P2Y1 receptor

Platelet aggregation is inhibited by MRS2179 [3]
Addition of MRS2179 (10 μM) to washed human platelets 30 s before ADP (1 μM) inhibited platelet aggregation and shape change (Fig. 1A), while MRS2179 alone did not induce shape change or aggregation even at high concentrations (up to 100 μM, data not shown). The nature of the inhibition was determined by generating a series of concentration–response curves for ADP in the presence of different concentrations of MRS2179. MRS2179 caused a parallel shift to the right of the concentration–response curve, but high concentrations of ADP could completely override high concentrations of MRS2179 (Fig. 1B). Schild analysis of the inhibition gave a pA2 value of 6.55±0.05 (n=5) and a slope of 0.64, which could be explained by the fact that we observed an integrated aggregation process involving the activation of two receptors (P2Y1 and P2cyc) and their transduction machinery. Identical results were obtained using washed rat platelets, MRS2179 inhibiting ADP-induced platelet aggregation with a parallel shift to the right of the concentration–response curve and a pA2 value of 6 (data not shown).

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MRS2179 affects only the P2Y1 receptor transduction pathway [3]
ADP induces simultaneous mobilization of intracellular Ca2+ stores and inhibition of adenylyl cyclase, through activation of the P2Y1 and P2cyc receptors, respectively. The intracellular Ca2+ rise induced in washed human platelets by 0.5 μM ADP was totally inhibited by 3 μM MRS2179, in the presence (Fig. 2A, left) or absence (data not shown) of 2 mM external Ca2+. MRS2179 modified the [Ca2+]i increase in response to 1 μM ADP in a dose-dependent manner with IC50=0.26 μM (Fig. 2A, right). A similar inhibition of ADP-induced [Ca2+]i rises was observed in washed rat and mouse platelets (data not shown).

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[33P]MRS2179 is a suitable radioligand to estimate numbers and affinities of P2Y1 sites [3]
Studies of the binding of ADP to its platelet receptors are currently performed using [33P]2MeSADP, a radioligand which binds to both P2Y1 and P2cyc Gachet et al., 1995, Hechler et al., 1998a, Léon et al., 1999b. MRS2179, on the other hand, appeared to us to be a good candidate for use as a P2Y1 specific radioligand. Firstly, in order to verify that MRS2179 displaced [33P]2MeSADP only from P2Y1 receptors, we compared its effects to those of A3P5P, a well known P2Y1 receptor antagonist Hechler et al., 1998b, Léon et al., 1999b. The specific binding of [33P]2MeSADP to washed human platelets was competitively and partially displaced by MRS2179 (Ki=111 nM) and A3P5P (Ki=0.25±0.06 μM, data not shown) at approximately 20% of [33P]2MeSADP sites (Fig. 3A). In preliminary assays, the binding of [33P]MRS2179 to washed platelets was tested at three different temperatures +4°C, +20°C, +37°C. The best condition was at 20°C and the binding was proportional to the concentration of platelets and maximal binding, stable for at least 30 min, was obtained after 30 s to 1 min at 20°C (data not shown). All subsequent saturation experiments were performed using a platelet concentration of 6×105/ml and an incubation time of 30 min at 20°C. The specific binding of [33P]MRS2179 to washed human platelets was saturable (Fig. 3B) with a linear Scatchard plot (insert), 134±8 binding sites per platelet and an affinity (Kd) of 109±18 nM (n=3). This result was confirmed by measuring the binding of [33P]MRS2179 to astrocytoma cells (1321 NI) transfected with the P2Y1 receptor or with the vector alone. In the cells transfected with P2Y1, which displayed normal pharmacological selectivity and signaling properties, the number of binding sites was 162,500±7500 per cell with a Kd of 138±8 nM (n=3), whereas the control cells showed no binding of [33P]MRS2179. A similar affinity was observed for the murine platelet P2Y1 receptor (data not shown).

