Indian Journal of Experimental Biology VoL 37, March 1999, pp. 228-233

Effect of sodium valproate and flunarizine administered alone and in combination on pentylenetetrazole model of absence seizures in rat

Prakash Shantilal, Joy David & Thangarn _ oseph*

Department of Pharmacology, St. John 's Medical Co llege, Bangalore 560 034,1ndia

Received l3 July 1998 : revised 4 January 1999

Sodium valproate (VPA) and flunarizine (FLU) administered individually and together were examined for their etlects on behavioural. and EEG changes in the pentylenetetrazole (PTZ) induced rat model of absence seizures. PTZ, 20 mglkg, ip, produced behavioural staring and immobility with concomitant, repetitive 7 to 9 l-Iz spike/wave discharges (SWDs) in CEG, monitored continuously for I hr and thereafter, intennittently for 4 hr, post-vehicle/drug. The number and duration (sec) of SWDslhr were the parameters used for evaluation of vehicle vs. drug effects in normal as well as rats made ep il eptogenic by repeated corti cal stimulation. VPA. 200 mg/kg, ip, produced a significant reduction in the number and durati n of SWDs at 20 min only in epileptogenic rats, declining to non-significant levels at 60 min, whereas FLU, 10 mglkg ip had no efrect on either parameter. The combination of \lPA and FLU produced a highly significant reduction of the number and durat ion of SWDsih for 60 min in nomlal and epileptogenic rats. The results provide evidence for a synergistic effect of VPA and FLU in experimental absence seizures and possible potential benefit in phannaco resistant seizures.

Absence seizures in humans are fundamentally different from any other kind of epi leptic manifestations. Class ical absence seizures (AS) which start and end abruptly and occur predominantly III ch i Idren, are characterised by temporary interruption of ongoing activity, star ing and unresponsiveness to external stimuli . Concurrently, EEG shows a generalised and bilaterally synchronous 3Hz spike/wave discharge(SWD), indicat ing that the EEG change is time locked to the behavioural absence!. An useful pharmacological an imal mode l displaying the characteristic behavioural and electrical correlates of AS, is induced by the administration' of non-convul sive low doses of pentylenetetrazole (PTZ), 20 mglkg, ip. to rats2.3. This model is also pharmacologically unique as SWDs are inhibited by sodium valproate (VPA) and ethosuximide (ESM) and exacerbated by phenytoin (PHT) and carbamazepine (CBZ) as seen clinicail/ .

In previous studies, we have shown that a low dose of t1unarizine (FLU), a Ca2

+ channel an tagonist, enhanced the anticonvu lsant effect of an ED50 dose of VPA to 100% in the maximal electroshock s~: izure

test as well as in the PTZ seizure test in mice4• We

also demonstrated that the combination of VPA and FLU showed a pharmaco logical synergistic effect in cortical seizure threshold in rats, which is a model of

' Correspondent author

clinical generalized tonic clonic se izures5,6 OLlr

findings suggest that low doses of FLU combi ned with VPA showed additive effects in experimenta l anticonvu lsant tests. Other workers have also shown th at FLU combined with phenytoin and carba­mazepine had an anticonvulsant effect in the PTZ seizure incidence/ latency test in mice7.Hence,in view of the anticonvulsant efficacy and low toxicity of FLU in animal as well as clinical studiesS

, the present study was designed to determine the interaction between FLU and VPA, beneficial or oth erwise, on behavioural and electrical correlates on PTZ induced absence se izures in normal and ep i leptogen ic rats.

Materials and Methods

Animals-Nlale Wistar rats 200 to 220g (n=8 per drug dose) were used for all the experiments and surgical procedures were carried Ollt under pentobarbitone anaesthesia, 30 mg/kg.

Drug Trealmenl--AII drugs were injected intraperitonea lly. Pentylenetetrazole, (PTZ, Sigma Laboratories), 20 mglkg was disso lved in sa line and Flunarizine (FLU, Torrent Pharmaceuticals), 10 mglkg was dissolved in 50% polyethylene glyco l and 50% distilled water. Sodium va lproate (VPA, Reckitt &.

