The developmental journey of therapies targeting purine receptors: from basic science to clinical trials

Since the discovery of ATP as an extracellular signalling molecule in 1972, purinergic signalling, mediated by extracellular purines and pyrimidines has been identified in virtually all mammalian tissues and is implicated in regulating fundamental cellular processes. In recent years, there has been an increasing focus on the pathophysiology and potential therapeutic interventions based on purinergic signalling. A vast range of compounds targeting purine receptors are in clinical development, and many more are in preclinical studies, which highlights the fast growth in this research field. As a tribute to Professor Geoffrey Burnstock’s legacy in purinergic signalling, we present here a brief review of compounds targeting purine receptors that are in different stages of clinical trials. The review highlights the 50-year journey from basic research on purinergic receptors to clinical applications of therapies targeting purine receptors.

The concept of purinergic signalling, first proposed in 1972 [1] refers to the role of extracellular nucleosides and nucleotides, most notably adenosine and adenosine 5′-triphosphate (ATP), as signalling molecules. It was initially viewed that these molecules act as neurotransmitters, but subsequent studies extended their roles to the regulation of numerous cellular processes, including cell proliferation, differentiation, migration, and apoptosis [2]. Since 1972, four subtypes of P1 (adenosine) receptors, seven subtypes of ATP-gated ion channels (P2X receptors) and eight subtypes of P2Y G protein-coupled receptors have been cloned and sequenced [3,4]. An increasing focus on the role of these receptors in disease and their therapeutic potential as pharmacological targets for drug development has been apparent in recent years [4,5]. Several decades following the early pioneering work of Professor Geoffrey Burnstock, the therapeutic potential of purinergic signalling is widely recognised, and translational studies have enabled therapeutic compounds to reach clinical trials. As a tribute to Professor Geoffrey Burnstock, we have prepared this brief review of compounds now in Phase I to Phase IV clinical trials and presented several case studies to describe the translational journey of therapies targeting purine receptors to clinical practice.

Compounds in clinical trials
Data from the clinical registries showed that 38 compounds, all agonists and antagonists targeting purine receptors, are currently in clinical development (Tables 1, 2, 3). Four compounds target the P2X 3 receptor, and five the P2X 7 receptor (Table 1). For P2Y receptors, three compounds target the P2Y 2 receptor and different generations of the same compound target the P2Y 12 receptor ( Table 2). Six compounds target the A 1 receptor, ten compounds the A 2A receptor, one the A 2B receptor , and one the A 3 receptor (Table 3, Fig. 1A). These compounds are in different stages of clinical trial. In Phase I, eleven compounds target P2X 7 , P2Y 2 , A 2A , and A 2B receptors. Seventeen compounds targeting P2X 3 , P2X 7 , P2Y 12 , A 1 , A 2A , and A 3 receptors are in Phase II. Only three compounds are in Phase III, targeting A 1 , A 2A , and A 3 receptors. Six ligands targeting P2Y 2 , P2Y 12 and A 2A receptors are in Phase IV under post-approval surveillance evaluation (Fig. 1A). The greater number of compounds targeting the A 2A and P2X 7 receptors highlights their potential (Significant) Indicates that the study has been successfully completed and primary outcomes are statistically significant (P < 0.05), (Not Significant) indicates that the study has been successfully completed, but primary outcomes are not statistically significant, (N/A) indicates that the study has been completed, but results have not been published, or primary outcomes are unknown. *BLT010t is a polyclonal sheep antibody that binds to the P2X7 receptor but is not an agonist or antagonist [11] [6] in pharmacotherapy ( Table 3). The A 2A and P2X 7 receptors have gained more attention, likely due to their involvement in inflammatory and immune processes [55]. For example, A 2A receptor agonists can suppress peripheral inflammation, and antagonists are used as adjuvant treatments for neuroinflammatory diseases [54]. P2X 3 , P2X 4 , P2X 7 receptors have been implicated in signalling pathways for pathological pain [65,66]. In particular, activation of P2X 3 subtype expressed in the central terminals of dorsal root ganglia increases nociception by sensitizing nerve fibers associated with the transmission of pain [65]. Blocking