Efficacy of zonisamide on interictal electroencephalography in familial spontaneous epileptic cats

Abstract

Objectives

The effectiveness of zonisamide (ZNS) against spontaneous epilepsy in cats has not yet been described. The purpose of this study was to investigate the effect of ZNS on interictal paroxysmal discharges (PDs) using scalp electroencephalography (EEG) in familial spontaneous epileptic cats (FSECs).

Methods

Eight FSECs were evaluated (six males and two females). Scalp EEG measurements were performed once a week for 3 weeks before ZNS administration (Pre-ZNS). Thereafter, administration of ZNS was started and an adjustment period was instituted until the drug in plasma achieved the steady state. When ZNS in plasma was confirmed to be within 10–40 μg/ml, scalp EEG measurements were performed once a week for 3 weeks (Post-ZNS). The number of PDs (counts/min) were compared between Pre-ZNS and Post-ZNS treatment.

Results

The median number of PDs for Pre-ZNS and Post-ZNS were 0.43/min (0.13–0.82/min) and 0.28/min (0.07–0.87/min), respectively. The number of PDs Post-ZNS was significantly reduced compared with Pre-ZNS (P = 0.02).

Conclusions and relevance

This study showed that ZNS, within the recommended therapeutic range suggested for use in humans and dogs (10–40 µg/ml), reduced the number of PDs recorded on EEG in FSECs that are considered a model for cats with idiopathic epilepsy. Although phenobarbital is the antiepileptic drug of choice for epileptic cats, the results of this research provide evidence to support the use of ZNS in cats with phenobarbital-resistant epilepsy or for cats that cannot use phenobarbital due to adverse side effects.

Introduction

Epilepsy is the most common functional brain disorder in dogs and cats. Its treatment is seizure management with antiepileptic drugs (AEDs). Recently, the International Veterinary Epilepsy Task Force (IVETF) and the American College of Veterinary Internal Medicine (ACVIM) recommended phenobarbital (PB), potassium bromide, imepitoin, levetiracetam and zonisamide (ZNS) for the treatment of epileptic dogs.^1,2 On the other hand, consensus on therapy for cats with epilepsy has not been reported, though PB has been recognised as the superior AED and the first line of therapy for epileptic cats.^3–6 Besides PB, several AEDs such as benzodiazepines, levetiracetam and gabapentin have also been used for the management of seizures in cats.^1,5,7

ZNS was developed in Japan in the 1980s, but its use as an AED in veterinary medicine is relatively new in Western countries. Its effectiveness has been reported in dogs with epilepsy^8–13 and, in Japan, it is frequently used like PB. ¹⁴ However, the effectiveness of ZNS in cats with spontaneous epilepsy has not been reported. Only one study has reported ZNS pharmacokinetics for cats ¹⁵ and there is one description of clinical experience in epileptic cats. ⁶

Familial spontaneous epileptic cats (FSECs) belong to a feline family line that was discovered in 2009 and maintained at Nippon Veterinary and Life Science University. ¹⁶ FSECs are considered a natural model of feline idiopathic epilepsy (suspected genetic epilepsy). FSECs have two seizure types: recurrent spontaneous limbic seizures (with or without generalisation) and vestibular stimulation-induced generalised seizures. Electroencephalography (EEG) and/or imaging studies of FSECs suggest that the epileptogenic zone lies in the mesial temporal lobe (amygdala and/or hippocampus), which is represented by interictal paroxysmal discharges (PDs) in the frontal-temporal regions on scalp EEG.^16–20

The purpose of this study was to investigate the effect of ZNS on interictal PDs using scalp EEG in FSECs. Our hypothesis is that the therapeutic concentration of ZNS suggested for use in epileptic humans and dogs (10–40 µg/ml), which has been confirmed as safe for use in cats, significantly decreases PDs in FSECs and thus will validate its use in cats with epilepsy.

Materials and methods

Ethics

This study was conducted with the approval of the Experimental Animals and Bioethics Committee of Nippon Veterinary and Life Science University (approval number: 27S-34, research leader: DH).

Animals

Nine FSECs were included in the study (six males and three females). We confirmed that all the included animals had two or more spontaneous epileptic seizures (tonic–clonic generalised seizures) prior to this study. The median body weight was 3.8 kg (2.8–5.2 kg) and the median age was 105 months (81–150 months). General physical and neurological examinations, complete blood count (CBC), serum biochemical analysis, urinalysis, visual evaluation of structural MRI (except minor hippocampal atrophy) and cerebrospinal fluid analysis in the interictal state were within reference intervals.

