Combination of cyclic nucleotide modulators with P2Y12 receptor antagonists as anti‐platelet therapy

Endothelium‐derived prostacyclin and nitric oxide elevate platelet cyclic nucleotide levels and maintain quiescence. We previously demonstrated that a synergistic relationship exists between cyclic nucleotides and P2Y12 receptor inhibition. A number of clinically approved drug classes can modulate cyclic nucleotide tone in platelets including activators of NO‐sensitive guanylyl cyclase (GC) and phosphodiesterase (PDE) inhibitors. However, the doses required to inhibit platelets produce numerous side effects including headache.


| INTRODUC TI ON
Platelets play a central role in cardiovascular disease, as they are integral to the development of acute thrombotic events. For this reason, anti-platelet therapy is prescribed for the secondary prevention of atherothrombotic events in patients with acute coronary syndromes or following percutaneous coronary intervention. 1,2 Aspirin, which irreversibly inhibits the cyclooxygenase enzyme and downstream thromboxane (Tx)A 2 production, 3,4 is often coadministered with a P2Y 12 receptor antagonist, such as clopidogrel or prasugrel, to produce dual anti-platelet therapy (DAPT). P2Y 12 receptor antagonists inhibit platelet aggregation by blocking the amplifying effects of adenosine diphosphate (ADP). 5,6 While such therapy is effective, recurrent events still occur 7,8 and alternative ways to prevent thrombosis continue to be required.
Vascular endothelial cells produce the short-lived autacoids prostaglandin I 2 (prostacyclin; PGI 2 ) and nitric oxide (NO) that relax blood vessels and inhibit platelets. PGI 2 binds to platelet PGI 2 (IP) receptors that in turn activate adenylyl cyclase (AC) to increase intracellular cyclic adenosine monophosphate (cAMP) levels. 9 In contrast, NO diffuses freely into platelets activating the α 1 β 1 isoform of guanylyl cyclase (GC-1, formerly known as soluble GC) 10 to increase intracellular cyclic guanosine monophosphate (cGMP) levels. 11 This intra-platelet elevation of levels of individual cyclic nucleotides is synergistic in maintaining basal platelet quiescence and preventing inappropriate platelet activation. 12 Drugs targeting the NO-cGMP pathway, such as organic nitrates, are long established clinically for treatment of heart failure and angina pectoris. 13 In recent years, drugs which directly activate or stimulate GC have been developed as potential vasodilators and have been clinically approved for the treatment of pulmonary vascular disease. 14 Similarly, drugs which modulate the cAMP pathway such as phosphodiesterase (PDE) inhibitors and PGI 2 analogues are approved for the treatment of peripheral and pulmonary vascular disease. However, the doses of cyclic nucleotide elevating drugs that produce anti-platelet effects are associated with side effects such as headache, nausea, and hypotension. 15,16 This is consistent with the doses required to inhibit platelets being the same as those that produce vasodilatation.
We have recently demonstrated that blockade of platelet P2Y 12 receptor further synergizes with PGI 2 and NO 17,18 to produce profound platelet inhibition. We therefore hypothesized that the actions of pharmacological agents acting upon cyclic nucleotides could be selectively amplified in platelets by combination with P2Y 12 receptor antagonists, thereby producing an enhanced anti-platelet effect of both drugs at doses which do not produce systemic vasodilator side effects. Here we report in vitro, ex vivo, and in vivo studies that support this hypothesis.

| Mouse strains
C57Bl/6 wild-type (WT) mice were purchased from Charles River UK. All mice were 8 to 12 weeks old (20-25 g) and housed for a minimum of 7 days before commencement of experiments. They K E Y W O R D S blood platelets, cyclic, nucleotides, pharmacology, purinergic P2Y receptor antagonists, thrombosis Essentials • A synergistic relationship exists between cyclic nucleotides and P2Y 12 receptor inhibition.
• Approved drugs that modulate cyclic nucleotide tone in platelets produce numerous side effects including headache.
• Low dose guanylyl cyclase activators synergize with P2Y 12 inhibition to produce a powerful anti-platelet effect without altering blood flow.
• This novel combination can provide a strong and focused anti-thrombotic regimen.
were housed on a 12-hour light-dark cycle, at a temperature of 22 to 24°C with access to water and food ad libitum. Animal procedures were conducted under UK Home Office project license authority (PPL/8422) in accordance with "The Animals (Scientific Procedures) Act 1986," EU directive 2010/63/EU, and were subject to local approval from Queen Mary University of London and Imperial College London Ethical Review Panels.