P2X 3 activities by selective P2X 3 antagonists shows promise in reducing pain associated with inflammatory, neuropathic, chronic, and cancer-induced conditions in preclinical studies [65]. P2X 7 receptors have also gained attention due to their role in regulating inflammation and the innate and adaptive immune responses [67]. Many studies have focused on manipulating the relation between P2X 7 receptor activation and subsequent release of inflammatory cytokines, which can promote cell proliferation or apoptotic cell death [55]. For example, the P2X 7 receptor is implicated as a mediator of cancer invasion and metastasis; hence, it has been investigated as a target to inhibit cancer progression [11,55]. As inflammation and immune processes are involved in most pathologies, sixteen different conditions were identified as targets for purine receptor-based therapies with P1 or P2 receptor ligands under clinical trial (Fig. 1B). Advanced cancer and complications associated with acute coronary syndrome (ACS) and/or cardiovascular disease (CD) appear to be the most targeted, with five different compounds in development for each condition. However, not all compounds were successful in providing significant clinical outcomes. It is not uncommon for compounds to result in less than optimal clinical efficacy when tested in humans despite displaying promising efficacy in preclinical animal studies [66]. For example, P2X 7 subtype-specific antagonist AZD9056 showed promising results in an in vivo rat model by suppressing symptoms of rheumatoid arthritis [68,69]. However, AZD9056 failed to show efficacy for rheumatoid arthritis in a Phase IIa study (NCT00520572). Another unsuccessful example is Rolofylline (KW-3902), an A 1 receptor antagonist investigated for preventing renal dysfunction. Although Rolofylline appeared to show potential benefits in improving kidney function in patients with congestive heart failure in a Phase II trial (NCT00159614) [70], it failed to show efficacy in Phase III clinical trials (NCT00354458, NCT00328692) [56] (Table 3). Although there is no definitive explanation for why some trials fail to show clinical efficacy, factors that may influence outcomes include inter-species differences in receptor function or 2017-003332-36 Completed September 2018 Significant [7] (Significant) Indicates that the study has been successfully completed and primary outcomes are statistically significant (P < 0.05), (Not significant) indicates that the study has been successfully completed, but primary outcomes are not statistically significant, (N/A) indicates that the study has been completed, but results have not been published, or primary outcomes are unknown pharmacology, variation in pharmacological profile of compounds for homomeric and heteromeric receptor complexes, and inter-individual variation in receptor function due to single nucleotide polymorphisms [65,66]. Furthermore, the ubiquitous nature of purinergic receptor expression throughout the body can potentially increase the risk of side effects that may outweigh clinical benefits [71]. However, there are many examples of successful drug development. BIL010t (formerly known as BSCT), a non-functional form of the P2X 7 receptor (nf-P2X 7 ) antibody used as topical therapy for basal cell carcinoma [11], and PBF-509, a potent A 2A receptor antagonist for the treatment of non-small cell lung cancer [60], were both well-tolerated and safe in Phase I/II trials [11] ( Table 3). The development of therapeutics generally follows an exhaustive translational research path from discovery to clinical trial. Approximately 38 compounds targeting purine receptors are at different stages of development, (Significant) Indicates that the study has been successfully completed and primary outcomes are statistically significant (P < 0.05), (Not significant) indicates that the study has been successfully completed, but primary outcomes are not statistically significant, (N/A) indicates that the study has been completed, but results have not been published, or primary outcomes are unknown. 1 3 and the most extensively studied are the P2Y 12 receptor-based antiplatelet agents clopidogrel, ticagrelor and prasugrel. These are all approved for treating complications associated with the ACS as percutaneous coronary intervention (PCI) [72]. Diquafosol (previously known as INS365), a P2Y 2 receptor agonist, is another approved drug that has been commercially successful in treating dry eye syndrome [73]. To illustrate the translational journey from basic science to clinical trials, we present case studies of diquafosol, and a family of P2Y 12 receptor antagonists.
