α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor

The response induced by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) (10 μM) is inhibited by GYKI 53655 (LY300168) hydrochloride, with an IC50 value of 5.9±0.1 μM. In HEK293 cells expressing recombinant G1uR4, GYKI 53655 hydrochloride inhibits the AMPA (10 μM) response with an IC50 value of 4.6±0.4 μM. 3 μM cyclothiazide was used to produce 79±2% inhibition (n=4 cells) with GYKI 53655 hydrochloride. At 30 μM, GYKI 53655 hydrochloride only suppresses the kainic acid-induced current by a small amount; at 100 μM, it suppresses the kainic acid-induced current by 12±2 (n=4) and 18±4 (n), respectively. GYKI 53655 hydrochloride, with an IC50 value of 1.5±0.1 μM, inhibits AMPA receptor-mediated responses in cerebellar Purkinje neurons[1].

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The activity and selectivity of the glutamate receptor antagonists belonging to the 2,3-benzodiazepine class of compounds have been examined at recombinant human non-NMDA glutamate receptors expressed in HEK293 cells and on native rat NMDA and non-NMDA receptors in vitro. The racemic 2,3-benzodiazepines GYKI52466, LY293606 (GYKI53405) and LY300168 (GYKI 53655 ) inhibited AMPA (10 microM)-mediated responses in recombinant human GluR1 receptors expressed in HEK293 cells with approximate IC50 values of 18 microM, 24 microM and 6 microM, respectively and AMPA (10 microM) responses in recombinant human GluR4 expressing HEK293 cells with approximate IC50 values of 22 microM, 28 microM and 5 microM, respectively. GYKI 52466, LY293606 and LY300168 were non-competitive antagonists of AMPA receptor-mediated responses in acutely isolated rat cerebellar Purkinje neurons with approximate IC50 values of 10 microM, 8 microM and 1.5 microM, respectively. The activity of racemic compounds LY293606 and LY300168 was established to reside in the (-) isomer of each compound. At a concentration of 100 microM, GYKI52466, LY293606 and LY300168 produced < 30% inhibition of kainate-activated currents evoked in HEK293 cells expressing either human homomeric GluR5 or GluR6 receptors or heteromeric GluR6+KA2 kainate receptors. The activity of the 2,3-benzodiazepines at 100 microM was weak at kainate receptors, but was stereoselective. Similar levels of inhibition were observed for kainate-induced currents in dorsal root ganglion neurons. Intact tissue preparations were also used to examine the stereoselective actions of the 2,3-benzodiazepines. In the cortical wedge preparation, the active isomer of LY300168, LY303070, produced a non-competitive antagonism of AMPA-evoked depolarizations with smaller changes in depolarizations induced by kainate and no effect on NMDA-dependent depolarizations. LY303070 was also effective in preventing 30 microM AMPA-induced depolarizations in isolated spinal cord dorsal roots with an approximate IC50 value of 1 microM. Synaptic transmission in the hemisected spinal cord preparation was stereoselectively antagonized by the active isomers of LY300168 and LY293606. In summary, these results indicate that 2,3-benzodiazepines are potent, selective and stereospecific antagonists of the AMPA subtype of the non-NMDA glutamate receptor. [1]

The half-recovery time for GYKI 53655 hydrochloride (4 mg/kg) is around 7 minutes, and it was found to have a transitory inhibitory effect on neuronal responses to iontophoretic therapy α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). Hydrochloride of GYKI 53655 (4 and 8 mg/kg) substantially reduces or stops the AMPA response. GYKI 53655 hydrochloride (2 to 8 mg/kg) suppressed AMPA in a dose-dependent manner, according to the results. GYKI 53655 hydrochloride decreased the AMPA response to a similar degree even at the highest dose examined [2]. At doses of GYKI 53655 hydrochloride more than 5.0 mg/kg, tonic cramps and death were totally avoided. GYKI 53655 hydrochloride has an ED50 value of 2.2 mg/kg ip. The export inhibitory impact of GYKI 53655 hydrochloride decreases to 20% after one hour, and its maximum effect lasts for three hours [3].