Colman, India) 200 mglkg, was dissolved ill distilled water. Drug were admini stered 10 min after the challenge dose of PTZ. FLU 10 mg/kg and VPA 200

SHANTILAL et aL SODIUM V ALPROTATE & FLUNARIZINE IN ABSENCE SEIZURES IN RATS 229

mglkg (hereinafter referred to as the 'combination'), was injected independently in the right and left lower abdominal regions. In view of the long half life of FLU9 the combination was given after a wash out period of 18 days. The same rats were used for control and dr.ug studies and controls received the corres­ponding vehicles.

Normal rats--Rats were chronically implanted with four extradural franto-parietal electrodes and one electrode on the frontal sinus serv ing as reference5

.

Baseline EEG recordings for 20 min, were done in conscious rats, one week after surgery in order to detect EEG evidence of abnormal spikes, sharp waves or spontaneous se izure activity. Rats with abnormal EEG activity were excluded from the study. During EEG recording sess ions, the rats were placed in a large circular perspex chamber of diameter 30 cm and continuously watched for behavioural changes.

Epileptogenic rats--A parallel group of rats, already chronically implanted with extradural electrodes (vide supra), were stimulated twice daily for 30 days via cortical leads, until severe and forceful clonic jerks of the forelimbs progressing to vigorous clonic activity occurred5

• These rats were potentially epileptogenic and prior to PTZ challenge, showed abnormal baseline EEG patterns, with spikes, polyspikes, dysrhthymia and hypersynchronous discharges. However, they had no visible spontaneous seizures.

Non-convulsive PTZ dosing and EEG recording­Normal (n=8) and epileptogenic rats (n=7) were habituated to the EEG recording chamber, with subdued background lighting and constant temperature

28° ± 2°e. A base line EEG was run for about 20 min' at a speed of 25 mm /sec. After administration of PTZ 20 mg/kg (as a dose of 10 mg/kg as suggested by Marescaux et af. 2, was insu fficient to produce reproducible spike/wave discharges, SWDs, in EEG, in our experiments). Behavioural changes were continuously monitored while the EEG was scanned for the occurrence of concurrent SWDs for I hr for quantitative assessment. Thereafter, recording was done intermittently at 15 min intervals for 3 to 4 hr, in order to qualitatively observe occurrence as ' well as cessation of SWDs in control and drug treated groups.

Behavioural and EEG effects-The behavioural effects seen after drugging were also evaluated8 and the quantitative analysis of the SWDs, described below followed the method described3

, with some modifications. The latency to the onset of the first SWD ( in min), the number and duration of SWDs/h

encountered after PTZ, as well as morphological aspects of the SWD patterns, was determined from the EEG recording.

Statistical Analysis-SWD Number and Duration­The ratio of SWDs post drug/SWDs pre drug for each animal was expressed as a percentage. The ratios were calculated for epochs of 20 min (I st, 2nd and 3rd), and for the overall 60 min period. This ensured normalization of the drug effect in each animal against its own baseline SWD number and reduced inter­animal variability. The effect of YPA, FLU and the combination of YPA and FLU over each epoch was compared using the paired t-test with Bonferoni correction6

. The mean cumulative SWDs of the tota l 60 min period for each group was calculated and were statistically compared using the one way analysis of variance (ANOY A) followed by Fisher's test of least signi ficance. Statistical sign ificance was val idated at P < 0.056

.

The duration of SWD for each animal during each 20 min epoch was measured from the EEG record and the cumulative duration for each epoch and for the total of 60 min was calculated. Normalization and statistical analyses were carried out as explained for analysis of number of SWDs, described above.

Results Behavioural effects of VPA , FLU and tire

combination in normal and epileptogenic rals---in normal and epileptogenic rats, FLU 10 mg/kg showed no behavioural changes when compared with vehicle treated controls. YPA 200 mglkg showed Illi Id sedation, ataxia, hypotonia and "wet dog shakes" (WDS). The WDS appeared within 5 min and lasted for about 45 min, while the other effects lasted for I to 2 hr. The combination produced mild sedation, reduced motor activity without ataxia. No specific behavioural changes occurred when PTZ was co-administered with the test drugs in both groups of rats.

