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.

Selective A3 Adenosine Receptor Antagonist Radioligand for Human and Rodent Species.

The A3 adenosine receptor (A3AR) is a target for pain, ischemia, and inflammatory disease therapy. Among the ligand tools available are selective agonists and antagonists, including radioligands, but most high-affinity non-nucleoside antagonists are limited in selectivity to primate species. We have explored the structure-activity relationship of a previously reported A3AR antagonist DPTN 9 (N-[4-(3,5-dimethylphenyl)-5-(4-pyridyl)-1,3-thiazol-2-yl]nicotinamide) for radiolabeling, including 3-halo derivatives (3-iodo, MRS7907), and characterized 9 as a high -affinity radioligand [3H]MRS7799. A3AR K d values were (nM): 0.55 (human), 3.74 (mouse), and 2.80 (rat). An extended methyl acrylate (MRS8074, 19) maintained higher affinity (18.9 nM) than a 3-((5-chlorothiophen-2-yl)ethynyl) derivative 20. Compound 9 had an excellent brain distribution in rats (brain/plasma ratio ∼1). Receptor docking predicted its orthosteric site binding by engaging residues that were previously found to be essential for AR binding. Thus the new radioligand promises to be a useful species-general antagonist tracer for receptor characterization and drug discovery.

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.

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.

Convergent synthesis of 2-thioether-substituted (N)-methanocarba-adenosines as purine receptor agonists.

A linear route has been used to prepare (N)-methanocarba-nucleoside derivatives, which serve as purine receptor ligands having a pre-established, receptor-preferred conformation. To introduce this rigid ribose substitute, a Mitsunobu reaction of a [3.1.0]bicyclohexane 5'-trityl intermediate 3 with a nucleobase is typically followed by functional group modifications. We herein report an efficient scalable convergent synthesis for 2-substituted (N)-methanocarba-adenosines, which were demonstrated to bind to the A3 adenosine receptor. The adenine moiety was pre-functionalized with 2-thioethers and other groups before coupling to the bicyclic precursor (3) as a key step to facilitate a high yield Mitsunobu product. This new approach provided the (N)-methanocarba-adenosines in moderate to good yield, which effectively increased the overall yield compared to a linear synthesis and conserved a key intermediate 3 (a product of nine sequential steps). The generality of this convergent synthesis, which is suitable as an optimized preclinical synthetic route, was demonstrated with various 2-thioether and 2-methoxy substituents.

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.

Development of a new permeability assay using low-efflux MDCKII cells.

Permeability is an important property of drug candidates. The Madin-Darby canine kidney cell line (MDCK) permeability assay is widely used and the primary concern of using MDCK cells is the presence of endogenous transporters of nonhuman origin. The canine P-glycoprotein (Pgp) can interfere with permeability and transporter studies, leading to less reliable data. A new cell line, MDCKII-LE (low efflux), has been developed by selecting a subpopulation of low-efflux cells from MDCKII-WT using an iterative fluorescence-activated cell sorting technique with calcein-AM as a Pgp and efflux substrate. MDCKII-LE cells are a subpopulation of MDCKII cells with over 200-fold lower canine Pgp mRNA level and fivefold lower protein level than MDCKII-WT. MDCKII-LE cells showed less functional efflux activity than MDCKII-WT based on efflux ratios. Notably, MDCKII-MDR1 showed about 1.5-fold decreased expression of endogenous canine Pgp, suggesting that using the net flux ratio might not completely cancel out the background endogenous transporter activities. MDCKII-LE cells offer clear advantages over the MDCKII-WT by providing less efflux transporter background signals and minimizing interference from canine Pgp. The MDCKII-LE apparent permeability values well differentiates compounds from high to medium/low human intestinal absorption and can be used for Biopharmaceutical Classification System. The MDCKII-LE permeability assay (4-in-1 cassette dosing) is high throughput with good precision, reproducibility, robustness, and cost-effective.

Species independence in brain tissue binding using brain homogenates.

Species independence of brain tissue binding was assessed with a large number of structurally diverse compounds using equilibrium dialysis with brain homogenates of seven species and strains (Wistar Han rat, Sprague-Dawley rat, CD-1 mouse, Hartley guinea pig, beagle dog, cynomolgus monkey, and human). The results showed that the fractions unbound of the seven species and strains were strongly correlated with correlation coefficients ranging from 0.93 to 0.99. The cross-species/strain correlations were not significantly different from the interassay correlation with the same species. The linear correlation between Wistar Han and other species had a slope close to 1 and an intercept near 0. Based on orthogonal statistical analysis, no correction is needed for extrapolation of fraction unbound from Wistar Han rat to the other species or strains. Hence, brain tissue binding of Wistar Han rat can be used to obtain binding of other species and strains in drug discovery.

High throughput ADME screening: practical considerations, impact on the portfolio and enabler of in silico ADME models.

Evaluation and optimization of drug metabolism and pharmacokinetic data plays an important role in drug discovery and development and several reliable in vitro ADME models are available. Recently higher throughput in vitro ADME screening facilities have been established in order to be able to evaluate an appreciable fraction of synthesized compounds. The ADME screening process can be dissected in five distinct steps: (1) plate management of compounds in need of in vitro ADME data, (2) optimization of the MS/MS method for the compounds, (3) in vitro ADME experiments and sample clean up, (4) collection and reduction of the raw LC-MS/MS data and (5) archival of the processed ADME data. All steps will be described in detail and the value of the data on drug discovery projects will be discussed as well. Finally, in vitro ADME screening can generate large quantities of data obtained under identical conditions to allow building of reliable in silico models.

Perspectives on bioanalytical mass spectrometry and automation in drug discovery.

The use of high speed synthesis technologies has resulted in a steady increase in the number of new chemical entities active in the drug discovery research stream. Large organizations can have thousands of chemical entities in various stages of testing and evaluation across numerous projects on a weekly basis. Qualitative and quantitative measurements made using LC/MS are integrated throughout this process from early stage lead generation through candidate nomination. Nearly all analytical processes and procedures in modern research organizations are automated to some degree. This includes both hardware and software automation. In this review we discuss bioanalytical mass spectrometry and automation as components of the analytical chemistry infrastructure in pharma. Analytical chemists are presented as members of distinct groups with similar skillsets that build automated systems, manage test compounds, assays and reagents, and deliver data to project teams. The ADME-screening process in drug discovery is used as a model to highlight the relationships between analytical tasks in drug discovery. Emerging software and process automation tools are described that can potentially address gaps and link analytical chemistry related tasks. The role of analytical chemists and groups in modern 'industrialized' drug discovery is also discussed.