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Increased susceptibility of mice to seizures after some anticonvulsant drugs
EUROPEAN JOURNAL OF PHARMACOLOGY 1 (1967) 369-377.
NORTH-HOLLAND PUBL. COMP.. AMSTERDAM
INCREASED SUSCEPTIBILITY OF MICE TO SEIZURES AFTER SOME ANTICONVULSANT DRUGS CHR. L. R U M K E Department o f Pharmacology and Neurobiochemistry, F r e e University, A m s t e r d a m , The Netherlands Accepted 12 May 1967 Chr. L. ROMKE, Increased susceptibility of mice to s e i z u r e s a f t e r some anticonvulsant drugs, European J. P h a r m a c o l . 1 (1967) 369-377. The effects of some antiepileptic drugs given one to four days beforehand, on the convulsant effects of bemegride, pentylenetetrazol and dioxone in mice were studied. Phenobarbital, methylphenobarbital, primidone and methoin were shown to enhance the effect of these convulsants, when given two days before t h e i r administration. A single dose of phenytoin was found to have an anticonvulsant effect in the f i r s t two days; a f t e r four days the CED50 of bemegride, pentylenetetrazol and dioxone was diminished. These enhanced convulsant effects after antiepileptics could be observed in c h e m o s h o c k - t e s t s with t h r e e s t r u c t u r a l l y different convulsants; however, they were not parallelled by an increased sensitivity to a s u p r a m a x i m a l electroshock. In r a t s phenobarbital did not i n c r e a s e the convulsant effect of bemegride or pentylenetetrazol, given two or t h r e e days l a t e r . The nature of the observed effects in mice could not be elucidated.
1. I N T R O D U C T I O N M a n y d r u g s a r e k n o w n to p r o l o n g t h e d u r a t i o n of h e x o b a r b i t a l o r h y d r o x y d i o n e n a r c o s i s in m i c e or rats when given shortly before the adminis t r a t i o n of t h e s e a n e s t h e t i c s . H o w e v e r , s e v e r a l of t h e s e d r u g s s h o r t e n t h e d u r a t i o n of t h e n a r c o sis when the time interval between the adminis t r a t i o n i s a b o u t two d a y s ( s e e , a m o n g s t o t h e r s , RiJmke a n d B o u t , 1960; R i i m k e , 1963). Similar effects with other drugs have been described by various authors: Conney and Burns (1960), a n d C o n n e y e t al. (1960) s h o w e d t h a t t h e d u r a t i o n of z o x a z o l a m i n e p a r a l y s i s in r a t s i s shortened by pretreatment with phenobarbital, phenylbutazone and orphenadrine. K a t o (1961) o b s e r v e d t h a t p r e m e d i c a t i o n of r a t s w i t h p h e n o barbital or meprobamate, one or two days beforehand, decreases meprobamate paralysis t i m e . I n b o t h c a s e s t h e a c t i v i t y of t h e l i v e r m i crosomal enzyme system metabolizing the drug w a s f o u n d to b e e n h a n c e d . C h r o n i c t r e a t m e n t of experimental animals with phenobarbital was s h o w n b y C u c i n e l l e t al. ( 1 9 6 5 ) t o c a u s e a c c e l e r a t e d m e t a b o l i s m of b i s h y d r o x y c o u m a r i n , diphenylhydantoin and antipyrin resulting in dim i n i s h e d e f f e c t s of t h e s e d r u g s . In t h e w o r k d e s c r i b e d here, antiepileptic
d r u g s w e r e g i v e n to m i c e a t d i f f e r e n t t i m e s b e f o r e t h e a d m i n i s t r a t i o n of b e m e g r i d e , p e n t y l e n e tetrazol and dioxone (see Maffii, Dezulian and S i l v e s t r i n i , 1961; M a f f i i a n d S e r r a l u n g a , 1961; M a f f i i , B i a n c h i , S c h i a t t i a n d S i l v e s t r i n i , 1961) to s e e w h e t h e r t h e t i m e i n t e r v a l i n f l u e n c e d t h e a c t i o n of t h e a n t i e p i l e p t i c s o n t h e c o n v u l s a n t e f f e c t of t h e s e d r u g s . T h e s a m e a n t i e p i l e p t i c s w e r e g i v e n a t t i m e i n t e r v a l s f r o m + 1 h o u r to s e v e r a l d a y s b e f o r e h a n d to s e e w h e t h e r t h e t i m e of a d m i n i s t r a t i o n i n f l u e n c e d t h e i r e f f e c t on t h e i n c i d e n c e of m a x i m a l e l e c t r o s h o c k s e i z u r e s i n m i c e , a n d on t h e r e a c t i o n - t i m e of m i c e in t h e hot p l a t e t e s t . In a d d i t i o n s o m e e x p e r i m e n t s w e r e d o n e o n r a t s to s t u d y t h e e f f e c t of p h e n o b a r b i t a l o n t h e i r s e n s i t i v i t y to b e m e g r i d e a n d p e n t y l e n e tetrazol on the second and third day after its administration. Preliminary r e p o r t s of s o m e of these experiments have been given (Rtimke, 1961a, 1961b, 1962).
2. M A T E R I A L S AND M E T H O D S M a l e a n d f e m a l e m i c e of t h e i n b r e d C P B - N s t r a i n w e r e u s e d in a l l e x p e r i m e n t s . The anim a l s in a n y o n e e x p e r i m e n t w e r e a l w a y s of t h e same sex and their weight range did not exceed
370
CHR.