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1. The effects of intravenous administration of two alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) antagonists were studied on responses of single neurones to iontophoretically applied excitatory amino acids. The tests were performed on spinal neurones in alpha-chloralose anaesthetized, spinalized rats. 2. Both the quinoxaline, NBQX (2-16 mg kg-1) and the 2,3-benzodiazepine, GYKI 53655 (2-8 mg kg-1) dose-dependently decreased responses to AMPA. 3. Both compounds were short acting, with half-recovery times of 15 min for NBQX and 7 min for GYKI 53655. 4. The selectivity for responses to AMPA over those to N-methyl-D-aspartate (NMDA) was significantly poorer for systemic NBQX than for either systemicGYKI 53655 or iontophoretic NBQX, suggesting that systemic NBQX may be converted to a less selective metabolite. 5. GYKI 53655 is therefore likely to be a more valuable tool than NBQX for the study of AMPA receptor-mediated processes in vivo. [2]

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In preliminary experiments, GYKI 53655 (4 mg kg-') was found to have a short-lasting depressant effect on neuronal responses to iontophoretic AMPA, with a half-recovery time of approximately 7 min (Figure la). We assumed that this was due to drug elimination, so for the rest of the experiments, bolus injections of GYKI 53655 were supplemented with a constant infusion at a rate of half the bolus dose given over 7 min. When tested on dorsal horn neurone responses to AMPA and NMDA, GYKI 53655 (4 and 8 mg kg-') substantially depressed or completely abolished AMPA responses, while NMDA-evoked firing was less affected (Figure lb). Pooled data from 14 such experiments show the dose-dependence of GYKI 53655 (2-8 mg kg-') in depressing responses to AMPA (Figure 2a). NBQX also dose-dependently decreased AMPA-evoked responses following either i.v. (2-16 mg kg-') or microiontophoretic administration (Figure 2b). Both antagonists reduced responses to AMPA significantly more than those to NMDA (P< 0.002 at all doses, based on a comparison of the percentage data). Nonetheless the results in Figure 2a and b indicate that, following i.v. administration, GYKI 53655 was more selective than NBQX. At the highest doses tested, GYKI 53655 and NBQX reduced AMPA responses to a comparable degree (23 ± 6% and 27 ± 6% control, respectively) whereas for NMDA the values were 77 ± 7% vs 59 ± 7% control respectively. Similar data that are more directly comparable were obtained by selecting those cells on which both antagonists were tested under directly comparable conditions (Figure 2c). On these cells the highest doses of each antagonist tested (NBQX 16 mg kg-' and GYKI 53655 8 mg kg-') again reduced AMPA responses to a very similar degree, but NBQX reduced NMDA responses significantly more than did GYKI 53655 (P<0.05,based on percentage data).
When administered by iontophoresis as opposed to intravenously, NBQX was a highly selective antagonist at the top currents tested (5-10 nA), reducing AMPA responses to 14±4% control (P< 0.001 vs control) whilst NMDA responses remained at 85 ± 4% control (nonetheless significantly less than control, P<0.01). Spontaneous firing rates were reduced by all antagonists, but significantly more by systemic GYKI 53655 and NBQX (respectively to 50 ± 13% and 36 ± 10% control at the highest doses tested on 10 cells having spontaneous activity) than by microiontophoretic NBQX (70 ± 7% control; 9 cells with spontaneous activity). [2]

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GYKI 52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine], a non-competitive AMPA [alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate] and kainate receptor antagonist and its two analogues, GYKI 53405 [1-(4-aminophenyl)-3-acetyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine] and GYKI 53655 [1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine] were investigated in two seizure models and in MgCl2 induced global cerebral ischaemia, as an acute neuroprotective model. The ED(50) values of GYKI 52466 for suppression of the tonic and clonic phases of sound-induced seizures were 3.6 and 4.3 mg/kg, respectively. The corresponding data for GYKI 53405 were 1.1 and 3.1 mg/kg, while ED(50) values of GYKI 53655 were 1.3 and 2.0 mg/kg, respectively. The inhibition of seizure evoked by maximal electroshock was also found to be remarkable: the ED(50) values of GYKI 52466 and its two analogues were 6.9, 2.6, and 2.2 mg/kg, respectively. All compounds prolonged the survival times in MgCl2 induced global cerebral ischaemia test in a dose-dependent fashion, with PD(50) (dose of 50% prolongation) values of 24.1, 8.3, and 8.2 mg/kg intraperitoneal, respectively. In audiogenic seizure model the duration of anticonvulsant action of 10 mg/kg GYKI 52466 and 5 mg/kg GYKI 53405, GYKI 53655 were examined, too. The effect of GYKI 52466 decreased to 50% after 2 h, while the analogues showed more than 80% seizure suppression 3 h after treatment. After 6 h the effect of GYKI 53655 decreased to zero, while the effect of GYKI 52466, remained on the 50% level. [3]

