Predominant role of spinal P2Y1 receptors in the development of neuropathic pain in rats.

The role of P2X2/3, P2X3, P2X4 or P2X7 and P2Y2, P2Y6, and P2Y12 receptors in neuropathic pain has been widely studied. In contrast, the role of P2Y1 receptors is scarcely studied. In this study we assessed the role of P2Y1 receptors in several neuropathic pain models in the rat. Furthermore, we analyzed the expression of P2Y1 receptors in the ipsilateral dorsal root ganglia (DRG) and dorsal part of the spinal cord during the development and maintenance of neuropathic pain. We also determined the effect of the P2Y1 receptor antagonist on the expression of P2Y1 receptors. Chronic constriction injury (CCI), spared nerve injury (SNI) or spinal nerve ligation (SNL) produced tactile allodynia from 1 to 14 days after nerve injury. CCI, SNI and SNL enhanced expression of P2Y1 receptors in DRG but not in the dorsal part of the spinal cord at 1-3 days after injury. Intrathecal injection of the selective P2Y1 receptor antagonist MRS2500, but not vehicle, reduced tactile allodynia in rats 1-3 days after CCI, SNI, or SNL. Moreover, intrathecal injection of MRS2500 (at day 1 or 3) reduced neuropathy-induced up-regulation of P2Y1 receptors expression. Intrathecal injection of MRS2500 lost most of the antiallodynic effect when injected 14 days after injury. At this time, MRS2500 did not modify nerve-injury-induced P2Y1 receptors up-regulation. Our results suggest that P2Y1 receptors are localized in DRG, are up-regulated by nerve injury and play a pronociceptive role in development and, to a lesser extent, maintenance of neuropathic pain.

ATP P2Y1 receptors control cognitive deficits and neurotoxicity but not glial modifications induced by brain ischemia in mice.

ATP is a pleiotropic cell-to-cell signaling molecule in the brain that functions through activation of the P2 receptors (P2R), encompassing ionotropic P2XR or metabotropic P2YR. Noxious brain insults increase the extracellular levels of ATP and previous studies have implicated different P2R, namely P2Y1R, in the control of ischemic brain damage, but it remains to be defined if P2Y1R antagonists also alleviate the behavioral impairments associated with brain ischemia. Furthermore, as P2Y1R can control neuronal and glial functions, we explored if P2Y1R antagonist-mediated protection would mainly involve neuronal and/or glial processes. Adult male mice subject to permanent middle cerebral artery occlusion (pMCAO) displayed an infarcted cortical area (2,3,5-triphenyltetrazolium chloride staining), decreased neurological score with decreased working and reference memory performance (Y-maze, object recognition and aversive memory), accompanied by neuronal damage (FluoroJade C), astrogliosis (glial fibrillary acidic protein) and microgliosis (CD11b). All of these changes were attenuated by intracerebroventricular pre-treatment (10 min before pMCAO) with the generic P2R antagonist 4-[(E)-{4-formyl-5-hydroxy-6-methyl-3-[(phosphono-oxy)methyl]pyridin-2-yl}diazenyl]benzene-1,3-disulfonic acid (PPADS, 0.5-1.0 nmol/µL). In contrast, the selective P2Y1R antagonist (1R*,2S*)-4-[2-Iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphono-oxy)bicycle[3.1.0] hexane-1-methanol dihydrogen phosphate ester (MRS2500, 1.0-2.0 nmol/µL) afforded equivalent behavioral benefits but only prevented neuronal damage but not astrogliosis or microgliosis upon pMCAO. These results indicated that P2Y1R-associated neuroprotection mainly occurred through neuronal mechanisms, whereas other P2R were also involved in the control of astrocytic reactivity upon brain injury.

Activation of the P2Y1 receptor induces apoptosis and inhibits proliferation of prostate cancer cells.

G protein-coupled receptors, the largest cell surface receptor family, have emerged as critical players in cell death and survival. High gene expression level of the G(q)-coupled P2Y(1) nucleotide receptor in PC-3 prostate cancer cells was demonstrated using real-time quantitative PCR and confirmed by Western blotting and confocal laser scanning microscopy. A selective P2Y(1) receptor agonist, the ADP analogue MRS2365, concentration-dependently induced intracellular calcium mobilization (EC(50) 5.28nM), which was diminished by P2Y(1) receptor-selective antagonist MRS2500. P2Y(1) receptor activation by MRS2365 induced apoptosis in assays of Caspase-3, LDH release, and annexin-V staining. The pro-apoptotic effect of MRS2365 was blocked by MRS2500, P2Y(1) siRNA, and an inhibitor of the MAP kinase pathway PD98059. MRS2365 significantly inhibited the proliferation of PC-3 cells, examined using a MTT assay. Thus, activation of the P2Y(1) receptor induced cell death and inhibited growth of human prostatic carcinoma PC-3 cells. Activation of the P2Y(1) receptor should be a novel and promising therapeutic strategy for prostate cancer.

MRS2500 [2-iodo-N6-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate], a potent, selective, and stable antagonist of the platelet P2Y1 receptor with strong antithrombotic activity in mice.

The platelet P2Y(1) ADP receptor is an attractive target for new antiplatelet drugs. However, because of the lack of strong and stable antagonists, only a few studies have suggested that pharmacological inhibition of the P2Y(1) receptor could efficiently inhibit experimental thrombosis in vivo. Our aim was to determine whether the newly described potent and selective P2Y(1) receptor antagonist MRS2500 [2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate] could inhibit platelet function ex vivo and experimental thrombosis in mice in vivo. MRS2500 was injected intravenously into mice, and its effect on ex vivo platelet aggregation and in several models of thrombosis in vivo was determined. MRS2500 displayed high potency and stable and selective P2Y(1) receptor inhibition ex vivo. Although MRS2500 injection resulted in only moderate prolongation of the bleeding time, it provided strong protection in systemic thromboembolism induced by infusion of a mixture of collagen and adrenaline. MRS2500 also potently inhibited localized arterial thrombosis in a model of laser-induced vessel wall injury with two degrees of severity. Moreover, combination of MRS2500 with clopidogrel, the irreversible inhibitor of the platelet P2Y(12) receptor for ADP, led to increased antithrombotic efficacy compared with each alone. These results add further evidence for a role of the P2Y(1) receptor in thrombosis and validate the concept that targeting the P2Y(1) receptor could be a relevant alternative or complement to current antiplatelet strategies.

2',3'-Bis(2-chloroethyl)aminophosphoryl-3'-amino-3'-deoxyadenosine: a cyclic nucleotide with antitumor activity.

The synthesis of the title compound from 3'-amino-3'-deoxyadenosine in 40% yield is reported. 3'-Amino-3'-deoxyadenosine was made by an improved synthesis in 12 steps from inexpensive D-xylose in 15% overall yield. Both isomers of the title compound, separated by column chromatography, possess confirmed activity against KB tumor cell cultures.