L. RIIMKE
6 g. All e x p e r i m e n t s w e r e p e r f o r m e d at 23oc. P h e n o b a r b i t a l - s o d i u m , t r i m e t h a d i o n e , phenyt o i n - s o d i u m and ethionine w e r e i n j e c t e d subcutaneously. P r i m i d o n e (Mysoline (R); I m p e r i a l C h e m i c a l I n d u s t r i e s Ltd.), m e t h y l p h e n o b a r b i t a l , me t ho i n and p h e n a c e m i d e w e r e given by g a v a g e * . The drugs to be i n j e c t e d and those to be given by mouth w e r e d i s s o l v e d o r suspended in n o r m a l s a l i n e so that each a n i m a l r e c e i v e d 0.2 m l p e r 10 g body weight. In each e x p e r i m e n t c o n t r o l s w e r e t r e a t e d s i m u l t a n e o u s l y with the s a m e v o l u m e of s al i n e, a d m i n i s t e r e d by the s a m e route as the drug to be studied. The activity of the convulsant a g e n t s a f t e r p r e m e d i c a f i o n and in c o n t r o l a n i m a l s was m e a s u r e d by d e t e r m i n i n g the CED50 and the MED. The f o r m e r value was defined in t h e s e e x p e r i m e n t s as the dose of the convulsant agent which c a u s e s at l e a s t a clonic convulsion in 50%of the a n i m a l s a f t e r an i n t r a v e n o u s injection lasting 5 se c into one of the tail veins. B e m e g r i d e (Megimide (R); A s p r o Nicholas (Ltd.), p e n t y l e n e t e t r a zol (Cardiazol (R)) and dioxone ( 5 , 5 - d i e t h y l - l , 3 o x a z i n - 2 , 4 - d i o n e ; Lepetit) w e r e diluted with s a line to give solutions containing 0.5 m g / m l b e m e g r i d e , 1.5 m g / m l p e n t y l e n e t e t r a z o l and 0.75 m g / ml dioxone. Convulsions w e r e o b s e r v e d 5-15 s e c a f t e r injecting the convulsant. The d e t e r m i n a t i o n of the CED50's in d r u g - a n d s a l ' n e p r e t r e a t e d a n i m a l s took p l a c e following a " s e m i - s e q u e n t i a l " design. A d e t a i l e d account of this method has been given e l s e w h e r e (Rtimke, 1961c). The d e sign and the s t a t i s t i c a l a n a l y s i s d e s c r i b e d in e x a m p l e 3 of the quoted publication was applied and the r a t i o of the CED50 in drug p r e t r e a t e d and in c o n t r o l a n i m a l s was calculated. An a n t i c o n v u l sant effect is r e f l e c t e d by a C E D 5 0 - r a t i o s i g n i f i cantly g r e a t e r than 1; a d e c r e a s i n g effect upon the s e i z u r e t h r e s h o l d is shown by a C E D 5 0 - r a t i o which is l e s s than 1. This d e c r e a s i n g effect is r e f e r r e d to as a ' p r o c o n v u l s a n t e f f e c t' throughout this paper. In each e x p e r i m e n t of this kind at l e a s t 20 d r u g - and 20 s a l i n e - p r e t r e a t e d a n i m a l s w e r e i n j e c t e d with one of the convulsant agents in di f f eren t doses. Most of the g i v e n C E D s 0 - r a tios have been obtained by a v e r a g i n g the r e s u l t s of two e x p e r i m e n t s , p e r f o r m e d on d if f e r e n t days. In all tables the n u m b e r of a n i m a l s (N) r e f e r s to the total n u m b e r of p r e t r e a t e d and c o n t r o l m i c e , which w e r e used in the e x p e r i m e n t s . In the s t a t i s t i c a l t e s t s 5% (two-sided) was chosen as the l e v e l of s i g n i f i c a n c e . The m i n i m a l e f f e c t i v e dose (MED) of the c o n * Thanks are due to the mentioned firms for supplying their drugs used in this experiment.
v u l san t agents was defined in t h e s e e x p e r i m e n t s as the m i n i m a l dose which c a u s e d at l e a s t a clonic convulsion with continuous i n t r a v e n o u s inj e c t i o n into one of the t a i l v e i n s at a constant r a t e of 0.30 ml p e r min f r o m a 1%o solution of b e m e g r i d e , a 2.5%o solution of p e n t y l e n e t e t r a z o l , or a 0.75%0 solution of dioxone. It was m e a s u r e d with a t u b e r c u l i n - s y r i n g e a c c u r a t e to 0.01 ml. In each e x p e r i m e n t the MED was obtained in 10 d r u g - and in 10 s a l i n e - p r e t r e a t e d a n i m a l s . Most of these e x p e r i m e n t s w e r e r e p e a t e d once o r twice on d i f f e r e n t days. Analysis of v a r i a n c e ( r a n d o m i z e d blocks) t e c h n i q u e s w e r e applied in the s t a t i s t i c a l a n a l y sis of the r e s u l t s of the M E D - e x p e r i m e n t s . The s t a t i s t i c a l a n a l y s i s was always b a s e d upon the l o g a r i t h m s of the o b s e r v a t i o n s . The r e s u l t s of the M E D - e x p e r i m e n t s have finally been e x p r e s s ed in the M E D - r a t i o (and its 95% confidence l i m its), which is the r a t i o of the ( g e o m e t r i c ) m e a n MED in p r e t r e a t e d a n i m a l s and the ( g e o m e t r i c ) m e a n MED in the c o n t r o l s (cf. Fingl and Mc Q u a r r i e , 1960; Swinyard et al., 1962). An an t i convulsant effect is sh o w n i n t h e s e t e s t s when the M E D - r a t i o e x c e e d s 1; a ratio, which is s m a l l e r than 1 i n d i cat es an e n h a n c e m e n t of c o n v u l s a n t ef f e c t (a p r o c o n v u l s a n t effect). An M E D - r a f i o is concluded to differ s i g n i f i c a n t l y f r o m 1 if its 95% confidence i n t e r v a l does not contain 1. The influence of d i f f e r e n t p r e t r e a t m e n t on the i n ci d en ce of s u p r a m a x i m a l e l e c t r o s h o c k s e i z u r e s was t e s t e d with an ap p ar at u s d e s c r i b e d by De Jongh (1957) which allows 10 m i c e to be t r e a t e d s i multaneously. An e l e c t r i c a l s t i m u l u s of 0.2 sec duration was given. The c u r r e n t was such that about 50% of the c o n t r o l a n i m a l s showed the e x t e n s o r component of a s e i z u r e . E a c h e x p e r i m e n t was p e r f o r m e d upon 4 groups of 5 + 5 d r u g - and s a l i n e - p r e t r e a t e d animals. Most experiments w e r e r e p e a t e d once with d i f f e r e n t a n i m a l s on a d i f f er en t day. The p e r c e n t a g e s g i v en in t a b l e s 6 and 7 r e f e r to the d i f f e r e n c e b et w een the p e r centage of all p r e t r e a t e d a n i m a l s r e a c t i n g with an e x t e n s o r s e i z u r e and the p e r c e n t a g e of r e acting c o n t r o l s . S t a t i s t i c a l s i g n i f i c a n c e of the d i f f e r e n c e between t h ese two p e r c e n t a g e s was d e t e r m i n e d in a c o m b i n e d 2 × 2 - t a b l e t e s t on the r e s u l t s f r o m all s e p a r a t e g r o u p s of 5 + 5 m i ce. A slight m o d i f i c a t i o n of E d d y ' s hot plate method (Eddy and L e i m b a c h , 1953) was u sed , to d e t e r m i n e the r e a c t i o n t i m e s in d r u g - and s a l i n e - p r e t r e a t e d animals. Most hot p l at e e x p e r i m en t s w e r e p e r f o r m e d twice on d i f f e r e n t days upon groups of m i c e which s h o r t l y a f t e r w a r d s w e r e to be t r e a t e d with a c h e m o - o r an e l e c t r o shock. The t e m p e r a t u r e of the plate was 56oc.