For details on the current use of diquafosol and its tolerance by patients, the reader is directed to a review by Keating et al., 2015 [75]. Here, we will focus on the discovery and translational journey. Basic science discoveries for the target receptor of diquafosol, P2Y 2 , were published in early 2000-2003. Early work identified P2Y 2 receptor mRNA expression to be highly conserved across species, from rhesus monkeys to white rabbits, with expression observed in the conjunctiva, cornea, ciliary body, lens, and pigmented epithelium [76]. Pharmacological and functional evidence of diquafosol action on P2Y 2 receptors has stemmed from several cell line and animal models, including non-transformed bovine ciliary epithelial cells [77], human non-pigmented ciliary epithelial cells [78], rabbit ciliary epithelial cells [79], and dry-eye rat model [74]. Earlier in 1999, diquafosol was developed as a P2Y 2 -specific agonist INS365 (note, the name subsequently changed to diquafosol), effective in airway clearance in sheep [80]. At that time, Inspire Pharmaceuticals had already announced its potential use for dry eye syndrome [81]. The effect of diquafosol on ocular tissues for dry eye treatment was published in 2001 [74]. These preclinical studies were important in demonstrating the mechanism of action of P2Y 2 receptor agonists on eye physiology. For example, diquafosol administration in rabbit conjunctiva led to stimulation of Cl − and water transport from the serosal to mucosal conjunctival epithelium [82]. The observed increase in tear fluid secretion was dependent on the dose of diquafosol with no significant change in tear fluid composition [82,83]. From a clinical viewpoint, a therapeutic effect of diquafosol was demonstrated using the rat dry-eye model, as the application of diquafosol (3.0% or 8.5%) resulted in a 1.5-fold transient increase in tear fluid secretion accompanied by a decline in corneal permeability (51% compared to controls). Both effects resulted in an overall improvement in ocular surface hydration [74]. Additionally, diquafosol improved ocular surface health and mitigated corneal epithelial damage caused by superficial punctate keratopathy. An instillation of diquafosol was associated with an increase in Periodic acid-Schiff (PAS)-stained positive goblet cells in the rat dry-eye model [74] and a significant decrease in corneal fluorescein staining scores compared to controls in a diabetic rat model [84].
Following substantial research and publications between 2000 and 2003, diquafosol was granted priority in the review process by the FDA in 2003 as a diquafosol tetrasodium ophthalmic formulation [85,86], driving a cascade of clinical trials. The translation of the potential therapeutic effects observed in the preclinical studies can be illustrated by two pivotal Phase IIb and Phase III clinical trials. Both were randomized, double-blind, multi-centre trials, with a primary outcome measure defined as improving corneal and conjunctival epithelial damage after 4 weeks [87,88]. In the Phase IIb (NCT01189032) study involving 286 participants, a greater dose-dependent reduction in fluorescein corneal staining scores in week 4 was reported in patients receiving either 1% or 3% diquafosol (DQS) [87]. However, when examining the maintenance of fluorescein corneal staining scores at week 6, only the 3% DQS group scores were significantly different from placebo controls (P = 0.005) [87]. Both 1% and 3% DQS groups showed a decrease in fluorescein corneal staining scores (1% DQS, P = 0.037; 3% DQS, P = 0.002) and rose bengal corneal staining scores (1% DQS, P = 0.007; 3% DQS, P = 0.003) compared to placebo at week 4 [87]. This study included subjective dry eye sensation symptom scores as a secondary outcome measure. Patients receiving DQS showed a significant improvement in this secondary outcome measure compared with the placebo (1% DQS, P = 0.003; 3% DQS, P = 0.033) [87]. Concerning dosage, 3% DQS was superior to 1% DQS in efficacy [87]. The efficacy of 3% DQS was also superior to 0.1% sodium hyaluronate ophthalmic solution [89]. Similar to the Phase IIb study, both fluorescein and rose bengal corneal staining scores showed significant improvement in both treatment groups at weeks 2 and 4 (P < 0.05), although the 3% DQS treatment group exhibited greater improvement in mucin coating on the ocular surface at week 4 than 0.1% sodium hyaluronate [89]. Similar improvement in subjective dry-eyerelated symptoms and corneal and conjunctival fluorescein staining scores was also observed in a larger Phase III study involving 3196 patients with dry eye disease conducted over two months (P < 0.001) [88]. Adverse reactions to 3% DQS treatment affected 6.3% of patients, including eye discharge, eye irritation, and eye pain [88]. Additional information regarding the effectiveness of 3% DQS as an intervention for dry-eye syndrome was delineated in a Phase IV study involving 580 patients with dry eye disease [90]. It was a prospective, multi-centre, open-label observational study conducted over 12 months [90]. Significant improvements in fluorescein corneal staining scores were observed at 3, 6, 9 and 12 months with DQS treatment (P < 0.001) [388]. Moreover, DQS treatment was associated with a significant reduction in other outcome measures such as Dry Eye-related Duality of Life Scores, ocular symptoms scores, and impact on daily life scores (P < 0.001) [90]. However, it should be noted that the open-label design of this study can be subject to bias in favor of DQS treatment in efficacy measures [90]. The study population included a higher proportion of elderly patients (42.8%, ≥ 70 years), which should also be taken into consideration [90]. DQS 3.0% ophthalmic solution was approved as an intervention for dry eye in 2010 by the Ministry of Health in Japan [91] and is now widely available in other countries, including South Korea, Indonesia, Malaysia, Philippines, Thailand, Vietnam, Cambodia and China [92]. The contributing factors to the success of diquafosol can be attributed to its localized direct delivery in the form of an 'eye-drop' and ease of assessment of the target tissue.

