DY-9760e, a calmodulin antagonist, reduces brain damage after permanent focal cerebral ischemia in cats

DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate), a calmodulin antagonist, provides protection against Ca(2+) overload-associated cytotoxicity and brain injury after cerebral ischemia in rats. In this study, we assessed the effect of DY-9760e on ischemic infarct volume in cats subjected to permanent focal cerebral ischemia. DY-9760e was infused for 6 h, beginning 5 min after occlusion of the middle cerebral artery. The infarct volume was measured at the end of drug infusion. DY-9760e, at the dose of 0.25 but not 0.1 mg/kg/h, significantly reduced cerebral infarct volume without affecting any physiological parameters, and its protective effect was mainly evident in the cerebral cortex, where the penumbra, a salvageable zone, exists. The present study demonstrates that DY-9760e protects against brain injury after focal ischemia in a gyrencephalic animal as well as in the rodents reported previously and suggests its therapeutic value for the treatment of acute stroke.     

DY-9760e, a novel calmodulin antagonist, reduces brain damage induced by transient focal cerebral ischemia.

Perturbations in Ca2+ homeostasis have been proposed to lead to neuronal damage after cerebral ischemia. DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1- (4-imidazolylethyl)-1H-indazole dihydrochloride 3.5 hydrate) is a novel calmodulin antagonist. In this study, we examined the effects of DY-9760e on brain damage in rats subjected to transient (1 h) focal cerebral ischemia. DY-9760e (0.25-1.00 mg kg(-1) h(-1)) was intravenously infused for 6 h, starting 1 h after middle cerebral artery occlusion. Treatment with DY-9760e 0.25, 0.50 and 1.00 mg kg(-1) h(-1) reduced infarct volume by 30, 42 (P < 0.05), and 60% (P < 0.05), respectively. Furthermore, the effect of DY-9760e on ischemia-induced fodrin breakdown was examined in the same model. Pronounced fodrin breakdown was observed in the cerebral cortex and striatum at 24 h after ischemia. DY-9760e caused potent suppression of fodrin breakdown in the cerebral cortex at the same doses as those that had a protective action against cerebral infarction. From these results DY-9760e may have a therapeutic effect against cerebral ischemic damage in the acute stage.     

Pharmacokinetics and disposition of DY-9760e, a novel calmodulin antagonist, in rats and monkeys

DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, CAS 162496-41-5) is a novel calmodulin antagonist that is being evaluated for the treatment of ischemia. The objective of this study was to characterize the pharmacokinetics and disposition of DY-9760e in rats and monkeys. After a 6 h continuous infusion at 1 mg/kg/h to male rats, the plasma concentration of unchanged DY-9760, as the anhydrous free base of DY-9760e, declined with a terminal half-life of 3.0 h. In monkeys, the plasma concentration of DY-9760 following a 4 h continuous infusion at 1 mg/kg/h declined with a terminal half-life of 3.8 h. Total clearance was 18.3 ml/min/kg in rats and 16.7 ml/min/kg in monkeys. The pharmacokinetics of DY-9760e was linear within a dose range from 1 mg/kg to 16 mg/kg in monkeys. After intravenous bolus administration of 14C-DY-9760e to rats, the radioactivity was widely distributed throughout the body except for the brain and testis. In the brain, which is the target organ of this compound, the concentrations of unchanged DY-9760 in rats were much lower than the corresponding plasma concentrations. These results indicated that the permeability of DY-9760 into the brain was restricted. In contrast, the brain concentrations of the N-dealkylated metabolite DY-9836 were approximately 2- to 3-fold higher than those observed in the plasma. The administered radioactivity was excreted mostly in the feces (95.2% in rats, 83.6% in monkeys), and the biliary excretion of the radioactivity in bile duct-cannulated rats was 86.2% within 48 h, part of which (11.1%) was re-absorbed. The urinary excretion of unchanged DY-9760 was less than 0.5% in both species. The metabolic profile characterized by thin-layer chromatography demonstrated that most of the radioactivity in the urine and bile referred to many polar metabolites. These results indicate that DY-9760e is eliminated mainly through hepatic metabolic clearance in both rats and monkeys.     