DECREASED SEIZURE THRESHOLD AFTER ANTICONVULSANTS T h e m e a s u r e m e n t s in d r u g - a n d s a l i n e - p r e t r e a t e d a n i m a l s w e r e m a d e in r a n d o m o r d e r . T h e o b s e r v e r d i d not k n o w w h e t h e r a n a n i m a l h a d b e e n given a drug or a saline premedication. The m e a s u r e d r e a c t i o n t i m e is the t i m e e l a p s i n g b e tween the f i r s t contact with the plate and the f i r s t l i c k i n g r e s p o n s e . In c o n t r o l s t h e a v e r a g e r e a c t i o n t i m e w a s a p p r o x i m a t e l y 10 s e c at t h e m o s t . T h e r e s u l t s of hot p l a t e r e a c t i o n t i m e d e t e r m i n a t i o n s in s e c f r o m 44 e x p e r i m e n t s w i t h 20 m i c e e a c h s h o w e d t h a t the d i s t r i b u t i o n of t h e r e a c t i o n t i m e s i s not n o r m a l . H o w e v e r , t h e d i s t r i b u t i o n of t h e l o g a r i t h m s of t h e s e r e a c t i o n t i m e s a p p r o x i m a t e s the n o r m a l d i s t r i b u t i o n v e r y w e l l . T h e r e f o r e t h e s t a t i s t i c a l a n a l y s i s of t h e r e s u l t s of t h e s e e x p e r i m e n t s w a s a l w a y s b a s e d on t h e l o g a r i t h m s of t h e r e a c t i o n t i m e s . T h e r e a c t i o n t i m e r a t i o i s t h e r a t i o of t h e ( g e o m e t r i c ) m e a n r e a c t i o n t i m e in p r e t r e a t e d a n i m a l s a n d t h e ( g e o m e t r i c ) m e a n r e a c t i o n t i m e in t h e c o n t r o l s . T h e d r u g s t u d i e d i s c o n c l u d e d to p r o l o n g t h e r e a c t i o n t i m e s i g n i f i c a n t l y if t h e r e a c t i o n t i m e r a t i o e x c e e d s 1, a n d if i t s 95% c o n f i d e n c e i n t e r v a l d o e s not c o n t a i n 1. T h e M E D - e x p e r i m e n t s in r a t s w e r e c a r r i e d out on m a l e a n d f e m a l e a n i m a l s of t h e i n b r e d TNO-WU-strain, weighing 150-200 grams. Bem e g r i d e a n d p e n t y l e n e t e t r a z o l w e r e g i v e n in a c o n t i n u o u s i n t r a v e n o u s i n f u s i o n into one of the t a i l v e i n s at a c o n s t a n t r a t e of 1 m l p e r m i n f r o m a 2.5 m g / m l , a n d a 7.5 m g / m l o r 3.75 m g / m l solution, respectively. T h e a n a l y s i s of v a r i a n c e of the r e s u l t s of t h e M E D - a n d hot p l a t e e x p e r i m e n t s w a s d o n e w i t h t h e h e l p of o n e of t h e E l e c t r o l o g i c a E L - X 1 o r E L - X 8 c o m p u t o r s of the M a t h e m a t i c a l C e n t r e a t Amsterdam.
Table 1 The influence of drugs given ½ to 120 hours ,beforehand on the CED50 of bemegride in mice. premedieation phenobarbital
primidone
100 100 100 100
methoin
s.c. s.c. s.c. s.c.
s.c. s.c. s.c. s.c. s.c. s.c. or. or. or. I or.
60 or. 60 or. 60 or. 60 or.
interval CED50 sig~ifin h r s ratio icance a~ ½ 1 2 12 24 48 72 96 120
0.98 0.86 1.10 1.30 4.20 1.48 0.71 0.77 0.94 0.97
1 24 48 72
1.57 1.02 0.89 1.00
+++
1½ 24 48 72
1.57 1.14 0.85 1.02
++~
+*+ +
trimethadione
500 500 500 500
s.c. s.c. s.c. s.c.
1½ 24 48 72
>3.00 1.15 1.06 1.32
phenytoin
50 50 50 100 50 100 50 50
s.c. s.c. s.c. s.c. s.c. s.c. s.c. s.c.