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Anticonvulsant activity [3]
All GYKI compounds blocked the sound-induced clonic and tonic convulsions in a dose-dependent manner. Tonic fit and death were completely prevented by GYKI 53405 and GYKI 53655 at doses over 2.5 and 5.0 mg/kg, respectively, while the corresponding value for GYKI 52466 was 10.0 mg/kg. The ED50 values for the inhibition of clonic and tonic extensor convulsions are summarised in Table 1. Similarly to earlier reports 11, 22 the compounds protected mice against tonic extensor seizures in the MES test in a dose-dependent fashion. The ED50 values of GYKI 53405 and GYKI 53655 were 2.6 and 2.2 mg/kg i.p., respectively, compared to 6.9 mg/kg for GYKI 52466 (Table 1).

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Acute neuroprotective effect [3]
All three GYKI compounds produced a dose-dependent prolongation of survival time in the MgCl2 induced global ischaemia model. GYKI 53405 and GYKI 53655 exerted similar anti-ischaemic effects (PD50 values were 8.3 and 8.2 mg/kg, i.p., respectively), while GYKI 52466 showed weaker activity (PD50 = 24.1 mg/kg, i.p.) in this test (Table 1). The latter compound produced significant increases of survival time in doses of 15 mg/kg and over [F(4,40) = 26.86; p < 0.001], while its two analogues GYKI 53405, and 53655 (Fig. 1) were effective in 7.5 mg/kg [F(4,43) = 34.93; p < 0.001 and F(4,43) = 52.02; p < 0.001], respectively.

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Time course of anticonvulsant activity in the AS test [3]
As for the time course of anticonvulsant activity (Fig. 2A) the effect of GYKI 52466 decreased to 50% after 2 h, while the other compounds showed more than 80% seizure suppression even after 3 h. The strong anticonvulsive effect of GYKI 53405 gradually disappeared from 3 to 8 h, while the effect of GYKI 53655 rapidly decreased to 20% from 3 to 4 h, then it disappeared, as well. Surprisingly, the effect of GYKI 52466 remained on the 50% level for up to 6 h. However, after 8 h, all GYKI compounds investigated were ineffective.
In exit inhibition (Fig. 2B), GYKI 52466 was completely protective for only 1 h, but about 70% protection sustained up to 6 h. The maximal effects of GYKI 53655 and 53405 lasted 3 and 4 h, respectively, then the exit inhibition effect of GYKI 53655 fall to 20% 1 h later. However, the effect of GYKI 53405 gradually decreased to zero from 4 to 8 h.

Acutely isolated cerebellar Purkinje neurons [1]
Cerebellar Purkinje neurons were isolated via modification of the method of Mintz et al. (1992). The cerebellar vermis of 6–11 day old rat pups (Sprague-Dawley) were removed and transferred to a buffer containing 82 mM Na2SO4, 30 mM K2SO4, 5 mM MgCl2, 1 mM HEPES, 1 mM glucose and 0.1% phenol red, pH 7. Tissue was cleaned, chopped and digested with buffer containing 1 mg/ml protease XXIII at 37°C for 6 min. After being digested and washed, the tissue was transferred to buffer supplemented wth 1 mg/ml BSA (Sigma) and 1 mg/ml trypsin inhibitor. Cells were dissociated by trituration and plated onto poly-l-lysine coated glass coverslips (50 μg/ml). Purkinje cells were identified morphologically by their large cell bodies (15–30 mm).

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Acutely isolated dorsal root ganglion neurons [1]
Dorsal root ganglion (DRG) neurons were isolated via modification of the method of Moises et al. (1994)(Bleakman et al., 1996). DRGs were dissected from the lumbar and thoracic regions of 4–7 day old rats and treated with collagenase (type II, 3 mg/ml) for 50 min at 37°C in a solution consisting of 82 mM Na2SO4, 30 mM K2SO4, 5 mM MgCl2, 1 mM HEPES and 1 mM glucose, pH 7.4. After centrifugation and removal of the enzyme solution, a solution of MEM with BSA (20 mg/ml) was added, and the tissue resuspended and centrifuged. This proceedure was repeated and the MEM/BSA solution replaced with an MEM solution supplemented with 1.5 mg/ml NaHCO3, 300 ng/ml nerve growth factor, 10 μg/ml penicillin/streptomycin and 10 mM glucose. Cells were mechanically dispersed with the use of a fine-tipped plastic Pasteur pipette and plated onto poly-l-lysine coated (50 μg/ml) glass coverslips. All recordings were performed within 36 hr of plating.

