Safety, Tolerability, and Pharmacodynamics of AZD3366 (Optimized Human CD39L3 Apyrase) Alone and in Combination With Ticagrelor and Acetylsalicylic Acid: A Phase 1, Randomized, Placebo-Controlled Study.

BACKGROUND: ADP and ATP are importantly involved in vascular and thrombotic homeostasis, via multiple receptor pathways. Blockade of ADP P2Y12 receptors inhibits platelet aggregation and represents an effective cardiovascular disease prevention strategy. AZD3366 (APT102), a long-acting recombinant form of an optimized CD39L3 human apyrase, has effectively reduced ATP, ADP, and platelet aggregation and provided tissue protection in preclinical models, features that could be very beneficial in treating patients with cardiovascular disease. METHODS AND RESULTS: We conducted this phase 1, first-in-human study of single ascending doses of intravenous AZD3366 or placebo, including doses added to dual antiplatelet therapy with ticagrelor and acetylsalicylic acid. The primary objective was safety and tolerability; secondary and exploratory objectives included pharmacokinetics, pharmacodynamics (measured as inhibition of platelet aggregation), adenosine diphosphatase (ADPase) activity, and ATP/ADP metabolism. In total, 104 participants were randomized. AZD3366 was generally well tolerated, with no major safety concerns observed. ADPase activity increased in a dose-dependent manner with a strong correlation to AZD3366 exposure. Inhibition of ADP-stimulated platelet aggregation was immediate, substantial, and durable. In addition, there was a prompt decrease in systemic ATP concentration and an increase in adenosine monophosphate concentrations, whereas ADP concentration appeared generally unaltered. At higher doses, there was a prolongation of capillary bleeding time without detectable changes in the ex vivo thromboelastometric parameters. CONCLUSIONS: AZD3366 was well tolerated in healthy participants and demonstrated substantial and durable inhibition of platelet aggregation after single dosing. Higher doses prolonged capillary bleeding time without detectable changes in ex vivo thromboelastometric parameters. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique Identifier: NCT04588727.

Modulation of CD39 and Exogenous APT102 Correct Immune Dysfunction in Experimental Colitis and Crohn's Disease.

BACKGROUND AND AIMS: CD39/ENTPD1 scavenges pro-inflammatory nucleotides, to ultimately generate immunosuppressive adenosine, which has a central role in immune homeostasis. Global deletion of Cd39 increases susceptibility to experimental colitis while single nucleotide polymorphisms within the human CD39 promoter, and aberrant patterns of expression during experimental hypoxia, predispose to Crohn's disease. We aimed to define the impact of transgenic human CD39 [hTG] overexpression in experimental colitis and to model therapeutic effects using the recombinant apyrase APT102 in vivo. We also determined the in vitro effects of APT102 on phenotypic and functional properties of regulatory T-lymphocytes derived from patients with Crohn's disease. METHODS: Colitis was induced by administration of dextran sulfate sodium in wild-type [WT] or hTG mice, and, in another model, by adoptive transfer of CD45RBhigh cells with or without WT or hTG regulatory T cells [Treg]. In additional experiments, mice were treated with APT102. The effects of APT102 on phenotype and function of Treg and type-1 regulatory T [Tr1] cells were also evaluated, after purification from peripheral blood and lamina propria of Crohn's disease patients [n = 38]. RESULTS: Overexpression of human CD39 attenuated experimental colitis and protected from the deleterious effects of systemic hypoxia, pharmacologically induced by deferoxamine. Administration of APT102 in vivo enhanced the beneficial effects of endogenous Cd39 boosted by the administration of the aryl hydrocarbon receptor [AhR] ligand unconjugated bilirubin [UCB]. Importantly, supplemental APT102 restored responsiveness to AhR stimulation by UCB in Treg and Tr1 cells, obtained from Crohn's disease patients. CONCLUSIONS: hCD39 overexpression ameliorated experimental colitis and prevented hypoxia-related damage in vivo. Exogenous administration of APT102 boosted AhR-mediated regulatory effects in vivo while enhancing Treg functions in Crohn's disease in vitro.

Involvement of ATP in noxious stimulus-evoked release of glutamate in rat medullary dorsal horn: a microdialysis study.

