It is hypothesized that activated leukocytes play key roles in sickle cell vaso-occlusion by adhering to inflamed venules and capturing circulating platelets and sickle red blood cells. GMI-1070 is a small molecule selectin antagonist which was recently reported to reverse acute vascular occlusion in a humanized sickle cell disease (SCD) mouse model (Chang et al, Blood 2010) presumably by inhibiting E-selectin and its effects on downstream signaling of leukocyte activation. Sickle cell patients express elevated levels of soluble E-selectin (Kato et al, Brit J Haem 2005) activated polymorphonuclear neutrophils (PMN) (Lum et al Amer J Hem 2004) and platelet/monocyte aggregates (PMA) (Wun et al Clin Lab Haem 2002). In this study, the activation state of leukocytes from whole blood samples of sickle cell patients not in crisis before and after infusion of GMI-1070 was evaluated ex vivo. Isolated PMN from normal, healthy volunteers were strongly activated by binding soluble E-selectin/hIg in vitro as determined by a 7-fold increase of the integrin MAC1 (CD11b) and an 8-fold increase in expression of the high affinity form of CD18 detected by antibody 327C. Addition of GMI-1070 completely blocked upregulation of MAC1 and 327C at 50μg/ml and showed pronounced inhibition (79% MAC1; 75% 327C) at 10μg/ml. These in vitro concentrations are consistent with blood levels of GMI-1070 found in sickle cell patients 4 and 8 hours after dosing. A phase 1/2 study was conducted on 10 adult subjects with SCD at steady state. GMI-1070 was given IV at 20mg/kg as a loading dose and at 10 hours a final dose of 10mg/kg was given. Blood samples were drawn from these adults pre-infusion and at 8, 24, and 48 hours after the initial infusion. In some subjects, a blood sample was also drawn at 4 hours post infusion. Activation of PMN's in whole blood samples from subjects was assessed by upregulation of MAC-1, expression of the high affinity CD18 and the loss of CD62L due to shedding of L-selectin determined by flow cytometric analysis of cell surface labeling with fluorescently conjugated antibodies. Of 4 subjects tested, 3 showed increased surface expression of L-selectin, 3 showed decreased expression of MAC-1, and 2 showed decreased expression of high affinity CD11b at the first time point tested (4 or 8hr) after dosing with GMI-1070 suggesting an inhibition of PMN activation in these patients. A functional consequence of monocyte activation is the formation of platelet/monocyte aggregates due to expression of high affinity integrins. Platelet-monocytes aggregates (PMA) in blood were detected using anti-CD11c for monocytes and anti-CD41a for platelets. Treatment of samples with lipopolysaccharide (LPS) was used for positive controls. Intracellular IL-1β was used as a marker of activated monocytes. In 5 patients out of 6 tested with this assay, PMA in the subject's blood were decreased at the first time point after dosing (8hr). These results are consistent with an effect of GMI-1070 on inhibition of activation given its IC50 value for E-selectin (4.3μM), the blood concentration in subjects after dosing, and the serum half life (7.7hr) in steady state sickle cell adults.
GMI-1070 significantly inhibited E-selectin-mediated activation of PMNs in vitro as determined by expression of the integrin MAC-1 and high affinity CD18 at 10μg/ml. Similar concentrations of GMI-1070 in sickle cell subjects' blood at 4 and 8 hours after dosing also resulted in a lowered activation state of PMNs identified by reduced expression of cell surface integrin molecules as well as the inhibition of shedding of L-selectin in some cases. A more functional measure of leukocyte activation is the aggregation of platelets on monocyte cell surfaces. In 5 of 6 subjects tested, GMI-1070 reduced PMA 8 hours after dosing. Thus, GMI-1070 not only inhibits E-selectin, but also blocks the expression of downstream integrin adhesion molecules that together play crucial roles in vaso-occlusion by promoting the adhesion to platelets and erythrocytes in the formation of occlusions that block blood flow. The effects of GMI-1070 on the activation state of leukocytes via the inhibition of functional adhesion molecules in steady state sickle cell subjects supports the further evaluation of treatment with GMI-1070 during vaso-occlusive episodes.
Simon:GlycoMimetics: Research Funding. Chase:GlycoMimetics:.Kuypers:GlycoMimetics Inc.: Research Funding. Styles:GlycoMimetics: Consultancy, clinical trial sponsorship. Wun:GlycoMimetics Inc.: Consultancy, clinical trial sponsorship. Thackray:GlycoMimetics: Employment, Equity Ownership. Magnani:GlycoMimetics: Employment, Equity Ownership. Off Label Use: The drug (GMI-1070) is not approved for any clinical indication.
Asterisk with author names denotes non-ASH members.