Indian Journal of Experimental Biology Vol. 39, October 200 1, pp. 998- 1001

Interaction of flunarizine with sodium valproate or ethosuximide in gamahydroxybutyrate induced absence seizures in rats

Kumaresan Subramanyam, Joy David & Than gam Joseph·

Department of Pharmacology, St. John' s Medical College, Bangalore 560 034, India

Received 15 February 2000; revised 23 May 2001

Sodi um valproate(VPA) , ethosuxi mide(ESM), 200 mg/kg ip and tlunari zine (FLU) 5 or 10 mg/kg ip were first admini stered independently to rats in order to study their effects on behav ioural and EEG aspects of spike and wave di scharges (SWDs) induced by y- hydroxybutyrate (GHB,IOO mg/kg ip). GHB treated rats show behavioural changes and concomitant repetiti ve EEG episodes of 7 to 9 Hz SWDs, mimicking human absence seizures (AS), and can be used as a pharmacological model. The number and duration of SWDs were calculated for I hr from the EEG and were parameters for drug evaluation. VPA and ESM at 200 mg/kg, significantly reduced SWD number and duration/hr, while FLU showed signifi cant reducti on only at 10 but not at 5 mg/kg. Combination of FLU, 10 mg/kg with either VPA or ES M showed significant reducti on of SWD number and duration, suggesting an additive effect of the anti-absence agents with the calcium channel blocker, FLU, on experimental absence seizures in rats.

The remarkable association of behavioural and EEG man ifestations of 3 Hz spike and wave discharges (SWDs) seen in patients, is the hallmark of absence seizures (AS). y-hydroxybutyrate (GHB; 100 mgfkg, ip) in rats produced characteristic behavioural and electrical con·el ates as manifested in human AS 1• This was associated with staring, immobility and unresponsiveness to external stimuli, with characteristic 7 to 9Hz SWDs in EEG. This rat model can be employed for pharmacological profiling of drugs with potential anti-absence activity2

.3 .

Combination of sodium valproate (VPA) and flunarizine (FLU) showed a pharmacological sy nergi stic effect in pentylenetetrazole(PTZ) induced rat model of AS 2

.

The objective of thi s study has been to determine the efficacy of anti-absence drugs, VPA or ESM as well as a Ca2

+ channel antagonist, FLU, independently on SWDs induced in the GHB treated rat model of AS. Thereafter, in order to confirm results obtained in the PTZ rat model of AS2

, FLU combined with either VPA or ESM, has been examined for additive or synergi stic effects in the GHB rat model.

Materials and Methods Male Wistar rats (200-220g) were employed and

surgical procedures were done under pentobarbitone

*Correspondent author

(35 mg/kg, ip) anaesthesia. They were kept under standard housing and lighting condi tions, (0800-2000 hrs) light and (2000 - 0800 hrs) dark, and at a temperature of (22° ± 2°C). Rats were fed ad libitum with food (Hindustan Lever pellets) and water except during experimental sessions. Eight rats per vehicle/drug were used and all experiments were conducted between 0900 - 1600 hrs each day .

Procedure for electrode implantation and baseline EEG recording- The methodology for placement of cortical electrodes and EEG recording has been previously described2

· Briefly, fronto-parietal mmJature screw electrodes were chronically implanted with a reference electrode on the frontal sinus. The coordinates for left to right frontal areas were 1.5 mm posterior to bregma and 4 mm lateral to the midline on either side; left to ri ght parietal was 4.5 mm posterior to bregma and 4 mm lateral on the left and right sides. Electrodes were fixed to the skull with dental cement. One week post operatively and prior to drug studies, baseline EEG's were done in order to detect evidence of abnormal spikes or spontaneous seizure activity. Rats with abnormal EEG activity were excluded from the study. During experimental EEG recording sessions, rats were placed in a large circular Perspex chamber, (30 em diam) and continuously observed for behavioural changes.

Behavioural changes- These were monitored (while concomitant EEGs were run continuously for 60 min) to determine drug induced changes of diminished

SUB RAM ANY AM et al : INTERACTION OF VPA, ESM, FLUE & SEIZURES IN RATS 999

motor activity, sedation, hypotonia and ataxia and were scored on a mild (1 + ), moderate (2 +) and marked (3 +) scale8

. Additional signs were noted down. Quantitative analysis of SWDs in the EEG­

Quantitative analysis of the SWDs followed the method described earlier2

. In order to facilitate recognition and estimation of the number and duration of SWDs, the EEG was recorded at a speed of 25 mm/sec continuously for 60 min . The SWDs had a frequency of 7 to 9 Hz, amplitude was 50 to 200 J..l V, and occurred repetitively for 60 min, declining thereafter. The following parameters of number and duration (in mins) of SWDs/hr were easily obtained post drug from the EEG records.