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Transfected cell lines [1]
Stable cell lines of HEK293 cells transfected with cDNA coding for the human GluR4(flip), GluR5(Q) and GluR6(Q) receptors were established as reported previously (Hoo et al., 1994; Korczak et al., 1995; Fletcher et al., 1995). The heteromeric cell line GluR6(Q) + KA2 was established by co-transfection of HEK293 cells with GluR6(Q) and KA2 cDNAs (Hoo et al., 1994; Kamboj et al., 1992), each incorporated into the mammalian expression vector pRc/CMV. Transfected cells were selected on the basis of G418 resistance, and expression of both genes confirmed by RT-PCR. The human GluR1 receptor (humGluR1) was isolated by hybridization of human fetal brain cDNA/λZAPII bacteriophage library (Stratagene) with radiolabelled rat GluR oligonucleotide probes. Stable cell lines were established by transfection of HEK293 cells with the mammalian expression vector pRc/CMV incorporating the cDNA for human GluR1. Transfected cells were selected on the basis of G418 resistance, and expression of the humGluR1 gene was confirmed by RT-PCR. For electrophysiological recordings, cells were dissociated by trituration and plated onto poly-l-lysine coated (10 μg/ml) glass coverslips.

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Electrophysiological recordings and drug application [1]
Whole-cell voltage clamp recordings were made from single cells with use of the tight seal whole cell configuration of the patch-clamp technique (Hamill et al., 1981). Glass fragments of coverslips with adherent cells were placed in a perfusion chamber and rinsed with buffer of composition 138 mM NaCl, 5 mM CaCl2, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES and 10 mM glucose, pH to 7.5 with NaOH (osmolality 315 mosm/kg). Pipette solutions contained 140 mM CsCl, 1 mM MgCl2, 14 mM diTris creatine phosphate, 50 U/ml creatine phosphokinase, 14 mM MgATP, 10 mM HEPES and 15 mM BAPTA, pH to 7.2 with CsOH (osmolality 295 mosm/kg). Experiments were performed at room temperature (20–22°C) and recorded on either a List EPC-7 or an Axopatch 1D amplifier. Pipette resistances were typically 1.5–2.5 MΩ. Drug application was via a series of perfusion lines to a multi-barrelled applicator (Biologic Inc.) and exchange of solutions under the present recording conditions was approximately 100 msec. Experiments using GluR6, GluR6/KA2 and GluR5 transfected cells were performed following removal of agonist-induced desensitization by preincubation of coverslips with 250 μg/ml concanavalin A. Agonist-induced desensitization at GluR1 and 4 transfected cells was removed by performing experiments in the presence of 100 μM cyclothiazide (Partin et al., 1993). For the acutely isolated cerebellar Purkinje neurons, drug application was by bath perfusion and occurred within approximately 15 sec. We have demonstrated previously that under such recording conditions and with the slow application of agonist, kainate produces non-desensitizing currents which are unaffected by concanavalin A (Bleakman et al., 1996) and thus likely to be mediated by an action of kainate at AMPA receptors in these cells. Curve fitting to data points was based upon the equation y = 100(Dn/(Dn + ic50n)), where the slope of the line n was fixed to a value of 1 and D is the antagonist concentration. Statistical significance was determined by one-way ANOVA followed by Student-Newman-Keuls test.

Animal preparation [2]
Experiments were performed on 27 male Wistar rats (260-350 g). Details of the experimental methods have been described elsewhere (Headley et al., 1987). Briefly, rats were anaesthetized with halothane in °2 and tracheal, carotid and jugular cannulae were inserted. The lumbo-thoracic spinal cord was exposed and cut at T9-Tl 1 and the animal was prepared for extracellular recordings of single dorsal horn neurone action potentials. Anaesthesia after surgery was maintained with a-chloralose (50mgkg-', i.v. initially, then 10mgkg-' as required). Arterial blood pressure was monitored continuously; systolic pressure remained above 100 mmHg. Core temperature was maintained close to 37'C. Extracellular recordings of single dorsal horn neurone action potentials were made with the central barrel of multibarrel glass micropipettes, filled with 3.5 M NaCl. Counts of evoked spike activity, in epochs related to the stimuli, were analysed on-line with a microcomputer. Cell discharge rates were monitored on a chart recorder. At the end of experiments, the rats were killed with an overdose of pentobarbitone.