1½ 24 48 48 72 72 96 120
1.01 1.46 1.00 1.16 1.09 0.94 0.87
1 24 48 48
0.95 1.16
phenaeemide
125 or. 125 or. 125 or. 500 or. +
++ ++÷
3.1. CED50-experirnents
do se mg/kg 0.01 0.1 1 10 100 100 100 100 100 100
sl nificanee: 3. E X P E R I M E N T S AND R E S U L T S
371
-, +~ +~
+ E+ + +
*
++
++ + +
1.02 1.80
1.05
+++
N 80 80 80 80 80 80 80 80 80 80 80 80 80 40 40 80 80 80 40 40 80 8o 80 80 40 4o 4o 4o 80 4o 4o 4o 4o 8o
P > 0.05 0.05 >~P > 0.01 0.01 >/P > 0.001 P ~< 0.001
on the convulsant a c -
tion o f bemegride T h e r e s u l t s of C E D 5 0 - e x p e r i m e n t s on t h e e f f e c t s of s e v e r a l d r u g s on t h e c o n v u l s a n t a c t i v i t y of b e m e g r i d e a r e s h o w n in t a b l e 1. In t h e s e e x p e r i m e n t s the t i m e interval b e t w e e n p r e m e d i c a tion and intravenous bemegride administration r a n g e d f r o m ½ to 120 h o u r s . P h e n o b a r b i t a l (10 m g / k g ) , p r i m i d o n e , m e t h oin, t r i m e t h a d i o n e and p h e n a c e m i d e had an a n t i 1 1 c o n v u l s a n t e f f e c t , if g i v e n 5 t o 15 h o u r s b e f o r e b e m e g r i d e . Of t h e s e d r u g s p h e n o b a r b i t a l (100 mg/kg), primidone and methoin had a proconvulsant effect when the i n t e r v a l b e t w e e n p r e t r e a t m e n t a n d b e m e g r i d e a d m i n i s t r a t i o n w a s 48
h r s . T h r e e d a y s a f t e r a s i n g l e d o s e of p h e n o b a r b i t a l (100 m g / k g ) t h e c o n v u l s a n t e f f e c t of b e m e gride was still enhanced. No e f f e c t u p o n t h e CED50 of b e m e g r i d e w a s o b s e r v e d w h e n p h e n y t o i n (50 m g / k g ) w a s g i v e n 1½ h r s b e f o r e h a n d . H o w e v e r , one d a y a f t e r a s i n g l e p h e n y t o i n i n j e c t i o n of 50 m g / k g a n d e v e n t w o d a y s a f t e r a s i n g l e i n j e c t i o n of 100 m g / k g t h e CED50 of b e m e g r i d e w a s i n c r e a s e d . A s i g n i f i c a n t d e c r e a s e of t h e C E D 5 0 (a p r o c o n v u l s a n t e f f e c t ) w a s f o u n d a f t e r a 96 h r s i n t e r v a l b e t w e e n p h e n y t o i n a d m i n i s t r a t i o n (50 m g / k g ) and i n j e c t i o n of b e m e g r i d e . A r e l a t i v e l y s m a l l d o s e of p h e n o b a r b i t o n e (0.1
372
CHR. L. RUMKE
m g / k g ) i n c r e a s e d t h e c o n v u l s a n t e f f e c t of b e m e g r i d e g i v e n 30 m i n l a t e r .
on the convulsant action of pentylenetetrazol
Table 3 The influence of drugs given ½ to 96 hours beforehand on the CED50 of dioxone in mice.
3.2. CED50-experiments
T h e r e s u l t s of C E D s 0 - e x p e r i m e n t s on t h e i n f l u e n c e of p r e t r e a t m e n t w i t h s e v e r a l d r u g s on t h e c o n v u l s a n t e f f e c t of p e n t y l e n e t e t r a z o l a r e s h o w n in t a b l e 2. Table 2 The influence of drugs given ½ to 120 hours beforehand on the CED50 of pentylenetetrazol in mice. Jremedication phenobarbital
!primidone methoin phenytoin
dose mg/kg 0.1 10 100 100 100 100
interval CED50 signifratio icance in h r s
s.c. s.c. s.c. s.c. s.c. s.c.
½ 1 24 48 72 96
1.12 2.06 2.29 0.85 0.89
N
1.00
-
80 80 40 80 80 80
~++ + ~+ -
100 or. 100 or.
1 48
1.37 0.91
+ +
40 80
60 or. 60 or.
1½ 48
1.46 0.88
+ ++
40 80
1½ 1½
1.08 0.93 1.21 1.34 0.95 0.90 0.68 1.00
÷ ÷+÷ +~ -
40 80 40 40 80 40 80 40
50 100 50 100 50 50 50 50
s.c. s.c. s.c. s.c. s.c. s.c. s.c. s.c.
24 24 48 72 96 120
Phenobarbital (10 m g / k g ) , p r i m i d o n e a n d m e t h o i n i n c r e a s e d t h e CED50 of p e n t y l e n e t e t r a z o l , w h e n g i v e n 1-1½ h r s b e f o r e h a n d . W h e n p h e n y t o i n w a s a d m i n i s t e r e d 1½ h o u r b e f o r e p e n t y l e n e t e t r a z o l , no s u c h e f f e c t w a s s e e n , b u t o n e day a f t e r a p h e n y t o i n i n j e c t i o n of 50 m g / k g o r 100 m g / k g t h e CED50 w a s i n c r e a s e d . When the interval b e t w e e n a single p h e n o b a r b i t a l (100 m g / k g ) , p r i m i d o n e o r m e t h o i n p r e m e d i c a t i o n w a s two d a y s , the C E D 5 0 ' s of p e n t y l e n e t e t r a z o l w e r e d e c r e a s e d . The s a m e enhanced convulsant effect was observed with p h e n y t o i n , ff the i n t e r v a l w a s 96 h r s . P h e n o b a r b i t o n e (0.1 m g / k g ) d i d n o t i n c r e a s e t h e c o n v u l s a n t e f f e c t w h e n g i v e n 30 m i n b e f o r e pentylenetetrazol. 3.3. CED50-experiments
on the convulsant ac-
tion of dioxone T a b l e 3 s u m m a r i z e s t h e r e s u l t s of C E D 5 0 e x p e r i m e n t s on t h e i n f l u e n c e of s e v e r a l d r u g s on t h e c o n v u l s a n t a c t i o n of d i o x o n e . T h e C E D 5 0 ' s of
premedication
dose mg/kg
phenobarbital
0.1 10 100 100 100 100
primidone
s.c. s.c. s.c. s.c. s.c. s.c.
interval CED50 signifin h r s ratio
N
21 1 24 48 72 96
1.00 1.29 1.74 0.85 0.81 0.98
÷+ +++ 4+ +++ -
40 40 40 80 80 40
100 or. 100 or.
