ITMI951155A1 - METHOD FOR MEASURING THE BINDING AFFINITY OF COMPONENTS OF PHARMACOLOGICAL INTEREST TO THE A2A ADENOSINE RECEPTOR THROUGH - Google Patents

Abstract

The invention relates to a method for evaluating the adenosine receptor binding affinity A2a of compounds of pharmacological interest. The invention also relates to reagents and a kit particularly suitable for the above mentioned purpose.

Description

Description of the industrial invention entitled: "METHOD FOR MEASURING THE AFFINITY OF ADENOSINE BINDING TO RECEPTOR A2a OF COMPOUNDS OF PHARMACOLOGICAL INTEREST THROUGH THE USE OF TRIZIATE LIGAND (3H) SCH 58261"

The invention relates to a method for evaluating the activity of binding to the A2a receptor of adenosine of compounds of pharmacological interest through the use of the tritiate ligand (3H) -SCH 58261. The invention also relates to particularly suitable reagents and a kit for the above-mentioned method.

PURPOSE OF THE INVENTION

Adenosine modulates a wide range of physiological functions by interacting with several receptor subtypes classified as Al, A2a, A2b and A3 [Pharmacol. Rev., 46, 143, (1994)]. While the availability of Al receptor ligands has led to rapid advances in the characterization of this receptor subtype, the pharmacology of the A2a-type adenosine receptor has been hampered by the lack of selective ligands [Med. Res. Rev., 12, 423 (1992)]. In the past, using different strategies to block interaction with Al receptors [Naunyn-Schniedeberg's Arch. Pharmacol. , 325, 218, (1984); Mol. Pharmacol-, 29, 331, (1986)], the nonselective (3H) 5 '-N-agone radium ligand (3H) 5' -N-et ilcarboxamnidoadenosine [(H) -NECA] has been used successfully to label the A2a receptor of 1 adenosine in rat striatal membranes [Mol. Pharmaool., 29, 331, (1986)]. However, it has also been described that (3H) NECA interacts with non-necector type binding proteins in both brain and peripheral tissues (Annu. Rev. Pharmaool. Toxicol., 27, 315, (1987)]. More recently, the compound 2- [p- (2-carboxyethyl) -phenethylanmin] -5'-N-ethylcarboxyadenosine (CGS 21680), a NBCA derivative with high affinity (Ki - 14 nM) and selectivity (Al / A2a ratio of about 180 times) for the A2a receptors of adenosine, has become the radioligand of choice for the study of this receptor subtype [J. Pharmaool. Exp. Ther., 251, 888, (1989)].

The development of antagonist A2a radioligands was hindered by the lack of selective compounds. Sdabene the compound (8- [4 [[[[2-aminoethyl) amino] carbcnyl] methyl] oxy] phenyl] -1,3-diprcpilxanthin (XAC) is a moderately selective Al antagonist, it has been used in labeled aonposite to characterize the adenosine receptor A2a in human platelet membranes [FEBS Lett., 199, 269, (1986)]. However, the specific binding of (3 H) XAC to the pyrin membranes was only 40% of the total binding. Similarly to NECA, PD 115119, a sulfuric derivative of 1,3-diethyl-8-phenylxanthine, is equivocal at the level of the A1 and A2 receptors. In the presence of 20 nM of 8-cyclopentyl-1,3-diprcpilxanthin (DPCPX), (3 H) PD 115119 interacted specifically with the striatal A2a receptors, but its radiostability was poor [Naunyn-Schmiedeberg's Arch. Pharmaool., 335 , 64), (1987)]. Recently, it has been described that the selective A2a antagonist (3H) - (E, 18% Z, 82%) - 8- (3, 4-dimethoxystyryl) -1,3-diprcpilxanthin (3H) -KF 17837S] is in able to interact directly with the adenosine receptor A2a in rat striated tissue, showing a specific bond of 60-70% [Mol. Pharmacol., 46, 817, (1995)]. However, although KF 17837S is described as a potent A2a antagonist (Ki -7.8 nM) and selective (Al / A2a - 49) in the original work [J. Med. Chan., 36, 3731, (1993)], substantial differences have been reported in A2a affinity (Ki values ranging from 30 to 60 nM) and selectivity (Al / A2a to 19) [J. Med. Chem., 36, 1333, (1993); Br. J. Pharmaool., 112, 659, (1994)]. Recently, the 5-amnino-7- (2-phenethyl) -2- (2-furyl-pyrazole- [4,3-e] -1,2,4-triazole [1,5-c-pyrimidine has been described as the first A2a antagonist ncn-xanthine potent (Ki = 2.3 nM) and selective (Al / A2a - 53) [Bioorg. Med. Chem. Lett., 4, 2539, (1994)]. The marked form of this compound appears particularly suitable for the characterization of the adenosine A2a receptor and for the identification of new compounds that interact with this receptor subtype.

