In-vitro characterization of YM872, a selective, potent and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist

The in-vitro pharmacological properties of (2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxal inyl)-acetic acid monohydrate, YM872, a novel and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor antagonist were investigated. YM872 is highly water soluble (83 mg mL(-1) in Britton-Robinson buffer) compared with 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX), 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride (YM90K) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). YM872 potently inhibits [3H]AMPA binding with a Ki (apparent equilibrium dissociation constant) value of 0.096 +/- 0.0024 microM. However, YM872 had very low affinity for other ionotropic glutamate receptors, as measured by competition with [3H]kainate (high-affinity kainate binding site, concentration resulting in half the maximum inhibition (IC50) = 4.6 +/- 0.14 microM), [3H]glutamate (N-methyl-D-aspartate (NMDA) receptor glutamate binding site, IC50 > 100 microM) and [3H]glycine (NMDA receptor glycine-binding site, IC50 > 100 microM). YM872 competitively antagonized kainate-induced currents in Xenopus laevis oocytes which express rat AMPA receptors, with a pA2 value of 6.97 +/- 0.01. In rat hippocampal primary cultures, YM872 blocked a 20-microM AMPA-induced increase of intracellular Ca2+ concentration with an IC50 value of 0.82 +/- 0.031 microM, and blocked 300-microM kainate-induced neurotoxicity with an IC50 value of 1.02 microM. These results show that YM872 is a potent and highly water-soluble AMPA antagonist with great potential for treatment of neurodegenerative disorders such as stroke.     

Chondroitin sulfate proteoglycans protect cultured rat's cortical and hippocampal neurons from delayed cell death induced by excitatory amino acids

Protective effects of chondroitin sulfate proteoglycans (CSPGs) from rat's brain against delayed cell death induced by excitatory amino acids were examined in cultured neurons of the rat. CSPGs reduced delayed neuronal death induced by 10 min exposure to glutamate at a concentration between 100 microM and 1 mM when lactate dehydrogenase activity of culture medium was assayed 24 h after the exposure. CSPGs also protected neuronal death induced by 200 microM N-methyl-D-aspartate (NMDA), kainate or 100 microM alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). CSPGs reduced death of cortical and hippocampal neurons even when they were administered at 2 h, but not 6 and 12 h, after the exposure to glutamate. These results indicate that CSPGs may have a neuroprotective action against acute noxious conditions in the brain.     

Synthesis of 7,8-(methylenedioxy)-1-phenyl-3,5-dihydro-4H-2, 3-benzodiazepin-4-ones as novel and potent noncompetitive AMPA receptor antagonists

A group of 7,8-(methylenedioxy)-1-phenyl-3,5-dihydro-4H-2, 3-benzodiazepin-4-ones was synthesized and assayed for antagonism of rat brain alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors expressed in Xenopus oocytes. The benzodiazepinones inhibited AMPA-activated membrane current responses in a manner consistent with noncompetitive, allosteric inhibition of the receptor-channel complex. The most potent compound in the series was 1-(4-aminophenyl)-7,8-(methylenedioxy)-3,5-dihydro-4H-2, 3-benzodiazepin-4-one (6), which had an IC50 of 2.7 microM. For comparison, the reference compound GYKI 52466 (2) had an IC50 of 6.9 microM. Compound 6 also had potent anticonvulsant activity in a mouse maximum electroshock-induced seizure (MES) assay: the ED50 was 2.8 mg/kg iv, whereas the ED50 for GYKI 52466 was 4.6 mg/kg iv. In contrast to a previous report, the 7,8-dimethoxy analogue of 6 was a low-potency AMPA antagonist (IC50 >100 microM) and weak anticonvulsant (ED50 >10 mg/kg iv). The benzodiazepinones described herein are potent noncompetitive AMPA receptor antagonists that could have therapeutic potential as anticonvulsants and neuroprotectants.     

