For all experiments described, stock solutions in Dimethyl sulfoxide (DMSO) (0.01–0.10 M) were made fresh on the day of the first experiment, visually inspected for solubility when diluted into ACSF at working concentrations, frozen, and then reused for multiple experiments and applied via bath perfusion. The working concentration of DMSO was kept below 0.01% for each solution. Bumetanide, carbamazepine, clobazam, clonazepam, ethosuximide, felbamate, midazolam, phenobarbital, phenytoin, sodium valproate, and stiripentol were obtained from Sigma. Eslicarbazepine, gabapentin, levetiracetam, rufinamide, tiagabine, and topiramate were obtained from TCI America (Portland, OR, U.S.A.). Lacosamide and ezogabine was obtained from Axon Medchem (Groningen, The Netherlands). Lamotrigine was obtained from AK Scientific (Union City, CA, U.S.A.).
Clobazam
Manufactured by Merck Group
Sourced in United States
Clobazam is a benzodiazepine medication used as an anticonvulsant. It is utilized in the treatment of epilepsy. Clobazam acts on the central nervous system to reduce seizure activity.
Automatically generated - may contain errors
Lab products found in correlation
Carbamazepine, by Merck Group (7 mentions)
Phenobarbital, by Merck Group (5 mentions)
Valproic acid, by Merck Group (5 mentions)
Clonazepam, by Merck Group (4 mentions)
Phenytoin, by Merck Group (4 mentions)
Levetiracetam, by Merck Group (4 mentions)
Ethosuximide, by Merck Group (3 mentions)
Stiripentol, by Merck Group (3 mentions)
Sodium valproate, by Merck Group (3 mentions)
Lamotrigine, by AK Scientific (3 mentions)
Eslicarbazepine, by Tokyo Chemical Industry (3 mentions)
Gabapentin, by Tokyo Chemical Industry (3 mentions)
Levetiracetam, by Tokyo Chemical Industry (3 mentions)
Rufinamide, by Tokyo Chemical Industry (3 mentions)
Tiagabine, by Tokyo Chemical Industry (3 mentions)
Topiramate, by Tokyo Chemical Industry (3 mentions)
Lacosamide, by Axon Medchem (3 mentions)
Lamotrigine, by Merck Group (3 mentions)
Topiramate, by Merck Group (3 mentions)
Ezogabine, by Merck Group (2 mentions)
10 protocols using clobazam
1
Antiepileptic Drug Stock Solution Protocol
2
Preparation of Anticonvulsant Compounds
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All compounds were prepared in 0.5% methylcellulose (Sigma; St. Louis, MO, USA) suspensions, with the exception of sodium valproate, which was prepared in saline (0.9% NaCl). Carbamazepine, clobazam, clonazepam, ethosuximide, ezogabine, phenobarbital, phenytoin, and sodium valproate (valproate) were obtained from Sigma. Eslicarbazepine, gabapentin, levetiracetam, rufinamide, tiagabine, and topiramate were obtained from TCI America. Lacosamide was obtained from Axon Medchem. Lamotrigine was obtained from AK Scientific.
3
Evaluation of Anticonvulsant Compounds
4
Cannabinoids and Anticonvulsant Drugs
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CBDV, CBG and Δ9‐THCV were purchased from THC Pharm GmbH (Frankfurt, Germany). CBGA was purchased from THC Pharm GmbH and Epichem (Bentley, Australia). CBGV was purchased from Toronto Research Chemicals Inc. (Ontario, Canada). CBDVA and CBGVA were synthesized by Professor Michael Kassiou at the University of Sydney (Australia). Clobazam and valproic acid were purchased from Sigma‐Aldrich Co. (St. Louis, USA). When available, analytical standards were purchased from Novachem Pty Ltd (Heidelberg West, Australia). For in vitro experiments, drugs were prepared as stocks in DMSO. For acute administration, drugs were prepared fresh on the day of the experiment. CBDV, CBG, CBGV and Δ9‐THCV were prepared in ethanol:Tween 80:saline (1:1:18). CBGV (100 mg·kg−1) was a suspension, with CBDV (60 mg·kg−1) and CBG (30 mg·kg−1) near solubility limits. CBDVA, CBGA, CBGVA and Clobazam were prepared as solutions in vegetable oil. Drugs were administered as an intraperitoneal injection in a volume of 10 ml·kg−1. ML‐186 was purchased from Enamine (Kyiv, Ukraine). CID16020046 and lysophosphatidylinositol were purchased from Tocris Biosciences (Bristol, United Kingdom) and Sigma‐Aldrich (St. Louis, USA).