Cytoprotective effect of 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e) against ischemia/reperfusion-induced injury in rat heart involves inhibition of fodrin breakdown and protein tyrosine nitration

We here assessed the effects of 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), a novel calmodulin antagonist, on infarct size in the rat heart subjected to ischemia/reperfusion. Rats were subjected to a 30-min coronary occlusion followed by a 24-h reperfusion. DY-9760e was intravenously infused for 20 min, starting at 20 min after coronary occlusion. Treatment with DY-9760e (10 mg/kg) significantly reduced the infarct size in the risk area assessed by Evans Blue/TTC (triphenyltetrazolium chloride) staining. DY-9760e treatment also ameliorated contractile dysfunction of the left ventricle 72 h after reperfusion. DY-9760e significantly inhibited fodrin breakdown and caspase-3 activation. The inhibitory effect of DY-9760e on the fodrin breakdown was prominent in the rim rather than in the center of the risk area. DY-9760e also blocked protein tyrosine nitration associated with infarction. These results suggest that the cardioprotective effect of DY-9760e involved inhibition of calpain/caspase activation and protein tyrosine nitration.     

Inhibition of nitric oxide production and protein tyrosine nitration contribute to neuroprotection by a novel calmodulin antagonist, DY-9760e, in the rat microsphere embolism

Microsphere embolism (ME)-induced ischemia model in rat resembles to multiple brain embolism in human with several clinical features. We here tested whether nitric oxide (NO) production contributes to the neuronal injury in the ME model. A novel calmodulin antagonist, DY-9760e, having a potent inhibitory effect on neuronal nitric oxide synthase (nNOS), reduced brain infarct size in the ME-induced brain ischemia. Consistent with our previous observation with gerbil ischemia/reperfusion model, DY-9760e completely inhibited NO production immediately after and 24 or 48 h after ME. Unlike the gerbil ischemia/reperfusion model, protein tyrosine nitration markedly increased 6-48 h after ME. DY-9760e treatment completely inhibited the marked increase in the protein tyrosine nitration at 24 h after ME. These results suggest that the inhibition of NO production and protein tyrosine nitration by DY-9760e contribute to its neuroprotective action in the ME-induced brain damage.     

DY-9760e, a novel calmodulin inhibitor, exhibits cardioprotective effects in the ischemic heart

DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride-3.5 hydrate) inhibits Ca(2+)/CaM-dependent nitric oxide synthase (NOS), thereby inhibiting nitric oxide (NO) production. In cardiomyocytes from ischemic rat heart NO and superoxide levels are increased causing protein tyrosine nitration. In hearts subjected to ischemia/reperfusion DY-9760e totally abolishes protein tyrosine nitration. Notably, DY-9760e also inhibits calpain and cas-pase-3 activation that occurs prior to apoptosis in cardiomyocytes. In ischemic hearts fodrin is the substrate for calpain. DY-9760e inhibits fodrin breakdown in the peri-infarct area rather than in the infarct core. In the ischemic rat brain DY-9760e inhibits caspase-3-induced proteolysis of calpastatin, an endogenous calpain inhibitor, suggesting that crosstalk between calpain and caspase-3 is mediated by calpastatin breakdown. Thus, DY-9760e rescues neurons and cardiomyocytes from ischemic injury by inhibiting crosstalk between calpain and caspase-3 as well as protein tyrosine nitration.     

Protective effect of DY-9760e, a calmodulin antagonist, against neuronal cell death

An excessive elevation of intracellular Ca(2+) levels is known to play a key role in the pathological events following cerebral ischemia. DY-9760e, 3-[2-[4-(3-chloro-2-methylphenylmethyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, is a potent calmodulin antagonist that attenuates brain damage in focal ischemia models. In the present study, we investigated the effect of DY-9760e on neuronal cell death induced by a variety of cell-toxic stimuli that increase intracellular Ca(2+). Cell death was induced by the exposure of primary cultured neurons to excitotoxic agents such as glutamate and N-methyl-D-aspartate, membrane-depolarizing agents such as veratridine and high KCl, or thapsigargin an endoplasmic reticulum Ca(2+)-ATPase inhibitor. Treatment with DY-9760e resulted in a dose-dependent prevention of neuronal cell death elicited by excitotoxicity, voltage-gated channel opening, and inhibition of endoplasmic reticulum Ca(2+)-ATPase. These results indicate that DY-9760e can rescue neurons from various types of cell-toxic stimuli, which may contribute to attenuation of brain injury after cerebral ischemia.     