Our electrophysiological studies have shown that both purinergic and glutamatergic receptors are involved in central sensitization of nociceptive neurons in the medullary dorsal horn (MDH). Here we assessed the effects of intrathecal administration of apyrase (a nucleotide degrading enzyme of endogenous adenosine 5-triphosphate [ATP]), a combination of apyrase and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, an adenosine A1 receptor antagonist), or 2,3-O-2,4,6-trinitrophenyl-adenosine triphosphate (TNP-ATP, a P2X1, P2X3, P2X2/3 receptor antagonist) on the release of glutamate in the rat MDH evoked by application of mustard oil (MO) to the molar tooth pulp. In vivo microdialysis was used to dialyse the MDH every 5 min, and included 3 basal samples, 6 samples after drug treatment and 12 samples following application of MO. Tooth pulp application of MO induced a significant increase in glutamate release in the MDH. Superfusion of apyrase or TNP-ATP alone significantly reduced the MO-induced glutamate release in the MDH, as compared to vehicle. Furthermore, the suppressive effects of apyrase on glutamate release were reduced by combining it with DPCPX. This study demonstrates that application of an inflammatory irritant to the tooth pulp induces glutamate release in the rat MDH in vivo that may be reduced by processes involving endogenous ATP and adenosine.

A study of the imbalance in B cell-expressed nucleoside triphosphate diphosphohydrolase 1-induced ADP degradation in graft injury during acute antibody-mediated rejection.

OBJECTIVE: To study the effects and mechanisms of the imbalance in B cell-expressed nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1)-induced ADP degradation on graft injury during acute antibody-mediated rejection (AMR). METHODS: The acute AMR animal model was established in male NTPDase 1-wild-type Balb/c nude mice. The levels of NTPDase 1 in B cells and NTPDase1 mRNA in grafted skin, changes in platelet activation markers and average platelet velocities were determined by luciferin/luciferase enzymatic, real-time fluorescent quantitative PCR, flow cytometry and inverted microscope. The pathological changes in grafted skin were observed by electron microscopy. The effects of pretreatment with different doses of exogenous NTPDase 1 on platelet activation and graft injury were studied. RESULTS: The expression of B-cell NTPDase 1 was significantly increased at 30 min after the induction of acute AMR and restored to baseline levels after 7 days. The levels of NTPDase 1 mRNA in grafted skin were decreased at 30 min after the induction of acute AMR. After the induction of acute AMR, the levels of platelet activation markers increased significantly, whereas the average platelet velocity significantly decreased. After pretreatment with exogenous NTPDase 1, the expression of platelet activation markers significantly decreased, the average velocity of platelets increased significantly, and the necrosis of grafted skin and inflammatory reaction significantly reduced. CONCLUSIONS: An imbalance in the NTPDase 1-induced degradation of extracellular ADP may be a major cause of graft injury in acute AMR. Pretreatment with exogenous NTPDase 1 may effectively inhibit platelet activation and protect grafted skin.

P2Y12 or P2Y1 inhibitors reduce platelet deposition in a microfluidic model of thrombosis while apyrase lacks efficacy under flow conditions.

Determination of the patient-specific response to antiplatelet agents facilitates proper dosing for both acute and chronic prophylaxis. "Closed" systems (with or without flow) may fail to predict pharmacological potency in situations where platelets rapidly accumulate under flow conditions at a site of thrombosis ("Open" systems). Using an 8-channel microfluidic flow assay of human whole blood with corn trypsin inhibitor (+/- PPACK) perfused over focal zones of collagen, dose-response curves were measured for pharmacological agents at a wall shear rate of 210 s(-1). The P2Y(1) inhibitor MRS 2179 (IC(50) = 0.233 +/- 0.132 microM) and P2Y(12) inhibitor 2-MeSAMP (IC(50) = 2.558 +/- 0.799 microM) were potent blockers of secondary platelet accumulation under flow, while the P2X(1) inhibitor (NF 449) and apyrase failed to reduce platelet accumulation. MRS 2179 and 2-MeSAMP had undetectable effects on initial platelet adhesion to collagen. Numerical simulation of convective-diffusive transport and apyrase-mediated catalytic degradation of ADP indicated that ultra-high concentrations of apyrase ( approximately 2000 U mL(-1)) would be required to have the same effect under flow as much lower concentrations (1 U mL(-1)) currently used in closed systems (aggregometry or cone-and-plate viscometer). This is the first evaluation of IC(50) values for P2Y(12) and P2Y(1) antagonists under controlled flow conditions. Evaluation of antiplatelet agents in open flow systems demonstrates that inhibition of either ADP by apyrase or antagonism of P2X(1) signaling had no inhibitory effect on platelet accumulation. This technique provides a platform for rapidly investigating effects of antithrombotic therapies simultaneously in a model injury system.

Intravenous apyrase administration reduces arterial thrombosis in a rabbit model of endothelial denudation in vivo.