ABSTRACT DEH, 'INVENTION

One aspect of the present invention is the synthesis of the labeled compound 5-antnino-7- [2— (2 ', 4', 53H) -phenethyl] -2- (2-furyl) -pyrazole- [4,3-e] -1,2,4-triazole [1,5-c] pyrimidine (hereinafter referred to as dog (3 H) Cnpost).

The type of marking indicated in the invention has the purpose of facilitating the measurement of the relative binding affinity values, preferably by introducing tritium (3 H) atoms, more preferably, located on the phenethyl group in positions 2 ', 41 and The second aspect of the invention is a method for determining the affinity for the adenosine receptor A2a of a test compound; said method consists in:

(a) preparing purified brain tissue of marble containing A2a receptors;

(b) add the (3⁄4) -Carpound to said marble brain tissue; (c) adding the test compound to said marble brain tissue; And

(d) measuring the amount of radioligand binded with said A2a receptors.

A test item can be synthesized and / or purified from natural infantry such as animal or plant tissues.

The third aspect of the invention is a kit for determining the A2a binding activity of a component under examination; said kit including:

(a) a purified brain tissue sample of marble containing A2a receptors; And

(b) a sufficient quantity of (H) -Compound to determine the level of affinity for the A2a receptor of said compound under examination.

DETAILED DESCRIPTION OF THE INVENTION

Due to the usefulness of having an A2a adenosine receptor antagonist radioligand, the (H) -Compound was synthesized. The studies described below were carried out in order to characterize the binding properties of the (3H) Compound to the A2a receptors of the rat striatum.

Synthesis of the 3H-COMPOUND

The (3H) -Compound was obtained by reduction, with tritium in the gaseous state and in the presence of 10% Pd / C (Dupont-New England Nuclear, Boston, MA, USA) of the precursor 5-anrnino-7- [2- (2 1, 4 ', 5'-trihr hemolysis ylethyl] -2- (2-furyl) -pyrazole [4,3-e] -1,2,4-triazole [1,5-c] -pirimidine.

The final product was purified by HPLC to give the desired labeled (3H) Carpox with radiochemical purity of 99% and specific activity of 68.6 Ci / mnol.

Preparation of the fabric

Male Sprague-Déwley rats (Charles-River, Calco) weighing 250-300 g breasts were sacrificed by decapitation and the striatum dissected on ice. The tissue, homogenized by a Polytrcn PTA 10 probe (adjustment of 5, 20 seconds) in 25 volumes (v / v) of tanpone 50 mM Tris-HCl, pH 7.4, was centrifuged at 48,000 x g for 10 minutes at 4eC and resuspended in Tris-HCl containing 2 units / ml of adenosine deaminase. After 30 minutes of incubation at 37 ° C, the membranes were centrifuged and the sediment stored at -70 ° C.

Bond essay

Sine binding saturation experiments were carried out in polypropylene tubes containing an aliquot of striatal membranes (100 µg of protein / assay) in incubation tubes (50 mM Tris-HCl, pH 7.4) and 11 different concentrations of (1 ⁄2) -Cnposite (0.0625-64 nM), in a final volume of 0.5 ml. Non-specific binding was established in the presence of 50 µM of RECA. After incubation at 25 ° C for 30 minutes, separation of the free ligand radium from that bound to the receptor was performed by rapid filtration through Whatman GF / B filters using the Brande1 cell harvesting device (Gaithersburg, MD, USA). The breast filters were washed twice with ice-cooled buffer {5 ml) and placed in bottles containing 5 ml of scintillation liquid (Ready Safe, Beckman Instruments, Rillertan, CA, USA). Radioactivity was measured using a Beckman LS-6000 liquid scintillation counter (Beckman Instruments, Rillertcn, CA, USA) with an efficiency of 50-60%. The protein concentration was determined by the method of Lcwry [J. Biol. Chem., 193, 265, (1951)] using standard acme bovine serum albumin.

In testing studies, different concentrations of different healthy adenosine receptor agonists and antagonists were added to the incubation tanpene containing 0.2 nM of (H) -Cn compound.

The sine binding parameters were estimated using the computerized program LIGAND [Anal. Biochent., 107, 220, (1980)].