P2Y 12 receptor antagonist: thienopyridines prasugrel and cangrelor
The discovery of P2Y 12 as the drug target for coronary diseases dates back to 1961, when ADP was found to play a functional role in platelet activation and aggregation [93]. The breakthrough came in 2001 when the P2Y 12 receptor was cloned and recognized as the molecular identity of the receptor responsible for triggering the potent ADP-induced antithrombotic activity [94]. Strikingly, the expression of P2Y 12 was found to be very specific to platelets and showed negligible expression in most other tissues [94]. The strong link between the P2Y 12 and ADP-induced platelet aggregation has set a rapid rise in investigations of P2Y 12 as a potential therapeutic target for blood clotting conditions and led to the development of thienopyridine prodrugs [95,96]. Thienopyridines are a family of closely related prodrugs. As prodrugs, thienopyridines need to be metabolized by a hepatic enzyme, Cytochrome P-450 (CYP), into the active metabolite to bind to the P2Y12 receptor irreversibly [97]. This blocks ADP binding, subsequently inhibiting platelet activation and aggregation [97]. The actions of thienopyridines are specific to P2Y 12 receptor and selectively interfere with platelet activation and aggregation induced by ADP [98,99]. Subsequent research has led to the development of at least three thienopyridine compounds in the market; firstgeneration ticlopidine (Ticlid ®), second-generation clopidogrel bisulfate (Plavix®) and third-generation prasugrel (Effient®) [100,101].
Thienopyridine antiplatelet drugs are indicated for managing and preventing complications after ACS and PCI, including ischemic complications, myocardial infarction 1 3 (MI), and stent thrombosis [102], albeit with slightly different properties. The first generation ticlopidine was replaced by the second generation clopidogrel due to a better tolerability profile but with similar efficacy. Soon dual antiplatelet therapy with aspirin and clopidogrel became the 'gold standard' for patients undergoing stenting and acute coronary syndromes [100,103]. However, reports of inter-individual variability in responsiveness to clopidogrel from in vitro studies sparked concerns, potentially explained by genetic polymorphisms and cytochrome P-450 polymorphisms, which can manifest as differential pharmacodynamic and therapeutic responses [104]. A heightened platelet reactivity (thienopyridine hypersensitivity reaction) or clopidogrel non-responsiveness (also referred to as clopidogrel resistance) was associated with a high risk of adverse ischemic events such as stent thrombosis [100,105]. This supported the development of the third-generation thienopyridine prasugrel, which exhibited a superior pharmacodynamic profile to clopidogrel with less interpatient variability and a more potent platelet aggregation response [100,103,105]. Pharmacological and functional evidence of prasugrel (CS-747, LY640315, Effient®), 5-[2-cyclopropyl-1-pyridin-2yl acetate] and P2Y 12 receptors have been attributed to preclinical research across a number of animal models including rats, beagle dogs, and cynomolgus monkeys [95,106,107]. One of the first studies to evaluate the therapeutic effect of prasugrel (CS-747) used a rat model, where single oral administration of prasugrel (0.3-3 mg/kg) produced a dose-related inhibition of ex vivo ADP-induced aggregation in washed platelets [95]. The same dose-dependent inhibition of platelet aggregation following oral administration of prasugrel was observed in a rat model where maximum inhibition was achieved 2-4 h after dosing [106]. A similar potent, dose-related inhibition of ADP-induced platelet aggregation was observed in beagle dogs (0.03-3 mg/kg/ day), cynomolgus monkeys (0.1-0.3 mg/kg/day), and rats (3 mg/kg/day) across a 14-day treatment period. Inhibition reached a plateau on days 3 and 5, suggesting a cumulative effect [107]. The order of potency (from high to low) in different animal models was: dogs, humans, monkeys, and rats [107]. When comparing the antiplatelet and antithrombotic potency of prasugrel with its predecessors, the potency of prasugrel exceeded that of clopidogrel and ticlopidine in a rat arterio-venous shunt model [95]. In an ex vivo rat model, clopidogrel exhibited a slower onset of action and antiplatelet potency 13 times lower than prasugrel [106]. A dose-dependency study of prasugrel (0.1-1 mg/kg/day, p.o.) in a rat carotid arterial thrombosis model demonstrated dose-related prolongation of the time to arterial occlusion. CS-747 (prasugrel) had approximately tenfold and 100-fold higher potency when compared to clopidogrel (1-20 mg/kg/ day, p.o.) and ticlopidine (30-300 mg/kg/day, p.o.), respectively [107].