Drugs- All drugs were given ip. y-Hydroxybutyrate (GHB, obtained as a solution from Sigma) was diluted with saline and used at a dose of 100 mg/kg. Two anti-absence drugs, sodium valproate (VPA, Reckitt and Colman India Ltd.) 200 mg/kg and ethosuximide (ESM, Parke Davis India Ltd.) 200 mg/kg were dissolved in distilled water. Flunarizine (FLU, Torrent Laboratories,) 5 and 10 mg/kg were dissolved in 50% polyethylene glycol and 50%

distilled water. An interval of 15 min following GHB was allowed for stabilization of the SWDs, before VPA, ESM, or FLU or VPA +FLU or ESM +FLU were administered.

Rationale for selection of drug doses - The doses selected for the study was based on the behavioural effects of various dose combination (Table 1) and only those doses/dose combinations showing minimal side effects were chosen.

Statistical analysis- The method for calculation of SWDs and statistical analysis described earlier 4

has been followed in these experiments. The ratio of number and duration of SWDs post drug I number and duration of SWDs pre drug for each animal was expressed as a percentage for the 60min period. This ensured normalization of the drug effect in each animal against its own baseline number and duration of SWDs and reduced inter-animal variability. The SWD number and duration for the entire 60 min period for each group was statistically compared using One Way ANOVA, followed by Fisher's test of least significance. Statistical significance was validated at P < 0.05.

Table 1 -Behavioural effects of VPA, ESM and FLU and combination of VPA or ESM with FLU in rats (n=6)

Drug/dose Motor activitl Sedation Hypotonia Ataxia (mg/kg, ip)

VPA 400 +++ +++ ++ +++"

300 ++ +++ ++ +++ 200 + ++ + + 100 ±

ESM 200 + +++ + + FLU 10

5

Combinations

VPA 200

+FLU 10 + ++ + + VPA 200

+FLU 5 + ++ + + ESM 200

+FLU 10 ++ +++ + + ESM 200

+FLU 5 + ++ + +

+ mild, ++ moderate, +++ marked (scores are means of all rats in a group) * loss of righting reflex in all rats "50% mortality in 24 h b dimini shed motor activity c excited, increased activity VPA = sodium valproate} ESM =ethosuximide (for Tables 1 & 2) FLU= flunarizine

1000 INDIAN 1 EXP BIOL, OCTOBER 200 1

Results Behavioural and EEG changes following GHB,

VPA,ESM, FLU and combinations of VPA or ESM with FLU- Behaviourally, GHB,100 mg/kg, produced initial excitation, piloerection and increased movement followed by quiescence. Table 1 shows the behavioural effects of VPA, ESM, FLU and combinations of VPA or ESM with FLU. Behavioural features of AS were•also abolished by both VPA and ESM and their combination with FLU.

EEG changes- GHB, 100 mg/kg produced bilaterally synchronous spike wave EEG discharges (SWDs). The SWDs had a frequency of 6 to 9Hz and the amplitude ranged from 50 to 200 flY , and were always accompanied by concomitant and characteristic behavioural signs of "absence", staring,

Table 2- Effect of YPA, ESM, FLU and the combination of YPA or ESM with FLU on number and duration of spike ­

wave discharges (SWD) [Values, expressed as percentages, are mean± SE with GHB

converted to 100% from 8 animals]

Drug SWD number SWD duration (mg I kg, ip)

VPA" 50.9 ± 7.6* 30.5 ± 8.7* 200 ESM" 34.2 ± 3.1 * 18.6 ±4.4* 200 FLU" 55.2 ± 2.8* 38.5 ± 10.3* 10 FLU" 57± 3.3 41.3 ± 8.8 5 YPA+FLUb 200 + 10 12.9 ± 1.8* 6.2 ± 1.5*

200 + 5 49.7 ± 9.7 32 ± 11.4

ESM+FLUc 200 + 10 5.6 ± 0.7* 3.2 ± 0.9* 200 + 5 3.8 ± 0.2· 5.9 ± 11.8

Significance was validated at * P<0.05 a= compared with controls; b = VPA+FLU compared with YPA alone and c = ESM+FLU compared with ESM alone Other abbreviations are same as in Table 1

immobility and unresponsiveness to light auditory or tactile stimuli. The latency (time to onset of the first SWD) was 5.2 ± 0.43 min. No significant EEG changes were observed following the anti-absence drugs or FLU.