EEG Changes in normal and epileptogenic rats following PTZ challenge---in normal rats (n=8), PTZ, 20 mg/kg, produced bilaterally synchronous spike/wave discharges (SWDs) in the EEG as shown in Fig 1. The latency (time to onset of first SWD) was 3±0.36 min. The number of SWDs in the first hour was 95.12±11.9. The SWDs occurred 1.6±0.2 times a

minute, with a frequency of 7 to 9 Hz, 50 to ISO /lY amplitude, and a mean duration of 1.9 ± 0.22 sec . The SWDs were always accompanied by behavioural signs of "absence" characterized by arrest of ongoing

230 INDIAN J EXP BIOL, MARCH 1999

Bregma

NI~I~~J"N

~U4~~v~ ~_~-~l~ 'HlW0.M~.~~fJ" 0 "~~~i~\M~WNrv~~~~tl1~'

t t ~ ~V 2.5 ems See

Fig. I--On the left, is the skull montage diagram showing the areas for placement of EEG leads with reference to Bregma. On the right, is EEG recording from channel I . left to right frontal (LF-RF),channel 2, left to right parietal (LP-RP). channel 3. le t! front al to len parietal (LF-LP) and channel 4. right frontal to right parietal (RF.RP).The spike/wave discharge (SWD) appears prominently in RF-RP at a frequen cy of 7Hz/sec and amp litude of 50 to 100 /lV . Calibration signal /lV/sec is shown . Note that EEG immediately returns to baseline foll owing SWD.

activity, staring and unrespons iveness to external stimuli. No visible or EEG evidence of clonic seizures were seen pre and post PTZ.

In epileptogenic rats (n=7), PTZ, 20 rflg/kg, produced 2.7 ± OA SWDs/min [a 68.8% increase compared to normal rats (P<0 .05)] ,while the latency, 2.8±0.18, mean duration of IA±O.08 min, frequency and amplitude were not significantly different from normal rats. The major difference compared to normal rats, was that although no visible se izures occurred prior to PTZ, several myoclonic jerks and mild to severe clonic s«izures were observed in all the rats after PTZ, suggesting that the epileptogenic rats had a lowered seizure threshold to an equivalent dose of

PTZ. Effect of VPA, FLU and the combination on SWD

after PTZ challenge in normal and epileptogenic rats-SWD Number as afunction of time - The effects of VPA, FLU and the combination as a function of t ime (20 min epochs) as compared to controls (SWD number post drug I SWD number pre drug), expressed as a percentage in normal and epileptogenic rats is shown in Fig 2. VPA reduced the % SWD number in both normal and epi leptogenic rats . The maximum reduction occurred in the first 20 min epoch in both groups, however this suppressant effect was s ignificant only 111 the epileptog{!n ic rats

(P < 0.05). The suppressant effect ofVPA declined in both groups and reached pre drug values at 60 min .

In the case of FLU, the SWD number rema ined above controls throughout the 60 m in test period in both normal and epileptogenic rats, the effects on the latter group· being more prominent. However, statistical s ignificance could not be obtained for any of these differences as the rats exhi bited variability in their responses, resulting in higher tandard errors.

When VP A. and FLU were given together a s ignificant (P <0.05) reduction in the number of SWDs was seen for the entire 60 min period, with respect to controls in both normal and epileptogenic rats , the maximum effect occurring at 60 min .

SWD number cumulative-The cumulative SWDs/hr over the 60 min test period in normal and epileptogenic rats are shown in Fig. 2 (bar diagrams). One way ANOV A revealed that the effects were significantly different between the three treatment groups F(df 3,31)=10.38, and F(df 3,27)=3 1.73, in normal and epileptogenic rats respective ly, (P < 0.001 ). While VPA reduced cumulativl! SWD number

(64.3±8.6 and 63A±9A % of controls in normal and epi leptogenic rats respectively), FLU did not

significantly influence the SWD number ( 121.7±24.7 %) in normal rats, as well as for epileptogenic rats

