Adenosine A1 receptor
The standard full A1-agonist CCPA and the A1-antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) are used in GTP shift assays to help clarify the pharmacological characteristics of capadenson. The binding assay on rat cortical brain membranes yields a K_i value of 4.2 nM for CCPA. This K_i value changes to 64 nM when 1 mM GTP is present. In light of this, the CCPA's GTP shift is 15. The GTP shift of DPCPX is 1, and its K_i values are almost the same when GTP is present as well. According to the binding assay, Capadenson has a K_i value of 24 nM. Capadenoson's GTP shifts by 5 when 1 mM GTP is present, as indicated by the K_i value shifting to 116 nM.

The standard full A1-agonist CCPA and the A1-antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) are used in GTP shift assays to help clarify the pharmacological characteristics of capadenson. The binding assay on rat cortical brain membranes yields a K_i value of 4.2 nM for CCPA. This K_i value changes to 64 nM when 1 mM GTP is present. In light of this, the CCPA's GTP shift is 15. The GTP shift of DPCPX is 1, and its K_i values are almost the same when GTP is present as well. According to the binding assay, Capadenson has a K_i value of 24 nM. Capadenoson's GTP shifts by 5 when 1 mM GTP is present, as indicated by the K_i value shifting to 116 nM.

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In isolated perfused hearts from Spontaneously Hypertensive Rats (SHR), capadenoson (6·10^-7 M) led to a significant concentration-dependent decrease in electrical field stimulation-induced norepinephrine (NE) release (S2/S1 ratio = 0.54±0.02), comparable to the reduction caused by the full agonist CCPA (10^-6 M). In hearts from Wistar rats, capadenoson at the same concentrations (6·10^-8 M and 6·10^-7 M) did not significantly affect stimulation-induced NE release (S2/S1 ratios ~1.0). Basal NE release under control conditions was significantly higher in SHR hearts (766±87 pmol/g) compared to Wistar hearts (173±18 pmol/g) [1]

In the in vivo trials, capadenoson is pretreated for five days at a dose of 0.15 mg/kg on Wistar rats and SHR. Day 5 involves a two-hour stress test (physical restraint). Four and five days before the restraint stress test, the average plasma concentration of Capadenoson, measured three hours after drug intake, was 7.63 µg/L on day four and day five, respectively[1].

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In SHR, pretreatment with capadenoson (0.15 mg/kg orally for 5 days) significantly blunted the heart rate increase induced by 2-hour physical restraint stress by approximately 45% (relative increase: 20±4% with drug vs. 36±4% with vehicle). In Wistar rats, the same treatment did not affect the stress-induced heart rate increase. Resting heart rate was not altered by capadenoson in either rat strain. Mean arterial pressure during stress increased in all groups, with no statistically significant difference between vehicle and capadenoson-treated animals in either strain, although a tendency towards a smaller pressure increase was noted in SHR. [1]

The human cortex is ready to produce membranes. We measure [³⁵S]GTPηS binding. In short, a 160 µL total volume and 2 hours at 25°C in a shaK_ing water bath are used to incubate 5 µg of membrane protein. A linear time course was observed for [³⁵S]GTPγS binding up to this incubation time, both in control and capadenoson-containing incubations. pH 7.4, 50 mM Tris/HCl, 2 mM triethanolamine, 1 mM EDTA, 5 mM MgCl₂, 10 µM GDP, 1 mM dithiothreitol, 100 mM NaCl, 0.2 units/mL adenosine deaminase, 0.2 nM [³⁵S]GTPγS, and 0.5% bovine serum albumin were all present in the binding buffer. In the presence of 10 µM GTPγS, non-specific binding is identified. After filtration of the samples through multiscreen FB glass fiber filters and two binding buffer washes, the incubations are ended. After drying and applying a scintillator coating, the radioactivity of the filters is measured. Using GraphPad Prism, nonlinear regression is used to analyze the binding curves of [³⁵S]GTPηS[1].

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The partial agonistic property of capadenoson at the adenosine A₁ receptor was determined using a [³⁵S]GTPγS binding assay. Human frontal cortex membranes (5 μg protein) were incubated in binding buffer (50 mM Tris/HCl pH 7.4, 2 mM triethanolamine, 1 mM EDTA, 5 mM MgCl₂, 10 mM GDP, 1 mM dithiothreitol, 100 mM NaCl, 0.2 U/ml adenosine deaminase, 0.2 nM [³⁵S]GTPγS, 0.5% BSA) for 2 hours at 25°C with increasing concentrations of capadenoson. The full agonist CCPA was used to determine maximal stimulation. Non-specific binding was defined with 10 μM GTPγS. Binding curves were analyzed by nonlinear regression. Capadenoson stimulated [³⁵S]GTPγS binding to 74±2% of CCPA's maximal effect [1]
A GTP shift assay was performed to further characterize receptor binding. Rat brain cortical membranes (10 μg protein) were incubated with 0.4 nM [³H]DPCPX and increasing concentrations of capadenoson in buffer (50 mM Tris-HCl, pH 7.4, 2 U/ml adenosine deaminase) in the presence or absence of 1 mM GTP for 20 min at 37°C. Non-specific binding was determined with 10 μM R-PIA. Binding was terminated by rapid filtration. The K_i value for capadenoson shifted from 24 nM (without GTP) to 116 nM (with 1 mM GTP), yielding a GTP shift factor of 5, indicative of partial agonism. [1]

