Adenosine receptor ( Ki = 0.46 nM; 3H-CHA binding to A1 receptors in rat whole brain membranes); A2 receptors (Ki = 340 nM; 3H-NECA binding to A2 receptors in rat striatal membranes)

(R)-N-(1-Methyl-2-phenylethyl) adenosine (R-PIA), an adenosine receptor agonist has both negative chronotropic activity and coronary vasodilator activity. These actions of R-PIA are proposed to be mediated by subtypes (A1 and A2) of adenosine receptors. DPCPX (PD 116948) is a xanthine derivative which is a highly selective A1 adenosine receptor ligand. In this study PD 116,948 selectively antagonized the negative chronotropic activity of R-PIA in the isolated rat heart. These results are consistent with, and add further support to the hypothesis that adenosine receptor agonists mediate their negative chronotropic activity via A1 receptors and their vasodilator activity via A2 receptors[1].

DPCPX (PD 116948) ( 0.1, 0.3 and 1.0 mg/kg i.v. ) generates notable dose-related increases in urine volume and urinary sodium, potassium, and chloride excretion (P<0.05)[3].

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For examination of the potency of the antagonists, adenosine was administered at a standard concentration of 5 ⁎ 10− 5 M, which caused well-preserved responses when administered repeatedly in the absence of any antagonist. Adenosine 5 ⁎ 10− 5 M administered in the presence of PPADS (1 ⁎ 10− 4 M) evoked slightly increased relaxatory responses in comparison with adenosine administered in its absence (− 15 ± 0.8% vs. − 19 ± 1%; p < 0.05). The presence of DPCPX (1 ⁎ 10− 5 M) substantially reduced the relaxatory response (to − 5 ± 1%; p < 0.001) and prolonged the latency (104 ± 4 s), and further, adenosine administration in the presence of both antagonists evoked similar responses as with only DPCPX present. However, neither PPADS (1 ⁎ 10− 4 M) nor DPCPX (1 ⁎ 10− 5 M) affected the relaxatory response of strip preparation from inflamed urinary bladder[4].

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The P1A1 antagonist DPCPX, however, abolished the difference of methacholine-evoked contractions between saline- and CYP-treated rats. ATP-evoked contractions were reduced in control after the DPCPX pre-treatment, but not in cystitis. The functional observations for DPCPX were supported by its suppression of CYP-induced submucosal thickening, muscarinic M5 receptor expression and, possibly, detrusor mast cell infiltration and the spread of urothelial MIF occurrence.[5]

8-Cyclopentyl-1,3-dipropylxanthine (PD 116,948; DPCPX) is a very potent, very A1-selective adenosine antagonist, with a Ki of 0.46 nM in 3H-CHA binding to A1 receptors in rat whole brain membranes and 340 nM in 3H-NECA binding to A2 receptors in rat striatal membranes. Its 740-fold A1-selectivity is the highest reported for an adenosine antagonist. 3H-PD 116,948 (117 Ci/mmol) was prepared by reduction of the diallyl analog. 3H-PD 116,948 bound to a single site in rat whole brain membranes, with a Bmax of 46 pmol/g wet weight and Kd of 0.42 nM. Nonspecific binding was extremely low, amounting to about 3% of total binding under standard conditions and less than 1% when higher tissue concentrations were used. Affinities of compounds for inhibition of 3H-PD 116,948 binding were highly consistent with an A1 adenosine receptor. Antagonists were equally potent in 3H-PD 116,948 binding and in 3H-CHA binding, while agonists were consistently about 12-fold more potent in 3H-CHA binding. Hill coefficients were 1.0 for antagonists and about 0.65 for agonists. 3H-PD 116,948 should be a useful antagonist ligand for adenosine A1 receptors[2].

60 h after the CYP/saline pre-treatment the rats were intraperitoneally injected with medetomidin (Domitor® vet., 0.2 ml) for anesthesia and then killed by an overdose of carbon dioxide. The urinary bladder was removed, cut and opened. Two or three strips (6 × 2 mm) were excised from the body of the organ. To prevent damaging the tissue by drying out, the organ was kept in Krebs bicarbonate solution at all times. For the contraction experiments, the strips were fastened between two steel rods, one of which was adjustable. The organ baths were filled with Krebs bicarbonate solution (deionized water, NaCl 118 mM, KCl 4.6 mM, KH2PO4 1.18 mM, MgSO4 1.16 mM, NaHCO3 25 mM, glucose 5.5 mM and CaCl2 1.27 mM). The Krebs solution was kept at 37 °C and gassed with 5% CO2 in O2 during the whole experiment to maintain a stable neutral pH and to provide the tissue with oxygen. The strips were stretched to a tension of 15 mN and left to equilibrate for 45 min. This resulted in gradual relaxation resulting in a stable tension of 7–9 mN. After 45 min, administration of potassium-containing Krebs solution (containing 124 mM K+; obtained by exchanging Na+ for equimolar amounts of K+) was performed and the resulting contraction was used as a viability referring response. The tension was then stabilized for each strip at 7–9 mN. This is a somewhat higher basal tension that is normally used (4–5 mN; Tobin and Sjogren, 1995). This tension turned out to amplify the relaxations. After 30 min of resting, medium potassium Krebs was administrated (50 mM K+; obtained by exchanging Na+ for equimolar amounts of K+) to pre-contract the tissue. An observation period of 20 min followed in order to establish a stable pre-contraction larger than 2.5 mN above the basal tension level. After 20 min of pre-contraction, adenosine was added at a standard concentration shown to evoke stable relaxatory responses of the rat urinary bladder strips (5*10− 5 M). This concentration was possible to dilute in water, whereas any higher concentration would have had to be diluted in dimethyl sulphoxide (DMSO). When the maximal relaxation of the tissue was obtained, usually after approximately 150 s, the organ baths were washed thoroughly and the strips were re-stretched to 7–9 mN and let to equilibrate for 30 min. Subsequently, an antagonist was added and the preparations were pre-contracted by medium potassium Krebs solution for a 20 min incubation period, after which the relaxatory response to adenosine (5*10− 5 M) was evaluated. This procedure was repeated for all antagonists used (i.e. PPADS, DPCPX and SCH 58261, MRS 1523, respectively). The agonist and all antagonists were added in the volume of 125 μl.[4]