Drugs AMPA, NMDA and NBQX were administered microiontophoretically from the side barrels of the multibarrel pipettes, from solutions of the sodium salts of AMPA (10 mM in 200 mM NaCI), NMDA (100 mm in 100 mM NaCl) and NBQX (1 mM in 50 mM NaCI), all at pH 7.5-8. Another barrel contained 200 mM NaCl for current balancing. AMPA and NMDA were ejected in regular cycles. NBQX, and also GYKI 53655, were administered intravenously. Antagonist effects are expressed quantitatively as percentages of control EAA responses, where control was taken as the mean of the last 3 pre-drug counts; mean values ± s.e.mean are indicated. No corrections for spontaneous activity were made. Tests were accepted only if AMPA and NMDA responses recovered by at least 50% of the initial antagonist effect. Statistical analysis was performed with the Wilcoxon matched pairs test on original spike count data (except where indicated).

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Cortical wedge preparation [1]
Rats, 150–200 g, were killed by cervical dislocation, decapitated and the brains rapidly removed to ice-cold artificial cerebrospinal fluid (aCSF). Transverse 450 μm sections were cut from a block containing the rostral forebrain. Wedge shaped slices were taken from the cingulate cortex and placed in a two-compartment chamber so that the grey matter was in one chamber and the white matter in the other, separated by a grease seal barrier. D.C. potentials were recorded between the two chambers and recorded continuously via Ag/AgCl electrodes to a chart recorder. The grey matter was superfused with aCSF, composition in mM: NaCl 124, NaHCO2 25.5, KCl 3.3, KH2PO4 1.2, CaCl2, 2.5, d-glucose 10, equilibrated with 5% CO2 in oxygen, to a pH of 7.4–7.5 at room temperature. The compartment with white matter was left in a static pool of aCSF containing 1 mM MgSO4. Compounds were tested as agonists by application to the grey matter side in 4 ml aliquots of the superfusing aCSF at not less than 20 min intervals. Compounds were tested as antagonists by continuous superfusion and 4 ml aliquots of standard agonist were added to this solution. Data are expressed as a percentage of the response evoked in the absence of antagonist.

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Spinal cord preparation [1]
Three to 5 day old rats were killed by decapitation. In Mg2+ containing aCSF, the spinal cord was removed from the vertebral column taking care to preserve the ventral and dorsal roots of the segment L4–L6. The cord was hemisected and placed on a Perspex base so that the dorsal root (DR) and the ventral roots (VR) rested on Ag/AgCl stimulating and recording electrodes, respectively. Grease seals were used to isolate these from the spinal cord itself. The cord was superfused continuously (1.5 ml/min) with an aCSF of composition (mM) NaCl 126, KCl 3, NaH2PO4 1.25, CaCl2 2.4, MgSO4 1.4 and d-glucose 10, equilibrated with 5% CO2 in O2 to maintain a pH of 7.4–7.5 at room temperature (20–22°C) (Paternain et al., 1995). To study the activity of the 2,3-benzodiazepines on depolarizations produced by AMPA and NMDA, the VR D.C. potential was recorded on a chart recorder in response to 3 ml aliquots of Mg2+-free aCSF containing AMPA (3 μM) or NMDA (30 μM) administered at not less than 20 min intervals. Effects of antagonists were assessed by superfusion for at least 20 min prior to agonist addition. Cumulative antagonist dose-response curves were normalized by expression of amplitude as a percentage of the control agonist response (Palmer and Lodge, 1993).

