Post-Kainic Acid Status Epilepticus-Induced Spontaneous Recurrent Seizures Model (rat) – Phase I
This test evaluates the efficacy of a 5-day treatment of the test compound administered via intraperitoneal or subcutaneous route in a rat model of post-kainic acid status epilepticus-induced recurrent spontaneous seizures. If the test compound shows promise, then it may be further studied in Phase II of the testing in the same animal model where the compound is formulated into food pellets and administered orally on a fixed schedule using an automated feeding system.
Status epilepticus (SE) is induced in adult male Sprague-Dawley CD rats (250-300 g) using a repeated low-dose kainic acid (KA) paradigm (Hellier et al., 1998). Rats are injected intraperitoneally (IP) or subcutaneously (SC) with an initial dose of 7.5 mg/kg KA (Tocris Bioscience, catalogue # 0222) to induce SE. If the animals do not develop a stage 4-5 seizure within an hour, an additional dose of 5 mg/kg KA is injected every 30 minutes up to 3 hours maximum and until the animal displays two Racine Stage 4-5 seizures. The Racine scale (Racine, 1972) defines wet dog shakes, facial/jaw clonus and head nodding as stage 1–2, forelimb clonus as stage 3, and rearing on hind legs and loss of balance as stages 4-5 seizures. The animals are observed, and seizures recorded for a minimum of 3.5 hours. Animals are required to display at least one stage 4-5 seizure every hour for 3.5 hours to be included in experiments. After 3.5 hours of behavioral monitoring, animals are given an injection of lactated Ringer’s solution subcutaneously to prevent dehydration and aid in recovery. Animals are then placed back in the holding facility in an individual cage with free access to food and water.
Approximately 10 weeks post-SE, rats are implanted with a TR50B electroencephalographic (EEG) wireless telemetry device (Kaha Telemetry Systems) under stereotaxic guidance and anesthesia. The body of the telemeter is implanted into the peritoneal space. The EEG cable is routed from the stomach to the head underneath the skin and the exposed wire tips are placed over the right cortex and secured via dental acrylic. The head is sutured shut, and then the excess wire is coiled in the peritoneal space and the stomach is sutured shut. One to two weeks after the surgery, which is approximately 12 weeks after the induction of SE when the frequency of spontaneous seizures have been shown to have stabilized, rats are placed in the EEG suites, and an initial seizure rate is acquired over a week. The 24 rats with the highest seizure burden are selected for further studies. The seizure burden is calculated as the sum total of all seizure scores divided by the number of days tested.
In Phase I of the study (Phase II of the study is described separately), rats are injected IP/SC with vehicle or drug, one to three times a day, based on the known pharmacokinetic profile of the drug being tested (if available) or the time-to-peak effect (TPE) of the drug in previously evaluated seizure models. It is anticipated that the lamotrigine-resistant amygdala kindling model will be used as a primary guide for determination of a treatment strategy in Phase I chronic monitoring studies. After 7 days of an evaluation period to establish baseline seizure rate, 24 animals are split into two groups of 8-12 rats each and they receive either a test drug or vehicle during a first 5-day treatment period. Following a 2-day washout period, animals are crossed over to the opposing treatment arm for a second 5-day period. Animals then enter a 2-day washout period and a new baseline is obtained before a new test drug is examined for efficacy. For compounds with a long half-life (e.g., 12 hours or greater) an optional one-week washout period is added. The screening component of the study is repeated up to a maximum of four times with a cohort of animals. Higher numbers of animals are enrolled at the start of the study as final group size may vary based on the power analysis and to account for attrition due to malfunctioning/removal of EEG implant, infection, and death.
Time synchronized video and EEG data are continuously recorded (24 h/7 d/wk) and the EEG data is reviewed daily, in a blinded fashion. Data channel order is randomly scrambled and unlabeled. A list of potential detected events is automatically generated overnight by an automated seizure detection algorithm. The reviewer goes through these detected listings in a sequential order and scores any positive detected events. Data is accumulated at the end of the paradigm and analyzed via a MATLAB GUI. Seizure burden and seizure freedom are the primary outcome measures. Seizure freedom is defined as zero seizures occurring between the first dose of drug and 12 hours following the final dose.
Prior to conducting this test, a sub-chronic tolerability assessment is performed to identify possible toxicity of drug and vehicle when given multiple times a day over multiple days in rats that have experienced KA-induced SE but has a low spontaneous seizure burden and hence not included in the above study. The study is conducted in four rats/group and compounds are dosed based on the results of prior screening tests and pharmacokinetics data if available. Compounds are administered intraperitonially at the recommended dose and dose interval for three days. On the final day of treatment, rats receive a functional observation battery, i.e., modified Irwin test assessment.