1 48
1.26 0.75
++ +
40 40
methoin
60 or. 60 or.
1 48
1.81 0.95
~+ +
40 80
phenytoin
50 s.c. 50 s.c.
24 96
1.70 0.86
+++ +
40 80
dioxone w e r e i n c r e a s e d by p r e t r e a t m e n t with p h e n o b a r b i t o n e (10 m g / k g ) , p r i m i d o n e a n d m e t h oin, g i v e n 1-1½ h r s e a r l i e r . When the interval between premedication with one d o s e of p h e n o b a r b i t o n e (100 m g / k g ) , p r i m i d o n e o r m e t h o i n w a s two d a y s , t h e C E D 5 0 ' s w e r e decreased. O n e d o s e of p h e n y t o i n , g i v e n o n e day e a r l i e r , had a m a r k e d anticonvulsant effect. However, four days after such a single dose the convulsant a c t i o n of d i o x o n e w a s e n h a n c e d . 3.4. E x p e r i m e n t s of the minimal effective dose (MED) of bemegride T h e r e s u l t s of t h e M E D - e x p e r i m e n t s on t h e i n f l u e n c e of s e v e r a l d r u g s on t h e c o n v u l s a n t e f f e c t of b e m e g r i d e a r e s h o w n in t a b l e 4. P h e n o b a r b i t a l (10 m g / k g ) , m e t h y l p h e n o b a r b i t a l (20 m g / k g ) , p r i m i d o n e , m e t h o i n a n d t r i m e t h a d i o n e i n c r e a s e d t h e M E D - r a f i o of b e m e g r i d e i.e. d e c r e a s e d i t s c o n v u l s a n t e f f e c t w h e n g i v e n 1 l ~ - 1 ~ h r s b e f o r e it. A s m a l l d o s e of p h e n o b a r b i t a l (0.1 m g / k g ) , a n d 20 m g / k g p h e n y t o i n d e c r e a s e d t h e M E D - r a tio significantly when given ½ hour b e f o r e b e m e g r i d e a d m i n i s t r a t i o n . In t h e s e d o s e s , t h e s e d r u g s m a y b e c o n c l u d e d to e n h a n c e t h e c o n v u l s a n t e f fect. T w o d a y s a f t e r a d m i n i s t r a t i o n of one l a r g e d o s e of p h e n o b a r b i t a l , m e t h y l p h e n o b a r b i t a l a n d p r i m i d o n e t h e c o n v u l s a n t e f f e c t of b e m e g r i d e w a s i n c r e a s e d . O n e day a f t e r a s i n g l e d o s e of 50 m g / k g p h e n y t o i n , a n d one a n d t w o d a y s a f t e r a s i n g l e d o s e of 100 m g / k g p h e n y t o i n t h e c o n v u l s a n t e f f e c t of b e m e g r i d e w a s s t i l l d e c r e a s e d . Two d a y s a f t e r a s i n g l e m e t h o i n , t r i m e t h a d i o n e or phenacemide pretreatment or four days after
D E C R E A S E D S E I Z U R E T H R E S H O L D A F T E R ANTICONVULSANTS Table 4 T h e i n f l u e n c e of d r u g s on t h e MED of b e m e g l : i d e in m i c e . MED-ratio with 95% interval
Table 5 The i n f l u e n c e of d r u g s on the MED of p e n t y l e n e t e t r a z o l and d i o x o n e in m i c e .
dose mg/kg
interval in h r s
0.1 s . c . 10 s . c . 100 s . c .
½ ½ 48
0.88(0.78-0.997) 40 1.37(1.19-1.58) 20 0.83(0.72-0.96) 40
methylphenobarbital
20 or. 200 or.
1 48
1.37(1.14-1.61) 0.83(0.70-0.97)
20 40
primidone
100 o r . 100 or.
1 48
1.37(1.12-1.68) 0.86(0.75-0.95)
20 60
60 or. 60 or.
11 48
1.78(1.43-2.22) 1.10(0.98-1.25)
20 60
s.c. s.c. s.c. s.c. s.c. s.c. s.c.
~1 24 24 48 48 96
0.85(0.77-0.93) 0.90(0.75-1.07) 1.28(1.14-1.44) 1.16(1.03-1.31) 1.02(0.87-1.18) 1.25(1.02-1.55) 0.99(0.85-1.15)
60 40 60 60 40 40 60
trimethadione
500 s . c . 500 s . c .
1½ 48
2.08{1.90-2.28) 1.00(0.76-1.31)
20 20
phenacemide
500 or.
48
0.88(0.76-1.02)
40
premedication phenobarbital
methoin phenytoin
20 50 50 100 50 100 50
phenytoin administration no significant t h e M E D of b e m e g r i d e w a s o b s e r v e d . 3.5.
E x p e r i m e n t s on the M E D ' s trazol and dioxone
N
effect on
of p e n t y l e n e t e -
The summary of the experiments on the MED's of pentylenetetrazol a n d d i o x o n e in t a b l e 5 s h o w s t h a t a s m a l l d o s e of p h e n o b a r b i t a l (0.1 mg/kg) does not significantly influence the MED's of pentylenetetrazol or dioxone. Methylphenobarbital (20 m g / k g ) , given 1 hour earlier, markedly inhibited the convulsant effects of pentylenetetrazol and dioxone. With pentylenetetrazol and dioxone one dose of phenobarbital (100 mg/kg) or methylphenobarbital (200 mg/kg) given 48 hours beforehand had a significant proconvulsant effect. O n e a n d t w o d a y s a f t e r 50 o r 1 0 0 m g / k g p h e n ytoin the convulsant effect of pentylenetetrazol was still reduced. No marked effect of phenytoin on the convulsant effect of pentylenetetrazol was observed after a four day interval. 3.6.