MRS2179 (50 mg/kg; ip) extends the duration of bleeding[3]
The bleeding time, which reflects in vivo primary haemostasis, was significantly prolonged in MRS2179-treated mice as compared to control mice, 30 s after injection of MRS2179 (50 mg/kg) (Fig. 5C). The mean bleeding time (±S.D.) was 595±189 s for MRS2179-treated mice (range 120–1200 s, n=4) and 83.5±5.7 s for control mice (range 68–110 s, n=6) and the difference between the two groups was statistically significant (P<0.01, unpaired t-test). The bleeding time was also prolonged in MRS2179-treated rats (data not shown).

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MRS2179 inhibits platelet aggregation ex vivo [3]
In order to test the stability of MRS2179 in the presence of ectonucleotidases, we measured the possible degradation products after a 2-h incubation with apyrase 0.1 U/ml. The HPLC profiles of MRS2179 before (Fig. 4A1) and after incubation (Fig. 4A2) were identical. Thus, MRS2179 is chemically stable in the presence of apyrase. Incubation of MRS2179 in citrated platelet-rich plasma for 2 h did not affect MRS2179 inhibitory activity regarding ADP-induced platelet aggregation (data not shown). Moreover, when MRS2179 (100 μM) was incubated for 2 h with washed platelets, ADP-induced aggregation was still inhibited (Fig. 4B), confirming the stability of MRS2179. MRS2179 was subsequently injected intravenously into anesthetized rat to study ex vivo ADP-induced aggregation. Blood samples were taken at various time points after injection of 50 mg/kg to follow the activity of the compound. In citrated platelet-rich plasma from MRS2179-treated rats, ADP-induced platelet aggregation was inhibited for ADP concentrations of up to 10 μM, 5 min after injection of MRS2179 (Fig. 5A,B). At this dose (50 mg/kg), there persisted an aggregation response to 30 μM ADP, which was nevertheless reduced as compared to the control (Fig. 5A,B).

Measurement of adenylyl cyclase activity [3]
A 450-μl aliquot of washed platelets resuspended in Tyrode's buffer containing 2 mM Ca2+ and 1 mM Mg2+ was stirred at 1100 rpm in an aggregometer cuvette and the following reagents were added at 30-s intervals: (i) 10 μM Prostaglandin E1, (ii) 1 μM AR-C66096MX or different concentrations of MRS-2179 and (iii) 5 μM ADP or vehicle (Tyrode's buffer containing no Ca2+ or Mg2+). The reaction was stopped 1 min later by addition of 50 μl of ice-cold 6.6 N perchloric acid. Perchloric acid extracts were centrifuged at 11,000×g for 5 min to eliminate protein precipitate and cyclic AMP was isolated from the supernatants using a mixture of trioctylamine and freon (28:22, vol/vol). The upper aqueous phase was lyophilized and the dry residue dissolved in the buffer provided with the commercial radioimmunoassay kit for cyclic AMP measurement.

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Binding studies [3]
Competitive binding of [33P]2MeSADP (850 Ci/mmol) to washed platelets at 37°C for 5 min was determined as described in earlier work (Gachet et al., 1995). Binding of [33P]MRS-2179 (2000 Ci/mmol) to washed human platelets in Tyrode's buffer containing 0.01% human serum albumin fatty acid free, was measured at 20°C for 30 min in 3 ml polypropylene tubes in a final volume of 1 ml and saturation was determined by isotopic dilution. The reaction was started by addition of washed platelets to the reaction mixture and all experiments were carried out in triplicate. Non specific binding, determined by incubation in the presence of 1 mM unlabeled A3P5P amounted to about 10–15% of total binding. Saturation and displacement experiments were performed using a single concentration of radiolabeled ligand, [33P]MRS2179 (0.5 nM, 200.000 dpm) or [33P]2MeSADP (0.2 nM, 200.000 dpm), in the presence of increasing concentrations of the appropriate unlabeled ligand. The reactions were terminated by addition of ice cold Tyrode's buffer and rapid filtration through Whatman GF/C glass fiber filters under vacuum, after which the tubes and filters were rinsed twice. Radioactivity bound to the platelets on the filters was measured by scintillation counting (Wallac 1409 β-counter, count rate (DPM/CPM±S.E.M.): 1.070±0.0018; Turku, Finland) and data were analysed with the program EBDA-LIGAND (Munson and Rodbard, 1980). The dissociation constant (Kd) of the radioligand and the inhibition constant for the drug (Ki) were calculated using the GraphPad software package.

