraC+DNR was significantly enhanced when combined with GMI-1687 (MST>47.5 days, P=0.0153 compared to AraC+DNR alone).

In summary, a highly potent innovative antagonist of E-selectin, GMI-1687, has been produced that demonstrates high bioavailability following SC administration. SC injection of GMI-1687 shows significant activity in preclinical models previously reported for parenteral administration of Uproleselan, but at approximately 250-fold lower dose. GMI-1687 is therefore well-positioned for potential use in outpatient treatment settings where an E-selectin antagonist has therapeutic relevance. IND-enabling studies with GMI-1687 are currently underway.