373
E x p e r i m e n t s on effects on the incidence of supramaximal electroshock seizures
Table 6 gives a summary of the results of the experiments on the influence of a single dose of some known antiepileptics given ½-96 hours earlier on the incidence of seizures after a supra-
premedication
dose mg/kg
interval in h r s
MED-ratio with 95% interval
N
pentylenetetrazol phenobarbital
! 0.1 s . c . 100 s . c .
½ 48
1.06(0.89-1.26) 0.80{0.70-0.91)
40 40
methylphenobarbital
20 or. 200 o r .
1 48
1.53(1.20-1.93) 0.79(0.69-0.90)
20 40
phenytoin
20 50 100 50 100 50
s.c. s.c. s.c. s.c. s.c. s.c.
½ 24 24 48 48 96
0.93(0.78-1.10) 1.32(1.13-1.55) 1.48(1.27-1.73) 1..19(1.02-1.39) 1.26(1.07-1.48) 1.07(0.95-1.21)
40 20 40 40 40 40
phenobarbital
0.1 s . c . 100 s . c .
½ 48
1.05(0.92-1.20) 0.81(0.69-0.95)
40 40
methylphenobarbital
20 or. 200 or.
1 48
1.40(1.18-1.65) 0.77(0.66-0.89)
40 40
dioxone
Table 6 The i n f l u e n c e of d r u g s g i v e n ½-96 h o u r s e a r l i e r on the i n c i d e n c e of s e i z u r e s a f t e r a s u p r a m a x i m a l e l e c t r o s h o c k in m i c e .
premedication phenobarbital
dose mg/kg
differe n%c e
N
-
80 80
35
+
40
Z-~-~
+++
2~ ½ 1 1 24 24 48 72 96
methylphenobarbital
20 s . c . 200 s . c .
1 48
-
primidone
100 or. 100 or.
1 48
-
60 o r . 60 or. 50 s . c . 100 s . c . 50 s . c .
phenytoin
significance
+12½
s.c. s.c. s.c. s.c. s.c. s.c. s.c. s.c. s.c.
methoin
0.01 0.1 10 50 50 100 100 100 100
interval in h r s
-
-
2½
40
+
+++ -
80 40 80 80
7~
-
80
12½
-
80
-
+
80
-
80
42½
+++
80
-
10
-
80
1 48
-
35
+++
80
-
15
-
80
24 48 96
-
65
++
40
-
35 0
~ -
80 80
25
-
2½
D i f f e r e n c e ~o: The d i f f e r e n c e b e t w e e n the p e r c e n t a g e of p r e t r e a t e d and of c o n t r o l a n i m a l s r e a c t i n g with a c o n v u l s i o n . S i g n i f i c a n c e : -: P > 0.05: +: P < 0.05; + + : P < 0.01; + + + : P < 0.001.
374
CHR. L. RUMKE
m a x i m a l e l e c t r o s h o c k . In t h i s t e s t one h o u r a f t e r phenobarbital, methylphenobarbital, primidone or methoin pretreatment a significant anticonvulsant effect was observed. This effect was also s e e n 24 h o u r s a f t e r p h e n o b a r b i t a l o r p h e n y t o i n a d m i n i s t r a t i o n . N o n e of t h e d r u g s s t u d i e d w a s f o u n d to i n c r e a s e t h e n u m b e r of c o n v u l s i o n s w h e n t h e i n t e r v a l b e t w e e n the a d m i n i s t r a t i o n a n d t h e e l e c t r o s h o c k t e s t w a s two d a y s . In t a b l e 7 t h e r e s u l t s a r e g i v e n of s o m e e x p e r i m e n t s in w h i c h t h e a n t i e p i l e p t i c s w e r e g i v e n on s i x c o n s e c u t i v e d a y s . T w o d a y s a f t e r t h e l a s t administration the electroshock test was perf o r m e d . No e f f e c t on t h e s e i z u r e t h r e s h o l d w a s o b s e r v e d in t h e s e e x p e r i m e n t s w i t h any of t h e drugs tested. Table 7 The incidence of s e i z u r e s after a supramaximal e l e c troshock in mice after six days of treatment with an anticonvulsant and two days of withdrawal. pretreatment phenobarbital phenobarbital primidone trimethadione phenytoin
daily dose mg/kg 50 75 50 250 50
s.c. s.c. or. s.c. s.c.
difference % -~ 16% ~ 5% 8% + 5% 8%
significance
N 100 50 50 40 50
Legend: see table 6.
3.7. E x p e r i m e n t s on the e f fe c t s on the hot plate
reaction time In t a b l e 8 t h e r e s u l t s of e x p e r i m e n t s on t h e i n f l u e n c e of s o m e d r u g s on t h e hot p l a t e r e a c t i o n t i m e h a v e b e e n s u m m a r i z e d . P h e n o b a r b i t a l (10 m g / k g ) a n d m e t h y l p h e n o b a r b i t a l (20 m g / k g ) g i v e n o n e h o u r b e f o r e t h e t e s t w e r e f o u n d to p r o l o n g t h e r e a c t i o n t i m e . One d a y a f t e r 50 m g / k g p h e n ytoin the reaction time was enhanced. These d r u g s h a d no e f f e c t in t h i s t e s t w h e n g i v e n two or more days earlier. 3.8. E x p e r i m e n l s with ethionine T h e i n f l u e n c e of e t h i o n i n e (250 m g / k g s . c . ) on t h e p r o c o n v u l s a n t e f f e c t of p h e n o b a r b i t a l w a s s t u d i e d in two g r o u p s of e x p e r i m e n t s . P h e n o b a r b i t a l (100 m g / k g s . c . ) w a s g i v e n 30 m i n a f t e r e t h i o n i n e a d m i n i s t r a t i o n . Two d a y s l a t e r the M E D of b e m e g r i d e w a s d e t e r m i n e d in the f i r s t g r o u p of e x p e r i m e n t s ; the MED of p e n t y l e n e t e t r a z o l w a s m e a s u r e d in the s e c o n d g r o u p . The f i r s t g r o u p c o n s i s t e d of t h r e e e x p e r i m e n t s and the s e c o n d g r o u p of two e x p e r i m e n t s , in e a c h of w h i c h f o u r s u b g r o u p s of 9 m i c e w e r e t r e a t e d .
