WRC-0470 is a selective agonist for the A₂A adenosine receptor. Its reported dissociation constant (KD) values for human recombinant adenosine receptors are: A₂A, 270 nM; A₁, 48 μM; A₂B, 430 μM; and A₃, 903 μM, demonstrating high selectivity for the A₂A subtype [1].

In human whole blood, binodenson (MRE-0470) (30-300 nM) operates in concert with Rolipram to decrease the oxidative activity of FMLP-stimulated polymorphonuclear leukocytes caused by tumor necrosis factor-alpha [1].

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In isolated human polymorphonuclear leukocytes (PMNL) adhering to a fibrinogen-coated surface, WRC-0470 (300 nM) alone did not significantly affect TNF-α-stimulated PMNL adherence. However, when combined with the type IV phosphodiesterase inhibitor rolipram (300 nM), it synergistically increased PMNL [cAMP] to 138% of control and significantly decreased TNF-α-enhanced adherence (P = .014). These effects were blocked by the selective A₂A antagonist ZM241385 (100 nM) [1].
In TNF-α-stimulated adherent human PMNL, WRC-0470 (30-300 nM) alone did not significantly inhibit superoxide release at 90 minutes. However, in combination with rolipram (100 nM), it synergistically and dose-dependently decreased superoxide release (P < .05). The combination of WRC-0470 (300 nM) and rolipram (300 nM) also prevented the rise in superoxide release over a 150-minute time course, an effect completely reversed by ZM241385 (100 nM) [1].
In human whole blood, WRC-0470 (300 nM) combined with rolipram (300 nM) synergistically decreased the oxidative activity of TNF-α-primed, FMLP-stimulated PMNL to levels lower than those observed in unprimed cells [1].
In TNF-α-stimulated adherent human PMNL, WRC-0470 (300 nM) combined with rolipram (300 nM) synergistically decreased degranulation, as measured by lysozyme release (from 430 ± 100 to 140 ± 41 ng/mL, P = .027). Neither compound alone had a significant effect [1].

Binodenson (infusion 0-0.9 μg/kg/h; adult Wistar rats; rat bacterial meningitis model), with or without rolipram (0-0.01 μg/kg/h), suppresses pleocytosis and lowers lipopolysaccharide-induced enhanced blood-brain barrier permeability (BBBP), indicating less neutrophil-induced damage [1].

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In a rat model of LPS-induced meningitis, continuous intravenous infusion of WRC-0470 (0-0.9 μg/kg/h) caused a dose-dependent inhibition of leukocyte pleocytosis into the cerebrospinal fluid, with 95% inhibition observed at 0.9 μg/kg/h (P < .05 vs. control). It also significantly reduced the LPS-induced increase in blood-brain barrier permeability (BBBP) at doses of 0.6 and 0.9 μg/kg/h (P < .05) [1].
In the same meningitis model, the combination of low doses of WRC-0470 (0.1 μg/kg/h) and rolipram (0.001 μg/kg/h) inhibited leukocyte migration (200 ± 70 WBC/μL) to a significantly greater extent than either WRC-0470 (600 ± 308 WBC/μL) or rolipram (1670 ± 1473 WBC/μL) alone (P < .05) [1].

Recombinant Adenosine Receptor Binding Assay: The potency and selectivity of compounds were evaluated by competition for radioligand binding to membranes derived from HEK293 or CHO-K1 cells stably expressing the four subtypes of recombinant human adenosine receptors. Dissociation constants (KD or Ki) were determined from these assays [1].