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AS test [3]
The experiments were performed according to the slightly modified method of De Sarro et al. Test compounds or vehicle (controls) were given i.p. to groups of 9–16 mice per doses. Fifteen minutes later mice were challenged with a 14-kHz sinusoidal tone at 120 dB in a covered glass cylinder (30 cm in diameter). Seizure response was assessed by two independent observers. The sound evoked behaviour was coded using the following scale: 0 = normal behaviour, 1 = wild running, 2 = clonus, 3 = tonic flexor seizure, 4 = tonic extensor seizure; on the basis of their concordant opinion of the observers. Sound effect was applied for 60 s, but it was interrupted earlier, when the observed animal showed tonic extensor seizure. The maximal response (0–4 score) evoked by discordant sound was recorded during the 60 s for each animal. Lethality was noted, as well. The ED50 values for the inhibition of clonic seizures and tonic extensor convulsions were determined by the method of Litchfield and Wilcoxon. In audiogenic seizure test the duration of the anticonvulsant action of 10 mg/kg GYKI 52466, and 5 mg/kg GYKI 53405 and GYKI 53655 were also examined. Percentage of seizure- and exit-inhibition compared to control group was calculated. In these experiments separate vehicle-treated and drug-treated groups were used at each pretreatment time (15 min–8 h).

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Global cerebral ischaemia induced by MgCl2 (acute neuroprotective model) [3]
Groups of 10 mice were treated i.p. with different doses of GYKI 53405 and GYKI 53655 (3.75, 7.5, 15, and 30 mg/kg), and GYKI 52466 (7.5, 15, 30, and 60 mg/kg), or vehicle. Thirty minutes later saturated solution of MgCl2 (5 ml/kg) was administered by i.v. injection resulting in cardiac arrest and subsequently complete global ischaemia of the brain. The increase in the survival time (interval between the lethal i.v. injection of MgCl2 and death, determined as the last observable gasp) was used to measure the neuroprotective effect, as described by Berga et al. Results were expressed as means ± SEM for the different treatment groups and statistical significance was assessed using analysis of variance followed by Duncan’s test. The percent changes in survival time were calculated comparing the treated groups to the MgCl2 controls. The doses prolonging the survival time by 50% (PD50 value) were calculated by linear regression analysis.

126757 mouse LD50 oral 97 mg/kg BEHAVIORAL: ALTERED SLEEP TIME (INCLUDING CHANGE IN RIGHTING REFLEX); BEHAVIORAL: MUSCLE WEAKNESS; LUNGS, THORAX, OR RESPIRATION: RESPIRATORY DEPRESSION United States Patent Document., #5519019
126757 mouse LD50 intraperitoneal 93 mg/kg BEHAVIORAL: ALTERED SLEEP TIME (INCLUDING CHANGE IN RIGHTING REFLEX); BEHAVIORAL: ATAXIA; LUNGS, THORAX, OR RESPIRATION: RESPIRATORY DEPRESSION United States Patent Document., #5519019

[1]. Activity of 2,3-benzodiazepines at native rat and recombinant human glutamate receptors in vitro: stereospecificity and selectivity profiles. Neuropharmacology. 1996;35(12):1689-702.

[2]. A comparison of intravenous NBQX and GYKI 53655 as AMPA antagonists in the rat spinal cord. Br J Pharmacol. 1994 Jul;112(3):843-6.

[3]. Comparison of anticonvulsive and acute neuroprotective activity of three 2,3-benzodiazepine compounds, GYKI 52466, GYKI 53405, and GYKI 53655. Brain Res Bull. 2001 Jun;55(3):387-91.

We determined the time courses of all the three GYKI compounds at equiactive doses in the AS test. Doses producing greater than 85% inhibitions against audiogenic stimulation (data not shown) were selected. At these doses, the protective effect of GYKI 52466 decreased to 50% at 2 h following administration, whereas the protective activities of the two analogues persisted for 4 and 3 h, respectively. Interestingly, the effect of the most potent analogue GYKI 53655 disappeared at 4 h, while the effect of GYKI 52466 was sustained at around 50% even 6 h later. The same phenomenon was found in exit inhibition, where the protective effect of GYKI 52466 rapidly decreased to the 70% level, but this protection sustained up to 6 h. The effects of the two analogues lasted a minimum of 3 h, but then their exit inhibition properties has been reduced. The effect of GYKI 53655 decreased rather quickly when compared to that of GYKI 53405, which decreased gradually from 4 to 8 h. Because De Sarro et al. and Yamaguchi et al. described short (max. 90 min) protective effect of GYKI 52466 in MES and AS test, respectively, the rapid weakening of the effect of this compound is not unexpected, however, the fact that about 50% seizure inhibition persisted after 6 h is somewhat surprising. A possible explanation could be that the compound is metabolised rather fast resulting brief maximal effect, and a metabolic product possessing the same or weaker anticonvulsant activity is responsible for the long lasting effect. Our results demonstrate that significant differences can be found between the GYKI compounds investigated in terms of efficacy and duration of action in various models. Their differences indicate that similar variability could be expected in terms of therapeutic indications and efficacy and that minor changes in the chemical structure of the compounds could result in significant improvement in their pharmacological action. [3]

