AMPA/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor

Stereoselectivity of 2,3-benzodiazepine compounds provides a unique way for the design of stereoisomers as more selective and more potent inhibitors as drug candidates for treatment of the neurological diseases involving excessive activity of AMPA receptors. Here we investigate a pair of enantiomers known as Talampanel and its (+) counterpart about their mechanism of inhibition and selectivity toward four AMPA receptor subunits or GluA1–4. We show that Talampanel is the eutomer with the endismic ratio being 14 for the closed-channel and 10 for the open-channel state of GluA2. Kinetic evidence supports that Talampanel is a noncompetitive inhibitor and it binds to the same site for those 2,3-benzodiazepine compounds with the C-4 methyl group on the diazepine ring. This site, which we term as the “M” site, recognizes preferentially those 2,3-benzodiazepine compounds with the C-4 methyl group being in the R configuration, as in the chemical structure of Talampanel. Given that Talampanel inhibits GluA1 and GluA2, but is virtually ineffective on the GluA3 and GluA4 AMPA receptor subunits, we hypothesize that the “M” site(s) on GluA1 and GluA2 to which Talampanel binds is different from that on GluA3 and GluA4. If the molecular properties of the AMPA receptors and Talampanel are used for selecting an inhibitor as a single drug candidate for controlling the activity of all AMPA receptors in vivo, Talampanel is not ideal. Our results further suggest that addition of longer acyl groups to the N-3 position should produce more potent 2,3-benzodiazepine inhibitors for the “M” site [3].

In a mouse model of ALS, Talampanel (oral; 5 mg/kg; once daily; 2 weeks) lowers the levels of calcium in motor neurons; however, as the disease progresses, its effectiveness diminishes [1].

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We tested the efficacy of treatment with Talampanel in a mutant SOD1 mouse model of ALS by measuring intracellular calcium levels and loss of spinal motor neurons. We intended to mimic the clinical study; hence, treatment was started when the clinical symptoms were already present. The data were compared with the results of similar treatment started at a presymptomatic stage. Transgenic and wild-type mice were treated either with talampanel or with vehicle, starting in presymptomatic or symptomatic stages. The density of motor neurons was determined by the physical disector, and their intracellular calcium level was assayed electron microscopically. Results showed that motor neurons in the SOD1 mice exhibited an elevated calcium level, which could be reduced, but not restored, with Talampanel only when the treatment was started presymptomatically. Treatment in either presymptomatic or symptomatic stages failed to rescue the motor neurons. We conclude that talampanel reduces motoneuronal calcium in a mouse model of ALS, but its efficacy declines as the disease progresses, suggesting that medication initiation in the earlier stages of the disease might be more effective. [1]

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Talampanel (IVAX) is a non-competitive AMPA-antagonist has a remarkable neuroprotection in different rodent stroke models. The focal cerebral ischemia in mice was induced by transient (60 min.) MCA occlusion and 48 h reperfusion and treated with talampanel (6 x 2 mg/kg, i.p.). The apoptotic and necrotic cells were analyzed by double immune histochemical staining on confocal laser microscope. The infarct size is decreased significantly by talampanel treatment (from 57.1+/-7.2mm(2) to 18.9+/-2.6 mm(2), p< 0.001). The number of TUNEL-positive cells localized mostly in the border zone of ischemic lesions is significantly decreased after Talampanel treatment (from 962+/-13.0 to 604+/-6.9, p < 0.01). A strong, significant reduction of caspase-3 active cells was visualized. Talampanel as a neuroprotective drug candidate has a significant effect in mice transient MCA occlusion model [2].

Cell Culture and Receptor Expression [3]
HEK-293S cells were cultured in a 37 °C, 5% CO2, humidified incubator, and in the Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units of penicillin/mL, and 0.1 mg streptomycin/mL. The DNA plasmids encoding all AMPA receptor subunits were prepared as previously described. The HEK-293S cells were transiently transfected to express each AMPA receptor by following a standard calcium phosphate method. The cells were also cotransfected with a plasmid encoding green fluorescent protein (GFP) as a transfection marker and a separate plasmid encoding large T-antigen to enhance the receptor expression at the single cell level. The weight ratio of the plasmid for GFP and the large T-antigen to that for an AMPA receptor was 1:1:10, respectively. The plasmid used for transient transfection of an AMPA receptor ranged from 5 to 15 μg per 35 mm dish. In general, the cells were used for recording 48 h after transfection.

