Lithium chloride (Wako Pure Chemical Industries, Osaka, Japan), pilocarpine (Wako Pure Chemical Industries), and scopolamine methyl bromide (Sigma-Aldrich, Tokyo, Japan) were dissolved in 0.9% sodium chloride solution. Diazepam (Wako Pure Chemical Industries), perampanel (Eisai Co., Ltd, Kashima, Japan), and GYKI52466 (Sigma-RBI, St Louis, MO) were prepared in 1:1:1 (v/v) distilled water, dimethyl sulfoxide, and polyethylene glycol 300.
Perampanel
Manufactured by Eisai
Sourced in Japan
Perampanel is a laboratory equipment product designed for use in scientific research and analysis. It is a selective, non-competitive AMPA-type glutamate receptor antagonist. The core function of Perampanel is to inhibit the activity of AMPA receptors, which play a role in excitatory neurotransmission.
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Lab products found in correlation
Diazepam, by Fujifilm (2 mentions)
Pilocarpine, by Fujifilm (1 mentions)
Scopolamine methyl bromide, by Merck Group (1 mentions)
Lithium chloride (licl), by Fujifilm (1 mentions)
Novolink max polymer detection system, by Leica (1 mentions)
Dulbecco s modified eagle medium dmem, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Mccoy s 5a modified medium, by Merck Group (1 mentions)
Pen strep solution, by Merck Group (1 mentions)
N methyl d aspartate nmda, by Merck Group (1 mentions)
Dizocilpine mk 801, by Merck Group (1 mentions)
Anti gst hrp, by GE Healthcare (1 mentions)
Anti flag hrp, by Merck Group (1 mentions)
Mycoplasma contamination, by Lonza (1 mentions)
Sw1783, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Lonza (1 mentions)
Plastic culture flasks, by Corning (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Yh 13, by Health Science Research Resources Bank (1 mentions)
9 protocols using perampanel
1
Pharmacological Compounds Preparation
2
Evaluating Anti-Cancer Compound Cytotoxicity
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Cell culture reagents such as McCoy’s 5A Modified Medium, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and a mixture of pen/strep solution were obtained from Millipore Sigma (Merck KGaA, Darmstadt, Germany). Other cell culture reagents were brought from Gibco (Thermo Fisher Scientific, Inc, Waltham, MA, USA). Fluphenazine (cat. no. F4765), latrepirdine (cat. no. D6196), 5-FU (cat. no. F6627), Thiazolyl Blue Tetrazolium Bromide (MTT, cat. no. M5655), Bovine Serum Albumin (BSA, cat. no. A8531), sulforhodamine B (SRB, cat. no. S1402), Entellan mounting medium (cat. no. 107960), and (3-Aminopropyl)triethoxysilane (silane, cat. no. A3648) were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Benztropine (cat. no. 16214), sertraline (cat. no. 14839), thioridazine (cat. no. 14400), fluoxetine (cat. no. 14418), artesunate (cat. no. 11817), and doxorubicin (cat. no. 15007) were acquired from Cayman Chemical (Ann Arbor, MI, USA). Chloroquine (cat. no. C6628) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). PTX (cat. no. 1097) was obtained from Tocris Bioscience (Bristol, UK). Perampanel was purchased from Eisai Co., Ltd. (Tokyo, Japan). Novolink Max-Polymer detection system was bought from Novocastra (Newcastle, UK).
3
Perampanel Pharmacological Evaluation
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Perampanel was obtained from Eisai Co., Ltd., methyl cellulose (400 cP) was purchased from WAKO, and TO-PRO-3 was purchased from lifetechnologies.
4
Perampanel Release Assay Protocol
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Perampanel [2-(2-oxo-1-phenyl-5-pyridin-2yl-1,2-dihydropyridin-3-yl) benzonitrile] was a kind gift by Eisai Co., Ltd. (Tokyo, Japan). Batch 173H2901 was used for this set of experiments. Perampanel powder was stored at 4 °C. All solutions were freshly prepared before use. The drug was dissolved in DMSO solution to obtain 10 mM stock solutions; further dilutions were made in the incubation medium or in a medium consisting of 56 mM KCl (see section: “Brainstem incubations ”).
Veratridine and capsaicin were purchased from Sigma (Sigma Chemicals Co., St. Louis, MO, USA), and dissolved in 100% ethanol at 10 mM concentration; subsequently, the standard solutions were diluted with incubation medium to reach the desired final concentrations.
Neither DMSO nor ethanol interfered with CGRP release when used at working concentrations (i.e 0.1% or less). Furthermore, none of the test drugs used interfered with CGRP assay.
Veratridine and capsaicin were purchased from Sigma (Sigma Chemicals Co., St. Louis, MO, USA), and dissolved in 100% ethanol at 10 mM concentration; subsequently, the standard solutions were diluted with incubation medium to reach the desired final concentrations.