Future time perspectives of the elderly; an empirical study rooted in theory

This study examined the efficacy of YM175 [disodium dihydrogen (cycloheptylamino) methylene-1, 1-bisphosphonate] in reducing alveolar bone loss caused by experimental periodontitis in beagle dogs. Thirty-six dogs were used and divided into 6 groups. Periodontitis was induced in 30 dogs (groups 2-6) by ligating the bilateral mandibular third and fourth premolar teeth with silk ligatures and by feeding a soft diet. Six dogs were sham-operated (group 1). Saline (placebo), flurbiprofen (0.02 mg/kg) and YM175 (0.01, 0.1 and 1.0 mg/kg) were administered to the dogs (groups 2-6) 5 d/wk for 25 wk. Radiographic and morphometric analyses were performed. In placebo-treated animals (group 2), the ligation caused a significant decrease in the alveolar bone height by 0.57 and 1.91 mm at 2 and 25 wk, respectively. YM175 (1.0 mg/kg) prevented the decrease in bone height by 47 and 31% at 2 and 25 wk. YM175 (0.1 mg/kg) and flurbiprofen tended to prevent bone loss after 15 wk. Although the ligation elicited no significant change in bone mineral density, it significantly decreased bone volume. YM175 (1.0 mg/kg) and flurbiprofen tended to increase the bone volume. The number of formative or resorptive Haversian canals and the bone turnover through the periosteal bone surface were increased by the ligation, indicating the increased turnover of the cortical bone. YM175 (1.0 mg/kg) reduced the increased bone turnover. The gingival index was maximally increased at 2 wk and was suppressed by YM175. These results suggest that YM175 prevents alveolar bone loss by reducing the increased alveolar bone turnover in dogs with periodontitis.     

Decahydroisoquinolines: novel competitive AMPA/kainate antagonists with neuroprotective effects in global cerebral ischaemia

In the present studies, we have evaluated the activity of a series of glutamate receptor antagonists from the decahydroisoquinoline group of compounds both in vitro and in vivo. Compound activity at alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate receptors was assessed using ligand binding to cloned iGluR2 and iGluR5 receptors and on responses evoked by AMPA and N-methyl-D-aspartate (NMDA) in the cortical wedge preparation. In vivo, compounds were examined for antagonist activity electrophysiologically in the rat spinal cord preparation and in the gerbil model of global cerebral ischaemia. Compounds tested were LY293558, which has been shown to protect in models of focal cerebral ischaemia, LY202157 (an NMDA antagonist), LY246492 (an NMDA and AMPA receptor antagonist), LY302679, LY292025, LY307190, LY280263, LY289178, LY289525, LY294486 (AMPA/kainate antagonists) and LY382884 (an iGluR5 selective antagonist). Results obtained support a role for AMPA receptors in cerebral ischemia. LY377770 (a mixed AMPA/iGluR5 antagonist and active isomer of LY294486) demonstrated good neuroprotection with a 2-h time window and may therefore be useful in the treatment of ischaemic conditions.     

Delayed treatment with YM90K, an AMPA receptor antagonist, protects against ischaemic damage after middle cerebral artery occlusion in rats

The neuroprotective effect of an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist YM90K [6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione monohydrochloride] has been examined in a rat middle cerebral artery occlusion model. Intravenous infusion of YM90K (2.5-20mgkg(-l)h(-l) for 4h) starting immediately after occlusion of the middle cerebral artery significantly reduced the cortical infarct volume 24h after occlusion compared with the control group. The protection at the highest dose was 39% (P < 0.05). Similar protective effects were observed when YM90K (20mgkg(-1)h(-1) for 4h) was delayed up to 2h after middle cerebral artery occlusion (45% reduction, P < 0.05). CNS1102 [N-(1-naphthyl)-N'-(3-ethylphenyl)-N'-methylguanidine hydrochloride], a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, also reduced the cortical infarct volume when 1.13mgkg(-1) was administered by intravenous bolus injection immediately after middle cerebral artery occlusion, followed by intravenous infusion at 0.785mgkg(-l)h(-1) for 4h (35% reduction, P<0.05). This neuroprotective effect was not observed when administration was delayed lh after middle cerebral artery occlusion. These results suggest that AMPA receptors might play a more important role than NMDA receptors in the late development of neuronal cell damage after focal cerebral ischaemia and that AMPA receptor blockade would be one beneficial strategy in treating acute stroke.     