The progress of prasugrel from the preclinical studies to clinical trials can be followed from the Phase II JUMBO-TIMI 26 study [108], which served as a feasibility study for one of the pivotal Phase III studies, TRITON-TIMI 38 [109]. The JUMBO-TIMI 26 study involved 904 patients and was the first to report on the use of prasugrel in patients undergoing elective or urgent PCI [97]. Patients were randomized into 1 of 3 prasugrel dosing regimens: low dose (40 mg loading dose (LD) and 7.5 mg maintenance dose (MD)), intermediatedose (60 mg LD and 10 mg MD), and a high-dose (60 mg LD and 10 mg MD). A control group treated with clopidogrel (300 mg LD and 75 mg MD) was also included [108]. Although patients receiving prasugrel had a slightly lower incidence of major adverse cardiac events, including myocardial infarction, stroke, recurrent myocardial ischemia requiring hospitalization, and thrombosis (7.2%) compared to patients receiving clopidogrel (9.4%), the difference was not statistically significant (P = 0.26) [108]. Patients receiving prasugrel had slightly lower rates (0.5%) of major bleeding, significant bleeding and transfusion events compared to the clopidogrel group (0.8%), although it also was not statistically significant (P = 0.590) [108]. TRITON-TIMI 38 (NCT00097591) was a double-blind, double-dummy, parallel-group, multi-centre, multinational Phase III clinical trial [109]. It involved 13,608 subjects with moderate-to high-risk ACS with planned PCI who were randomized to receive either clopidogrel (300 mg LD and 75 mg MD) or prasugrel (60 mg LD and 10 mg MD) daily for 6-15 months [109]. The primary outcome measure was defined as a combination of cardiovascular death, nonfatal MI, or urgent target vessel revascularization at 30 days [109]. Patients receiving prasugrel reported fewer cardiovascular events (primary outcome measure composite) of 9.9% compared to 12.1% reported from patients receiving clopidogrel (Hazard Ratio (HR), 0.81; 95% Confidence Interval (CI), 0.73-0.090; P < 0.001) [109]. This was driven by a significant reduction in ischemic events among patients receiving prasugrel including myocardial infarction (9.7% for clopidogrel vs. 7.4% for prasugrel; P < 0.001), urgent target vessel revascularization (3.7% for clopidogrel vs. 7.4% for prasugrel; P < 0.001), and stent thrombosis (2.4% clopidogrel versus 1.1% prasugrel; P < 0.001), but there was no significant difference in the rate of stroke [109]. However, prasugrel treatment was associated with an increase in the rate of major bleeding (2.4% prasugrel vs. 1.8% clopidogrel; HR, 1.32; 95% CI, 1.03-1.68; P = 0.03) and life-threatening bleeding (1.4% vs. 0.9%; P = 0.01) events, inclusive of nonfatal bleeding (1.1% vs. 0.9%; HR, 1.25; P = 0.23) and fatal bleeding (0.4% vs. 0.1%; P = 0.002) [109]. There was no significant difference in overall mortality between treatment groups [109]. Notably, in a different Phase III study, TRILOGY-ACS trial (NCT00699998), the more serious or life-threatening bleeding events observed with the prasugrel group in the TRITON study were not observed [110]. This particular trial had a long follow-up of up to 2.5 years, and the risk of the major bleeding event was observed to be similarly low in the two treatment groups, prasugrel (10 mg daily) and clopidogrel (75 mg daily) [110,111]. As for the efficacy, at 30 months, no significant difference was observed in the rate of death from cardiovascular causes, MI or stroke among patients under the age of 75 (13.9% in the prasugrel group and 16% in the clopidogrel group; HR 0.91; 95% CI 0.79-1.05; P = 0.21) [110]. However, a lower risk of multiple recurrent ischemic events was observed among patients under the age of 75 receiving prasugrel (6%) compared to the clopidogrel group (13%) after 12 months of treatment (HR 0.94; 95% CI 0.79-0.86; P = 0.018), consistent with findings from the TRITON study [109,110]. In July 2009, prasugrel gained FDA approval as an intervention for reducing thrombotic cardiovascular events in patients with ACS managed with PCI, with a warning indicating a higher risk of bleeding events [112]. Several years after prasugrel became available for clinical use, some concerns were raised in subsequent analysis from the TRILOGY-ACS trial regarding the long-term effects of prasugrel and clopidogrel [113]. In 2020, the manufacturer, Eli Lilly and Co., discontinued production of prasugrel as a business decision; hence it is no longer available in Canada [114] and New Zealand [115]. After many years of success, the gap left by the withdrawal of prasugrel further prompted the use and investigation of reversible non-thienopyridine agents ticagrelor and, most recently, cangrelor.