Results

After incubation at 25 ° C and pH 7.4, 0.2 nM of (1⁄2) -Cnpound was licked to rat striatal manhranas with a specific binding of 92% which increased linearly with respect to the protein concentration over a range of 50 to 300 pg of protein / assay. The presence of 10 nM of MgCl2 or of 100 μΜ of guanosine triphosphate (GTP) in the test mixture did not significantly modify the percentage of specific binding.

The kinetics of the reaction showed that the bond of (3 H) Compound reached equilibrium after about 5 minutes and was stable for at least 4 hours. Compound (3H) binding was rapidly removed by the addition of 50 μΜ of NBCA. The association and dissociation rate constants were as follows: = 0.85 / min and K-1 - 0.62 min from a T1 / 2 = 1.12 min. From these experiments a value of the dissociation constant (Kd) equal to 0.54 nM was calculated.

Saturation experiments showed that Compound (3H) bound to a single class of receptors in rat striatal membranes, with Kd values and an apparent number of receptors (B max) 'of 0.70 nM and 971 fmol / mg of protein, respectively.

In the c appetition experiments, numerous adenosine receptor agonists inhibited binding of (H) ~ Cnposed to rat striatal membranes with the following order of potency: NECA> CGS 21680> 2-phenylaminoadenosine (CV 1808)> R-N ^ -2- f eni lisoprcpiladenosine (R-PIA)> cyclohexyladenosine (CHA)> S-N6-2-phenylisopyladenosine (S-PIA). The non-selective agonist NECA was shown to be the most potent compound with nanomolar affinity (Ki = 61 nM). Furthermore, the selective R-PIA Al agnist appeared about 8 times more potent than its stereoisanero S-PIA, thus demonstrating the stereoselectivity of the (1⁄2) -Compound bond. The ability of various adenosine receptor antagonists, xanthines and ncn-xanthines, to caipeter with the {H) -Compound in binding to striatal A2a receptors was also examined. Their power order showed: CGS 15943> Compound> XAC - KF 17837> DPCPX. CGS 15943 was the most potent compound in inhibiting the binding of (3H) Compound with a Ki value of 0.38 nM.

The (3H) C compound interacted with the agonists and adencsin antagonists with a similar order of potency to that observed using (3 H) CGS 21680 as a radioligand, and demonstrated a selective interaction with the A2a receptor [J. Pharmacol. Exp. Iher., 251, 888, (1989)].

Conclude it

In conclusion, (3 H) Carpox, by directly labeling the striatal A2a adenosine receptor, proves to be an excellent means of studying the A2a receptor subtype of adenosine in the brain of maitmifera. Clear advantages over other A2a antagonist radioligands proposed for this purpose are the high receptor affinity and the very low non-specific binding. A kit containing the components necessary to perform the dog assays described above, as well as a method for using such a kit, can be considered essential for evaluating the interaction of a test compound with the A2a adenosine receptors in marble brain tissues. Furthermore, the (3 H) Conposta possesses the characteristics to become a useful tool for the study of A2a receptors distributed in peripheral tissues, such as vascular preparations, platelets and neutrophils, in which their presence has been clearly demonstrated [TiPS, 14, 360 , (1993)].

Claims (6)

CLAIMS 1. The method for determining the affinity to the adenosine receptor A2a of compounds of pharmacological interest, which includes: a) preparing purified mammalian brain tissue containing A2a receptors; b) add the labeled form of the compound 5-anriine-7- (2-phenethyl) -2- (2-furyl) -pyrazole- [4,3-e] -1, 2,4-triazole [1,5- c] pyrimidine to said mammalian brain tissue; c) adding the compound under examination to said mammalian brain tissue; d) measuring the amount of radioligand complexed with said A2a receptors. The method according to claim 1, wherein the marking form exists in an enriched layer of radioactive atoms. Method according to claim 2, wherein the marking form consists of a layer enriched with tritium atoms. 4. The method of claim 1 wherein said marble brain tissue is rat brain tissue. 5. Kit for determining the binding affinity for the A2a receptor of a test compound, which includes: a) a purified brain tissue sample of marble containing A2a receptors; e b) a sufficient amount of arched form of the compound 5-amin-7- (2-phenethyl) -2- (2-furyl) -pyrazole- [4,3-e] -1,2,4-triazole [1, 5-c] pyrimidine to determine the affinity for the A2a receptor of said compound under examination. 6. Kit according to claim 5 containing 5-amino-7- [2- (21, 4 1, 5 '3H -phenethyl] -2- (2-furyl) -pyrazole- [4, 3-e] -1, 2, 4-triazole [1, 5-c] pyrimidine.