GLS-409, a Synergistic Inhibitor of Both P2Y1 and P2Y12: Efficacy in a Canine Model of Recurrent Coronary Thrombosis and Reversibility of Human Platelet Inhibition

Abstract

Introduction. Dual antiplatelet therapy with aspirin and an adenosine diphosphate (ADP) P2Y12 receptor inhibitor is a mainstay of pharmacological therapy in acute coronary syndromes. GLS-409 is a novel diadenosine tetraphosphate derivative that inhibits both P2Y1 and P2Y12platelet ADP receptors.Aims. 1) Determine the minimal dose of GLS-409 for in vivo inhibition of recurrent coronary thrombosis in a canine model. 2) Characterize the effects of the minimal antithrombotic dose of GLS-409 on the bleeding time. 3) Determine the extent and kinetics of the reversibility of platelet inhibition by GLS-409.Methods. (1) Canine Model of Recurrent Coronary Thrombosis. Recurrent platelet-mediated coronary thrombosis was initiated in anesthetized dogs by application of a stenosis at a site of vessel injury. At 1 h post-injury + stenosis, dogs were randomized to receive either GLS-409 or matched volumes of vehicle (saline). Three doses of GLS-409 were assessed: A) 0.054 mg/kg bolus + 0.00018 mg/kg/min infusion maintained for 2 hours; B) 1/10th bolus + 1/10th infusion; C) 1/100th bolus + 1/100th infusion. Coronary blood flow was monitored throughout the 2 hours post-treatment. Primary endpoints were: (i) area of the flow-time profile (index of coronary patency, expressed as % of baseline flow); and (ii) template bleeding time. (2) Reversibility of platelet inhibition following 30 minutes exposure to GLS-409. Sodium citrate 3.2% anticoagulated whole blood from healthy human volunteers (n=9) was treated with 80% inhibitory dose of GLS-409 for 30 minutes and assessed for recovery of response to ADP by flow cytometric analysis of platelet surface P-selectin and activated GPIIb-IIIa (reported by binding of the activation-dependent monoclonal antibody, PAC-1), at specified times before and after 300-fold dilution in drug-free, platelet-poor plasma.Results. (1) All dogs developed spontaneous episodes of recurrent thrombosis following coronary injury + stenosis. In the saline control group, there was no change in coronary patency during the 2 hour treatment period versus the pretreatment phase. Administration of GLS-409 at Doses A and B was associated with significant increases in % flow-time area, while Dose C had no significant effect on patency (Figure A). GLS-409 at Doses A and B which effectively inhibited recurrent coronary thrombosis, had no effect on the template bleeding time.(2) Incubation of human whole blood with GLS-409 for 30 min at room temperature, produced strong, concentration-dependent inhibition of the ADP-stimulated increases in platelet surface P-selectin and activated GPIIb-IIIa; 80% inhibition was obtained with 1.56 nM GLS-409. Blood exposed for 30 minutes to the 80% inhibitory concentration of GLS-409, then diluted 300-fold in drug-free, platelet-poor plasma (thereby reducing GLS-409 to 0.0052 nM, final concentration) showed a time-dependent recovery of reactivity to ADP as measured by both platelet surface P-selectin and activated GPIIb-IIIa (Figure B). However, while ADP-stimulated activated GPIIb-IIIa fully recovered within 60 min to levels seen in vehicle-treated samples, ADP-stimulated P-selectin response recovered to only 75% of the response seen in the vehicle-treated control (Figure B).Conclusions. (1) GLS-409 improved coronary patency in this canine model of recurrent coronary thrombosis without an accompanying increase in the bleeding time. (2) The time required after exposure of platelets to GLS-409 in whole blood for 50% recovery of ADP-stimulated PAC-1 reactivity (~26 min) is longer than published (J Thromb Haemost . 2017. 10.1111/jth.13627) recovery times after exposure to cangrelor (1.8 min), ticagrelor (4.4 min), and ticagrelor active metabolite (6.3 min), suggesting a slower off-rate for GLS-409. Incomplete reversibility of ADP-stimulated P-selectin following exposure to GLS-409 is similar to published results for cangrelor, but different from ticagrelor, both of which are selective for P2Y12. This relatively slow reversibility of GLS-409 inhibition offers another explanation, in addition to its dual specificity for P2Y1 and P2Y12, for the presently-described high potency of GLS-409 observed in the animal model. These properties suggest that GLS-409 could become a useful addition to the current drugs used in acute coronary syndromes. Further evaluation of the potential clinical utility of GLS-409 is warranted. [Display omitted] DisclosuresMichelson:Ionis: Research Funding; Sysmex: Research Funding; Baxalta: Research Funding; GLSynthesis: Research Funding; Eisai: Research Funding; Elsevier: Patents & Royalties; Ironwood: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Instrumentation Laboratory: Consultancy; Pfizer: Research Funding. Yanachkov:GLSynthesis: Employment. Yanachkova:GLSynthesis: Employment. Wright:GLSynthesis: Employment. Przyklenk:GLSynthesis: Research Funding. Frelinger:Pfizer: Research Funding; Ionis: Research Funding; Sysmex: Research Funding; Baxalta: Research Funding; GLSynthesis: Research Funding; Ironwood: Research Funding.