Table 8 The influence of drugs given 21 to 96 hours e a r l i e r on the reaction time of mice in the hot plate test. premeditation phenobarbital
dose mg/kg 0.01 0.1 10 100 100 100 100
s.c. s.c. s.c. s.c. s.c. s.c. s.c.
interval in hrs
ratio with 95% interval
N
½ ½ 1 24 48 72 96
1.01(0.84-1.22) 1.12(0.99-1.27) 1.28(1.13-1.45) 1.14(0.98-1.32) 0.90(0.79-1.03) 0.88(0.76-1.02) 0.97(0.86-1.10)
80 80 80 80 80 80 80
methylphenobarbital
20 or. 200 or.
1 48
1.17(1.05-1.31) 1.09(0.98-1.21)
80 80
primidone
100 or. 100 or. 100 or.
1 24 48
0.99(0.82-1.20) 1.01(0.86-1.20) 1.01(0.86-1.17)
80 80 80
60 or. 60 or.
1 48
1.09(0.93-1.28) 80 1.09(0.95-1.26) ~80
500 s . c . 500 s.c.
24 48
0.98(0.83-1.15) 0.94(0.80-1.11)
80 80
50 s.c. 50 s.c.
24 48
1.18(1.05-1.32) 1.06(1.91-1.23)
80 80
methoin trimethadione phenytoin
Ratio: The ratio of the (geometric) mean hot plate r e action time in p r e t r e a t e d animals and the (geometric) mean reaction time in controls.
S u b g r o u p s I and II r e c e i v e d a ( c o n t r o l ) s a l i n e p r e t r e a t m e n t w h e n e t h i o n i n e w a s g i v e n to s u b g r o u p s III a n d IV. T h i r t y m i n u t e s l a t e r g r o u p s I a n d I I I w e r e i n j e c t e d w i t h s a l i n e and g r o u p s I I a n d IV w e r e t r e a t e d w i t h p h e n o b a r b i t a l . Two d a y s l a t e r the MED of b e m e g r i d e and p e n t y l e n e t e t r a z o l , r e s p e c t i v e l y , w a s d e t e r m i n e d in all 4 g r o u p s of 9 m i c e . The r e s u l t s of t h e e x p e r i m e n t s a r e g i v e n in t a b l e 9. A n a l y s i s of v a r i a n c e of t h e r e s u l t s of t h e s e e x p e r i m e n t s s h o w s , that the p r o c o n v u l s a n t eff e c t of p h e n o b a r b i t a l i s a b o l i s h e d by e t h i o n i n e pretreatment. After ethionine pretreatment, p h e n o b a r b i t a l , g i v e n 48 h o u r s e a r l i e r , e x e r t s a m a r k e d a n t i c o n v u l s a n t e f f e c t . C o m p a r i s o n of s u b g r o u p s I and III in the b e m e g r i d e e x p e r i m e n t s i n d i c a t e s , t h a t e t h i o n i n e a l o n e , g i v e n two d a y s earlier e n h a n c e d the e f f e c t of t h e c o n v u l s a n t d r u g s . In o t h e r ( u n p u b l i s h e d ) e x p e r i m e n t s t h i s find has been corroborated. 3.9. E x p e r i m e n t s in rats T a b l e l 0 s u m m a r i z e s t h e r e s u l t s of s o m e e x p e r i m e n t s in r a t s on t h e i n f l u e n c e of p h e n o b a r b i t a l on t h e M E D of b e m e g r i d e a n d p e n t y l e n e t e t r a zol. ( T h e s e e x p e r i m e n t s h a v e b e e n p e r f o r m e d in c o o p e r a t i o n w i t h M r . E. S l a g e r ) . One d o s e of
DECREASED SEIZURE THRESHOLD AFTER ANTICONVULSANTS
375
Table 9 The influence of ethionine on the proconvulsant effect of phenobarbital in mice. Mean MED with 95% interval 1st p r e t r e a t m e n t I II III IV
2nd p r e t r e a t m e n t
saline saline ethionine ethionine
saline phenobarbital saline phenobarbital
Ethionine (250 m g / k g s.c.) m g / k g s.c.). The MED's of l a t e r . They a r e e x p r e s s e d lution of pentylenetetrazol,
MED-ratio with 95% interval
dose mg/kg
interval in h r s
100 s.c.s.c. 100
72 96
0.95(0.89-1.01} 40 80 1.01(0.91-1.12)
phenobarbital
100 100 s.c.
48 72
0.94(0.81-1.11) 0.97(0.84-1.12)
30 28
phenobarbital
100 s.c. 100
48 72
0.90(0.78-1.04) 0.99(0.84-1.17)
24 20
N
bemegride 10
pentylenetetrazol
41.8(38.3-49.8) 33.5(30.7-36.6) 35.9(32.9-39.2) 41.1(37.7-44.9)
45.2(41.1-49.6) 39.5(35.9-43.4) 42.2(38.4-46.3) 54.4(49.5-59.9)
was given 30 min before phenobarbital a d m i n i s t r a t i o n (100 bemegride and pentylenetetrazol were determined 48 hours in 0.01 ml of a 0.1% solution of bemegride, and a 0.25070 sorespectively.
Table 10 The influence of phenobarbital on the MED of b e m e gride and pentylenetetrazol in rats. premedieation
bemegride
,0
.11,
pentylenetet×azol
] 1) In c o n t r a s t to the other e x p e r i m e n t s , in which only one phenobarbital dose was given, phenobarbital was a d m i n i s t e r e d here on 7 consecutive days. MED's were d e t e r m i n e d 48 or 72 h r s after the end of this treatment. p h e n o b a r b i t a l (100 m g / k g s . c . ) d i d n o t d e c r e a s e t h e M E D of b e m e g r i d e s i g n i f i c a n t l y , d e t e r m i n e d 2, 3 o r 4 d a y s a f t e r i t s a d m i n i s t r a t i o n . T h e M E D of p e n t y l e n e t e t r a z o l w a s not s i g n i f i c a n t l y d e creased when given 2-3 days after a single dose of p h e n o b a r b i t a l , nor after seven consecutive d a i l y d o s e s of p h e n o b a r b i t a l .