5
Anticonvulsant Compound Preparation and Dosing
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Valproate (VPA), topiramate (TPM), stiripentol (STP), cannabidiol (CBD), clobazam (CLB), levetiracetam (LEV), carbamazepine (CBZ), and lamotrigine (LTG) were purchased from Sigma-Aldrich. Phenytoin (PHT) was from Acros Organics. (±)-Fenfluramine [(±)-FFA] was a gift from Prof. Berten Ceulemans (University of Antwerp, Belgium). The enantiomers of FFA and norfenfluramine (norFFA) were provided by Zogenix International (Emeryville, USA). Compounds were dissolved in dimethylsulfoxide (DMSO), and diluted in embryo medium to achieve a final DMSO concentration of 0.1% w/v. Embryo medium with 0.1% w/v DMSO served as a vehicle control (VHC).
6
Analytical Reference Standards for Neuropharmaceuticals
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Reference standards for carbamazepine (purity 99%), phenytoin (certified reference material), clonazepam, lacosamide (certified reference material), phenobarbital (purity ≥ 95%), levetiracetam (purity ≥ 98%), lamotrigine (purity ≥ 98%), oxcarbazepine (purity ≥ 98%), clozapine (purity ≥ 98%), clobazam (purity ≥ 98%), valproic acid (purity ≥ 98%), and topiramate (purity ≥ 98%) were purchased from Sigma-Aldrich (Steinheim, Germany); methanol (LC–MS grade), ammonium acetate (purity ≥ 99.0%), and ammonium hydroxide solution (ACS reagent, 28.0–30.0% NH3 basis) from Sigma-Aldrich (Riedel de Haёn, Germany); acetonitrile (LC–MS grade) from J.T. Baker (Avantor Performance Materials, Corporate Parkway Center Valley, PA, USA).
7
Anticonvulsant Compounds Preparation Protocol
8
Anticonvulsant Drug Dosing and Administration
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The drugs that were used were: valproic acid (VA, 300 mg/kg in saline; Sigma-Aldrich), clobazam (CLB, 10 mg/kg in sesame oil; EDQM), stiripentol (STP, 150 mg/kg in sesame oil; Angene Chemical), a combination of clobazam and stiripentol (CLB + STP, 5 mg/kg + 100 mg/kg in sesame oil), carbamazepine (CBZ, 20 mg/kg in 30% polyethylene glycol 400; Alomone Labs) and lamotrigine (LTG, 10 mg/kg in 30% polyethylene glycol 400; Alomone Labs). Drug dosages were chosen to reach therapeutic-relevant concentrations following acute administration (Hawkins et al., 2017 (link)). All drugs were administered as a single IP injection in a volume of 10 ml/kg.
9
Acute Administration of Antiepileptic Drugs
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Carbamazepine, clobazam, lamotrigine, levetiracetam, phenobarbital, topiramate, and valproic acid were purchased from Sigma‐Aldrich Co. (St. Louis, MO). Stiripentol (Sigma‐Aldrich Co.; Cayman Chemical, Ann Arbor, MI) and phenytoin sodium solution (X‐Gen Pharmaceuticals, Horseheads, NV) were also used. For acute administration, drugs were prepared as solutions with the following vehicles: A‐ saline (levetiracetam, phenobarbital, topiramate, valproic acid); B‐ 0.5% methylcellulose in water (phenytoin); C‐ 5% hydroxypropyl‐β‐cyclodextrin (Carbamazepine, lamotrigine); and D‐ vegetable oil (clobazam, Stiripentol). All drugs were administered as a single intraperitoneal (ip) injection in a volume of 10 mL/kg. The following acute doses resulted in plasma concentrations within the human therapeutic range (Table 1 ): 20 mg/kg Carbamazepine, 5 mg/kg clobazam, 20 mg/kg lamotrigine, 10 mg/kg levetiracetam, 15 mg/kg phenobarbital, 15 mg/kg phenytoin, 40 mg/kg topiramate, 25 mg/kg Stiripentol, 75 mg/kg valproic acid, and 0.1–10 mg/kg clobazam plus 25 mg/kg Stiripentol. Acute toxicity was not observed with any drugs at the administered doses.
10
Cucurbitacin E and I Quantification
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The cucurbitacin E (≥ 95%) and cucurbitacin I (≥ 95%) standards, clobazam (internal standard) (Figure 2), and dimethylsulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA). All solvents used were of HPLC grade. Methanol, acetonitrile, sodium chloride, monobasic sodium phosphate, and dibasic sodium phosphate were purchased from Merck (Darmstadt, Germany). Isopropanol was purchased from Fisher Scientific (Fair Lawn, NJ, USA). The water used in the experiments was purified with the Synergy ® UV water purification system (Millipore, Belford, MA, USA).
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