Study protocol

A summary of the study protocol is shown in Figure 1. Scalp EEG measurement was performed once a week for 3 weeks (for a total of three times) before ZNS administration (Pre-ZNS). To confirm a 0 μg/ml plasma concentration of ZNS, a blood sample was collected at the time of the third EEG measurement of Pre-ZNS. Thereafter, administration of ZNS (Consave; DS Pharma Animal Health) was initiated at 2.5 mg/kg q12h PO and an adjustment period of at least 1 week was instituted until the steady state was achieved. After the adjustment period, the plasma concentration of ZNS was confirmed to be within 10–40 µg/ml. The dose of ZNS in cats with plasma ZNS concentrations below 10 μg/ml was increased and re-measured after a re-adjustment period to confirm it reached the therapeutic range. All cats were administered the adjusted dose until the end of the study. After ZNS reached the plasma therapeutic concentration, scalp EEG measurement was performed once a week for 3 weeks (for a total of three times) (Post-ZNS). Body weight, physiological and neurological examinations, CBC, biochemical examination and urinalysis were acquired before administration of ZNS, after the adjustment period and at the end of the experiment, respectively. In addition, all the subject animals included in this study were under continuous video monitoring to track the development of seizures and behaviour.

Figure 1.

Summary of the study protocol. Electroencephalogram (EEG) recordings were performed three times in both the Pre-ZNS and Post-ZNS phase. ▼: EEG recording point. Solid line shows the extrapolated concentration of ZNS

Blood samples for ZNS concentration measurement were collected from the jugular vein within 12–14 h of the latest administration. ZNS concentration was measured by high-performance liquid chromatography (Marupi Lifetech).

Electroencephalogram measurement

EEG measurement was performed under sedation using medetomidine (Domitol: Nippon Pharmaceutical Industry). All cats were fasted for 12 h before EEG measurement and then sedated by intramuscular administration of 30 µg/kg of medetomidine. ²¹ If the sedation was insufficient 10 mins after the initial administration, 10 µg/kg were additionally administered. During EEG measurements, body temperature (rectal) was maintained between 37–38°C using hot water bags.

EEG measurement was performed using a digital electroencephalograph (Neurofax EEG–1200: Nihon Kohden) with synchronised video recording for the detection of motion artefacts. The recording electrodes were placed on both sides symmetrically in the frontal (F3, F4), parietal (C3, C4), temporal (T3, T4) and occipital (O1, O2) regions and three midline regions longitudinally (Fz, Cz, Pz) (Figure 2).^22–24 The referential electrode was placed on the nose tip and the ground electrode was placed on the back of the cranial neck (approximately the level of the spinous process of the axis). In addition, electrocardiographic electrodes were located in the dorsal chest and left axillary regions for electrocardiogram (ECG) recording. All electrodes were subdermal stainless wire electrodes. The thin (0.2 mm in diameter) and short-cut (approximately 3.0 cm) stainless wire (Unique Medical) was inserted into the skin with a 23 G needle as an applicator. The needle was then removed and the remaining wire was clipped with an alligator-clip electrode (Nihon Kohden). Real-time EEG monitoring montages were the average reference derivation (AV derivation) for each electrode (Figure 3). Other recording/monitoring conditions included: sampling frequency = 1000 Hz; high frequency filter = 60 Hz; time constant = 0.1; sensitivity = 5–10 μV/mm; AC cut-off notch filter = ON; and tracing speed = 10 s/view. The median recording time was 38 mins (26–50 mins).

Figure 2.

Electrode positioning. The frontal (F3, F4), central (C3, C4), temporal (T3, T4) and occipital (O1, O2) pairs are positioned symmetrically. Three longitudinal electrodes (Fz, Cz, Pz) are positioned on the midline and centre of F, C and O, respectively. Referential electrodes (R) are positioned on the tip of the nose and the ground electrode (G) is on approximately C2 spine

Figure 3.