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Whole-Cell Current Recording [3]
Glutamate-induced whole-cell current from transfected HEK-293S cells was recorded on an Axopatch 200B instrument at a cutoff frequency of 2–20 kHz by a built-in, four-pole low-pass Bessel filter. The whole-cell current traces were digitized at a 5–50 kHz sampling frequency using a Digidata 1322A. All recordings were at −60 mV and room temperature. The pClamp 8 software was used for data acquisition. A recording electrode was made from glass capillary and had a resistance of ∼3 MΩ when filled with the electrode solution. The electrode solution was composed of (in mM) 110 CsF, 30 CsCl, 4 NaCl, 0.5 CaCl2, 5 EGTA, and 10 HEPES (pH 7.4 adjusted by CsOH). The external solution contained (in mM) 150 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES (pH 7.4 adjusted by NaOH). All chemicals were from commercial sources.

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Laser-Pulse Photolysis Measurement [3]
The use of the laser-pulse photolysis technique to measure the channel-opening kinetics has been described previously. In this experiment, a caged glutamate (e.g., 4-methoxy-7-nitroindolinyl-caged-l-glutamate) was dissolved in the external buffer and applied to a cell using a flow device. A single laser pulse at 355 nm generated from a pulsed Q-switched Nd:YAG laser, with a pulse length of 8 ns and energy output in the range of 200–1000 μJ, was applied to an HEK-293S cell via optical fiber. To determine the concentration of glutamate generated photolytically by laser photolysis, we calibrated the receptor response in the same cell by applying two solutions of free glutamate with known concentrations before and after laser flash, with reference to the dose–response relation. These measurements also allowed us to monitor any damage to the receptors and/or the cell for successive laser experiments with the same cell. A flow device was used to deliver free glutamate and/or caged glutamate solutions in the absence and presence of inhibitor. The time resolution of this flow device, determined by the rise time of the whole-cell current response (10–90%) to saturating glutamate concentrations, was 1.0 ± 0.2 ms, an average of the measurement from >100 cells expressing the same receptor. Furthermore, we used an 8 s time protocol in preincubating all of the 2,3-benzodiazepine compounds in both flow and laser photolysis experiments in order to observe and record full inhibition by these inhibitors, a phenomenon we previously observed with other 2,3-benzodiazepine compounds.

Animal/Disease Models: Female mutant SOD1 Tg mouse[1]
Doses: 5 mg/kg
Route of Administration: Oral; 5 mg/kg; one time/day; 2 weeks
Experimental Results: Significant reduction in calcium levels only at 12 weeks of age Effect.

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Hemizygous Tg mice, expressing mutant human SOD1 with a G93A substitution, obtained originally from Jackson Laboratories, were bred and maintained in a C57BL/ 6JOlaHsd mice strain. The animals were housed at a temperature of 21°C under a 12-h dark/light cycle. Food (standard pellets) and water were supplied ad libitum. Female mutant SOD1 Tg mice were treated orally with Talampanel (5 mg/kg body weight dissolved in 0.1 ml Tween 80) or with vehicle only once a day for two weeks. The oral 5 mg/kg dose of Talampanel was selected on the basis of no observed adverse effect on body weight gain (TEVA, personal communication) and on a pilot study in our laboratory showing that this dose was effective in ameliorating mitochondrial functionality in mutant SOD1 Tg mice (unpublished data). Animals were treated at the same hour in the morning every day, without anaesthesia, by direct administration into the lower oesophagus by using an appropriate stainless steel gavage needle with a round tip. Treatments were started at 10 or 17 weeks of age, i.e. ages corresponding to the presymptomatic stage and to the onset of the motor dysfunction. Age-matched female non-Tg mice that were treated similarly were used as controls. [1]