P2Y1 receptor-promoted stimulation of inositol phosphate formation by adenine nucleotide analogues was measured in turkey erythrocyte membranes as previously described. The K0.5 values were averaged from 3–8 independently determined concentration–effect curves for each compound. Briefly, 1 mL of washed turkey erythrocytes was incubated in inositol-free medium with 0.5 mCi of 2-[3H]myo-inositol (20 Ci/mmol; American Radiolabelled Chemicals, Inc., St. Louis, MO) for 18–24 h in a humidified atmosphere of 95% air/5% CO2 at 37 °C. Erythrocyte ghosts were prepared by rapid lysis in hypo-tonic buffer (5 mM sodium phosphate, pH 7.4, 5 mM MgCl2, 1 mM EGTA) as described.36 Phospholipase C activity was measured in 25 μL of [3H]inositol-labeled ghosts (approximately 175 μg of protein, 200–500000 cpm/assay) in a medium containing 424 μM CaCl2, 0.91 mM MgSO4, 2 mM EGTA, 115 mM KCl, 5 mM KH2PO4, and 10 mM Hepes, pH 7.0. Assays (200 μL final volume) contained 1 μM GTPγS and the indicated concentrations of nucleotide analogues. Ghosts were incubated at 30 °C for 5 min, and total [3H]inositol phosphates were quantified by anion-exchange chromatography as previously described [1].

Animal/Disease Models: CL57BLr6 mice[3]
Doses: 50 mg/kg
Route of Administration: Injection into the jugular vein of mice
Experimental Results: The bleeding time, which reflects in vivo primary haemostasis, was Dramatically prolonged in MRS-2179-treated mice, 30 s after injection of MRS2179.

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Ex vivo studies [3]
Male Wistar rats weighing 300 g were anesthetized by intraperitoneal injection of 200 μl xylazine base (0.2 mg/kg) and ketamine (1 mg/kg). At time zero, MRS-2179 (50 mg/kg) or vehicle was injected into the penis vein. Blood (6.3 ml) was drawn 5 min later from the abdominal aorta into syringes containing 0.7 ml 3.15% sodium citrate and immediately centrifuged (70 s at 1570×g) at room temperature. Citrated platelet-rich plasma (cPRP) was removed and platelets were adjusted to 5×105/μl with platelet-poor plasma (PPP). Platelet aggregation was measured in citrated platelet-rich plasma from control and MRS-2179-treated rats as described above.

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In vivo studies [3]
The bleeding time was measured 1 min after injection of MRS-2179 (50 mg/kg) or vehicle into the jugular vein of mice. CL57BL/6 mice were bred at Iffa Credo. Male mice weighing 20–30 g were anesthetized by intraperitoneal injection of 150 μl of a mixture of 0.2% xylazine base and 1% ketamine in physiological saline. The mice tail was amputated 3 mm from the tip and was immediately immersed in isotonic 0.9% NaCl buffer at 37°C. The bleeding time was defined as the time required for arrest of bleeding.

[1]. Synthesis, biological activity, and molecular modeling of ribose-modified deoxyadenosine bisphosphate analogues as P2Y(1) receptor ligands. J Med Chem. 2000;43(5):829-842.

[2]. von Kügelgen I. Pharmacological profiles of cloned mammalian P2Y-receptor subtypes. Pharmacol Ther. 2006;110(3):415-432.