Screenshot of an electroencephalogram (EEG) recording in a familial spontaneous epileptic cat (Pre-ZNS period). Each EEG trace shows referential derivation using average potential reference (AV). A spike was identified at F4 and T4 dominantly and comparatively small sharp waves were also observed on T3 or C3/Cz. The small window shows synchronised recorded video to check facial movement. Channel 12 (X1-X2) traced electrocardiogram

EEG reading

A veterinarian (YH) with more than 2 years of EEG reading experience visually assessed the recorded digital EEG data in a blinded manner (for individual information and timing of the measurement). During EEG reading, the high frequency filter, time constant, sensitivity, tracing speed and montage could be freely adjusted according to the need of the reader. The reader counted the number of PDs in a stable recorded 10 min period except for the first 5 mins at the start and at the end of the recording. PDs included spikes, polyspikes, sharp waves and spike-and-waves. When a waveform considered as a PD was observed, the reader confirmed that it was not an artefact from EEG, ECG and video recording. Finally, for each recording of each individual, the average number of PDs per minute was calculated (PDs count/min) and used for statistical analysis.

Statistical analysis

The Wilcoxon-signed rank sum test was used for statistical analysis. P <0.05 values were considered statistically significant.

Results

One cat did not show clear PDs in any of the three Pre-ZNS EEG readings, and thus was excluded from the study. Eventually, eight cats (six males and two females) and a total of 24 EEG recordings Pre-ZNS (three times in eight cats) and Post-ZNS (three times in eight cats) were evaluated.

We confirmed that the plasma concentration of ZNS was 0 μg/ml before ZNS administration. The median ZNS concentration 1 week after starting the initial dose (2.5 mg/kg q12h) of ZNS was 5.9 (2.1–8.3) μg/ml. In all cases, therefore, dosage was increased to 5 mg/kg q12h and after the re-adjustment period, the median ZNS concentration reached 13.45 (11.9–24.4) μg/ml. There was no significant change in body weight and there were no abnormalities at physical and neurological examinations in CBC, serum biochemistry and urinalysis before and after adjustment of ZNS and at the end of the study (data not shown). Additionally, clinical side effects such as anorexia, vomiting and diarrhoea were not observed during the study.

Recorded PDs were dominant spikes or sharp waves in the frontal and temporal region, although some variations were observed in the left/right or part of head depending on the individual (Figure 3). Detailed results are shown in Table 1. The median number of PDs for Pre-ZNS and Post-ZNS were 0.43/min (0.13–0.82/min) and 0.28/min (0.07–0.87), respectively. PD numbers in Post-ZNS were significantly reduced compared with Pre-ZNS (P = 0.02).

Table 1.

Concentrations of zonisamide (ZNS) and paroxysmal discharge (PD) numbers Pre- and Post-ZNS

	ZNS concentration (µg/ml)	Pre-ZNS (PDs/min)	Post-ZNS (PDs/min)
FSEC1	16.5	0.444	0.211
FSEC2	11.9	0.586	0.469
FSEC3	13.8	0.611	0.600
FSEC4	12.8	0.229	0.156
FSEC5	24.4	0.495	0.311
FSEC6	13.0	0.589	0.378
FSEC7	13.1	0.266	0.192
FSEC8	18.9	0.356	0.200
Mean	15.6	0.447	0.315
Median	13.5	0.433	0.283
Maximum	24.4	0.824	0.867
Minimum	11.9	0.133	0.067
SD	4.0	0.202	0.196

ZNS concentration is indicated at a dosage of 5 mg/kg q12h. PD numbers Pre- and Post-ZNS for individuals (FSECs 1–8) are expressed as the mean of three measurements

FSEC = familial spontaneous epileptic cat

Discussion

Here, we showed that the ZNS plasma concentration within the therapeutic range suggested for use in epileptic humans and dogs (10–40 µg/ml) causes a significant PD reduction on interictal scalp EEG in FSECs that are considered a model of feline idiopathic epilepsy. Although we did not aim to measure the therapeutic effects of ZNS (ie, suppression of epileptic seizures) and epilepsy treatment does not aim to improve EEG findings, the result of this study may provide the scientific basis to support the use of ZNS for the treatment of spontaneous epilepsy in cats.

Although the mechanism of action of ZNS has not yet been fully elucidated, ZNS appears to block the propagation of seizure activities from the cortex to subcortical areas of the brain. In humans and dogs, three action mechanisms have mainly been described. ²⁵ The first involves the inhibition of voltage-dependent sodium and T-type calcium channels related to excessive excitation of neurons. The second is a neuroprotective effect caused by a free radical scavenging action. The third involves the up-regulation of excitatory amino acid transport carrier-1 and down-regulation of gamma-aminobutryric acid transport carrier-1 and the correction of the imbalance of excitability and inhibition related to indirect suppression of ryanodine receptor and suppression of glutamate release in nerve cells with abnormal excitation. ²⁶ Although ZNS is currently positioned as the second line AED for epileptic dogs in veterinary medicine, it is reported that its effectiveness as monotherapy for idiopathic epilepsy in dogs is approximately 60–75%.^11,14

