Pentylenetetrazole

One-channel video-EEG was recorded differentially between the reference (right olfactory bulb) and active (left occipital lobe) electrodes. Baseline data were continuously acquired over a period of 8 days, which included day and night cycles. Along with EEG sampled at 1000 Hz, the implanted transmitters also continuously measured core-body temperature at 200 Hz and locomotor activity at a sampling rate of 200 Hz.
All mouse cages were assigned to respective PhysioTel RPC receiver plates that transmitted data in real time from telemetry transmitters to a computer via the data exchange matrix using Dataquest ART software (Data Sciences International, St. Paul, MN). The recording and seizure induction times were standardized for all groups, and the high definition videos (30 frames/sec) were time-registered with the EEG.
At the end of baseline EEG acquisition, all animals were provoked with a convulsive dose (40 mg/kg; i.p.) of pentylenetetrazol (PTZ; Sigma-Aldrich, Co., St. Louis, MO), a GABA_A receptor antagonist, to measure seizure susceptibility. Historically in our laboratory, this dose has been sufficient to induce seizure in more than 50% of healthy rodents [34 (link), 35 (link)].

The published cryo-EM structure (PDB: 6DW0) of the GABA A receptor in complex with GABA was used as a starting template [34 (link)]. The 6DW0 was prepared using a protein preparation wizard (Schrödinger, New York, NY, USA), where the bond orders were assigned and the structure was minimized. Each of the structures: chloroquine, erythromycin, metronidazole, minocycline, ofloxacin, (-)-epigallocatechin gallate, flumazenil, hydroxychloroquine, pentylenetetrazol, clarithromycin, and rifampicin, were built and optimized in a Spartan ‘20 Parallel Suite (Wavefunction, Irvine, CA, USA). Tautomers were generated for each ligand, and the dominant ionization states of acidic and basic functional groups were used, assuming pH 7.4 (Schrödinger, New York, NY, USA). QikProp was used to calculate the ADME properties of the 11 ligands tested (Schrödinger, New York, NY, USA). A receptor grid generator was used to place 10 Å × 10 Å × 10  Å boxes covering the whole receptor (Schrödinger, New York, NY, USA). All 11 ligands were run for each of the grid boxes generated in Glide SP, using flexible docking with enhanced conformational sampling by four-fold, and an expanded sampling for the selection of initial poses (Schrödinger, New York, NY, USA). The binding site for each ligand was chosen based on the most negative glide score. Erythromycin was not found to bind to any of the sites, and thus it was not carried forward. For each ligand–receptor complex, a minimization was run to reduce steric clashing between the ligand–receptor complex (Schrödinger, New York, NY, USA) using the VGSB solvation model. MMGBSA was run on each ligand–receptor complex to determine their relative affinities.

The anticonvulsant effect of the newly synthesized compounds was investigated by tests comprising pentylenetetrazole, thiosemicarbazide convulsions, and maximal electroshock (MES). Outbred mice (weight 18–22 g) were used for the study. In the case of convulsions induced by PTZ, it was injected subcutaneously at 90 mg/kg, which induced convulsions in 95% of animals (CD 95%). Each animal is placed into an individual plastic cage for observation lasting 1 h. Seizures and clonic convulsions were recorded. Substances were administered intraperitoneally (i.p.) at doses of 10–200 mg/kg in suspension with carboxymethylcellulose and Tween-80 45 min before administration of PTZ and applying electrical stimulation. The control animals were administered as an emulsifier. Every dose of each test compound was studied in six animals.
The MES test is used as an animal model for the generalized tonic seizures of epilepsy. The parameters of MES were: 50 mA, duration of 0.2 s, and an oscillation frequency of 50 imp/s. The anticonvulsant properties of compounds were assessed by their prevention of the tonic-extensor phase of convulsions.
Thiosemicarbazide, an antimetabolite of GABA inhibitor (glutamic acid decarboxylase) in the brain, is administered subcutaneously to mice at a dose of 18 mg/kg as a 0.5% solution, which causes clonic convulsions in animals. Anti-thiosemicarbazide activity was evaluated based on the latency time of the onset of seizures. Compounds were administered intraperitoneally at doses of 100 mg/kg in suspension with carboxymethylcellulose and Tween-80 45 min before administration of thiosemicarbazide.
The comparative drug ethosuximide was administered in doses of 200 mg/kg and diazepam in doses of 2 mg/kg.