Neuroprotective Roles of the Adenosine A3 Receptor Agonist AST-004 in Mouse Model of Traumatic Brain Injury

Abstract

Background: Traumatic brain injury (TBI) remains one of the greatest public health concerns with increasing morbidity and mortality rates worldwide. Our group reported stimulation of astrocyte mitochondrial metabolism by P2Y1 receptor agonists significantly reduced cerebral edema and reactive gliosis in a TBI model. Subsequent data on the pharmacokinetics (PK) and rapid metabolism of these compounds suggested neuroprotection was likely mediated by a metabolite, AST-004, which binding data indicated was an adenosine A3 receptor (A3R) agonist. Methods: The neuroprotective efficacy of AST-004 was tested in a controlled closed cortical injury (CCCI) model of TBI in mice. Results: Twenty-four (24) hours post-injury, mice subjected to CCCI and treated with AST-004 (0.22mg/kg) exhibited significantly less secondary brain injury. These effects were quantified with less cell death (PSVue794 fluorescence) and loss of blood brain barrier breakdown (Evans Blue extravasation assay), compared to vehicle treated TBI mice. TBI treated mice also exhibited significantly reduced neuroinflammatory markers, glial-fibrillary acidic protein (GFAP, astrogliosis) and ionized Ca2+ binding adaptor molecule 1 (Iba1, microgliosis), both at the mRNA (gRT-PCR) and protein (western blot and immunofluorescence) levels, respectively. Four (4) weeks post-injury, AST-004 treated TBI mice presented significantly reduced impairment of long-term memory. Spatial memory was assessed with a contextual fear conditioning behavior assay (freezing behavior after shock). Finally, AST-004 treatments were found to increase in vivo ATP production in astrocytes (gfap-targeted luciferase activity), consistent with the proposed mechanism of action. Conclusions: These data reveal AST-004 as a novel A3R agonist that increases astrocyte energy production and enhances their neuroprotective efficacy after brain injury.

Adenosine A1R/A3R Agonist AST-004 Reduces Brain Infarction in Mouse and Rat Models of Acute Ischemic Stroke

“…AST-004 is a lower affinity A1R/A3R agonist that exhibits significant efficacy as shown here and reported previously 14,26 . The cerebroprotective benefits of AST-004 were completely blocked by the A3R antagonist MRS1523, suggesting A1R agonism is not required.…”

“…Subsequent work showed these phosphorylated nucleotides were rapidly metabolized in vivo and that the active cerebroprotective compounds were likely the metabolites of MRS2365 and 2MeSADP, AST-004 and 2-methylthioadenosine, respectively 12 . We confirmed AST-004 treatments were cerebroprotective after TBI in mice 26 and tMCAO in non-human primates 14 . Binding studies showed AST-004 was primarily an A3R agonist with some affinity for A1Rs 12 .…”

“…Receptor binding models indicate significant efficacy is observed at estimated brain receptor occupancy levels as low as 5% in a non-human primate model of stroke 14 . In light of these results, we tested AST-004 efficacy at doses an order of magnitude lower and higher than previously reported in mice 26 . All AST-004 concentrations were cerebroprotective against stroke, and the lower and higher doses were not significantly different from the mid-dose in mice.…”

“…In light of these results, we tested AST-004 efficacy at doses an order of magnitude lower and higher than previously reported in mice 26 . All AST-004 concentrations were cerebroprotective against stroke, and the lower and higher doses were not significantly different from the mid-dose in mice.…”

“…We also found increased levels of A3R mRNA following stroke, revealing an intrinsic protective response that could be exploited for treatment. A3R agonists have been reported as cerebroprotective against stroke since the mid-1990s [15][16][17][18][19][20][21][22][23][24][25] , and most recently for TBI 26,27 . Together, these studies confirm that certain A3R agonists, such as AST-004, may be exciting new treatment options that need to be developed for clinical evaluation.…”