Cangrelor (also known as AR-C69931MX) is a nonthienopyridine ATP analogue that has different actions to thienopyridines, and is a class of selective antagonists of the P2Y 12 receptor [116]. Similar to thienopyridine, cangrelor is a direct-acting antagonist of the P2Y 12 receptor; however, unlike thienopyridines, the inhibitory action of cangrelor is reversible [116]. It is delivered intravenously and is characterized by a rapid onset of action with a fast offset of effects due to its short plasma half-life of 3 to 6 min [116]. Furthermore, due to the drug's short half-life, the platelet function returns to normal within 30 to 60 min after intravenous infusion of Cangrelor [116]. This fast termination of action makes Cangrelor an attractive compound by reducing the risk of potential ischemic or thrombotic complications [116][117][118]. In a Phase II BRIDGE trial (Bridging Anti-Platelet Therapy With Intravenous Agent Cangrelor In Patients Undergoing Cardiac Surgery, NCT00767507), a greater proportion of patients with ACS treated with Cangrelor (0.75 μg/kg per minute) had low levels of platelet reactivity during the treatment period compared with placebo (P < 0.001), with a low risk of thrombotic events [117]. However, in a large-scale international Phase III trial, CHAMPION (Cangrelor Versus Standard Therapy to Achieve Optimal Management of Platelet Inhibition) PCI trial (NCT00305162) reported a contradictory outcome that cangrelor (bolus of 30 μg/kg plus an infusion at 4 μg/kg per minute) was not superior to clopidogrel (loading dose of 600 mg) in reducing the primary composite death, myocardial infarction, or ischemiadriven revascularization at 48 h (P = 0.59) in patients with ACS before PCI [118]. This study was terminated due to insufficient evidence of the clinical effectiveness of cangrelor [6]. Another separate Phase III trial, CHAM-PION PHOENIX (NCT01156571) reported that cangrelor significantly reduced the rate of myocardial infarction, ischemia-driven revascularization, or stent thrombosis at 48 h (adjusted odds ratio [OR] with Cangrelor, 0.78; 95% CI, 0.66 to 0.93; P = 0.005) with a lower rate of stent thrombosis compared to clopidogrel (OR, 0.62; 95% CI, 0.43 to 0.90; P = 0.01) [119]. Although several ongoing studies will likely provide additional insight into the clinical use of cangrelor (Table 2), further evaluations on the safety and efficacy of cangrelor are needed.

Conclusion
The development of therapies targeting purine receptors has been a long journey that began with basic research characterising P1 and P2 receptors in health and disease, and this led to the identification of potential therapeutic targets for different conditions. Through the insights gained from preclinical studies and with the increasing interest from clinical researchers, the development of therapies targeting purine receptors became a reality. With 38 therapeutic compounds currently in clinical trials, we should expect more to emerge over the next few decades. Furthermore, building on our knowledge of purinergic signalling in various tissues and the existence of many drug candidates in the pipeline, repurposing the existing drugs as alternative pathways for drug development should also be considered. Substantial progress made in the last two decades is a true reflection of Professor Geoffrey Burnstock's legacy that has established the field of purinergic signalling and paved the way for the development of purine receptor targeting therapies for several diseases and clinical conditions. Data availability Clinical trial information included in this publication are available in a public repository specified in the manuscript.

Declarations
Conflicts of interest S. Han declares that she has no conflict of interest. H. Suzuki-Kerr declares that he/she has no conflict of interest. S. M. Vlajkovic declares that he/she has no conflict of interest. P. R. Thorne declares that he/she has no conflict of interest.
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. troponin-negative acute coronary syndrome patients undergoing ad hoc percutaneous coronary intervention. J Am Heart Assoc 6(4