[3]. Inhibition of platelet function by administration of MRS2179, a P2Y1 receptor antagonist. Eur J Pharmacol. 2001;412(3):213-221.

The structure-activity relationships of adenosine-3', 5'-bisphosphates as P2Y(1) receptor antagonists have been explored, revealing the potency-enhancing effects of the N(6)-methyl group and the ability to substitute the ribose moiety (Nandanan et al. J. Med. Chem. 1999, 42, 1625-1638). We have introduced constrained carbocyclic rings (to explore the role of sugar puckering), non-glycosyl bonds to the adenine moiety, and a phosphate group shift. The biological activity of each analogue at P2Y(1) receptors was characterized by measuring its capacity to stimulate phospholipase C in turkey erythrocyte membranes (agonist effect) and to inhibit its stimulation elicited by 30 nM 2-methylthioadenosine-5'-diphosphate (antagonist effect). Addition of the N(6)-methyl group in several cases converted pure agonists to antagonists. A carbocyclic N(6)-methyl-2'-deoxyadenosine bisphosphate analogue was a pure P2Y(1) receptor antagonist and equipotent to the ribose analogue (MRS-2179). In the series of ring-constrained methanocarba derivatives where a fused cyclopropane moiety constrained the pseudosugar ring of the nucleoside to either a Northern (N) or Southern (S) conformation, as defined in the pseudorotational cycle, the 6-NH(2) (N)-analogue was a pure agonist of EC(50) 155 nM and 86-fold more potent than the corresponding (S)-isomer. The 2-chloro-N(6)-methyl-(N)-methanocarba analogue was an antagonist of IC(50) 51.6 nM. Thus, the ribose ring (N)-conformation appeared to be favored in recognition at P2Y(1) receptors. A cyclobutyl analogue was an antagonist with IC(50) of 805 nM, while morpholine ring-containing analogues were nearly inactive. Anhydrohexitol ring-modified bisphosphate derivatives displayed micromolar potency as agonists (6-NH(2)) or antagonists (N(6)-methyl). A molecular model of the energy-minimized structures of the potent antagonists suggested that the two phosphate groups may occupy common regions. The (N)- and (S)-methanocarba agonist analogues were docked into the putative binding site of the previously reported P2Y(1) receptor model. [1]

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Membrane-bound P2-receptors mediate the actions of extracellular nucleotides in cell-to-cell signalling. P2X-receptors are ligand-gated ion channels, whereas P2Y-receptors belong to the superfamily of G-protein-coupled receptors (GPCRs). So far, the P2Y family is composed out of 8 human subtypes that have been cloned and functionally defined; species orthologues have been found in many vertebrates. P2Y1-, P2Y2-, P2Y4-, P2Y6-, and P2Y11-receptors all couple to stimulation of phospholipase C. The P2Y11-receptor mediates in addition a stimulation of adenylate cyclase. In contrast, activation of the P2Y12-, P2Y13-, and P2Y14-receptors causes an inhibition of adenylate cyclase activity. The expression of P2Y1-receptors is widespread. The receptor is involved in blood platelet aggregation, vasodilatation and neuromodulation. It is activated by ADP and ADP analogues including 2-methylthio-ADP (2-MeSADP). 2'-Deoxy-N6-methyladenosine-3',5'-bisphosphate (MRS-2179) and 2-chloro-N6-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate (MRS2279) are potent and selective antagonists. P2Y2 transcripts are abundantly distributed. One important example for its functional role is the control of chloride ion fluxes in airway epithelia. The P2Y2-receptor is activated by UTP and ATP and blocked by suramin. The P2Y2-agonist diquafosol is used for the treatment of the dry eye disease. P2Y4-receptors are expressed in the placenta and in epithelia. The human P2Y4-receptor has a strong preference for UTP as agonist, whereas the rat P2Y4-receptor is activated about equally by UTP and ATP. The P2Y4-receptor is not blocked by suramin. The P2Y6-receptor has a widespread distribution including heart, blood vessels, and brain. The receptor prefers UDP as agonist and is selectively blocked by 1,2-di-(4-isothiocyanatophenyl)ethane (MRS2567). The P2Y11-receptor may play a role in the differentiation of immunocytes. The human P2Y11-receptor is activated by ATP as naturally occurring agonist and it is blocked by suramin and reactive blue 2 (RB2). The P2Y12-receptor plays a crucial role in platelet aggregation as well as in inhibition of neuronal cells. It is activated by ADP and very potently by 2-methylthio-ADP. Nucleotide antagonists including N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP (=cangrelor; AR-C69931MX), the nucleoside analogue AZD6140, as well as active metabolites of the thienopyridine compounds clopidogrel and prasugrel block the receptor. These P2Y12-antagonists are used in pharmacotherapy to inhibit platelet aggregation. The P2Y13-receptor is expressed in immunocytes and neuronal cells and is again activated by ADP and 2-methylthio-ADP. The 2-chloro-5-nitro pyridoxal-phosphate analogue 6-(2'-chloro-5'-nitro-azophenyl)-pyridoxal-alpha5-phosphate (MRS2211) is a selective antagonist. mRNA encoding for the human P2Y14-receptor is found in many tissues. However, a physiological role of the receptor has not yet been established. UDP-glucose and related analogues act as agonists; antagonists are not known. Finally, UDP has been reported to act on receptors for cysteinyl leukotrienes as an additional agonist--indicating a dual agonist specificity of these receptors. [2]