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Erdő's method as a rodent stroke model with male mice (C57Bl6/J/Charles River) was used. Adult male C57BL/6J mice (six control and six Talampanel treated) weighing 25–30 g were used in the experiments.
Originally, 10–10 animals have been operated, in animals selected for morphology, blood-flow reduction was detected by laser Doppler cortical flow monitoring. Focal cerebral ischemia was induced under halothane anesthesia (1% halothane in 30% oxygen and 70% N2O) by transient (60 min) thread occlusion of MCA. Body temperature was continuously detected by rectal temperature control. After a midline neck incision, the left common and external carotid arteries were isolated and legated. A microvascular clip was temporarily placed on the internal carotid artery. An 8-0 nylon monofilament coated with silicon resin (150–200 μm in thickness) was introduced through a small incision into the common carotid artery and advanced 9 mm distal to the carotid bifurcation for occlusion of MCA. Reperfusion was initiated after 60 min by withdrawal of the thread. Animals were sacrificed after 48-h recirculation. Talampanel was suspended in saline containing 0.5% Tween-80. The mice received 6 × 2 mg/kg, 0.1 ml, i.p. Talampanel. Injections were given first time at 15 min after the termination of occlusion and then in every 15 min altogether 6 times. Control animals received vehicle.
Ischemic lesions were defined on Nissle stained histological sections after five consecutive slides (0.5 mm in between). The average lesion size was calculated and statistically analyzed.
Penumbra area was defined as the border zone between infracted and normal looking brain tissue, as we described earlier [11]. We analyzed the effect of Talampanel on the apoptotic/necrotic neuronal death by double fluorescence stainin [2].

Absorption, Distribution and Excretion
Absorption is rapid, reaching peak plasma concentration within 1-3 hours. Biological Half-Life 3-6 hours

The oral LD50 of 164509 in mice is 100 mg/kg. Behavioral effects include altered sleep duration (including changes in the righting reflex); muscle weakness; and respiratory depression in the lungs, chest, or respiration. (US Patent No. 5519019). The intraperitoneal LD50 of 164509 in mice is 73500 μg/kg. Behavioral effects include altered sleep duration (including changes in the righting reflex); ataxia; and respiratory depression in the lungs, chest, or respiration. (US Patent No. 5519019).

[1]. Talampanel reduces the level of motoneuronal calcium in transgenic mutant SOD1 mice only if applied presymptomatically. Amyotroph Lateral Scler. 2011 Sep;12(5):340-4.

[2]. Talampanel a non-competitive AMPA-antagonist attenuates caspase-3 dependent apoptosis in mouse brain after transient focal cerebral ischemia. Brain Res Bull. 2006 Jul 31;70(3):260-2.

[3]. Mechanism of Inhibition of the GluA2 AMPA Receptor Channel Opening by Talampanel and Its Enantiomer: The Stereochemistry of the 4-Methyl Group on the Diazepine Ring of 2,3-Benzodiazepine Derivatives. ACS Chem Neurosci. 2013 Jan 25;4(4):635–644.

Talampanel belongs to the benzodioxane class of compounds. Talampanel is a substance under investigation for the treatment of brain tumors and other brain diseases, such as epilepsy and Parkinson's disease. It is an AMPA receptor antagonist. Talampanel is a synthetic derivative of the dioxane-pentene benzodiazepine class with antiepileptic activity. Talampanel antagonizes the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) isoform of the glutamate excitatory amino acid receptor and may inhibit glioma growth by interfering with neurotransmitters involved in brain tumor growth. The drug may also have a protective effect against traumatic brain injury. Drug Indications For the treatment of epilepsy. Mechanism of Action Talampanel is a potent, non-competitive, selective glutamine AMPA receptor antagonist. Primate studies have shown that administration of talampanel to Parkinson's disease monkeys significantly reduced levodopa-induced motor dysfunction by up to 40%. Taraparib alone does not alter the severity of Parkinson's disease symptoms. However, when used in combination with levodopa, it can enhance the anti-Parkinson's effect of levodopa by increasing motor activity.

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Pharmacodynamics
Traparib is a potent and selective α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist and a potential novel antiepileptic drug (AED). The dosing strategy for traparib may depend on other AEDs administered concurrently, as enzyme-inducible AEDs enhance its metabolism, while valproic acid (VPA) inhibits it. Taraparib is well tolerated, although adverse events have occurred at lower doses compared to healthy subjects, likely due to the additive effect of other concurrently administered AEDs.