The role of adenine nucleotides on vascular and platelet functions has long been established. Apyrase (CD39) takes part of a family of ecto-enzymes that hydrolyze adenosine diphosphate and adenosine triphosphate. The participation of apyrase in the thromboregulatory system is under study. An in vivo experimental model of acute arterial thrombosis was used to test the hypothesis that administering a soluble form of potato apyrase could prevent thrombus formation. Twenty-five white New Zealand male rabbits suffered balloon aortic endothelium denudation and, after 15 days, they were submitted to a thrombosis-triggering protocol with a procoagulant (Russel's viper venom) and epinephrine. After the thrombosis-triggering protocol, 12 animals received two soluble apyrase administrations intravenously (with 90 min intervals), while 13 control animals received no apyrase. Three hours after the triggering protocol, the animals were killed and the rate and area of arterial thrombosis were analyzed. The rate of thrombosis in the apyrase group was significantly lower than that of the control group (16.7 versus 69%, respectively; P = 0.015), as was the area of thrombosis (1.7 +/- 4.3 versus 21.7 +/- 37.4 mm2, respectively; P = 0.008). Our results confirm that apyrase participates in homeostasis through a potent anti-thrombotic effect.

In vitro verification of antithrombotic effect of recombinant soluble nucleotide triphosphate diphosphohydrolase 1.

PURPOSE: Ecto-ADPase (NTPDase1 or CD39) has been identified on endothelial cells and found to be antithrombogenic, with actions resulting from degradation of adenosine diphosphate (ADP), a thrombogenic molecule secreted by activated platelets at sites of vascular injury. Reasoning that the ADPase activity of CD39 might provide clinical use as an antithrombotic agent, the authors investigate the comparative ability of the agent to inhibit platelet and fibrin deposition. MATERIALS AND METHODS: With use of an in vitro perfusion system, fresh, heparinized human blood was passed over expanded polytetrafluoroethylene grafts at hemodynamic conditions similar to those observed in the human arterial circulation. Three different concentrations of CD39 (30 mcg/mL, 100 mcg/mL, and 300 mcg/mL) were compared with abciximab (4 mcg/mL) and heparin controls. The deposition of radiolabeled platelets and fibrinogen was measured in five perfusions for each treatment group. RESULTS: The addition of soluble CD39 to heparinized blood inhibited platelet deposition to an extent greater than that of heparin alone (P =.04). Effects were similar to those observed with abciximab. The addition of CD39 to heparinized blood did not result in augmentation of fibrin inhibition beyond that observed with heparin alone. The fibrin inhibitory effects of CD39 appeared to be less significant than the fibrin inhibition observed with abciximab, but this difference did not attain statistical significance. CONCLUSION: These results suggest that recombinant CD39 holds potential as antithrombotic agent, with the potential to achieve antiplatelet effects similar to that observed with abciximab.

Apyrase administration prolongs discordant xenograft survival.

Platelet thrombi and vascular inflammation are prominent features of discordant xenograft rejection. The purinergic nucleotides ATP and ADP, which are secreted from platelets and released by injured endothelial cells (EC), are important mediators of these reactions. Quiescent EC express the ectoenzyme ATP-diphosphohydrolase (ATPDase; an apyrase), which exerts an important thromboregulatory function by hydrolyzing both ATP and ADP. We have shown that ATPDase activity is rapidly lost from the surface of the EC following ischemia-reperfusion injury and during xenograft rejection. The aim of this study was to supplement ATPDase activity within xenografts by infusion of soluble apyrases, and thereby validate the importance of local ATPDase activity in the modulation of xenograft rejection. Lewis rats underwent heterotopic cardiac xenografting from guinea pigs and apyrase was administered intravenously (200 U/kg) as a single dose to evaluate effects on hyperacute rejection (HAR). This initial dose was followed by a continuous apyrase infusion (8.0 U/kg/hr) directly into the graft aorta in combination with systemic cobra venom factor (CVF) administration to deplete complement when delayed xenograft rejection (DXR) was studied. Functional apyrase levels in vivo were assessed by the capacity of blood samples taken at the time of surgery and rejection to inhibit platelet aggregation in vitro. Apyrase administration significantly prolonged graft survival in HAR and DXR. Functional assays showed inhibition of platelet aggregation suggesting effective systemic antiaggregatory effects of the administered apyrases. Histologic studies showed that apyrase administration abrogated local platelet aggregation and activation in HAR and DXR. Our data demonstrate that local administration of apyrase prolonged discordant xenograft survival. These observations emphasize the potential importance of purinergic mediators in platelet activation during xenograft rejection.