Experimental design

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 [16]) were treated with 30 mg/kg imepitoin twice daily for 8 weeks.

Medication

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.

Clinical trial

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 [16]). 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).

Statistics

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).

Imepitoin was well tolerated by healthy cats

No serious adverse events were observed in any of the dose groups. Behavioural changes or sedation were not noted during the course of the study. Repeated oral administration of imepitoin to clinically healthy male and female cats at doses of 30, 40 and 80 mg imepitoin/kg body weight twice daily for 30 days, led to occasional emesis after at least 1 week of treatment. Emesis was intermittently observed until test day 22 of treatment, possibly indicating a transient effect. However, as emesis was more commonly observed in verum groups, it is considered test-item related. Salivation was the most frequent finding in the 40 and 80 mg/kg groups, while it was observed in only one placebo cat.

At the highest doses (40 and 80 mg/kg), a slight reduction in food consumption was observed, especially at the beginning of the study, with subsequently slightly lower body weights compared to controls. No effect on body weight or food consumption was observed in the placebo and 30 mg/kg group.

Urine analysis revealed no clinically evident effects of the treatment. Haematology and clinical chemistry revealed no relevant changes. No changes in liver enzymes or other indicators of hepatic malfunction were observed.

Pharmacokinetics of imepitoin in cats is similar to dogs

Pharmacokinetics was evaluated in the 30 mg/kg experiment, and plasma samples were taken at 0, 0.5, 1, 3, 6 and 24 h post first treatment (second treatment 8-12 h after first treatment). Therefore, no sampling points were taken at the absorption and distribution phase of the second dose and the pharmacokinetic evaluation was based on the plasma concentration data of the first absorption/distribution phase (0, 0.5, 1, 3, 6 h post dose) and the 24 h value (end of the second absorption/distribution phase). No relevant gender effect was observed, and accordingly male and female data (n = 3 per gender) were combined for the pharmacokinetic analysis.

After 14 and 29 days of dosing a decrease in Cmax (about 50%) and an about 60% reduction in exposure (AUC) compared with day 1 data was observed (Table 1; Fig. 1). The observed vomitus may have led to the fact that individual animals had not received the full target dose on days 14 and 29 which consequently resulted in a lower systemic exposure and lower Cmax values and may have been the reason for the observed differences.

	Day
	1			14			29
	Cmax	AUClast	Tmax	Cmax	AUClast	Tmax	Cmax	AUClast	Tmax
Dose 30 mg/kg	9990	39,640	3.0	2940	11,720	1.0	1590	6932	3.0
	5180	35,870	1.0	1840	7615	1.0	5600	47,560	1.0
	5980	46,780	3.0	1910	9726	0.5	934	4242	0.5
	7220	35,030	3.0	4380	9883	1.0	1400	4115	0.5
	6830	47,970	1.0	6540	22,620	1.0	6300	26,190	1.0
	5880	35,790	3.0	3240	15,850	1.0	7500	20,800	1.0
N	6	6	6	6	6	6	6	6	6
Mean	6850	40,200	2.3	3480	12,900	0.92	3890	18,300	1.2
SD	1700	5810	1.0	1770	5510	0.20	2900	17,000	0.93

Safety under field conditions

A total of 8 cats with newly diagnosed epilepsy with no known underlying cause were recruited and received at least a single dose of the appropriate drug treatment. One cat was lost to follow-up, as neither the owner nor the referring veterinarian provided further information.

Represented breeds were Domestic Shorthair (DSH, n = 2) and European Shorthair (ESH, n = 6). Mean age was 6.3 years (SD 5.8; Median 4; Range 1 to 15). Half of the cats were female (n = 4), and all cats were neutered (n = 8). Mean body weight at inclusion was 4.5 kg (SD 0.76 Range 3.0–5.0 kg). The mean initial dose was 27.92 mg/kg (SD 5.41; Median 30 mg/kg) twice daily. Four cats had concomitant diseases, and one of these cats had multiple diseases (hyperthyroidism, fibrosarcoma, toothache and developed feline infectious peritonitis after the observation period). In the other cats the diseases were not considered to likely cause neurological signs.

At least one adverse event was observed in five of the seven evaluable cats. Most adverse events reported were mild and occurred transiently or intermittently. Lethargy was observed in two cases, which resolved completely by lowering the dose to 20 mg/kg twice daily and keeping the dose at this level. Two cats showed emesis intermittently, and decreased appetite was reported two times. In addition, ataxia, polydipsia, increased appetite, increased salivation, decreased appetite and decreased drinking were reported one time each.

Efficacy under field conditions

In total, 4 out of 8 cats achieved seizure freedom at the end of the study, while 1/8 experienced a partial therapeutic success, and 2/8 continued seizuring without therapeutic success. One cat was lost to follow-up, and accordingly 1/8 was considered undetermined and accordingly treatment failure (Fig. 2).

The monthly seizure frequency at baseline was 57.71 ± 73.77 seizures/month (mean ± SD; median 20; range 2 to 200), which reduced significantly under treatment to 19.43 ± 37.15 seizures/month (median 1.5; range 0 to 100; p = 0.0313 in Wilcoxon matched-pairs signed rank test compared to baseline). The response ratio was −68.42 ± 38.52 (mean ± SD). Out of the four cats which achieved seizure freedom at the end of the 8 week observation period, the mean reported time of seizure freedom until they were lost to follow up in the extended follow-up period was 16.4 weeks (SD 11.4; range 8.0–30.4 weeks). In none of these cats further seizures were reported until lost to follow-up.

One cat had 3 seizures during the first 4 weeks of treatment, and had no further seizures for the next 5 weeks. As it was lost to follow-up then and did not meet 8 weeks of seizure freedom, it was classified partial success.

In the two cats that continued seizuring without therapeutic success, one cat had continuous seizure activity during the first 2 weeks of imepitoin treatment, so phenobarbital (1 mg/kg twice daily, serum level 34.5 μmol/ml) was added to imepitoin in the third week after inclusion. Seizure frequency decreased during the following weeks, and from week 7 after inclusion onwards, no further seizures were observed. Seizure freedom remained for 4 weeks. Afterwards no information was available.

Following an initial improvement in the first week, the other cat experienced highly frequent focal seizures in the second week of imepitoin treatment. Subsequently, the owners wished to change treatment, and levetiracetam (20 mg/kg 3 times daily) was added to imepitoin treatment. A slight improvement in seizure frequency was observed, however even with increasing the dose of levetiracetam (to 30 mg/kg 3 times daily), sufficient seizure control was not achieved. In week six after inclusion, imepitoin was gradually replaced by phenobarbital. After switching to phenobarbital and levetiracetam, no information on the further course of disease was available.