Rats: In total, 18 SHR (body weight 200–50 g, all female) and 14 Wistar rats participated in the experiments to assess the exocytotic, stimulation-induced NE release during electrical field stimulation. Rats are given an intraperitoneal injection of pentobarbital (0.5 mL/100 mg body weight) to K_ill them. Their hearts are then quickly removed and placed in a cold Krebs-Henseleit solution (KHL). They are immediately put on a Langendorff device for KHL retrograde perfusion. By using 5% CO₂/95% O₂, the temperature is brought down to 37°C, the pH is brought up to 7.4, and the perfusion rate is maintained at 10 mL/min. Desipramine is added to the perfusion buffer at a concentration of 10⁻⁷ M via an inflow line. Following a 20-minute equilibration period, two metal paddles placed next to the beating heart are used to apply an electrical field for one minute (5V, 6 Hz). One minute prior to, during, and three minutes following the stimulation, we collected the efflux in plastic tubes. They are kept at -20°C until analysis and quickly frozen in liquid nitrogen. The cumulative release brought on by the electrical stimulation is used to compute the NE release. The study drug Capadenoson is added via separate perfusion lines for 30 minutes after the first stimulation (S1) at concentrations of 30 µg/L (6×10⁻⁸ M), 300 µg/L (6×10⁻⁷ M), or 2-chloro-N6-cyclopentyladenosine (CCPA, 10−6 M). To find out how the medications affect NE release in comparison to the first stimulation, a second stimulation (S2) is carried out after this point. By determining the ratio of NE release brought on by the first and second stimuli (S2/S1 ratio), the impact of each pharmaceutical intervention is examined.

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For in vivo stress experiments, female Wistar rats and SHR (200-220 g) were chronically implanted with radiotelemetry transmitters for cardiovascular monitoring at least one month prior to experiments. Capadenoson was administered orally at a dose of 0.15 mg/kg/day for 5 days. The drug was formulated by solubilizing the extrudate in a vehicle containing 10% dimethyl sulfoxide, 30% polyethylene glycol, and 60% physiologic (0.9%) sodium chloride. Control animals received the vehicle only. On day 5, two hours after drug/vehicle administration (at 11:00), animals were subjected to physical restraint stress in transparent tubes for 2 hours. Heart rate and mean arterial pressure were continuously recorded via telemetry before, during, and after the restraint period. [1]

On days 4 and 5 of the treatment regimen, plasma capadenosone concentrations were measured 3 hours after oral administration of capadenosone (0.15 mg/kg). The concentrations remained stable over these two days, averaging 7.63 μg/L. [1]

At the test doses (1.0 and 2.0 mg/kg, intraperitoneal), capadenoson induced mild hypothermia and bradycardia, but did not cause severe hypothermia (T_b as low as 21°C) as observed in some animals with the full agonist CHA. Its bradycardia effect was also milder than that induced by CHA. This study suggests that capadenoson, due to its partial agonist effect, has limited effects on the cardiovascular system (reducing the risk of severe bradycardia/hypotension) compared to the full agonist, and therefore its advantage may be more pronounced when a target body temperature of around 36°C is required. [2]

[1]. Selective attenuation of norepinephrine release and stress-induced heart rate increase by partial adenosine A1 agonism. PLoS One. 2011 Mar 28;6(3):e18048.

[2]. Optimization of Thermolytic Response to A1 Adenosine Receptor Agonists in Rats. J Pharmacol Exp Ther. 2017 Sep;362(3):424-430.

Capadenoson is being investigated in the clinical trial NCT00518921 (Capadenoson for angina). Capadenoson (BAY 68-4986) is a selective partial agonist of adenosine A1 receptors. Researchers have investigated its potential to inhibit the release of norepinephrine from cardiac sympathetic nerve endings presynaptically, particularly in cases of high sympathetic tone. Studies have shown that partial A1 receptor agonism may selectively inhibit stress- or exercise-induced tachycardia without affecting resting heart rate, providing a potential therapeutic approach different from postsynaptic β-blockers. The differential effect observed between normotensive Wistar rats and hypertensive SHR rats is thought to be due to differences in baseline endogenous adenosine receptor occupancy or sympathetic tone. [1]

C25H18CLN5O2S2

520.0257

519.059

C, 57.74; H, 3.49; Cl, 6.82; N, 13.47; O, 6.15; S, 12.33

544417-40-5

9936489

Solid powder

1.5±0.1 g/cm3

787.9±70.0 °C at 760 mmHg

430.3±35.7 °C

0.0±2.9 mmHg at 25°C

1.735

6.15

2

9

8

35

774

0

ClC1C([H])=C([H])C(=C([H])C=1[H])C1=NC(=C([H])S1)C([H])([H])SC1=C(C#N)C(=C(C#N)C(N([H])[H])=N1)C1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])C([H])([H])O[H]

CITWCLNVRIKQAF-UHFFFAOYSA-N

InChI=1S/C25H18ClN5O2S2/c26-17-5-1-16(2-6-17)24-30-18(13-34-24)14-35-25-21(12-28)22(20(11-27)23(29)31-25)15-3-7-19(8-4-15)33-10-9-32/h1-8,13,32H,9-10,14H2,(H2,29,31)

2-amino-6-[[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methylsulfanyl]-4-[4-(2-hydroxyethoxy)phenyl]pyridine-3,5-dicarbonitrile

BAY 68-4986; BAY-68-4986; BAY68-4986

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: ~50 mg/mL (~96.1 mM)

Solubility in Formulation 1: 2.5 mg/mL (4.81 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), suspension solution; with heating and sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (4.81 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 5%DMSO + 40%PEG300 + 5%Tween 80 + 50%ddH2O: 2.5mg/ml (4.81mM)

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