Effect of VPA, ESM, FLU, VPA + FLU and ESM + FLU on GHB induced SWD number and duration­One Way ANOVA showed that the effects were

significantly different between the five treatment groups F = (df 5, 42 =52.34) (P< O.OS).Table 2 shows the effect of VPA and ESM, 200 mg/kg, produced significant reduction of SWD number and duration for 60 min (P < 0.05). FLU showed significant reduction of SWD number and duration at 10 but not at 5 mglkg. The combination of VPA, 200 mg/kg with FLU 10 mg/kg, produced a significant reduction of number and duration at 60 min (P < 0.05), but combined with 5 mg/kg it was not significant. Likewise, the combination of ESM and FLU at 10 mg/kg also produced a significant reduction of SWD number and duration (P < 0.05) and when combined with FLU 5 mg/kg, significant reduction of SWD number, but not duration was seen. At equivalent doses, ESM showed a greater quantitative reduction of SWD number than VPA. In combination with FLU at 5 mg/kg, ESM showed significant reduction of SWD number, but not VP A.

The results obtained in GHB rat model of AS, showed that when FLU 10 mg/kg was combined with either VPA or ESM, 200 mg/kg, significant reduction of both SWD number and duration was obtained.

Discussion This study attempts to pharmacologically validate

the y-hydroxybutyrate (GHB) induced rat model of absence seizures (AS), which shows electro-clinical analogies with human AS. The clinically useful anti­absence drugs, VPA or ESM show activity in this model, while phenytoin (PHY) and carbamazepine

Table 3- Specific mechanisms of activity of anti-absence agents and flunarizine, a Ca2+ channel blocker, on ion channels and GABA

Drug Ion channels Neurotransmitter

Na+ Ca2+(T) GABA

Sodium valproate + +J.. ++i

Ethosuximide +++J..

Flunarizine + J..++

Degree of anticonvulsant activity : + mild; ++ moderate; +++ moderate to marked. i or J.. signifies that ion channeVneurotransmitter function is increased or decreased (modified from Meldrum, 8 S, Epilepsia, 37 (1996) (Supp/6) S 4.)

SUBRAMANY AM et al : INTERACTION OF VPA, ESM, FLUE & SEIZURES IN RATS 1001

(CBZ) exacerbate symptoms of AS in rats4; a feature

which has been shown in clinical studies 1• The

objective of this study was to provide evidence to substantiate the additive effects of either VPA or ESM in GHB induced AS in rats, when simultaneously administered with a low dose of FLU, confirming similar effects previously obtained in pentylenetetrazol induced rat model of AS 2

.

The results of this study showed that GHB, 100 mg/kg produced reproducible and quantifiable SWDs, the number and duration being comparable to that obtained previously3

. When the calcium channel antagonist, FLU, 10 mg/kg was administered with the anti-absence agents, VPA or ESM at 200 mg/kg, the combinations produced a greater reduction of SWD number and duration than that obtained with either VPA or ESM per se, suggesting the possibility of a positive additive/ or synergistic effect.

The mechanism of action of VP A has been attributed to its voltage dependent blockade of Na+ channels (like PHY and CBZ) as well as by enhancing GABA mediated inhibition 5

. VPA also reduces the T type Ca2+ current, especially blocking low frequency (3Hz) stimulation of thalamic neurones, representing a third distinct mechanism of action6

•7

. ESM exerts its anticonvulsant action by specifically blocking T and L type Ca2+ channels in thalamic neurons6

, but does not influence the Na+ channel or GABA mediated inhibition. FLU is a Ca2+ antagonist possessing distinct anti-electroshock and anti-pentylenetetrazol activity in mice and rats8 and exerts its anticonvulsant action by specifically blocking T and L type Ca2+ channels in thalamic neurons9

. FLU has an analogous mechanism of action like VPA on Na+ channels, but does not influence gabergic inhibition9

. It is clear that many putative mechanisms of pathological neuronal excitability are calcium dependent 10

. FLU may be additive to both VPA and ESM in counteracting these calcium dependent pathways. The accumulated mechanisms of action of the various drugs employed are shown in Table 3.

The SWD patterns observed in EEG which are associated with typical behavioural features of AS in animals and humans, are generated by the rhythmic oscillation of thalamocortical neuronal circuits in the brain . The T type channel and associated low voltage calcium currents, (which generate SWD rhythms) are selectively blocked by ESM resulting in arrest of SWD rhythms 11

• As FLU also blocks T type calcium channels, it is tempting to speculate that the combination of ESM (or VPA) with FLU may exert

their anticonvulsant act1v1ty at a common target of action, resulting in a synergistic effect at Ca2+ channels and/or Na+ channels in the brains of rats pretreated with GHB. However, a viable pharma­cological explanation for the enhanced effect obtained in this study with the combination of anti-absence agents and calcium channel blockers must await further elucidation.

In conclusion, the results obtained in this study further confirm the synergistic effect of either VPA or ESM in combination with FLU in another experimental model and sustains previous evidence that FLU may be a valuable additive in pharmaco­resistant epilepsy.

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