Neither DMSO nor ethanol interfered with CGRP release when used at working concentrations (i.e 0.1% or less). Furthermore, none of the test drugs used interfered with CGRP assay.
5
Antibody and Reagent Characterization
6
Glioma Cell Lines and Inhibitor Treatments
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The human glioblastoma cell lines U87 (#HTB-14), U138 (#HTB-16), and A172 (#CRL-1620) and the grade III astrocytoma cell line SW1783 (#HTB-13) were from ATCC. All glioma cell lines were grown in DMEM medium (Lonza, Basel, Switzerland), supplemented with 10% FBS (Gibco, Life Technologies, Carlsbad, CA, SUA). Cells were routinely checked for mycoplasma contamination (Lonza, Basel, Switzerland), used within 20 passages from thawing from a frozen stock. For in vitro studies, perampanel (Eisai Co., Ibaraki, Japan) and temozolomide (Schering Plough Corporation, Kenilworth, NJ, USA) were dissolved in dimethylsulfoxide (DMSO, Sigma-Aldrich, St. Louis, MI, USA). Final DMSO concentration in medium never exceeded 0.25%.
7
Comparative Analysis of Human Glioma Cell Lines
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Human malignant glioma cell lines A-172 (cat. no. JCRB0228; lot no. 021999), AM-38 (cat. no. IFO50492; lot no. 12082003), T98G (cat. no. IFO50303; lot no. 1007), U-251MG (cat. no. IFO50288; lot no. 12132002) and YH-13 (cat. no. IFO50493; lot no. 1164) were purchased from Health Science Research Resources Bank. The human glioblastoma U-138MG cell line (cat. no. HTB-16; lot no. 1104428) was purchased from the American Type Culture Collection.
Cells were cultured in Dulbecco's modified Eagle's minimum essential medium (DMEM) (Nissui Pharmaceutical, Co., Ltd.) supplemented with 5% fetal calf serum (Thermo Fisher Scientific, Inc.) using plastic culture flasks (Corning, Inc.) in a 37°C humidified incubator with an atmosphere containing 5% CO2.
Perampanel was gifted by Eisai Co., Ltd. and TMZ was purchased from Tokyo Chemical Industry Co., Ltd.
Cells were cultured in Dulbecco's modified Eagle's minimum essential medium (DMEM) (Nissui Pharmaceutical, Co., Ltd.) supplemented with 5% fetal calf serum (Thermo Fisher Scientific, Inc.) using plastic culture flasks (Corning, Inc.) in a 37°C humidified incubator with an atmosphere containing 5% CO2.
Perampanel was gifted by Eisai Co., Ltd. and TMZ was purchased from Tokyo Chemical Industry Co., Ltd.
8
Competitive Displacement Assay for E2730 Binding
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For the competitive displacement assay, rat synaptosomal membrane extract (0.2 mg protein/tube) was incubated for 120 minutes at 4°C with 20 nM [3H]E2730 and the following ASMs: carbamazepine (FUJIFILM Wako Pure Chemical Corporation; 100 μM); lamotrigine (Sigma‐Aldrich, MO; 300 μM); diazepam (FUJIFILM Wako Pure Chemical Corporation; 10 μM); gabapentin (Tokyo Chemical Industry; 300 μM); ethosuximide (Tokyo Chemical Industry; 3000 μM); perampanel (Eisai Co., Ltd.; 30 μM), retigabine (LKT Laboratories; 100 μM); levetiracetam (Tokyo Chemical Industry; 1000 μM); acetazolamide (Sigma‐Aldrich; 300 μM); sodium valproate (FUJIFILM Wako Pure Chemical Corporation; 3000 μM); zonisamide (FUJIFILM Wako Pure Chemical Corporation; 300 μM); and topiramate (Sigma‐Aldrich; 300 μM). Unlabeled E2730 (0.1–10 μM) was also assessed to confirm a representative displacement. Non‐specific binding was defined as the residual binding observed in the presence of 1 mM unlabeled E2730. Radioactivity of the synaptosome‐bound radioligand was measured in the same way as described above.
9
Synthesis and Administration of TETS, Perampanel, and Diazepam
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TETS was synthesized by a modification of the procedure of Esser et al. (1991). Solutions of TETS were made in 100% DMSO at a concentration of 1 mg/ml. Further dilutions were made in 10% DMSO in 0.9% saline. TETS was injected intraperitoneally at a dose of either 0.14 mg/kg or 0.2 mg/kg, or administered by oral gavage at a dose of 0.4 mg/kg. Perampanel was supplied by Eisai Inc. (Ibaraki Prefecture, Japan) and diazepam was purchased from Sigma-Aldrich (St Louis, MO). Perampanel and diazepam were prepared as suspensions in 1% Tween 80 in sterile saline and were administered intraperitoneally. Drug solutions for intraperitoneal or oral dosing were administered in a volume equaling 10-ml/kg body weight.
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