(167.2±20.2 % ). When FLU and VPA were given

SHANTILAL e/ at.: SODI UM V ALPROTATE & FLUNARIZINE IN ABSENCE SEIZURES IN RATS 231

NORMAL RATS

140 ----------- 160

120 120

100 % -

80 S W 80

60 D ~ 40

___ =LU

40

% * --..- JPA+FLU

N 20 * S * * U 0 W 0 1.1

Control VPA FLU VPA+FLU D 20 40 60 B E R

N EPILEPTOGENIC RATS U C 1.1 250

__ VPA U 200

B

~ 1.1

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150 A 120 T I 80 * 100 ....:.-- V

*~ E 50 40

* * * * • 0 0

20 TIME (MINS ) 40 60 Control VPA FLU VPA+FLU

Fig. 2-Number of spike/wave discharges (SWDs) as a percentage is represented as a function of time. On th e left, the line diagrams represent the number of SWDs at 20. 40 and 60 min and on the ri ght. is the cumulative SWD number for 60 min· P < 0.05 . Abbreviat ions: VPA: sod ium valproate. FLU : flunarizine

NORMAL RATS

140 120

120 100 r--

100 80

00 % __ VPA S 60 *

ro ___ FLU

W r--......- VPA ... FlU D 40

40

20 20 * * D % * s 0 R a -,

W 20 40 60 U CONTROL VPA FLU VPA + FLU

D A T I

D EPILEPTOGENIC RATS 0

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0 M 120

N 150 U

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**~ T

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20 40 60 CONTROL VPA FLU VPA+ TIME(MINS) FLU

Fig. 3-Duration of spike/wave di scharg.::s (SWDs) as a percentage is represented as a fun ction of tinK On the kli, the lin.:: diagrams represent the duration of SWDs at 20,40 and 60 min and on th.:: right. is the cumul ative SWD duration for 60 mins· I' <: 0.05 . Abbreviations are same as Fig. 2.

232 INDI AN J EXP BIOL, MARCH 1999

together, FLU potentiated the se izure suppressant effect of YPA, reducing the se izure number to 3.9± 1.6 and 16.3±3 .7 % in normal and epileptogenic rats respectively (P <0.05) .

SWD duration as a jllnctioll o/ tillle--As observed with SWD number, YPA likewise reduced SWD duration in both normal and epileptogeni c rats, maximum effect being in the first 20 min (F ig 3). This effect was statistically significant only in epileptogenic ralts (P <0.05). The seizure durati on dec lined thereafter, reaching pre drug levels at 60 min .

[n normal rats, FLU margi nally red uced SWD duration at 20 min , increased at 40 m in and the duration tended to return to predrug va lues at 60 min . However, in epileptogenic rats, SWD duration was increased throughout 60 min , although stati stical significance could not be obtained due to hi gh variabilities in responses.

The combination of YPA and FLU produced marked reductions in SWD durati on at 20,40 and 60 min, in both groups, all differences being stati stica lly significant (P < 0.05) with respect to controls.

SWD duration cumulative-The cumulat ive SWD duration over the 60 min period in normal and epileptogenic rats are shown in Fig. 3 (bar diagrams). The SWD duration was significantly different between the treatment groups as revealed by one way ANOY A (F df3 ,31 = 12.68; and F df 3, 27=30.09; for normal and epileptogenic rats respectively, (P < 0.00 I). While YPA reduced cumulative SWD duration in normal and epileptogenic rats respectively (P < 0.05 with respect to controls), FLU did not significantly influence the SWD duration (\05.9±26 %) in normal rats but the reverse was true for epileptogenic rats (190.4±25 .2 %). When YPA and FLU were given together, FLU potentiated the seizure suppressant effect of Y PA III

normal and epileptogenic rats (P < 0.05).

Discussion Many genetic and pharmacological models of

Absence Seizures(AS) have been described2..1. Each model has unique advantages and disadvantages for studying the diverse mechanisms that appear to underlie typical AS. Our objective in thi s study was to provide supporting evidence for newer phanna­cological approaches in the management of AS. Another goa l was to address the problem of pharmacoresistant epilepsy, as a part of our continuing

d . . 1 '1 5 6 P "[I I en eavor III expenmenta epI epsy . , -. le pentye-netetrazole (PTZ) rat model of AS used in our study,

shows electro-clinical analogies with human AS, and the dose/effi cacy pharmacological responSIveness mimics the clinica[ situation2

.