Human PMNL Purification: Human PMNL were purified from normal heparinized venous blood using a one-step ficoll-hypaque separation procedure, resulting in ~98% PMNL with >95% viability [1].
PMNL [cAMP] and Adherence Assay: PMNL were incubated in fibrinogen-coated tissue culture plates with test compounds (TNF-α, WRC-0470, rolipram, ZM241385) for 45 minutes. After incubation, HCl was added to extract cAMP, which was measured by radioimmunoassay. The wells were then washed, and the remaining adherent cell monolayer was digested with NaOH/SDS. Protein content was measured to determine relative PMNL adherence [1].
PMNL Superoxide Release Assay (Adherent Cells): PMNL were incubated in fibrinogen-coated wells with test compounds, cytochrome c, and catalase. Superoxide dismutase (SOD)-inhibitable reduction of cytochrome c was measured spectrophotometrically at 550 nm over time to quantify superoxide release [1].
PMNL Oxidative Activity (Human Whole Blood): Heparinized human whole blood was incubated with dihydrorhodamine 123, test compounds, and TNF-α, then stimulated with FMLP. Red blood cells were lysed, and the remaining leukocytes were analyzed by flow cytometry. PMNL were gated by forward and side scatter, and their mean fluorescence intensity (reflecting oxidative activity) was measured [1].
PMNL Degranulation Assay (Adherent Cells): PMNL were incubated in fibrinogen-coated wells with test compounds for 120 minutes. Cell-free supernatants were collected and assayed for lysozyme activity by measuring the lysis of a Micrococcus lysodeikticus suspension spectrophotometrically at 540 nm [1].

Rat Bacterial Meningitis Model:** Adult Wistar rats were anesthetized with ketamine and xylazine. Meningitis was induced via intracisternal inoculation of LPS (200 ng) from *E. coli*. Test compounds (WRC-0470 and/or rolipram) were infused intravenously over the duration of the experiment using a Harvard pump. At 4 hours post-inoculation, cerebrospinal fluid (CSF) and blood were sampled. CSF white blood cell (WBC) counts were determined by hemocytometer. For assessment of blood-brain barrier permeability (BBBP), rats received an intravenous injection of ¹²⁵I-labeled BSA at the time of intracisternal inoculation. The percentage of BBBP was calculated as (cpm in CSF / cpm in blood) × 100 [1].
* **Determination of WRC-0470 Plasma Concentrations:** A sensitive radioreceptor assay was developed using the high-affinity binding of WRC-0470 to recombinant A₂A receptors. Plasma samples were first purified on C18 Sepaks to remove interfering compounds. The eluted samples were then tested in the radioligand binding assay, and drug concentrations were derived from a standard curve [1].

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Rat Bacterial Meningitis Model: Adult Wistar rats were anesthetized with ketamine and xylazine. Meningitis was induced via intracisternal inoculation of LPS (200 ng) from E. coli. Test compounds (WRC-0470 and/or rolipram) were infused intravenously over the duration of the experiment using a Harvard pump. At 4 hours post-inoculation, cerebrospinal fluid (CSF) and blood were sampled. CSF white blood cell (WBC) counts were determined by hemocytometer. For assessment of blood-brain barrier permeability (BBBP), rats received an intravenous injection of ¹²⁵I-labeled BSA at the time of intracisternal inoculation. The percentage of BBBP was calculated as (cpm in CSF / cpm in blood) × 100 [1].
Determination of WRC-0470 Plasma Concentrations: A sensitive radioreceptor assay was developed using the high-affinity binding of WRC-0470 to recombinant A₂A receptors. Plasma samples were first purified on C18 Sepaks to remove interfering compounds. The eluted samples were then tested in the radioligand binding assay, and drug concentrations were derived from a standard curve [1].

Biological Half-Life
10 ± 4 minutes

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In rats, a steady-state plasma concentration of 0.8 ± 0.17 μM was reached with an intravenous infusion rate of 0.300 μg/kg/h. An infusion rate of 0.6 μg/kg/h produced a plasma level of 2.33 ± 0.29 μM [1].

The study notes that the anti-inflammatory effects of WRC-0470 in the rat meningitis model were observed at infusion rates 10² to >10³-fold lower than dosages required to induce hemodynamic responses (such as tachycardia and lowered blood pressure) typically associated with A₂A agonists [1].

