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.

HspB1 Overexpression Improves Life Span and Stress Resistance in an Invertebrate Model.

To explore the role of the small heat shock protein beta 1 (HspB1, also known as Hsp25 in rodents and Hsp27 in humans) in longevity, we created a Caenorhabiditis elegans model with a high level of ubiquitous expression of the naked mole-rat HspB1 protein. The worms showed increased life span under multiple conditions and also increased resistance to heat stress. RNAi experiments suggest that HspB1-induced life extension is dependent on the transcription factors skn-1 (Nrf2) and hsf-1 (Hsf1). RNAseq from HspB1 worms showed an enrichment in several skn-1 target genes, including collagen proteins and lysosomal genes. Expression of HspB1 also improved functional outcomes regulated by SKN-1, specifically oxidative stress resistance and pharyngeal integrity. This work is the first to link a small heat shock protein with collagen function, suggesting a novel role for HspB1 as a hub between canonical heat response signaling and SKN-1 transcription.

Adenosine A1R/A3R agonist AST-004 reduces brain infarction in mouse and rat models of acute ischemic stroke.

Acute ischemic stroke (AIS) is the second leading cause of death globally. No Food and Drug Administration (FDA) approved therapies exist that target cerebroprotection following stroke. Our group recently reported significant cerebroprotection with the adenosine A1/A3 receptor agonist, AST-004, in a transient stroke model in non-human primates (NHP) and in a preclinical mouse model of traumatic brain injury (TBI). However, the specific receptor pathway activated was only inferred based on in vitro binding studies. The current study investigated the underlying mechanism of AST-004 cerebroprotection in two independent models of AIS: permanent photothrombotic stroke in mice and transient middle cerebral artery occlusion (MCAO) in rats. AST-004 treatments across a range of doses were cerebroprotective and efficacy could be blocked by A3R antagonism, indicating a mechanism of action that does not require A1R agonism. The high affinity A3R agonist MRS5698 was also cerebroprotective following stroke, but not the A3R agonist Cl-IB-MECA under our experimental conditions. AST-004 efficacy was blocked by the astrocyte specific mitochondrial toxin fluoroacetate, confirming an underlying mechanism of cerebroprotection that was dependent on astrocyte mitochondrial metabolism. An increase in A3R mRNA levels following stroke suggested an intrinsic cerebroprotective response that was mediated by A3R signaling. Together, these studies confirm that certain A3R agonists, such as AST-004, may be exciting new therapeutic avenues to develop for AIS.

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

Traumatic brain injury (TBI) remains one of the greatest public health concerns with increasing morbidity and mortality rates worldwide. Our group reported that 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 that neuroprotection was likely mediated by a metabolite, AST-004, which binding data indicated was an adenosine A3 receptor (A3R) agonist. The neuroprotective efficacy of AST-004 was tested in a control closed cortical injury (CCCI) model of TBI in mice. Twenty-four (24) hours post-injury, mice subjected to CCCI and treated with AST-004 (0.22 mg/kg, injected 30 min post-trauma) 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 (qRT-PCR) and protein (Western blot and immunofluorescence) levels, respectively. Four (4) weeks post-injury, both male and female TBI mice presented a significant reduction in freezing behavior during contextual fear conditioning (after foot shock). AST-004 treatment prevented this TBI-induced impairment in male mice, but did not significantly affect impairment in female mice. Impairment of spatial memory, assessed 24 and 48 h after the initial fear conditioning, was also reduced in AST-004-treated TBI-male mice. Female TBI mice did not exhibit memory impairment 24 and 48 h after contextual fear conditioning and similarly, AST-004-treated female TBI mice were comparable to sham mice. 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. These data reveal AST-004 as a novel A3R agonist that increases astrocyte energy production and enhances their neuroprotective efficacy after brain injury.