First-in-Human Phase I Clinical Trial of the Adenosine A1R/A3R Agonist AST-004 in Healthy Subjects.

BACKGROUND: AST-004 is a small-molecule adenosine A1/A3 receptor agonist that has exhibited significant cerebroprotective efficacy in preclinical models of acute ischemic stroke and traumatic brain injury. The primary objectives of this clinical phase I first-in-human study were to evaluate the safety and tolerability profile of single ascending intravenous doses in healthy subjects. The secondary objectives were to characterize the single-dose pharmacokinetic profiles in plasma, cerebrospinal fluid (CSF), and urine. METHODS: In part 1 of the study, AST-004 was administered in ascending dose cohorts of 5, 25, 50, 75, and 100 mg, with 6 subjects in each cohort receiving the study drug and 2 receiving placebo. In part 2, all 12 subjects received a 100 mg IV infusion of the study drug followed by a single CSF collection per subject via lumbar puncture at 20, 40, or 60 minutes after infusion. RESULTS: A total of 42 subjects received AST-004, with no severe or serious adverse events observed. Twelve of these subjects experienced a treatment-emergent adverse event, the most frequent across groups being headache. In part 2, pharmacokinetic analyses confirmed that AST-004 was distributed in the CSF, with the CSF-to-plasma ratio increasing over the 3 timepoints sampled. The mean half-life was 1.1 to 1.4 hours for doses of 25 to 100 mg, and the geometric mean maximum plasma concentration obtained in the highest dosing cohort (100 mg) was 2232±428 ng/mL. CONCLUSIONS: AST-004 was safe and well-tolerated at plasma concentrations 3 to 8× higher than those associated with significant efficacy in astrocyte's preclinical primate stroke efficacy studies, with CSF concentrations highest at the 60-minute collection timepoint, the last timepoint tested. This study supports additional clinical investigations, including evaluation of an extended infusion to support the phase 2 program in stroke and traumatic brain injury.

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.

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.

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.

Behavioral responses of beluga whales (Delphinapterus leucas) to environmental variation in an Arctic estuary.

Some Arctic estuaries serve as substrate rubbing sites for beluga whales (Delphinapterus leucas) in the summer, representing a specialized resource for the species. Understanding how environmental variation affects the species' behavior is essential to management of these habitats in coming years as the climate changes. Spatiotemporal and environmental variables were recorded for behavioral observations, during which focal groups of whales in an estuary were video-recorded for enumeration and behavioral analysis. Multiple polynomial linear regression models were optimized to identify the effects of spatiotemporal and environmental conditions on group size, composition, and the frequency of behaviors being performed. Results suggest that belugas take advantage of environmental variation to express behaviors that 1) protect young, e.g., bringing calves close to shore during cloudier days, obscuring visualization from terrestrial predators; 2) avoid predation, e.g., rubbing against substrates at higher Beaufort sea states to obscure visualization, and resting during low tides while swimming on outgoing tides to avoid stranding; and 3) optimize bioenergetic resources, e.g., swimming during lower Beaufort sea states and clearer days. Predictive models like the ones presented in this study can inform conservation management strategies as environmental conditions change in future years.

Stable isotopic analysis of porcine, bovine, and ovine heparins.

The assessment of provenance of heparin is becoming a major concern for the pharmaceutical industry and its regulatory bodies. Batch-specific [carbon (δ(13) C), nitrogen (δ(15) N), oxygen (δ(18) O), sulfur (δ(34) S), and hydrogen (δD)] stable isotopic compositions of five different animal-derived heparins were performed. Measurements readily allowed their differentiation into groups and/or subgroups based on their isotopic provenance. Principle component analysis showed that a bivariate plot of δ(13) C and δ(18) O is the best single, bivariate plot that results in the maximum discrimination ability when only two stable isotopes are used to describe the variation in the data set. Stable isotopic analyses revealed that (1) stable isotope measurements on these highly sulfated polysaccharide (molecular weight ∼15 kDa) natural products ("biologics") were feasible; (2) in bivariate plots, the δ(13) C versus δ(18) O plot reveals a well-defined relationship for source differentiation of hogs raised in the United States from hogs raised in Europe and China; (3) the δD versus δ(18) O plot revealed the most well-defined relationship for source differentiation based on the hydrologic environmental isotopes of water (D/H and (18) O/(16) O); and (4) the δ(15) N versus δ(18) O and δ(34) S versus δ(18) O relationships are both very similar, possibly reflecting the food sources used by the different heparin producers.