Cardioprotective effects of the novel adenosine A1/A2 receptor agonist AMP 579 in a porcine model of myocardial infarction

This study examined the cardioprotective effects and pharmacology of the novel adenosine A1/A2 receptor agonist ([1S-[1a,2b,3b, 4a(S*)]]-4-[7-[[2-(3-chloro-2-thienyl)-1-methylpropyl]amino]-3H-imida zo[4,5-b] pyridyl-3-yl] cyclopentane carboxamide) (AMP 579), in a model of myocardial infarction. Experiments were performed in pentobarbital-anesthetized pigs in which myocardial infarction was induced by a 40-min occlusion of the left anterior descending coronary artery, followed by 3 hr of reperfusion. This procedure resulted in approximately 20% of the left ventricle being made ischemic in all test groups. In untreated animals, an infarct size equal to 56 +/- 5% of the ischemic area was observed. Preconditioning, with two cycles of 5 min of ischemia followed by 10-min reperfusion, resulted in a 70% reduction in infarct size (17 +/- 5%) relative to risk area. Administration of AMP 579 30 min before ischemia (3 microg/kg i.v. followed by 0.3 microg/kg/min i.v. through 1 hr of reperfusion) did not change blood pressure, HR or coronary blood flow but resulted in marked cardioprotection: a 98% reduction in infarct size (1 +/- 1%) relative to risk area. Moreover, whereas approximately 90% of control pigs suffered ventricular fibrillation during ischemia, no fibrillation was observed in animals treated with AMP 579. Further experiments determined the effects of AMP 579 when administered 30 min after the onset of myocardial ischemia, 10 min before reperfusion. Two doses were studied: a low hemodynamically silent dose (3 microg/kg + 0.3 microg/kg/min through 1 hr of reperfusion) and a 10-fold higher dose that did cause reductions in blood pressure and HR. Both doses of AMP 579 produced a comparable cardioprotective effect, reducing infarct size to approximately 50% of that observed in control animals. The cardioprotective effect of AMP 579 was a consequence of adenosine receptor stimulation, because it was completely inhibited by pretreatment with the specific adenosine receptor antagonist CGS 15943 (1 mg/kg i.v.). However, the selective A1 receptor agonist GR 79236 (3 microg/kg + 0.3 microg/kg/min i.v.) did not reduce infarct size, which suggests that under these experimental conditions, stimulation of adenosine A2 receptors is important for the cardioprotective effect of AMP 579. The adenosine-regulating agent acadesine (5 mg/kg + 0.5 mg/kg/min i.v.) also failed to reduce infarct size. In conclusion, the novel adenosine A1/A2 receptor agonist AMP 579 produces marked cardioprotection whether administered before myocardial ischemia or reperfusion. Cardioprotection is not dependent on changes in afterload or myocardial oxygen demand and is a consequence of adenosine receptor stimulation. The pharmacological profile of AMP 579 in this model is consistent with its potential utility in the treatment of acute myocardial infarction.     

Glutamate receptors in the rat medial prefrontal cortex regulate set-shifting ability.

The authors examined set-shifting abilities in rats injected with antagonists of N-methyl-D-aspartate (NMDA) receptors (MK801) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (LY293558) into the medial prefrontal cortex (mPFC). Set-shifting was assessed with a maze-based task requiring a switch between brightness and texture discrimination strategies. Intra-mPFC injection of MK801 prior to training on the 2nd discrimination impaired discrimination strategy acquisition. The MK801-induced deficit was due to increased perseverative responding. AMPA receptor blockade also impaired acquisition of the 2nd discrimination, these impairments were due to more general cognitive deficits. Results suggest that, within the mPFC, both AMPA and NMDA receptors are necessary for set-shifting, and that NNMA receptor hypofunction impairs the capacity to modify existing knowledge or to inhibit responses that are no longer appropriate. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A crucial role of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype of glutamate receptors in piriform and perirhinal cortex for the initiation and propagation of limbic motor seizures

The role of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors in the initiation and propagation of limbic motor seizures in rats was examined by the intracerebral and systemic administration of 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (f) quinoxaline (NBQX), a selective antagonist of the AMPA subtype of glutamate receptor. Limbic motor seizures were evoked focally by the application of the gamma-aminobutyric acid receptor antagonist, bicuculline, into area tempestas, an epileptogenic site in the deep anterior piriform cortex. Before eliciting seizures, NBQX was applied focally into either 1) area tempestas or 2) perirhinal or posterior piriform cortex ipsilateral to the area tempestas from which seizures were evoked. In addition, pretreatment with i.p. NBQX was evaluated for anticonvulsant actions against area tempestas-evoked clonic or systemically evoked tonic seizures. In all conditions, a dose-dependent decrease in the severity of seizures was obtained with NBQX. With focal intracerebral administration, a dose of 500 pmol of NBQX consistently protected against limbic motor seizures, with partial protection achieved with 100 pmol. After i.p. administration, 2.5 and 5.0 mg/kg significantly protected the rats from both limbic motor seizures and tonic extensor seizures. No overt disturbance of spontaneous behavior was associated with the anticonvulsant doses of NBQX. Moreover, both forebrain substrates of limbic motor seizures and hindbrain substrates of tonic extensor seizures were highly susceptible to disruption by NBQX. The results indicate that AMPA subtype of glutamate receptors are crucial mediators of seizure propagation via perirhinal and piriform cortics.     