The post-ischemic administration of 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), a novel calmodulin antagonist, prevents delayed neuronal death in gerbil hippocampus

The novel calmodulin (CaM) antagonist DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate) with an apparent neuroprotective effect in vivo preferentially inhibits neuronal nitric oxide synthase (nNOS), Ca2+/CaM-dependent protein kinase IIalpha (CaMKIIalpha), and calcineurin in vitro. In the present study, we investigated the molecular mechanism underlying its neuroprotective effect with the gerbil transient forebrain ischemia model, by focusing on its inhibition of these Ca2+/CaM-dependent enzymes. Post-ischemic DY-9760e treatment (5 mg/kg, i.p.) immediately after 5-min ischemia significantly reduced the delayed neuronal death in the hippocampal CA1 region. CaMKIIalpha was transiently autophosphorylated immediately after reperfusion with concomitant sustained decrease in its total amounts in the Triton X-100-soluble fractions. Calcineurin activity, accessed by the phosphorylation state of dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32) at Thr34, was elevated at 6 h after reperfusion. Post-treatment of DY-9760e had no effects on both CaMKIIalpha and DARPP-32 phosphorylation at 6 h after reperfusion. However, DY-9760e significantly inhibited nitrotyrosine formation, as a biomarker of NO, and in turn, peroxynitrite (ONOO-) production. These results suggest that DY-9760e primarily inhibits Ca2+/CaM-dependent neuronal NOS, without any effects on CaMKII and calcineurin, and the inhibition of NO production possibly accounts for its neuroprotective action in brain ischemic injury.     

Neuroprotection by LY341122, a novel inhibitor of lipid peroxidation, against focal ischemic brain damage in rats

LY341122 (2-(3, 5-di-t-butyl-4-hydroxyphenyl)-4-(2-(4-methylethylaminomethyl-ph enylox y)ethyl)oxazole) is a potent inhibitor of lipid peroxidation which has been shown to protect against global ischemia and traumatic brain injury in rats. The purpose of this study was to examine the effect of LY341122 on ischemic injury in a highly reproducible model of focal cerebral ischemia in rats. Male Sprague-Dawley rats were anesthetized with halothane and subjected to 120 min of temporary middle cerebral artery occlusion by retrograde insertion of an intraluminal nylon suture coated with poly-L-lysine. The drug (LY341122, n=19) or vehicle (phosphate-buffered saline (PBS), n=10) was administered i.v. (as a 5 or 10 mg/kg bolus followed by a 5 or 10 mg/kg/h infusion for 20 h, respectively, starting 1 or 2 h after the onset of middle cerebral artery occlusion). Neurological status was evaluated during middle cerebral artery occlusion (60 min) and daily for 3 days thereafter. Three days after ischemia, brains were perfusion-fixed and infarct volumes and brain edema were determined. LY341122 significantly improved the neurological score compared to vehicle at 24, 48 and 72 h after middle cerebral artery occlusion. Treatment with LY341122 significantly reduced total infarct volume in all treated groups compared to vehicle rats. Cortical infarct volume was significantly reduced by LY341122 treatment in the 10 mg/kg (1 h) and LY341122 10 mg/kg (2 h) groups compared to vehicle rats (14.7+/-9.5 vs. 106.8+/-20.9 mm(3), and 36.9+/-20.1 vs. 106. 8+/-20.9 mm(3), respectively (mean+/-S.E.M.)). Striatal infarct volume was also significantly reduced by treatment with LY341122 in the 10 mg/kg (1 h) group compared to vehicle (23.7+/-3.4 vs. 68. 2+/-6.7 mm(3)). These results demonstrate the neuroprotective efficacy of LY341122 in focal cerebral ischemia.     