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The substances used in the pre-treatment of the rats were: the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) ( Bruns et al., 1987 , Haleen et al., 1987 ), the selective A2B adenosine receptor antagonist 4-(2,3,6,7-tetrahydro-2,6-dioxo-1-propyl-1H-purin-8-yl)-benzenesulfonic acid (PSB1115) ( Muller et al., 1998 ) and the non-selective P2 purinergic antagonist suramin ( Dunn and Blakeley, 1988 , Soto et al., 1997 ). The concentrations used were as follows and based on pilot experiments and/or on previous studies; DPCPX; 1 mg/kg i.p. ( Kadowaki et al., 2003 , Wang et al., 2005 ), PSB1115; 1 mg/kg i.p. ( Savegnago et al., 2008 ), suramin; 10 mg/kg. The antagonists were dissolved in saline, except for DPCPX which, due to limitations in solubility, was dissolved in pure dimethyl sulfoxide (DMSO; 1 ml/kg), a volume previously shown to be too small to induce any immunosuppressive effects ( Watson et al., 1985 ). In addition, a small number of rats (n = 4 in each group) were pre-treated with pure DMSO (1 ml/kg, as control). In order to induce cystitis, cyclophosphamide (CYP; 100 mg/kg i.p.) in combination with the analgesic buprenorphine (Temgesic®; 10 μg/kg s.c.) was administered 60 h prior to sacrifice, ensuring peak inflammation at the time of the experiment ( Giglio et al., 2005 ). Control groups instead received an injection of saline (0.9%; 1 ml/kg i.p.). On the sixth day the rats were anesthetized with medetomidine (Domitor®Vet., 1 mg/kg i.p.) and subsequently euthanized by CO2 asphyxiation. The “pre-treatment and treatment” protocol was designed to match each “active” CYP-treatment group with a saline treated group. Thus, the control group and experimental group (saline–saline and saline–CYP, DPCPX–saline and DPCPX–CYP, PSB1115–saline and PSB1115–CYP and eventually suramin–saline and suramin–CYP) were treated in parallel. In the text, “pre-treatment” refers to the five day saline- or drug administration, whereas “treatment” refers to the single administration of either saline or CYP on day 3 ( Fig. 1 ). [5]

[1]. PD 116,948, a Highly Selective A1 Adenosine Receptor Antagonist. Life Sci. 1987 Feb 9;40(6):555-61.

[2]. Binding of the A1-selective Adenosine Antagonist 8-cyclopentyl-1,3-dipropylxanthine to Rat Brain Membranes. Naunyn Schmiedebergs Arch Pharmacol. 1987 Jan;335(1):59-63.

[3]. Br J Pharmacol. 1989 Jul; 97(Suppl): 496P–535P.

[4]. Functional and morphological examinations of P1A1 purinoceptors in the normal and inflamed urinary bladder of the rat. Auton Neurosci . 2011 Jan 20;159(1-2):26-31.
[5]. Adenosine receptor antagonism suppresses functional and histological inflammatory changes in the rat urinary bladder. Auton Neurosci . 2012 Nov 2;171(1-2):49-57.

DPCPX is an oxopurine that is 7H-xanthine substituted at positions 1 and 3 by propyl groups and at position 8 by a cyclohexyl group. It has a role as an adenosine A1 receptor antagonist and an EC 3.1.4.* (phosphoric diester hydrolase) inhibitor. It is functionally related to a 7H-xanthine.
CPX has been used in trials studying the treatment of Cystic Fibrosis.

C16H24N4O2

304.38736

304.19

C, 63.13; H, 7.95; N, 18.41; O, 10.51

102146-07-6

1329

White to off-white solid powder

1.195g/cm3

535.1ºC at 760mmHg

178°C

277.4ºC

1.59E-11mmHg at 25°C

1.564

2.363

1

3

5

22

436

0

CCCN1C2=C(C(=O)N(CCC)C1=O)N=C(C3CCCC3)N2

FFBDFADSZUINTG-UHFFFAOYSA-N

InChI=1S/C16H24N4O2/c1-3-9-19-14-12(15(21)20(10-4-2)16(19)22)17-13(18-14)11-7-5-6-8-11/h11H,3-10H2,1-2H3,(H,17,18)

8-cyclopentyl-1,3-dipropyl-7H-purine-2,6-dione

DPC-PX; DP-CPX; 8-Cyclopentyl-1,3-dipropylxanthine; DPCPX; 102146-07-6; 8-cyclopentyl-1,3-dipropyl-1H-purine-2,6(3H,7H)-dione; 1,3-dipropyl-8-cyclopentylxanthine; 1,3-Dpcpx; PD-116948; PD-116,948;

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: 10~31 mg/mL (32.9~101.8 mM)
Ethanol: ~5 mg/mL

Solubility in Formulation 1: ≥ 1 mg/mL (3.29 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 10.0 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.)