Three independent trials were conducted. Two tolerability studies were performed in healthy laboratory cats and one efficacy study was conducted in epileptic cats. In one of the tolerability studies pharmacokinetics were also evaluated. All three studies utilized different doses of imepitoin.
Study 1: In the first randomized, controlled, blinded GLP (good laboratory practice) laboratory study, six cats (3 male, 3 female) received orally 30 mg/kg imepitoin twice daily over 30 days. Six untreated cats (3 male, 3 female) served as controls.
Study 2: The aim of the second, randomized, controlled, blinded GLP laboratory study was to examine safety in higher doses of 40 mg/kg and 80 mg/kg twice daily, while placebo-treated animals served as control. Each of the three parallel groups consisted of six cats (3 male, 3 female), and the treatment duration was 30 days.
Study 3: To evaluate tolerability under field conditions in epileptic cats, a small, single-arm, open label, uncontrolled clinical pilot trial was performed in two centers. In addition, efficacy parameters were assessed. Eight cats suffering from idiopathic epilepsy (TIER II confidence level ) were treated with 30 mg/kg imepitoin twice daily for 8 weeks.
Imepitoin (Pexion®, Boehringer Ingelheim, Ingelheim, Germany) was used in 100 mg or 400 mg tablets, each divisible in two equal parts. For placebo, visually identical tablets without active ingredient were used. In the laboratory studies, medication was provided twice daily, approximately 12 h apart, using a tablet applicator (BUSTER; Jergen Kruuse A/S, Langeskov, Denmark). In the clinical trial, owners provided tablets twice daily in the morning and in the evening.
Laboratory trials in healthy cats
Intact domestic short hair cats (Liberty Research Inc., Waverly, NY, USA), aged one to 3 years, were kept under a 12 h light/dark cycle at room temperature (15 °C – 26 °C). Drinking water was offered ad libitum, and food once daily. Food and water consumption was monitored once daily during the study period and at baseline. Environmental enrichment (toys, shelves, etc.) was provided to all cats. In the first study (30 mg/kg), cats were housed in groups (3 cats of same gender), while in the second study (40/80 mg/kg) cats were kept individually for the study period.
Cats were observed daily for occurrence of adverse events and general wellbeing, and a physical examination was performed before treatment start, at day 7 (only in 40/80 mg/kg study), day 14 and day 30. This included a general check of all body systems; body temperature measurement, behavior and nervous system evaluation, cardiovascular and respiratory assessment, and visual examination of the eyes. Body weight was measured once a week.
Blood samples were taken for hematology and clinical chemistry 1 day before first dosing, at day 15 in study 1 (30 mg/kg study) and in all cats at day 30. An automated complete blood count, including differential blood count (ADVIA hematology system, Siemens Healthcare Diagnostics, Eschborn, Germany) and coagulation assessment was performed. Clinical chemistry included a comprehensive metabolic panel (study 1: HECKMAN Synchron CX7; Heckman Coulter, Inc., Fullerton, USA or study 2: KONELAB 30i, Thermo Fisher Scientific, Dreieich, Germany) and additionally cholesterol, triglycerides, phosphate and glutamate dehydrogenase (GLDH). Urine was analyzed with test strips (study 1: Urispec® 9+ Leuko Plus; Henry Schein Inc., Melville, USA or study 2: Combur 9® test, Roche Diagnostics, Mannheim, Germany) and microscopically 1 day before dosing, at day 15 (only study 1) and at day 30. In the 30 mg/kg study, pharmacokinetics were assessed at day 1, 15 and 29 collecting blood 0 h (i.e. prior to treatment) and 30 min, 1, 3, 6 and 24 h after the first treatment. To obtain at least 100 μL K3EDTA-plasma per animal and sampling time, sufficient whole blood was collected from the vena cephalica, saphena or jugularis. Pharmacokinetic parameters were calculated with Phoenix WinNonlin (Version 6.4; Pharsight/Certara, St. Louis MO 63101; USA) applying a noncompartmental analysis. To validate an adequate exposure to the test substance, in the 40 and 80 mg/kg study, blood was collected at day 1 and 28 at timepoints 0, 1, 3 and 24 h after first dosing.
The study was conducted as a multicenter clinical field trial, observing cats with newly diagnosed idiopathic epilepsy for 8 weeks of treatment with 30 mg/kg imepitoin twice daily. If cats stayed on imepitoin monotherapy after this 8 weeks observation period, investigators attempted to follow the cat for as long as possible.
Privately owned cats with epilepsy were included, if they had a history of at least two generalized seizures or focal seizures in the last 2 weeks prior to inclusion, with at least two of the following signs: drooling, facial twitching, tremor, rapid running, mydriasis, hypersalivation, disorientation, or impaired consciousness. Furthermore, the minimum age had to be 9 months and a signed Owner-Informed-Consent had to be obtained. All cats had an MRI scan to rule out structural intracranial lesions as cause for seizures (TIER II ). Owners were asked to describe the seizures of their cat, and the investigator graded them as focal or generalized.
A cat was excluded, if it had been on oral antiepileptic treatment for more than 3 consecutive days within the last 6 months prior to inclusion, was treated with antiepileptic drugs within the last 24 h prior to inclusion, was known or suspected to be pregnant or lactating, had a known or suspected concomitant disease that may be accompanied by or result in neurological symptoms (e.g. renal or liver failure, diabetes mellitus) which might interfere with interpretation of the study results, or had a life-threatening disease which may prevent completion of the study (e.g. congestive heart failure).
At the end of the study, the investigator conducted a clinical examination to determine the general health status of the cat and asked the owners for their observations. In addition, owners were asked to note observations of possible adverse events in the epilepsy seizure diary or to contact the investigator immediately, if adverse events occurred. All adverse events were recorded and classified according to the Veterinary Dictionary for Drug Related Affairs (VeDDRA).
Clinical efficacy assessment
During the study, owners were asked to observe their cat for occurrence of seizures and to keep a record of the occurrence of seizures (both focal and generalized) in a patient diary. After 8 weeks, the investigator reviewed the patient diary with the owner and recorded the total number of seizures occurring during the study period, the time to first seizure and the characteristics of the seizures.
The primary measure of efficacy was the achievement of seizure freedom . Animals with at least 8 subsequent weeks of seizure freedom and no reported seizure afterwards were counted as treatment success, at least 50% reduction in seizure frequency as partial success and no sufficient improvement or lost to follow-up as treatment failure. Seizures per individual treatment week were recorded for each patient and plotted over time, modifying a proposal for human epilepsy trials . In addition, monthly seizure frequency (MSF) before and during treatment was calculated by dividing the number of seizures by the number of weeks under observation and multiplying the result by 4. The response ratio, defined as the ratio [(T − B)/(T + B)] × 100 where B = Baseline Seizure Rate and T = Treatment Seizure Rate, was used to obtain a ‘symmetrized’ percent change of seizure activity . For all calculations, generalized and partial seizures were taken into account.
According to the nature of the data and the purpose of the study, we applied appropriate descriptive statistics. Data from the clinical trial were analyzed by two-sided Wilcoxon matched-pairs signed rank test using GraphPad Prism 6.05 (GraphPad Software, Inc., La Jolla, CA, USA; conventionally p < 0.05 was considered significant).