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

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; first-generation 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 (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)), intermediate-dose (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, non-fatal 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 non-thienopyridine 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 – 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 ischemia-driven 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, CHAMPION 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.

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 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 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 ). Fig. 1 Summary of therapeutic compounds in clinical trials targeting purine receptors for specific diseases/conditions. ( A ) Therapeutic compounds targeting purine receptors in clinical development from Phase I–IV. ( B ) Purine receptors targeted for specific diseases/conditions in clinical development from Phase I–IV, created with BioRender.com Summary of therapeutic compounds in clinical trials targeting purine receptors for specific diseases/conditions. ( A ) Therapeutic compounds targeting purine receptors in clinical development from Phase I–IV. ( B ) Purine receptors targeted for specific diseases/conditions in clinical development from Phase I–IV, created with BioRender.com 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, 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.

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.