Evaluating parameters of osseointegrated dental implants using finite element analysis--a two-dimensional comparative study examining the effects of implant diameter, implant shape, and load direction

The hexachlorophene-induced cytotoxic brain oedema is an experimental model of brain damage, suitable for testing cerebroprotective substances (Andreas 1993). In order to examine whether glutamate receptors are involved in mediating functional disorders due to neurotoxic brain damage, we have studied the protective effects of several competitive and non-competitive antagonists using adult male Wistar rats in a simple "ladder-test" for assessing coordinative motor behaviour. Hexachlorophene-induced brain damage was verified by histological examination of the cerebellum with vacuolation of white matter, astrocyte hypertrophy and astrocyte proliferation taken as signs of neurotoxic injury. The non-competitive N-methyl-D-aspartate (NMDA) antagonist dizocilpine maleate (MK-801) decreased the motor disturbance on the first and second day of the "ladder-test" when applied in the doses 0.1 mg/kg and 0.2 mg/kg intraperitoneally for 3 weeks during the hexachlorophene treatment. Acute MK-801 administration (0.1 mg/kg intraperitoneally) after 3 weeks hexachlorophene exposure improved the coordinative motor response only on the first day. When testing the competitive NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP-5) in the dose 1.0 mg/kg intraperitoneally the motor disturbance was lowered significantly earlier than in spontaneous remission. Similar effects were observed with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in the dose of 0.8 mg/kg intraperitoneally, an antagonist interacting both with the strychnine-insensitive binding site for glycine within the NMDA receptor complex and with the kainate(+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor complex. Concurrent MK-801 administration decreased the vacuolation of white matter. The results suggest that NMDA receptors and non-NMDA receptors are involved in development of functional disorders induced by hexachlorophene.     

Diagnostic concordance of telecytology and conventional cytology for evaluating breast aspirates

We examined the effect of a humanized anti-glycoprotein IIb/IIIa monoclonal antibody, YM337, on thrombolysis with tissue-type plasminogen activator in a copper coil-induced coronary thrombosis model in rhesus monkeys. Fifty minutes after the formation of an occlusive thrombus, a test drug was administered by either i.v. bolus injection followed by continuous infusion (YM337, 0.25 mg/kg + 1.5 microg/kg/min) or i.v. bolus injection (aspirin, 17 mg/kg). Sixty minutes after induction of the occlusive thrombus, thrombolysis was initiated with tPA at a total dose of 0.5 mg/kg intravenously administered over 60 min, with 10% given as an initial bolus. The median time to reperfusion was significantly shortened by YM337 [saline, 60 min (n = 5); aspirin, 45 min (n = 5); YM337, 30 min (n = 5)]. The incidence of reocclusion was significantly decreased by YM337 (saline, 4/4; aspirin, 5/5; YM337, 1/5), and the median time to reocclusion was significantly prolonged by YM337 [saline, 30 min (n = 4); aspirin, 30 min (n = 5); YM337, 180 min (n = 5)]. YM337 significantly reduced the thrombus protein content at the end of experiment. ADP-induced platelet aggregation was completely inhibited by YM337. These results suggest that YM337 may be of clinical value as an adjunctive agent in thrombolytic therapy for patients with acute myocardial infarction.     