Combination of low dose ethanol and caffeine protects brain from damage produced by focal ischemia in rats

Caffeine and ethanol are two commonly overused psychoactive dietary components. The purpose of this study was to assess the effects of acute, chronic, oral (p.o.) and intravenous (i.v.) caffeine, ethanol and their combination on infarct volume following focal ischemia in rats. Rats received treatment either p.o. 3 h and 1 h before, or by i.v. infusion for 2.5 h beginning 30-180 min after, ischemia. There were six acute treatment groups. (1) oral dH2O (control); (2) oral caffeine (10 mg/kg); (3) oral ethanol (0.65 g/kg total); (4) oral ethanol plus caffeine; (5) intravenous saline; and (6) intravenous ethanol (0.65 g/kg) plus caffeine (10 mg/kg) in saline. A 7th group received oral ethanol plus caffeine for three weeks prior to ischemia. After 3 h of left MCA/CCA occlusion and 24 h reperfusion, infarct volume was determined. Control animal infarct volume was 102.4+/-42.0 mm3. Oral caffeine alone had no effect (122.4+/-30.2 mm3). Oral ethanol alone exacerbated infarct volume (177.2+/-27.8 mm3). Oral caffeine plus ethanol almost entirely eliminated the damage (17.89+/-10.41 mm3). When i.v. treatment with ethanol plus caffeine was initiated at 30, 60, 90 and 120 minutes post-ischemia the infarct volume was reduced by 71.7%, 49.8%, 64.8% and 47.1%, respectively. Chronic daily oral ethanol plus caffeine prior to ischemia eliminated the neuroprotection seen with acute treatment. These studies indicate that ethanol, which by itself aggravates cerebral ischemia, and caffeine, when combined together immediately before or for 2 h after focal stroke, reduces ischemic damage.     

白三烯拮抗剂ONO-1078对小鼠持续性局灶性脑缺血的作用

目的在小鼠模型研究白三烯拮抗剂{4-氧-8-[对-(4-苯丁氧基)苯甲酰氨基]-2-(5-四唑基)-4H-1-苯并吡喃半水合物(ONO-1078)}对脑缺血的保护作用.方法以大脑中动脉阻塞法诱导持续性局灶性脑缺血.在脑缺血前30min和缺血后60min腹腔注射ONO-1078、尼莫地平或生理盐水(对照).脑缺血24h后,观察神经症状,并测定脑湿重和脑梗死体积.结果ONO-1078(0.01,0.03,0.1mgkg-1)和尼莫地平(0.2 mg*kg-1)减少脑梗死体积;ONO-1078(0.1 mg kg-1)减轻脑湿重,尼莫地平无此作用;ONO-1078和尼莫地平对神经症状无明显作用.结论ONO-1078对持续性局灶性脑缺血有保护作用,提示白三烯拮抗剂可作为急性脑缺血的一种新治疗药.     

Montelukast, a cysteinyl leukotriene receptor-1 antagonist, dose- and time-dependently protects against focal cerebral ischemia in mice

Our previous studies showed that cysteinyl leukotriene receptor-1 (CysLT1) antagonist pranlukast has a neuroprotective effect on cerebral ischemia in rats and mice. However, whether the neuroprotective effect of pranlukast is its special action or a common action of CysLT1 receptor antagonists remains to be clarified. This study was performed to determine whether montelukast, another CysLT1 receptor antagonist, has the neuroprotective effect on focal cerebral ischemia in mice, and to observe its dose- and time-dependent properties. Permanent focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). Montelukast was injected intraperitoneally either as multiple doses (once a day for 3 days and 30 min before MCAO) or as a single dose (at 30 min before, 30 min after, or 1 h after MCAO), respectively, and pranlukast and edaravone were used as controls. The neurological deficits, infarct volumes, brain edema, neuron density, and Evans blue extravasation in the brain were determined 24 h after MCAO. Pretreatments with multiple doses or a single dose of montelukast (0.1 and 1.0 mg/kg) before MCAO significantly attenuated all the ischemic insults. Post-treatment with a single dose of montelukast (0.1 and 1.0 mg/kg) at 30 min after MCAO also significantly decreased brain edema and infarct volume, but not neurological deficits. However, post-treatment with a single dose of montelukast at 1 h after MCAO had no significant effect. Pranlukast showed the same effects as montelukast, but edaravone attenuated the ischemic insults only with multiple doses before MCAO. Thus, montelukast has a dose- and time-dependent neuroprotective effect on permanent focal cerebral ischemia in mice, with an effective dose range of 0.1-1.0 mg/kg and a therapeutic window of 30 min. These findings further support the therapeutic potential of CysLT1 receptor antagonists in the treatment of cerebral ischemia at earlier phases.     