| Mouse dosing and obtaining blood
To maximize clinical relevance in vivo we replaced PAM with prasugrel and BAY-70 with cinaciguat (BAY 58-2667, kind gift from Bayer AG), as both are approved for human administration. However, given the short half-life of intra-platelet cyclic nucleotides we coadminis- intraperitoneal was administered 2 hours prior to blood collection or arterial injury. Mice were subsequently anesthetized with ketamine (Narketan, 100 mg/kg; Vetoquinol) and xylazine (Rompun, 10 mg/ kg; Bayer, Germany) intraperitoneal cinaciguat (0.3 mg/kg) or vehicle (2% DMSO) intravenous was administered 10 minutes prior to blood collection or arterial injury. Blood was collected from the inferior vena cava into trisodium citrate (0.32%).

| Platelet adhesion under physiological flow
Flow chamber slides (VI0.1 µ-slide, Ibidi) were coated with Horm collagen (100 µg/mL) followed by blocking with bovine serum albumin (BSA, 4%, Sigma). Whole blood, treated with mepacrine (10 µmol/L, Sigma) to label platelets, was perfused across the coated surface at 1000 s −1 for 5 minutes. Post-flow images were taken (four per experiment) at x40 magnification using a TE-2000S, Nikon Eclipse inverted microscope connected to a RT slider CCD camera (Diagnostic Instruments Inc). Images were analyzed using Image J (NIH).

| Whole blood aggregation
Aggregation was conducted as we have previously described. 19 Half-area 96-well microtiter plates (Greiner Bio-One, UK) were pre-coated with hydrogenated gelatin (0.75% w/v; Sigma) in phosphate-buffered saline to block nonspecific activation of blood.
At the time of experiment whole blood was placed into each well and aggregation stimulated by placing the plate on a heated plate shaker (Bioshake IQ, Q Instruments) at 37°C, mixing at 1200 rpm, for Labelled, diluted blood was then analyzed using a FACSCalibur flow cytometer (BD Biosciences).

| Mouse ferric chloride arterial injury
In mice anesthetized with ketamine and xylazine, the carotid artery was exposed and isolated from surrounding tissues to permit a ferric chloride (FeCl3, 10% solution, Sigma) soaked filter paper to be applied for 3 minutes. The carotid artery was then flooded with saline and the filter paper removed. A Doppler flow probe (Transonic) was then placed around the artery and flow monitored for up to 30 minutes. The time to stable occlusion (defined as flow 0.0 ± 0.2 mL/min for 1 minute) was recorded.

| Mouse tail bleeding assay
Anesthetized mice were maintained at 37°C and their tail was transected with a scalpel blade at 1 mm from its end. The tail was immersed immediately in warm saline (37°C) and time recorded until it stopped bleeding for 30 seconds.

| Statistics and data analysis
Parametric data presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using Prism 6.0 (GraphPad software). Significance was determined by one-way analysis of variance (ANOVA) with Dunnett's post-hoc test unless otherwise stated, and data sets considered different if P < .05. For non-parametric data significance was determined by Mantel-Cox test and with Holm-Sidak correction for multiple comparisons where necessary. Flow cytometry data was analyzed using FlowJo v7.4 (Tree Star). For analysis, the "single platelet" population was gated based on side scatter and anti-platelet immunoreactivity (fluorescence intensity).

| BAY-70 potentiates the inhibitory action of PAM in washed platelets and PRP in a concentrationdependent manner
Aggregation responses to thrombin were first established in WP ( Figure 1A A similar pattern was seen in experiments examining collageninduced aggregation ( Figure 1B). Interestingly, in these conditions BAY-70 achieved a 65 ± 4% decrease at the far lower concentration of 10 nmol/L in the presence of PAM, compared to a 17 ± 5% decrease without PAM (P < .05).
In experiments using PRP, thrombin was replaced by the activator peptide TRAP-6. As in WP, PAM 3 µmol/L had little effect on TRAP-6 induced aggregation (77 ± 10% vehicle; 70 ± 8% PAM; Figure 1C,D). Similarly, BAY-70 potentiated the effect of PAM against both TRAP-6 and collagen. Aggregation ( Figure 1D) conducted in the presence of 10 µmol/L BAY-70 plus PAM were inhibited by 55 ± 10% and 76 ± 11% when induced by TRAP-6 and collagen, respectively, compared to 15 ± 3% and 28 ± 5%, respectively, when PAM was not present (P < .05 versus BAY-70 plus PAM). In a separate mechanistic study ( Figure S1 in supporting information) we found that PAR4, as compared to PAR1, agonism was more sensitive to P2Y 12 antagonism in the presence of BAY-70.

