Lack of Interactions Between Alteplase/Tenecteplase and the Adenosine A1R/A3R Agonist AST-004.

BACKGROUND: AST-004, a small molecule agonist of the adenosine A1 and A3 receptors, is a potential cerebroprotectant for patients with acute stroke and is currently in clinical trials. Drug-drug interactions are critically important to assess in the context of acute stroke care. Lytic therapy with tPA (tissue-type plasminogen activator)-induced plasmin formation (alteplase) is the only available pharmacotherapy for acute stroke. Consequently, it is imperative to evaluate potential interactions between AST-004 and tPAs such as alteplase and tenecteplase. METHODS: The interactions between AST-004 and tPAs were evaluated in 3 ways in preparation for AST-004 phase II trials. First, the metabolic stability of AST-004 was determined in the presence of alteplase and plasmin. Second, the potential for AST-004 to influence the thrombolytic efficacy of alteplase and tenecteplase was evaluated with an in vitro assay system utilizing a fluorogenic substrate of plasmin. Finally, the potential for AST-004 to influence the thrombolytic efficacy of alteplase was also determined with an in vitro thrombolysis assay of human blood thrombi. RESULTS: Neither alteplase nor plasmin affected the stability of AST-004 in vitro. In 2 different in vitro systems, AST-004 had no effect on the ability of alteplase or tenecteplase to generate plasmin, and AST-004 had no effect on the thrombolytic efficacy of alteplase to lyse blood clots in human blood. CONCLUSIONS: These studies indicate that there will be no interactions between AST-004 and tPAs such as alteplase or tenecteplase in patients with stroke undergoing thrombolytic therapy.

Adenosine A1R/A3R (Adenosine A1 and A3 Receptor) Agonist AST-004 Reduces Brain Infarction in a Nonhuman Primate Model of Stroke.

BACKGROUND AND PURPOSE: Treatment with A1R/A3R (adenosine A1 and A3 receptor) agonists in rodent models of acute ischemic stroke results in significantly reduced lesion volume, indicating activation of adenosine A1R or A3R is cerebroprotective. However, dosing and timing required for cerebroprotection has yet to be established, and whether adenosine A1R/A3R activation will lead to cerebroprotection in a gyrencephalic species has yet to be determined. METHODS: The current study used clinical study intervention timelines in a nonhuman primate model of transient, 4-hour middle cerebral artery occlusion to investigate a potential cerebroprotective effect of the dual adenosine A1R/A3R agonist AST-004. Bolus and then 22 hours intravenous infusion of AST-004 was initiated 2 hours after transient middle cerebral artery occlusion. Primary outcome measures included lesion volume, lesion growth kinetics, penumbra volume as well as initial pharmacokinetic-pharmacodynamic relationships measured up to 5 days after transient middle cerebral artery occlusion. Secondary outcome measures included physiological parameters and neurological function. RESULTS: Administration of AST-004 resulted in rapid and statistically significant decreases in lesion growth rate and total lesion volume. In addition, penumbra volume decline over time was significantly less under AST-004 treatment compared with vehicle treatment. These changes correlated with unbound AST-004 concentrations in the plasma and cerebrospinal fluid as well as estimated brain A1R and A3R occupancy. No relevant changes in physiological parameters were observed during AST-004 treatment. CONCLUSIONS: These findings suggest that administration of AST-004 and combined A1R/A3R agonism in the brain are efficacious pharmacological interventions in acute ischemic stroke and warrant further clinical evaluation.

Nucleotide P2Y1 receptor agonists are in vitro and in vivo prodrugs of A1/A3 adenosine receptor agonists: implications for roles of P2Y1 and A1/A3 receptors in physiology and pathology.

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y1 receptor (P2Y1R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A3 and A1ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y1R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.