Effects of LY215490, a competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, on the micturition reflex in the rat

The effects of glutamate receptor antagonists on urinary bladder and external urethral sphincter- (EUS) electromyogram (EMG) activity were evaluated in unanesthetized decerebrate rats. In normal rats, LY215490, an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, in small i.v. doses (1-3 mg/kg) decreased bladder contraction amplitude (BC-Amp) by 29% and EUS-EMG by 41%; whereas a large dose (10 mg/kg) completely abolished bladder and EUS-EMG activity. LY215490 injected intrathecally in small doses (0.01-0.1 microg) decreased BC-Amp by 20% and EUS-EMG by 62%; whereas large doses (1-10 microg) completely abolished bladder and EUS-EMG activity. LY215490 (0.1 microg i.t.) increased bladder capacity by 28% and decreased voiding efficiency by 44%. Combined i.t. administration of small doses of LY215490 (0.1 microg) and MK-801 (1 microg), an N-methyl-D-aspartate (NMDA) receptor antagonist, which individually had little effect on BC-Amp, markedly suppressed bladder activity. In chronic spinal rats, LY215490 (10 mg/kg i.v.) abolished EUS-EMG activity and decreased BC-Amp by 41%. Intrathecal injections of LY215490 were also less effective in chronic spinal rats; a 10-microg dose producing only a partial block (53%) of BC-Amp, but complete block of EUS-EMG. In chronic spinal rats, MK-801 (1 mg/kg i.v.) abolished EUS-EMG activity and decreased BC-Amp by 36%. Pretreatment with MK-801 (1 mg/kg i.v.) did not enhance the effect of LY215490 on bladder activity in chronic spinal rats. These data suggest that AMPA glutamate receptors have a major role in the excitatory pathways controlling bladder and EUS activity in spinal cord intact rats. However, in chronic spinal rats, AMPA and NMDA receptors are essential for EUS reflexes, but are responsible for only a part of reflex bladder activity.     

Glutamate receptor binding sites in MPTP-treated mice

Changes in excitatory amino acid (EAA) neurotransmission are thought to play an important role in the development of parkinsonian symptoms. We examined EAA receptor binding sites in substantia nigra, striatum, globus pallidus, and cortex at 2 weeks and 2 months after MPTP (1-methyl-4-phenyl-1,2,3,6-tetra-hydroxypyridine) injection in C57bl6 mice. At 2 weeks striatal dopamine content in MPTP-treated mice was reduced to 7% of control and N-methyl-D-aspartate (NMDA)-sensitive [3H]glutamate and [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) binding sites were decreased in substantia nigra to 57 and 76% of control, respectively. In globus pallidus only [3H]AMPA binding sites were decreased to 80% of control; no significant changes were found in striatum or cortex. [3H]Kainate binding sites remained unchanged. At 2 months striatal dopamine content was reduced to 31% and no changes in EAA binding sites could be detected in any of the structures examined. [3H]Mazindol binding to striatal monoamine-uptake sites was decreased to 17% of control at 2 weeks versus 37% at 2 months. Our data indicate that modulation of NMDA and AMPA binding sites in substantia nigra and globus pallidus, the major projection areas of the subthalamic nucleus, takes place only after severe impairment of the nigrostriatal system.     

Reconstruction of the centrosome cycle from cryoelectron micrographs

Single and repeated intravenous toxicity studies of Pamiteplase (genetical recombination) YM866, a novel recombinant human tissue-type plasminogen activator, were conducted. No animal died from toxic effects of YM866 after single administration to F344 rats, squirrel monkeys and cynomolgus monkeys. Male and female F344 rats were given YM866 intravenously for 4 weeks at doses of 0 (vehicle), 0.1, 0.3 and 1 mg/kg/day. An increase in platelet count, slight decreases in hemoglobin and hematocrit, increases in plasma phospholipids, total cholesterol and total protein, and liver weight were observed at 1.0 mg/kg. Histopathology revealed no changes in any organ except for hemorrhage at the injection sites. These changes recovered after 4 weeks of withdrawal. Male and female squirrel monkeys were given YM866 intravenously for 4 weeks at doses of 0 (saline), 0 (vehicle), 0.1, 0.3 and 1 mg/kg/day. Prolongation of coagulation time at the injection site was observed at 0.3 mg/kg or more. Subcutaneous hemorrhage and a transient decrease in locomotor activity were observed at 1 mg/kg. Prolongation of coagulation time at the injection site was considered to be related to the pharmacological action of YM866. The results show that the approximate single lethal dose of YM866 is more than 60 mg/kg in rats, and more than 10 mg/kg in squirrel monkeys and cynomolgus monkeys. The no-toxic-effect level of YM866 after repeated administration for 4 weeks in rats and squirrel monkeys is considered to be 0.3 mg/kg.     