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The effects of a potent P2Y1 receptor antagonist, N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS-2179) on adenosine-5'-diphosphate (ADP)-induced platelet aggregation in vitro, ex vivo and on the bleeding time in vivo were determined. In suspensions of washed platelets, MRS-2179 inhibited ADP-induced platelet shape change, aggregation and Ca2+ rise but had no effect on ADP-induced inhibition of adenylyl cyclase. Binding studies using the new radioligand [33P]MRS-2179 showed that washed human platelets displayed 134+/-8 binding sites per platelet with an affinity (Kd) of 109+/-18 nM. Finally, intravenous injection of MRS-2179 resulted in inhibition of rat platelet aggregation in response to ADP and prolonged the bleeding time, in rats or mice, as compared to controls. These results suggest this potent P2Y1 receptor antagonist to be a promising tool to evaluate the in vivo effects of pharmacologically targeting the P2Y1 receptor with a view to antithrombotic therapy.[3]

C11H13N5NA4O9P2

513.16

512.977

C, 25.75; H, 2.55; N, 13.65; Na, 17.92; O, 28.06; P, 12.07

1454889-37-2

MRS2179 tetrasodium hydrate; 101204-49-3

90479745

Typically exists as solid at room temperature

1

13

5

31

594

3

[Na+].[Na+].[Na+].[Na+].CNC1N=CN=C2N([C@@H]3C[C@H](OP([O-])(=O)[O-])[C@@H](COP([O-])(=O)[O-])O3)C=NC=12

XLPQPYQWGFCKEY-IDAKGYGSSA-J

InChI=1S/C11H17N5O9P2.4Na/c1-12-10-9-11(14-4-13-10)16(5-15-9)8-2-6(25-27(20,21)22)7(24-8)3-23-26(17,18)19;;;;/h4-8H,2-3H2,1H3,(H,12,13,14)(H2,17,18,19)(H2,20,21,22);;;;/q;4*+1/p-4/t6-,7+,8-;;;;/m0..../s1

tetrasodium;[(2R,3S,5S)-5-[6-(methylamino)purin-9-yl]-2-(phosphonatooxymethyl)oxolan-3-yl] phosphate

MRS 2179 tetrasodium salt; 1454889-37-2; MRS2179TetrasodiumSalt; tetrasodium;[(2R,3S,5S)-5-[6-(methylamino)purin-9-yl]-2-(phosphonatooxymethyl)oxolan-3-yl] phosphate; MRS 2179 tetrasodium;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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