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In this study GYKI 53655 , a more potent derivative of GYKI 52466 (Tarnawa et al., 1993) was more selective than NBQX in reducing responses to AMPA vs NMDA. Even GYKI 53655, however, did reduce NMDA responses to some extent. The reduction of background activity (which presumably results from antagonism of AMPA receptormediated spontaneous inputs onto the cells) reflects a decreased excitability that would in turn reduce responses to NMDA as well as those to AMPA; this effect is therefore likely to contribute to this minor degree of non-selectivity. Such a mechanism cannot, however, explain the difference in the degree of selectivity between systemic NBQX and either iontophoretic NBQX or systemic GYKI 53655 (see data in Figure 2), particularly when the two agents were compared on the same cells. Since NBQX is evidently metabolized rapidly (the halfrecovery time under these conditions was 15 min), it seems likely that a metabolic product may underlie the observed low selectivity of NBQX after systemic administration. GYKI 53655, whilst more selective, was even shorter-acting (half recovery time 7 min), which would limit the possible therapeutic use of this compound. Disubstituted reduced analogues of GYKI 52466 are longer lasting (Tarnawa et al., 1993); development in, this direction may yield more useful compounds. In conclusion, the present work has demonstrated that both NBQX and GYKI 53655 can be effective AMPA antagonists following systemic administration. Care should, however, be taken in the interpretation of results obtained with systemic NBQX since its antagonist profile appears to be less selective than that observed either in vitro or following localized administration in vivo. This seems likely to be due to conversion of NBQX to a less selective metabolite. GGYKI 53655 therefore seems to be a more valuable tool than NBQX for the investigation of AMPA receptor-mediated processes in vivo. The authors are grateful to the Wellcome Trust and the Medical Research Council for financial support, and to Dr D. Lodge (Eli Lilly & Co., U.K.) and Dr Leimner (Merz + Co., Germany) for gifts of GYKI 53655 and NBQX, respectively. [2]

C19H21CLN4O3

388.848043203354

388.13

C, 58.69; H, 5.44; Cl, 9.12; N, 14.41; O, 12.34

143692-48-2

143692-18-6 (Free Base);143692-48-2 (HCl);

126757

Light yellow to yellow solid powder

563.6ºC at 760 mmHg

294.7ºC

5.11E-13mmHg at 25°C

3.297

3

5

1

27

561

0

Cl.O1COC2C1=CC1=C(C=2)C(C2C=CC(=CC=2)N)=NN(C(NC)=O)C(C)C1

ASLCSBBDVWPSQT-UHFFFAOYSA-N

InChI=1S/C19H20N4O3.ClH/c1-11-7-13-8-16-17(26-10-25-16)9-15(13)18(22-23(11)19(24)21-2)12-3-5-14(20)6-4-12;/h3-6,8-9,11H,7,10,20H2,1-2H3,(H,21,24);1H

5-(4-aminophenyl)-N,8-dimethyl-8,9-dihydro-[1,3]dioxolo[4,5-h][2,3]benzodiazepine-7-carboxamide;hydrochloride

91HGG22IDM; ly-300168 monohydrochloride; 1-(4-Aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride; 7H-1,3-DIOXOLO(4,5-H)(2,3)BENZODIAZEPINE-7-CARBOXAMIDE, 5-(4-AMINOPHENYL)-8,9-DIHYDRO-N,8-DIMETHYL-, HYDROCHLORIDE (1:1); 7H-1,3-Dioxolo[4,5-h][2,3]benzodiazepine-7-carboxamide, 5-(4-aminophenyl)-8,9-dihydro-N,8-dimethyl-, hydrochloride (1:1); LY-300168 MONOHYDROCHLORIDE, (+-)-; 692-678-5; 143692-48-2;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 160 mg/mL (~411.47 mM)
H2O : ~8 mg/mL (~20.57 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)