[1]. Neutrophil A2A adenosine receptor inhibits inflammation in a rat model of meningitis: synergy with the type IV phosphodiesterase inhibitor, rolipram. J Infect Dis. 1999;180(5):1550-1560.

[2]. Pharmacological stress thallium scintigraphy with 2-cyclohexylmethylidenehydrazinoadenosine (WRC-0470). A novel, short-acting adenosine A2A receptor agonist. Circulation. 1996;94(7):1726-1732.

Binodenoson is a pharmacological stress test agent that specifically targets the A2A receptor, the only adenosine receptor essential for increasing blood flow to the heart. This specificity allows Binodenoson to deliver a more effective dose of the drug with fewer side effects in a single injection compared to existing therapies, which typically require 15-20 minutes of infusion. Drug Indications For use in cardiac pharmacological stress SPECT imaging, used to diagnose coronary artery disease. Mechanism of Action Binodenoson is a highly selective adenosine A2A receptor agonist. The adenosine A2A receptor is essential for increasing blood flow to the heart, and Binodenoson's specificity to this particular receptor allows it to deliver a more effective dose of the drug with fewer side effects in a single injection compared to existing therapies, which typically require 15-20 minutes of infusion. Cardiac stress testing allows physicians to determine the presence of cardiovascular disease by examining blood flow to the heart.

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Pharmacodynamics
Binodenoson is a novel diagnostic agent for cardiovascular disease. It is an adenosine A2A receptor agonist and is currently being developed for cardiac pharmacological stress SPECT imaging, which is used to diagnose coronary artery disease.

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Background: WRC-0470 is a selective A₂A adenosine receptor agonist. It was selected from a series of 2-substituted adenosine analogs for evaluation based on its affinity and selectivity for the A₂A receptor. In this study, it was used to investigate the anti-inflammatory effects of A₂A receptor activation on human neutrophils and in a rat model of meningitis [1].
Mechanism of Action: WRC-0470 binds to A₂A adenosine receptors on neutrophils. These receptors are coupled to Gs protein, which activates adenylyl cyclase, leading to an increase in intracellular cAMP. Elevated [cAMP] inhibits several pro-inflammatory functions of neutrophils, including adherence, superoxide release, and degranulation. The study demonstrates synergy with the type IV phosphodiesterase inhibitor rolipram, which prevents cAMP breakdown, thereby potentiating the effects of WRC-0470 [1].
Therapeutic Potential: The study suggests that combining a low dose of a selective A₂A agonist like WRC-0470 with a low dose of a type IV PDE inhibitor like rolipram could be a feasible therapeutic strategy for treating inflammatory diseases like meningitis, while minimizing the adverse side effects (e.g., hemodynamic effects) associated with higher doses of either drug alone [1].

C17H25N7O4

391.432

391.197

144348-08-3

9576912

White to off-white solid powder

1.76g/cm3

765.6ºC at 760mmHg

416.8ºC

0mmHg at 25°C

1.812

0.652

5

10

5

28

550

4

C1(/C=N/NC2=NC3=C(N=CN3C3OC(CO)C(O)C3O)C(N)=N2)CCCCC1

XJFMHMFFBSOEPR-DNZQAUTHSA-N

InChI=1S/C17H25N7O4/c18-14-11-15(22-17(21-14)23-20-6-9-4-2-1-3-5-9)24(8-19-11)16-13(27)12(26)10(7-25)28-16/h6,8-10,12-13,16,25-27H,1-5,7H2,(H3,18,21,22,23)/b20-6+/t10-,12-,13-,16-/m1/s1

(2R,3R,4S,5R)-2-[6-amino-2-[(2E)-2-(cyclohexylmethylidene)hydrazinyl]purin-9-yl]-5-(hydroxymethyl)oxolane-3,4-diol

Binodenoson WRC-0470 WRC 0470

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~319.35 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.31 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.31 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.31 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)