Inhibitory effect of sulfobutyl ether beta-cyclodextrin on DY-9760e-induced cellular damage: In vitro and in vivo studies

The effects of water-soluble beta-cyclodextrin derivatives (beta-CyDs), such as 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) and sulfobutyl ether beta-cyclodextrin (SBE7-beta-CyD) on cytotoxicity of DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate) toward human umbilical vein endothelial cells (HUVECs) in vitro and vascular damage of the auricular vein of rabbits by DY-9760e in vivo were investigated. The spectroscopic study revealed that of the four beta-CyDs SBE7-beta-CyD forms the most stable inclusion complex in phosphate-buffered saline, probably because of a synergetic effect of hydrophobic and electrostatic interactions. beta-CyDs inhibited DY-9760e-induced cell death toward HUVECs in an order of G(2)-beta-CyD < beta-CyD < HP-beta-CyD < SBE7-beta-CyD, which was consistent with the order of the magnitude of stability constants. When the DY-9760e solution was infused into the auricular vein of rabbits for 24 h, SBE7-beta-CyD suppressed a DY-9760e-induced irritation such as thrombus, desquamation of the endothelium vasculitis, and perivasculitis. The present data indicated that SBE7-beta-CyD formed an inclusion complex with DY-9760e in a buffer solution and possessed the protective effect on DY-9760e-induced cytotoxicity toward HUVECs and vascular damage in rabbits. These results suggested potential use of SBE7-beta-CyD as a parenteral carrier for DY-9760e.     

LF 16-0687 Ms,a bradykinin B2 receptor antagonist,reduces ischemic brain injury in a murine model of transient focal cerebral ischemia

1. Bradykinin promotes neuronal damage and brain edema through the activation of the B(2) receptor. The neuroprotective effect of LF 16-0687 Ms, a B(2) receptor antagonist, has been described when given prior to induction of transient focal cerebral ischemia in rat, but there are no data regarding the consequence of a treatment when given after injury. Therefore, in a murine model of transient middle cerebral artery occlusion (MCAO), we evaluated the effect of LF 16-0687 Ms given prior to and/or after the onset of ischemia on neurological deficit, infarct volume and inflammatory responses including cerebral edema, blood-brain barrier (BBB) disruption and neutrophil accumulation. 2. LF 16-0687 Ms (1, 2 and 4 mg kg(-1)) administered 0.5 h before and, 1.25 and 6 h after MCAO, decreased the infarct volume by a maximum of 33% and significantly improved the neurological recovery. 3. When given at 0.25 and 6.25 h after MCAO, LF 16-0687 Ms (1.5, 3 and 6 mg kg(-1)) decreased the infarct volume by a maximum of 25% and improved the neurological score. 4. Post-treatment with LF 16-0687 Ms (1.5 mg kg(-1)) significantly decreased brain edema (-28%), BBB disruption (-60%) and neutrophil accumulation (-65%) induced by ischemia. Physiological parameters were not modified by LF 16-0687 Ms. 5. These data emphasize the role of bradykinin B(2) receptor in the development of infarct lesion, neurological deficit and inflammatory responses resulting from transient focal cerebral ischemia. Therefore, B(2) receptor antagonist might represent a new therapeutic approach in the pharmacological treatment of stroke.     

In vivo neuroprotective effects of ACEA 1021 confirmed by magnetic resonance imaging in ischemic stroke