The therapeutic use of ZNS has not been reported in cats except for a pharmacokinetic study ¹⁵ and clinical experience in a review article. ⁶ According to Dewey, ⁶ the use of ZNS as an add-on drug to PB has been reported in two cats diagnosed with idiopathic epilepsy. One cat had decreased appetite and treatment had to be discontinued; however, the other cat experienced a substantial reduction in seizure frequency and had no side effects or blood work abnormalities attributable to ZNS therapy after approximately 1 year of treatment. However, this article provided no description of dosage and blood concentration of ZNS.

According to Hasegawa et al, ¹⁵ the maximum ZNS (at 10 mg/kg/day) plasma concentration, time to maximum plasma concentration and plasma apparent elimination half-life (t1/2) were 13.1 μg/ml, 4 h and 33 h, respectively. It was also reported that half of the animals presented side effects when the dose was 20 mg/kg/day and/or the plasma concentration more than 40 μg/ml. In general, during repeated administration, the time to reach a steady-state concentration is more than three to five times its t1/2 value. Taking the t1/2 of ZNS in cats into consideration, the plasma ZNS concentration was estimated to reach the steady state in 7 days. Therefore, in this study the plasma concentration was measured after 1 week or more of administration.

Although in some textbooks^13,27,28 ZNS administration for cats was described to be once a day, in this study we carried out administration every 12 h to reduce the risk of the peak concentration of a single administration exceeding the safety zone (40 μg/ml) and diurnal variation of the blood concentration depending on the ZNS dose. In addition, because some textbooks^13,27,28 indicate 5–10 mg/kg q24h for cats and because of our limited clinical experience of the use of ZNS, we started with a 2.5 mg/kg dose q12h (= 5 mg/kg/day). However, since the plasma concentration did not reach 10 µg/ml, we elevated the dose to 5.0 mg/kg q12h. Although 5.0 mg/kg q12h (= 10 mg/kg/day) may be a comparatively high dose for cats, there were no side effects. For these reasons, we recommend ZNS for cats to be administered q12h.

In the present study, suppression of PDs with no obvious clinical side effects was observed at ZNS doses of 5.0 mg/kg q12h and plasma concentrations from 11.9–24.4 μg/ml. These findings suggest that the therapeutic concentration of ZNS suggested for use in epileptic humans and dogs (10–40 µg/ml) can also be adapted to cats. It is impossible to clarify the lower limit of the therapeutic range for cats from the results of the present study, but taking into account the results of a previous toxicity study, ¹⁵ 10–40 μg/ml of ZNS may be a reasonable range for cats. In this study, because the minimum concentration was set at 10 μg/ml, EEG evaluation at 2.5 mg/kg q12h (median concentration was 5.9 μg/ml) was not performed. If evaluation of the EEG was performed at this point, it would have been possible to assess its effect on PD at lower doses.

One of the limitations of our study was that we excluded one cat that had no PD in Pre-ZNS. This might introduce selection bias and lead to our positive results. In general, although most epileptic patients have PDs, there are a certain number of individuals that do not display any, especially during interictal and/or scalp EEG. In other words, it is important to know that interictal PDs are not always detectable.

The main limitation of this study is that the efficacy of ZNS in controlling epileptic seizures was not evaluated. The epilepsy of the FSECs included in this study was relatively mild and the seizure frequency was low, although there were individual differences. In addition, although we had previously confirmed spontaneous seizures in the individuals included in this study, it was difficult to target the frequency of spontaneous seizures because of the limited study period and the number of individuals that could be included. Indeed, none of the animals presented clinical seizures during the study period. However, although spontaneous seizures in FSECs are low in frequency, they can be induced by vestibular stimulation. Therefore, in the future it may be possible to investigate the effectiveness of ZNS and/or other AEDs for epileptic seizures induced by the vestibular stimulation.

Conclusions

This study showed that the therapeutic range of ZNS suggested for use in epileptic humans and dogs (10–40 µg/ml) reduced the number of PDs on EEG in FSECs that are considered a model to study idiopathic epilepsy in cats. Although PB is the drug of choice for epileptic cats, the results of this research provide evidence to support the use of ZNS in cats with PB-resistant epilepsy or in cats that cannot use PB due to side effects.