The activation of poly(ADP-ribose) polymerase-1 (PARP-1) after exposure to nitric oxide or oxygen-free radicals can lead to cell injury via severe, irreversible depletion of NAD. Genetic deletion or pharmacological inhibition of PARP-1 attenuates brain injury after focal ischemia and neurotoxicity in several neurodegenerative models in animals. FR247304 (5-chloro-2-[3-(4-phenyl-3,6-dihydro-1(2H)-pyridinyl)propyl]-4(3H)-quinazolinone) is a novel PARP-1 inhibitor that has recently been identified through structure-based drug design. In an enzyme kinetic analysis, FR247304 exhibits potent and competitive inhibition of PARP-1 activity, with a K i value of 35 nM. Here, we show that prevention of PARP activation by FR247304 treatment protects against both reactive oxygen species-induced PC12 cell injury in vitro and ischemic brain injury in vivo. In cell death model, treatment with FR247304 (10 Ϫ8 -10 Ϫ5 M) significantly reduced NAD depletion by PARP-1 inhibition and attenuated cell death after hydrogen peroxide (100 M) exposure. After 90 min of middle cerebral artery occlusion in rats, poly(ADP-ribosy)lation and NAD depletion were markedly increased in the cortex and striatum from 1 h after reperfusion. The increased poly(ADPribose) immunoreactivity and NAD depletion were attenuated by FR247304 (32 mg/kg i.p.) treatment, and FR247304 significantly decreased ischemic brain damage measured at 24 h after reperfusion. Whereas other PARP inhibitors such as 3-aminobenzamide and PJ34 [N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylactamide] showed similar neuroprotective actions, they were less potent in in vitro assays and less efficacious in an in vivo model compared with FR247304. These results indicate that the novel PARP-1 inhibitor FR247304 exerts its neuroprotective efficacy in in vitro and in vivo experimental models of cerebral ischemia via potent PARP-1 inhibition and also suggest that FR247304 or its derivatives could be attractive therapeutic candidates for stroke and neurodegenerative disease.Activation of nuclear enzyme poly(ADP-ribose) polymerase (PARP) promotes cell death through processes involving energy depletion. Reactive oxygen species (ROS)-mediated damage of DNA can activate PARP (Szabo et al., 1996;Eliasson et al., 1997) and consumes NAD and consequently ATP, culminating in cell dysfunction or necrosis (Ha and Snyder, 1999). In addition, PARP plays a central role in the caspase-independent apoptosis pathway mediated by apoptosis-inducing factor. Translocation of apoptosis-inducing factor from the mitochondria to the nucleus is dependent on PARP activation in neurons treated with various DNA-damaging stimuli, including hydrogen peroxide (Yu et al., 2002). This cellular suicide mechanism of both necrosis and apoptosis by PARP activation has been implicated in the pathogenesis of ischemic brain injury and neurodegenerative disorders, and PARP inhibitors have been shown to be effective in animal models of stroke, traumatic brain injury and Parkinson's disease (Cosi et al., 1996;Endres et al...

Emerging evidence has shown that tumor suppressor p53 expression is enhanced in response to brain ischemia/hypoxia and that p53 plays a critical role in the cell death pathway in such an acute neurological insult. However the mechanism remains unclear. Recently it was reported that Peg3/Pw1, originally identified as a paternally expressed gene, plays a pivotal role in the p53-mediated cell death pathway in mouse fibroblast cell lines. In this study, we found that Peg3/Pw1 expression is enhanced in peri-ischemic neurons in rat stroke model by in situ hybridization analysis, where p53 expression was also induced by immunohistochemical analysis. Moreover, we found that p53 was co-localized with Peg3/Pw1 in brain ischemia/hypoxia by double staining analysis. In human neuroblastoma-derived SK-N-SH cells, Peg3/Pw1 mRNA expression is enhanced remarkably at 24 h post-hypoxia, when p53 protein expression was also enhanced at high levels. Subcellular localization of Peg3/Pw1 was observed in the nucleus. Adenovirus-mediated high dose p53 overexpression induced Peg3/Pw1 mRNA expression. Overexpression of Peg3/Pw1 reduced cell viability under hypoxic conditions, whereas that of the C-terminal-deleted mutant and anti-sense Peg3/Pw1 inhibited hypoxia-induced cell death. These results suggest that Peg3/Pw1 is involved in the p53-mediated cell death pathway as a downstream effector of p53 in brain ischemia/hypoxia.

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The anti-platelet properties of FK419 ((S)-2-acetylamino-3-[(R)- [1-[3-(piperidin-4-yl)propionyl]piperidin-3-ylcarbonyl] amino]propionic acid), a novel non-peptide GPIIb/IIIa antagonist, were compared in a variety of experimental settings, both in vitro and in vivo, with other GPIIb/IIIa antagonists including xemilofiban, lamifiban, tirofiban, and FK633. Receptor binding studies suggested that FK419 had potent GPIIb/IIIa antagonistic activity that is comparable with those of reference antagonists. FK419 effectively inhibited human platelet aggregation, regardless of agonist stimuli (IC 50 ¼ 35-170 nM). FK419 demonstrated in vitro species-dependent anti-platelet activity, with higher potency in human than in dog, guinea pig, or rat tissue, and dose-dependently inhibited ex vivo platelet aggregation in dogs and guinea pigs. In contrast to other antagonists, FK419 minimally affected template bleeding time at doses that completely inhibited platelet aggregation in canines. These results demonstrate that FK419 is a novel, potent, and selective GPIIb/IIIa antagonist that safely inhibits platelet aggregation in vivo, suggesting that it may be a promising anti-platelet agent for thrombotic diseases. Drug Dev.

Platelet activation and subsequent aggregation play a key role in the pathogenesis of ischemic brain damage. Recent studies revealed that enhanced platelet activation is also observed after ischemia, suggesting that secondary thrombus formation might participate in the development of cerebral infarction. The binding of platelet glycoprotein GPIIb/IIIa (integrin ␣ IIb ␤3) to fibrinogen is the final common pathway in platelet aggregation. Therefore, GPIIb/IIIa antagonists might be useful in acute ischemic stroke as well as in the secondary prevention of ischemic stroke. In the present study, we evaluated the effect of three compounds, FK419 ((S)-2-acetylamino-3-[(R)- [1-[3-(piperidin-4-yl) propionyl] piperidin-3-ylcarbonyl] amino] propionic acid trihydrate), a novel nonpeptide GPIIb/IIIa antagonist, ozagrel, a selective thromboxane A 2 synthase inhibitor, and argatroban, a thrombin inhibitor, on middle cerebral artery (MCA) patency and ischemic brain damage using photochemically induced MCA thrombosis model in guinea pigs. FK419, ozagrel, or argatroban was administered 5 min after the termination of photoirradiation. FK419 dose-dependently improved MCA patency by decreasing the total occlusion time, time to continuous reperfusion, and the number of cyclic flow reductions, at doses that inhibited ADP-induced platelet aggregation ex vivo. In contrast, ozagrel only improved total occlusion time, and argatroban showed no improvement in MCA patency. FK419 also reduced ischemic brain damage in a dose-dependent fashion, whereas ozagrel and argatroban did not. Finally, FK419 ameliorated neurological deficits, whereas ozagrel and argatroban did not. These results indicate that FK419, a GPIIb/IIIa antagonist, ameliorates ischemic brain damage by improving MCA patency after occlusion and that FK419 is a promising candidate for the treatment of acute ischemic stroke.