GLS-409, an Antagonist of Both P2Y1 and P2Y12, Potently Inhibits Canine Coronary Artery Thrombosis and Reversibly Inhibits Human Platelet Activation

Abstract

Dual antiplatelet therapy with aspirin and an adenosine diphosphate (ADP) P2Y₁₂ receptor antagonist reduces ischemic events in patients with acute coronary syndrome. Previous evidence from our group, obtained in a preclinical model of recurrent platelet-mediated thrombosis, demonstrated that GLS-409, a diadenosine tetraphosphate derivative that inhibits both P2Y₁ and P2Y₁₂ ADP receptors, may be a novel and promising antiplatelet drug candidate. However, the salutary antiplatelet effects of GLS-409 were accompanied by a trend toward an unfavorable increase in bleeding. The goals of this study were to: 1) provide proof-of-concept that the efficacy of GLS-409 may be maintained at lower dose(s), not accompanied by an increased propensity to bleeding; and 2) establish the extent and kinetics of the reversibility of human platelet inhibition by the agent. Lower doses of GLS-409 were identified that inhibited in vivo recurrent coronary thrombosis with no increase in bleeding time. Human platelet inhibition by GLS-409 was reversible, with rapid recovery of platelet reactivity to ADP, as measured by platelet surface activated GPIIb-IIIa and platelet surface P-selectin. These data support the concept that GLS-409 warrants further, larger-scale investigation as a novel, potential therapy in acute coronary syndromes.

Figure 1

Effect of GLS-409 and vehicle control (saline) on coronary patency as measured by % flow-time area in a canine model of recurrent arterial thrombosis. % Flow-time area, quantified before and after treatment, in cohorts treated with saline (Control n = 3) and GLS-409 doses 1, 2 and 3. Dose 1: 0.054 mg/kg bolus + 0.00018 mg/kg/min infusion maintained for 2 hours (same bolus + 1/10 of the infused dose administered in the initial GLS-409 study, n = 3). Dose 2: 0.0054 mg/kg bolus + 0.00018 mg/kg/min infusion for 2 hours (1/10 of the bolus + 1/10 of the infused dose administered in the initial study, n = 5). Dose 3: 0.00054 mg/kg bolus + 0.000018 mg/kg/min infusion for 2 hours (1/100 of the bolus + 1/100 of the infused dose administered in the initial study: n = 5). Insert: for purposes of comparison, data from the current study are plotted together with results obtained previously with high-dose GLS-409 (0.054 mg/kg IV bolus followed by a continuous intravenous infusion of 0.0018 mg/kg/min) and matched (historical) controls. Data are mean ± SEM, *p < 0.05 versus pretreatment and p < 0.05 versus controls.

Figure 2

Effect of GLS-409 and vehicle control (saline) on coronary patency as measured by zero flow duration in a canine model of recurrent arterial thrombosis. % Zero flow duration (mean ± SEM), quantified before and after treatment, in cohorts treated with saline (Control) and GLS-409 doses 1, 2 and 3 (see Fig. 1 legend for details). p = 0.14 (ns) for group-time interaction. Insert: for purposes of comparison, data from the current study are plotted together with results obtained previously with high-dose GLS-409 (0.054 mg/kg IV bolus followed by a continuous intravenous infusion of 0.0018 mg/kg/min) and matched (historical) controls.

Figure 3

Effect of GLS-409 and vehicle control (saline) on the template bleeding time. Bleeding time (seconds), measured immediately before treatment and at the end of the 2-hour treatment period, for cohorts treated with saline (Control) and GLS-409 Doses 1, 2 and 3 (see Fig. 1 legend for details). Insert: for purposes of comparison, results obtained previously with high-dose GLS-409 (0.054 mg/kg IV bolus followed by a continuous intravenous infusion of 0.0018 mg/kg/min) and matched (historical) controls are shown.

Figure 4

GLS-409 concentration-dependent inhibition of human platelet reactivity to ADP as measured by platelet surface activated GPIIb-IIIa and platelet surface P-selectin expression. Sodium citrate 3.2% anticoagulated whole blood from healthy volunteers was treated in vitro with different concentrations of GLS-409 (0.003–12.5 nM) or vehicle. (A) Platelet surface activated GPIIb-IIIa, detected with the activation-dependent monoclonal antibody PAC1 (MFI, mean fluorescence intensity) and (B) Normalized MFI of PAC1. IC₅₀ = 0.17 nm (95% CI, 0.13–0.22), R² = 0.97. (C) MFI and (D) normalized MFI for platelet surface P-selectin. IC₅₀ = 0.13 nM (95% CI, 0.065–0.25), R² = 0.84. Data were analyzed using fitting of non-linear regression. Results are mean ± SEM, n = 3.

Figure 5

Reversibility of human platelet inhibition by GLS-409: recovery of platelet reactivity to ADP. Sodium citrate 3.2% anticoagulated whole blood from healthy volunteers was treated with vehicle (saline) or the IC₈₀ dose (1.56 nm) of GLS-409 for 30 minutes at room temperature. Treated blood was diluted 300-fold in drug-free filtered platelet-poor plasma and then incubated at room temperature for different lengths of time (0–90 min), followed by ADP 5 µM stimulation and measurement of platelet surface activated GPIIb-IIIa and platelet surface P-selectin. (A) MFI and (B) normalized MFI of PAC1, as a percentage of maximal response at each point, for platelet surface activated GPIIb-IIIa. (C) MFI and (D) normalized MFI, as a percentage of maximal response at each point, for platelet surface P-selectin. In panels B and D, data are expressed as the percentage of the control (vehicle treatment) at the same time point, and data were analyzed using non-linear fit of dissociation: one-phase exponential decay in GraphPad Prism. Data are mean ± SEM, n = 9.