Ischemia-reperfusion induced microvascular dysfunction in skeletal muscle: application of intravital video microscopy

Video microscopy of red cell flow in capillaries at the surface of skeletal muscle provided the opportunity to quantitate ischemia-reperfusion (I-R) induced microcirculatory changes, in vivo. Extensor Digitorum Longus (EDL) muscles of 22 male Wistar rats (300-400 g), anesthetized with sodium pentobarbital (Somnotol, 65 mg kg,-1 IP), were used to measure the number of perfused capillaries (CDper: mm-1) crossing lines drawn perpendicular to the muscle axis, and red blood cell velocity (VRBC: mm/s) within individual capillaries from controls (n = 6), and after 2 hr (n = 4), 3 hr (n = 4), and 4 hr (n = 5) of no-flow ischemia with the muscle temperature maintained at its normal value of 32 degrees C. Ischemia was induced by tightening a tourniquet placed around the limb above the EDL muscle. Measurements were made after 30, 60, and 90 min of reperfusion. To test the usefulness of this skeletal muscle model for evaluating proposed interventions in I-R, the effect of hypothermia (24 degrees C) on the microcirculation following 4 hr ischemia (n = 3) was measured. Edema formation was estimated from the wet/dry weight ratio of the ischemic and contralateral control EDL muscles. Capillary perfusion at the surface of the control muscles was remarkably stable over the 5 hr period studied, while significant changes occurred following the ischemic periods. Significantly lower CDper was measured 30 min following all periods of normothermic ischemia. However, unlike the 2 and 4 hr ischemic periods 3 hr normothermic ischemia resulted in a progressive decline in CDper throughout the reperfusion period. VRBC showed evidence of a hyperemic response following 2 hr normothermic ischemia (control: 0.12 mm/s +/- 0.19 compared to 0.26 mm/s +/- 0.03 following 90 min reperfusion; mean +/- sem). However, no such hyperemia was measured following either 3 or 4 hr normothermic ischemia (i.e., 3 hr control: 0.24 mm/s +/- 0.01 compared to 0.07 mm s +/- 0.003 following 90 min reperfusion). In fact, VRBC was essentially zero 90 min following 4 hr normothermic ischemia (0.01 mm/s +/- 0.01). However, when the muscle was allowed to cool to 24 degrees C during 4 hr ischemia no significant change in either VRBC or CDper was measured compared to pre-ischemic controls. Evidence of edema was found after 3 and 4 hr normothermic ischemia. This study establishes a skeletal muscle model of I-R, which may be useful in testing hypotheses regarding mechanisms of I-R injury, and effectiveness of proposed treatments of I-R.     

Antiasthmatic activity of the second-generation phosphodiesterase 4 (PDE4) inhibitor SB 207499 (Ariflo) in the guinea pig

We evaluated the airway activity of the novel phosphodiesterase type 4 inhibitor SB 207499 [Ariflo; c-4-cyano-4-(3-cyclopentyloxy-4-methoxyp henyl-r-1-cyclohexane carboxylic acid)], in the guinea pig. Ovalbumin (OA)-induced contractions of guinea pig isolated tracheal strips were inhibited by SB 207499 with an EC50 of 1 microM but had little or no effect on exogenous agonist-induced contraction, which suggests that its effect on OA-induced contraction in vitro is primarily due to inhibition of mediator release from mast cells. In anesthetized guinea pigs, SB 207499 inhibited OA-induced bronchoconstriction with i.v. and p.o. ID50 values of 1.7 and 17 mg/kg, respectively. At 1, 3 and 6 hr after SB 207499 (30 mg/kg p.o.), OA-induced bronchospasm was inhibited by 92%, 70% and 58%, respectively, corresponding to elevated plasma concentrations of 1.62 +/- 0.19, 1.65 +/- 0.29 and 0. 93 +/- 0.24 microg/ml, respectively, of SB 207499. SB 207499 also inhibited house dust mite-induced bronchoconstriction (ID50 = 0.9 mg/kg i.v. and 8.9 mg/kg p.o.). In contrast to its lack of bronchorelaxant activity in vitro, SB 207499 inhibited bronchospasm induced by i.v. leukotriene D4 (LTD4) [ID50 = 3 mg/kg i.v.]. The bronchorelaxant effect of i.v.-administered SB 207499 was at least additive with that of salbutamol in reversing infused histamine-enhanced airway tone, but it did not alter base line or enhance salbutamol-induced cardiovascular effects. In conscious guinea pigs, SB 207499 (10 or 30 mg/kg p.o.), 1 hr before antigen or LTD4 challenge, markedly reduced bronchospasm and subsequent eosinophil influx as measured by bronchoalveolar lavage 24 hr after provocation. SB 207499 administered after OA or LTD4 challenge also reduced airway eosinophilia measured at 24 hr after OA challenge or 96 hr after LTD4 challenge. These results, coupled with the broad anti-inflammatory activity of SB 207499 previously described (Barnett et al., 1998), suggest that SB 207499 will be useful in the treatment of asthma and other inflammatory disorders.     