The neuroprotective activity of ACEA 1021 (5-nitro-6,7-dichloro-1,4-dihydro-2,3-quinoxalinedione; licostinel), a selective antagonist at the strychnine-insensitive glycine site associated with the NMDA receptor complex, has been investigated in various models of focal cerebral ischemia. In isoflurane-anaesthesised Wistar rats with permanent ipsilateral carotid artery ligation and transient middle cerebral artery occlusion (duration of occlusion, 2 h) followed by reperfusion (24 h), intravenous administration of ACEA 1021 (bolus: 10 mg/kg, 15 min after the onset of middle cerebral artery occlusion; infusion: 7 mg/kg/h for 6 h beginning 30 min after occlusion of the artery) produced a 32% reduction in infarct volume. Similarly, in Sprague-Dawley rats with transient middle cerebral artery occlusion (2 h) followed by 24 h of reperfusion, identical treatment with ACEA 1021 decreased infarct size by 39%. Magnetic resonance imaging (MRI) confirmed these effects in the transient model, in that infarct volume observed using apparent diffusion coefficient (ADC) maps was significantly smaller after 24 h in the ACEA 1021-treated rats compared with Tris-treated controls. Furthermore, the increase in perfusion signal intensity after reperfusion was more pronounced in the ACEA 1021-treated rats than in controls. In Fisher 344 rats with permanent occlusion of the middle cerebral artery, ACEA 1021 induced a dose-related decrease in infarct volume, which was associated with an improvement in neurological outcome as measured by the rope suspension procedure. Administration of the same dose regimen, as above, in Fisher rats with permanent middle cerebral artery occlusion reduced infarct volume by 68%. This dose was as effective when administration was delayed for 2 h. In mice with permanent middle cerebral artery occlusion, ACEA 1021 (5 mg/kg, i.v., 5 min after occlusion; 30 mg/kg, s.c., 1 and 4 h post-middle cerebral artery occlusion) decreased infarct size by 42%. The consistent anti-ischemic effects of ACEA 1021 make it a valuable compound for exploratory stroke research.     

白三烯受体拮抗剂ONO—1078对大鼠局灶性脑缺血的神经保护作用

AIM: To determine whether ONO-1078 (pranlukast), a potent leukotriene receptor antagonist, has neuroprotective effect on focal cerebral ischemia in the rat. METHODS: Focal cerebral ischemia was induced by 30 min of middle cerebral artery (MCA) occlusion and followed by 24 h reperfusion. ONO-1078 (0.003-1.0 mg/kg) or vehicle (saline 1 mL/kg) was ip injected 30 min before MCA occlusion and 2 h after reperfusion. The neurological score, infarct volume, neuron density (in cortex, hippocampus, and striatum), brain edema, and albumin exudation around the vessels were determined 24 h after reperfusion. RESULTS: ONO-1078 slightly improved the neurological deficiency, and dramatically decreased infarct volume and neuron loss which showed a bell shaped dose response effect with highest effect at doses of 0.01-0.3 mg/kg. Enlargement of the ischemic hemisphere and albumin exudation were inhibited at doses of 0.01-1.0 mg/kg. CONCLUSION: ONO-1078 has the protective effect on focal cerebral ischemia in rats, which is partially attributed to the inhibition of brain edema. This may represent a novel approach to the treatment of acute cerebral ischemia with cysteinyl leukotriene receptor antagonists.     

Effect of NXY-059 on infarct volume after transient or permanent middle cerebral artery occlusion in the rat; studies on dose, plasma concentration and therapeutic time window.

1. The efficacy of the free radical trapping agent NXY-059 in reducing the infarct volume following both transient and permanent focal ischaemia has been examined in rats. 2. In the transient ischaemia model, rats were subjected to a 2 h occlusion of the middle cerebral artery (MCA). Intravenous infusion of NXY-059 (1, 10 and 30 mg kg(-1) h) for 21.75 h starting 2.25 h after the occlusion, produced a dose-dependent decrease in both neurological impairment and the histologically measured infarct volume (a mean 59% decrease at 10 mg kg(-1) h). 3. In the permanent ischaemia model, animals were injected (s.c.) with a loading dose of NXY-059 of 32.5, 53.8 or 75.4 mg kg(-1) and osmotic minipumps were implanted which had been primed to deliver respectively 30, 50 or 70 mg kg(-1) h. When treatment was initiated 5 min after MCA occlusion there was a dose dependent protection of both cortical and sub-cortical tissue (cortex: 63% at the mid-range dose). Protection was related linearly to plasma concentration (plasma unbound NXY-059 concentration at 1 h: 37+/-16 micromol l(-1) at the mid-range dose). 4. When the mid range dose was administered between 5 min - 4 h after MCA occlusion, a marked and statistically significant protection was seen at all time points (44% protection in cortex at 4 h). 5. These data demonstrate the substantial neuroprotective efficacy of NXY-059 at plasma concentrations that can be achieved clinically and indicate that NXY-059 also has a therapeutic window of opportunity that is clinically relevant.     