The resu its of th is study showed that PTZ, 20 mglkg in normal rats, produced SWDs, the number and duration/hour being comparable to that obtained prev iousl/, while the behavioural aspects were also identica l. There was a 68% increase (P < 0.05 ) in the number of SWDs/hour occurring in rats made epileptogenic by repeated direct cortical stimulation4

compared to normal , suggesting that the seizure threshold was lowered in epileptic rats. The phannacolog i ca ~ responses to Y (lA , 200 mglkg in both normal and epileptic rats, were com parable to that previously obta ined by Marescaux et aI. , 1984.

The Ca2+ channel antagonist, flunarizine (FLU),

readily crosses the blood brai n barrier and is thought to inhibit the entry of Ca2t into neurones primarily under pathophysiological conditions, such as ischaemia or se izure act ivity, but has no effect on normal Ca2

+

homeostas isi3. I-[ence, it has been proposed as a

promi sing cand ndate, paIticularly as add-on therapy for phannacoresistant epilepsy'4. In several controlled clinical trials, FLU has been shown to decrease seizure frequency in therapy resistant patients, bei ng treated with establi shed antiepil eptic drugs l 4

. FLU has been shown to have anticonvulsant acti vity in the maximal electroshock and metrazo l induced 'seizures in rats9

.

FLU 20 mglkg with YPA 200 mgikg did show an inhibitory effect on SWD di scharges,but we hav(! previously shown that thi s dose combination has side effects of ataxia and hypotonia6 For this reason we reported only the lower dose of FLU, 10mglkg in combination with Y PA , as this showed potentiation in the absence of adverse effects,an aspect which IS

deemed important for cl in ical extrapolation of pharmacological data.

A combination of FLU with YPA yielded promising results in experimental model s of generalized se izures4

,6. The natural outcome of our earlier studies was to determ ine whether a combination of YPA and FLU would demonstrate similar/superior efficacy in the PTZ model of AS. Using this model , YPA combined with FLU showed a marked and highly significant reduction in the number and duration of SWDs in normal as well as epileptogenic rats, suggest ing that this drug interaction also has a synergistic effect in AS.

There is a poss ibility that FLU may exeIt its anticonvul sant effect by interactin g with voltage

SHANTILAL 1'1 ai. ~ODIUM VALPROATE & PLUNAR IZINE IN ABSENCE SEIZURES IN RATS 233

dependent Na; channels IX I ~ (like VPA) in addition to the selecti ve blockade of Ca2

' channel s. Moreover, the dose of FLU employed in th is study produced its anticonvulsant effects with no adverse effects. Nuglisch el 0/

2 °, have shown that an oral dose of FLU, 40 mg/kg produced no changes on mean al1erial blood pressure in rats.

Two general hypotheses have been proposed to explain the mechanism of action of VPA I·i::. bl ocking of voltage dependent Na ' channels and enhancing GABA mediated inhibition. FL U possesses a similar anticonvulsant profil e to carbamazepi ne and phenytoin and this suggests the poss ibility of an ana logous mechanism of action on a ' channel s. FLU does not increase GABAergic inhibition, but it read ily crosses the blood brain barri er and also bl ocks T type ca lcium channel s. However, it is not known which of these actions contributes to its ant iconvul san t actions. For the present, the pharmacologica l mechani sm underlying the enhanced effect of these two drugs must await further elucidation.

The pharmacological rationale of combining FLU with VPA, was to take advantage of thei r differing mechanisms of action to produce a supra-add iti ve effect and converge to a common CNS target in a synergist ic manner. However, these are specu lat ive explanations and definitive ev idence must await further elucidati on .

In conc lusion, FLU has been shown to potenti ate the act ivity of VPA against PTZ induced absence se izures in 11 0rmal and epileptogenic rats, at we ll tolerated dose combinations employed in thi s study.

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