In vivo and in vitro evaluation of AMPA receptor antagonists in rat hippocampal neurones and cultured mouse cortical neurones

The effects of four glutamate receptor antagonists on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)- and N-methyl-D-aspartate (NMDA)-responses were evaluated using both in vitro and in vivo electrophysiological techniques: whole cell patch-clamp recordings from cultured mouse cortical neurones and microiontophoresis in the rat hippocampus. The compounds tested were NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline), GYKI 52466 (1-(4-amino-phenyl)-4-methyl-7,8-methyl-endioxyl-5H-2,3-benzodiaze pine), PNQX (pyrido[3, 4-f]quinoxaline-2,3-dione, 1,4,7,8,9,10-hexahydro-9-methyl-6-nitro-, methanesulfonate), NS377 (7-ethyl-5-phenyl-1,6,7,8-tetrahydro-1,7-diaza-as-indacene-2 ,3-dione), and MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenz(a,d)cycloheptene-5,10-imine hydrogen maleate). In vitro, the IC50 values (in microM) for inhibition of AMPA-evoked inward currents were approximately 0.4 for NBQX, approximately 7.5 for GYKI 52466, approximately 1 for PNQX and approximately 15 for NS377. PNQX and NS377 also inhibited NMDA-induced currents with IC50 values at approximately 5 and approximately 18 microM, respectively, while NBQX at 60 microM and GYKI 52466 at 100 microM had only weak effects. The ED50 values in micromol/kg i.v. for inhibition of AMPA-evoked hippocampal neuronal spike activity in vivo were approximately 32 for NBQX, approximately 19 for GYKI 52466, approximately 17 for PNQX and approximately 11 for NS377 with efficacy values (maximal inhibition) between 71% and 81%. The ED50 values (in [Lmol/kg i.v.) and efficacy values for inhibition of NMDA-evoked hippocampal neuronal spike activity were approximately 28 with an efficacy of 61% for NBQX, approximately 16 with 35% for PNQX and approximately 6 with 61% for NS377. GYKI 52466 did not significantly affect NMDA responses, whereas MK-801 showed NMDA specificity in vivo.     

NMDA and non-NMDA receptor antagonists protect against excitotoxic injury in the rat spinal cord

The neuroprotective properties of the N-methyl-D-aspartate (NMDA) antagonist dizocilpine (MK-801) and the non-NMDA antagonists 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX) and alpha-methyl-4-carboxyphenylglycine (MCPG) were evaluated against neuronal injury produced by the intraspinal injection of NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). Forty-nine animals were divided into eight groups in order to evaluate the effects of different drug combinations: (a) NMDA; (b) NMDA + MCPG; (c) NMDA + NBQX; (d) NMDA + MK-801; (e) AMPA; (f) AMPA + MCPG; (g) AMPA + MK-801; and (h) AMPA + NBQX. Drugs were microinjected into spinal segments T12-L3 through a micropipette attached to a Hamilton microliter syringe. Spinal cords were evaluated after a survival period of 48 h at which time NMDA and AMPA were found to produce morphological changes over the concentration ranges of 125-500 mM and 75-500 microM, respectively. Neuronal loss following injections of NMDA + MK-801 or AMPA + NBQX was significantly less than that following injections of NMDA or AMPA alone. By contrast, neuronal loss following co-injections of NMDA or AMPA with inappropriate antagonists, i.e., NMDA + NBQX/MCPG or AMPA + MCPG/MK-801, was not significantly different from that produced by NMDA or AMPA. The results suggest that elevations in spinal levels of glutamate followed by prolonged activation of NMDA and AMPA receptor subtypes initiate an excitotoxic cascade resulting in neuronal injury. Blockade of NMDA and AMPA effects by MK-801 and NBQX respectively confirms the well documented neuroprotective effects of these drugs and lends support to the potential importance of NMDA and especially AMPA receptor antagonists as therapeutic agents in the treatment of acute spinal cord injury.