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The proven effect of traparib in reducing calcium ion concentration in motor neurons before symptom onset suggests that the theoretical basis for this treatment is largely correct. However, no efficacy was observed during the symptomatic phase, suggesting that the drug’s efficacy would gradually be lost as the disease progresses. Although talaparib effectively reduced intracellular calcium levels in the pre-symptomatic phase, it had no effect on the rescue of surviving motor neurons (MNs), which may suggest that talaparib actually played its intended role but was masked by the increasingly potent pathogenic mechanism. Therefore, the ultimate failure of talaparib treatment may be at least in part due to the late timing of administration relative to disease progression and/or the lack of adjuvant therapy with other drugs to combat other components of the pathogenic mechanism. These results highlight the importance of evaluating the efficacy of drugs in the pre-symptomatic and symptomatic phases in preclinical animal studies and underscore the need for early diagnostic biomarkers for amyotrophic lateral sclerosis (ALS) that may aid in clinical trials and promise earlier and more effective treatment. [1] In conclusion, in our experiments, Talampanel significantly inhibited the expression of caspase-3 in ischemic brain tissue, indicating a significant anti-apoptotic effect. These results are consistent with those of Erdő and colleagues and suggest that Talampanel may play a potential role in stroke treatment. [2] Chirality can enhance the selectivity of biomolecules in vivo and may even enhance their specificity. For example, the mammalian olfactory system can distinguish (+)- and (−)-carvone by similar caraway seed and peppermint reactions, respectively, although the only difference between the two enantiomers is the configuration of the methyl vinyl group. d-glutamate has a lower affinity for glutamate receptor activation and its binding results in less receptor activation. Chirality can also enhance the selectivity and even specificity of inhibitor and drug design. This is particularly advantageous for developing better 2,3-benzodiazepines to more precisely control AMPA receptor activity in vivo. The results of this study suggest that any effective 2,3-benzodiazepine inhibitor containing a C-4 methyl group on the diaza ring, such as BDZ-d or Talampanel, must have an R configuration to effectively bind to non-competitive receptor sites, or “M” sites. Our study further suggests that adding a longer group at the N-3 site should produce a more effective "M" site inhibitor. Based on the fact that BDZ-d inhibits GluA1 and GluA2 but is almost ineffective against GluA3 and GluA4, we speculate that the "M" sites on GluA1 and GluA2 are different from those on GluA3 and GluA4, which likely reflects the different combination of amino acid residues that make up the "M" site. If the molecular characteristics of AMPA receptor and BDZ-d or Talampanel are considered when selecting a single inhibitor as a candidate drug for ALS clinical trials, then Talampanel is not an ideal choice. [3]

C19H19N3O3

337.37246

337.142

C, 67.64; H, 5.68; N, 12.46; O, 14.23

161832-65-1

164509

White to yellow solid powder

1.4±0.1 g/cm3

528.9±60.0 °C at 760 mmHg

273.7±32.9 °C

0.0±1.4 mmHg at 25°C

1.675

1.23

1

5

1

25

544

1

C[C@@H]1CC2=CC3=C(C=C2C(=NN1C(=O)C)C4=CC=C(C=C4)N)OCO3

JACAAXNEHGBPOQ-LLVKDONJSA-N

InChI=1S/C19H19N3O3/c1-11-7-14-8-17-18(25-10-24-17)9-16(14)19(21-22(11)12(2)23)13-3-5-15(20)6-4-13/h3-6,8-9,11H,7,10,20H2,1-2H3/t11-/m1/s1

(8R)-7-Acetyl-5-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxolo[4,5-h][2,3]benzodiazepine

LY300164, LY 300164, LY-300164, Talampanel; GYKI 53,773; Talampanel (INN); Talampanel [INN]; LY 300,164; LY-300,164; GYKI-53,773; ...; 161832-65-1; GYKI53773,GYKI 53405, GYKI-53405, LY293606, LY-293606, LY 293606,

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ≥ 100 mg/mL (~296.41 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.41 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.41 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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