| BAY-70 potentiates the anti-thrombotic action of PAM in whole blood
Similar data were obtained in whole blood assays. In studies of whole blood platelet adhesion under flow, BAY-70 and PAM when used individually produced similar reductions in platelet coverage from 9.8 ± 2.6% (vehicle) to 5.4 ± 1.1% and 5.5 ± 0.9%, respectively.
Combination of BAY-70 and PAM together caused a further reduction of coverage to 2.3 ± 0.5% (P < .05 versus vehicle; Figure 2A,B).

| Cyclic nucleotide modulators potentiate the inhibitory action of prasugrel in vivo
Having established that the GC-1 activator BAY-70 potentiates the anti-platelet effect of PAM we next sought to investigate if this can occur and is relevant in vivo.
Following in vivo drug administration, ex vivo platelet aggregation studies demonstrated no significant inhibitory effects of either prasugrel alone or the combination of two cyclic nucleotide elevating drugs-the GC-1 activator cinaciguat plus the PDE inhibitor dipyridamole ( Figure 3A-C) at the selected doses. In contrast, platelets in blood from mice that had received the combination prasugrel with cinaciguat plus dipyridamole demonstrated significantly lower aggregatory responses than those from vehicle treated animals (collagen, 22 ± 9% versus 53 ± 9%; PAR-4 peptide, 7 ± 4% versus 37 ± 9%; U46619, 13 ± 6% versus 48 ± 11%; P < .05 for all).
In the FeCl3-induced arterial thrombosis injury model prasugrel alone (time to occlusion, 438 ± 87 seconds) or cinaciguat plus dipyridamole (482 ± 110 seconds) had no effect relative to vehicle which is an early event in the pathophysiology of cardiovascular disease. 28 During endothelial dysfunction NO and PGI 2 production will be reduced, leading to reduced intra-platelet cyclic nucleotide tone and so increased platelet reactivity. Therefore, it may well be those patients with the greatest level of endothelial dysfunction that get the smallest benefit from adequate P2Y 12 inhibition. Indeed, we recently reported that the P2Y 12 inhibitor ticagrelor attenuated platelet function more potently in samples from well-trained middle-aged men with a superior vascular function compared to matched untrained men with a reduced vascular function. 29 A logical extension of the argument above is that drug therapy to increase intra-platelet cyclic nucleotides would boost the anti-platelet effect of P2Y 12 antagonists. There exist a number of agents available to modulate cyclic nucleotide levels; however, we chose to focus prophylaxis. These GC-1 activators directly act upon NO-sensitive GC to stimulate cGMP production without the requirement of NO or the heme moiety, 31 the separation of which can occur during endothelial dysfunction and oxidative stress. 14 Our initial in vitro experiments were completed using the compound BAY-70 as a pharmacological tool, which is effective at raising cGMP levels. 32 Notably it has also previously been reported that BAY-70, in the micro-molar range, can inhibit washed platelet activation. 33 In our experiments in washed platelet preparations, nano-molar concentrations were sufficient to inhibit platelet activation in the presence of PAM.
For the in vivo studies we chose to use the related compound cinaciguat. Cinaciguat has previously been studied in a phase IIb clinical trial in patients with acute decompensated heart failure, 15 and therefore has potentially more clinical relevance. We also  own. This means that it may be possible to achieve therapeutic effectiveness using lower doses than those currently prescribed for individual use and so reducing drug-associated side effects.
In conclusion, our study builds upon our previous observations of a synergistic relationship between P2Y 12 receptor inhibition and platelet cyclic nucleotide levels to identify a novel potential anti-platelet drug regimen. We demonstrate the principle of a combination of low doses of approved drugs targeted at cyclic nucleotide modulation, combined with P2Y 12 inhibition, as a realistic and powerful therapeutic regimen. While more work and optimization will be required to clinically translate this in human F I G U R E 4 Summary of the pathophysiological rationale for the efficacy of combined cyclic nucleotide modulators with P2Y 12 receptor antagonists as anti-platelet therapy. A, In the healthy circulation endothelial-derived mediators nitric oxide (NO) and prostaglandin I 2 (PGI 2 ) act upon platelets to raise cyclic nucleotide (cAMP and cGMP) levels that in turn maintain platelets in a quiescent state. B, During established cardiovascular disease, concurrent endothelial dysfunction results in reduced production of NO and PGI 2 , lowering intra-platelet cyclic nucleotide tone and decreasing the threshold for activation. C, A synergic relationship exists between intra-platelet cyclic nucleotides and P2Y 12 receptor blockade such that pharmacological modulators of cyclic nucleotides, to compensate for reduced endothelial cell function, combined with P2Y 12 receptor antagonist produces a focused anti-platelet effects at low doses of each associated with reduced drug side effects