Neuroprotective effects of an AMPA receptor antagonist YM872 in a rat transient middle cerebral artery occlusion model

The neuroprotective effects of YM872 ([2,3-dioxo-7-(1H-imidazol-1-yl)6-nitro-1,2,3,4-tetrahydro-1-quinoxal inyl]acetic acid monohydrate), a novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist with high water solubility, were examined in rats with transient middle cerebral artery (MCA) occlusion. The right MCA of male SD rats was occluded for 3 h using the intraluminal suture occlusion method. YM872 significantly reduced the infarct volume 24 hours after occlusion, at dosages of 20 and 40 mg/kg/h (iv infusion) when given for 4 h immediately after occlusion. Furthermore, delayed administration of YM872 (20 mg/kg/h iv infusion for 4 h, starting 2 or 3 h after the occlusion) also reduced the infarct volume and the neurological deficits measured at 24 h. Additionally, the therapeutic efficacy of YM872 persisted for at least seven days after MCA occlusion in animals treated with YM872 for 4 h starting 2 h after MCA occlusion. These data demonstrate that AMPA receptors contribute to the development of neuronal damage after reperfusion as well as during ischemia in the focal ischemia models and that the acute effect of the blockade of AMPA receptors persists over a long time period. YM872 shows promise as an effective treatment for patients suffering from acute stroke.     

Inhibition of neuronal nitric oxide synthase activity by 3-[2-[4-(3-chloro-2-methylphenyl)- 1-piperazinyl]ethyl]-5, 6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), a novel neuroprotective agent, in vitro and in cultured neuroblastoma cells in situ

DY-9760e, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5, 6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, a novel calmodulin (CaM) antagonist, possesses neuroprotective activity. In the current study, we examined the effects of DY-9760e on nitric oxide synthase (NOS) activities in vitro and on calcium ionophore-induced NO production in situ. DY-9760e inhibited both neuronal NOS and endothelial NOS activities without affecting inducible NOS activity. It also inhibited purified neuronal NOS activity with a potency similar to that seen for purified CaM kinase II activity in vitro. Furthermore, DY-9760e significantly inhibited Ca(2+) ionophore (A23187)-induced NO production in mouse N1E-115 neuroblastoma cells, at a concentration of less than 1 microM. In contrast, no apparent inhibitory effect on Ca(2+)/CaM-dependent protein kinase II activity was observed in cultured hippocampal neurons up to 5 microM. These results suggest that the inhibitory effect of DY-9760e on CaM-dependent NOS activities underlies neuroprotective effects of the agent.     

The broad-spectrum cation channel blocker pinokalant (LOE 908 MS) reduces brain infarct volume in rats: a temperature-controlled histological study

Activation of cation channels conducting Ca2+, Na+ and K+ is involved in the pathogenesis of infarction in experimental focal cerebral ischaemia. Pinokalant (LOE 908 MS) is a novel broad-spectrum inhibitor of several subtypes of such channels and has previously been shown to improve the metabolic and electrophysiologic status of the ischemic penumbra and to reduce lesion size on magnetic resonance images in the acute phase following middle cerebral artery occlusion in rats. The purpose of the present study was to investigate whether these beneficial effects of pinokalant are translated into permanent neuroprotection in terms of a reduction in infarct size one week after middle cerebral artery occlusion in rats. Halothane-anaesthetized male Wistar rats subjected to permanent distal middle cerebral artery occlusion were randomly assigned to one of two treatment groups: 1) Control (vehicle intravenous loading dose followed by infusion); 2) Pinokalant (0.5 mg/kg intravenous loading dose followed by infusion of 1.25 mg/kg/hr). Infusions started 30 min. after middle cerebral artery occlusion and were continued for 24 hr. Body temperature and mean arterial blood pressure were monitored by telemetry during this period and the spontaneous temperature after course in control rats established in other experiments was imitated. Seven days later histological brain sections were prepared and the infarct volumes measured. Body temperature did not differ between the groups. Mean arterial blood pressure was slightly higher in the pinokalant group. Pinokalant treatment significantly reduced cortical infarct volume from 33.8+/-15.8 mm3 to 24.5+/-13.1 mm3 (control group versus pinokalant group, P=0.017, t-test). Taking the effective drug plasma concentration established in other experiments into account revealed that in rats with plasma concentrations within the therapeutic interval, infarct volumes were further reduced to 17.9+/-7.5 mm3 (P<0.005).