3-4-dihydro-5-(4-(1-piperidinyl)butoxy)-1(2h)-isoquinolinone and Brain-Ischemia

Excessive activation of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme catalyzing the transfer of ADP-ribose units from NAD to acceptor proteins, induces cellular energy failure by NAD and ATP depletion and has been proposed to play a causative role in a number of pathological conditions, including ischemia/reperfusion injury. In this study, we used an in vitro enzyme activity assay to characterize a series of newly synthesized isoquinolinone derivatives as potential PARP-1 inhibitors. Several compounds displayed powerful inhibitory activity: thieno[2,3-c]isoquinolin-5-one (TIQ-A) displayed a submicromolar IC50 of 0.45 +/- 0.1 microM, whereas the 5-hydroxy and 5-methoxy TIQ-A derivatives had IC50 values of 0.39 +/- 0.19 and 0.21 +/- 0.12 microM, respectively. We then examined the neuroprotective effects of the newly characterized compounds in cultured mouse cortical cells exposed to 60 min of oxygen and glucose deprivation (OGD). When PARP-1 inhibitors were present in the incubation medium during OGD and the subsequent 24-h recovery period, they significantly attenuated neuronal injury. TIQ-A provided neuroprotection even when added to the culture 30 min after OGD and was able to reduce the early activation of PARP induced by OGD as detected by flow cytometry. When the IC50 values observed in the PARP-1 activity assay for selected compounds were compared with their IC50 values for the neuroprotective activity, a significant correlation (r = 0.93, P < 0.01) was observed. Our results suggest that TIQ-A and its derivatives are a new class of neuroprotectants that may be helpful in studies aimed at understanding the involvement of PARP-1 in physiology and pathology.

Topics: Animals; Brain Ischemia; Cells, Cultured; Disease Models, Animal; Isoquinolines; Mice; Neuroprotective Agents; Phenanthrenes; Piperidines; Poly(ADP-ribose) Polymerase Inhibitors; Thiophenes

The single-cell gel electrophoresis (comet assay) was used to evaluate the possibility of detecting single-strand breaks of brain DNA in the early phase of ischemia. Four hours after occlusion of the middle cerebral artery (MCAO) in rats, the percentage of DNA migrating into the comet tail (indicating the presence of breaks) increased from 11.4 +/- 4.70 to 34.7 +/- 9.2 (means +/- SD) in the caudate and from 9.9 +/- 4.3 to 42.8 +/- 14.1 in the cortex. Interestingly, a subpopulation of cells exhibiting higher resistance to the ischemic insult was present in the caudate putamen, but not in the cortex. Administration of MK801, an N-methyl-d-aspartate (NMDA) glutamate receptor antagonist, (1 mg/kg subcutaneously, 10 minutes before MCAO), reduced the ischemia-induced DNA breaks and the infarct volume, suggesting that excessive stimulation of NMDA receptors contributes to the formation of both DNA damage and infarct volume. In contrast, DPQ, an inhibitor of poly(ADP-ribose) polymerase (PARP) (10 mg/kg intraperitoneally, 2 hours before and 1 hour after MCAO), reduced the infarct volume but not DNA damage, suggesting that the neuroprotective actions of PARP inhibitors occur at a later step of the processes leading to postischemic neuronal death.

Topics: Animals; Brain Ischemia; Comet Assay; Dizocilpine Maleate; DNA; DNA Damage; DNA, Single-Stranded; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Infarction, Middle Cerebral Artery; Isoquinolines; Male; Neuroprotective Agents; Piperidines; Poly(ADP-ribose) Polymerase Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate

An excessive activation of poly(ADP-ribose) polymerase (PARP) has been proposed to play a key role in post-ischemic neuronal death. We examined the neuroprotective effects of the PARP inhibitors benzamide, 6(5H)-phenanthridinone, and 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinone in three rodent models of cerebral ischemia. Increasing concentrations of the three PARP inhibitors attenuated neuronal injury induced by 60 min oxygen-glucose deprivation (OGD) in mixed cortical cell cultures, but were unable to reduce CA1 pyramidal cell loss in organotypic hippocampal slices exposed to 30 min OGD or in gerbils following 5 min bilateral carotid occlusion. We then examined the necrotic and apoptotic features of OGD-induced neurodegeneration in cortical cells and hippocampal slices using biochemical and morphological approaches. Cortical cells exposed to OGD released lactate dehydrogenase into the medium and displayed ultrastructural features of necrotic cell death, whereas no caspase-3 activation nor morphological characteristics of apoptosis were observed at any time point after OGD. In contrast, a marked increase in caspase-3 activity was observed in organotypic hippocampal slices after OGD, together with fluorescence and electron microscope evidence of apoptotic neuronal death in the CA1 subregion. Moreover, the caspase inhibitor Z-VAD-FMK reduced OGD-induced CA1 pyramidal cell loss. These findings suggest that PARP overactivation may be an important mechanism leading to post-ischemic neurodegeneration of the necrotic but not of the apoptotic type.

Topics: Animals; Apoptosis; Benzamides; Brain Ischemia; Caspase 3; Caspases; Cell Death; Cell Line; Cerebral Cortex; Enzyme Inhibitors; Gerbillinae; In Vitro Techniques; Isoquinolines; Microscopy, Electron; Microscopy, Fluorescence; Necrosis; Neurons; Neuroprotective Agents; Phenanthrenes; Piperidines; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Pyramidal Cells; Rats; Rats, Wistar

Poly(adenosine 5'-diphosphoribose) synthetase (PARS) has been described as an important candidate for mediation of neurotoxicity by nitric oxide. In the current study, we demonstrate for the first time that in vivo administration of a potent PARS inhibitor, 3,4-dihydro 5-[4-1(1-piperidinyl) butoxy]-1(2H)-isoquinolinone, leads to a significant reduction of infarct volume in a focal cerebral ischemia model in the rat. Focal cerebral ischemia was produced by cauterization of the right distal middle cerebral artery (MCA) with bilateral temporary common carotid artery occlusion for 90 minutes. 3,4-Dihydro 5[4-(1-piperidinyl) butoxy]-1(2H)-isoquinolinone was dissolved in dimethyl sulfoxide and injected intraperitoneally. Animals were treated 2 hours before MCA occlusion (control, n = 14; 5 mg/kg, n = 7; 10 mg/kg, n = 7; 20 mg/kg, n = 7; 40 mg/kg, n = 7), and 2 hours after MCA occlusion (same doses as before treatment). Twenty-four hours after MCA occlusion, the total infarct volume was measured using 2,3,5-triphenyltetrazolium chloride. Inhibition of PARS leads to a significant decrease in the damaged volume in the 5 mg/kg-treated group (106.7 +/- 23.2 mm3; mean +/- SD, P < 0.002), the 10 mg/kg-treated group (76.4 +/- 16.8 mm3, P < 0.001), and the 20 mg/kg-treated group (110.2 +/- 42.0 mm3, P < 0.02) compared with the control group (165.2 +/- 34.0 mm3). The substantial reduction in infarct volume indicates that the activation of PARS may play an important role in the pathogenesis of brain damage in cerebral ischemia through intracellular energy depletion.

Topics: Animals; Brain Ischemia; Carotid Artery, Common; Cerebral Arteries; Cerebrovascular Circulation; Constriction, Pathologic; Enzyme Inhibitors; Isoquinolines; Male; Neuroprotective Agents; Piperidines; Poly(ADP-ribose) Polymerase Inhibitors; Rats; Rats, Inbred Strains