4. DISCUSSION In m i c e p h e n o b a r b i t a l , m e t h y l p h e n o b a r b i t a l , p r i m i d o n e a n d m e t h o i n h a v e b e e n s h o w n to d e crease t h e s e i z u r e t h r e s h o l d w h e n g i v e n two days before the administration of b e m e g r i d e , pentylenetetrazol o r d i o x o n e . In o u r e x p e r i m e n t s , a s i n g l e d o s e of p h e n y t o i n w a s f o u n d to h a v e a n a n t i c o n v u l s a n t e f f e c t i n t h e f i r s t two days after its administration; however, after
four days a decreasing effect upon the seizure t h r e s h o l d w a s o b s e r v e d in t h e C E D 5 0 e x p e r i ments with bemegride, pentylenetetrazol or dio x o n e , b u t n o t in t h e M E D - e x p e r i m e n t s with b e m e g r i d e o r p e n t y l e n e t e t r a z o l . An e x p l a n a t i o n f o r t h i s d i s c r e p a n c y h a s n o t b e e n found. T h e s e p r o c o n v u l s a n t e f f e c t s c o u l d b e o b s e r v e d in c h e m oshock-tests with three structurally different convulsants. The antiepileptics studied never inc r e a s e d t h e i n c i d e n c e of s e i z u r e s a f t e r a s u p r a m a x i m a l e l e c t r o s h o c k . Two d a y s a f t e r o n e d o s e of p h e n o b a r b i t a l (100 m g / k g s . c . ) t h e r a t i o of t h e L D 5 0 of b e m e g r i d e ( i . v . ) a f t e r p h e n o b a r b i t a l p r e t r e a t m e n t a n d the LD50 in c o n t r o l s w a s 0.64. T h u s , w i t h a two d a y i n t e r v a l , not o n l y t h e c o n v u l s a n t e f f e c t of b e m e g r i d e b u t a l s o i t s l e t h a l e f f e c t is e n h a n c e d ( u n p u b l i s h e d e x p e r i m e n t s ) . In r a t s p h e n o b a r b i t a l d i d not i n c r e a s e t h e c o n v u l s a n t e f f e c t of b e m e g r i d e o r p e n t y l e n e t e t r a z o l w h e n g i v e n two o r t h r e e d a y s a f t e r i t s a d m i n i s tration. The reason for this difference between t h e r e a c t i o n of r a t s a n d m i c e i s a s y e t o b s c u r e . T h e n a t u r e of t h e p r o c o n v u l s a n t e f f e c t in m i c e c a n n o t a s y e t b e e x p l a i n e d . S m a l l d o s e s of p h e n o b a r b i t a l a r e k n o w n to h a v e a s t i m u l a t o r y e f f e c t , t h e e x i s t e n c e of w h i c h c o u l d b e c o n f i r m e d in s o m e of o u r e x p e r i m e n t s . Moreover, in t h e MED-experiments, o n e d o s e of 20 m g / k g p h e n y t o i n w a s f o u n d to i n c r e a s e t h e c o n v u l s a n t a c t i o n of b e m e g r i d e . A s i m i l a r o b s e r v a t i o n w a s m a d e b y N i c h o l l s (1961). N e v e r t h e l e s s , t h e p r o c o n v u l s a n t e f f e c t s in t h e e x p e r i m e n t s d e s c r i b e d h e r e cannot be completely explained by this stimulat o r y e f f e c t of a s m a l l d o s e . One d a y a f t e r 100 mg/kg phenobarbital there is still a marked ant i c o n v u l s a n t e f f e c t . It i s u n l i k e l y t h a t two, t h r e e a n d f o u r d a y s a f t e r a d m i n i s t r a t i o n of t h i s d o s e the blood level remains between the narrow limits at which a proconvulsant (stimulatory) effect is exerted. T h e h o t p l a t e t e s t w a s p e r f o r m e d to i n v e s t i -
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g a t e , w h e t h e r d e c r e a s e d r e a c t i o n t i m e s , 48 h r s after giving phenobarbital and the other drugs r e f l e c t e d s o m e k i n d of g e n e r a l e x c i t a t i o n of t h e a n i m a l s . No s u c h e f f e c t w a s o b s e r v e d . Phenobarbital is known to increase the activity of s o m e l i v e r m i c r o s o m a l e n z y m e s y s t e m s i n v o l v e d in t h e m e t a b o l i s m of m a n y d r u g s , a n d e t h i o n i n e i s k n o w n to p r e v e n t t h i s i n c r e a s e d a c t i v i t y if g i v e n s h o r t l y b e f o r e p h e n o b a r b i t a l . Therefore, some experiments with ethionine w e r e d o n e to d i s c o v e r w h e t h e r t h e e n h a n c e d s e n s i t i v i t y to b e m e g r i d e a n d p e n t y l e n e t e t r a z o l , two days after phenobarbital administration, is corr e l a t e d w i t h t h e i n d u c t i o n of e n h a n c e d l i v e r m i crosomal enzyme activity. These experiments s h o w e d t h a t the p r o c o n v u l s a n t e f f e c t of p h e n o b a r b i t a l w a s a b o l i s h e d by e t h i o n i n e g i v e n 30 m i n earlier. After ethionine pretreatment phenobarbital still exerts a marked anticonvulsant effect for bemegride and pentylenetetrazol, when this i s t e s t e d two d a y s l a t e r . M o r e o v e r , two d a y s a f t e r e t h i o n i n e a l o n e t h e s e n s i t i v i t y of m i c e to t h e s e c o n v u l s a n t s i s e n h a n c e d . H o w e v e r , t h e way in w h i c h i n c r e a s e d l i v e r m i c r o s o m a l e n z y m e a c t i v i t y m i g h t c a u s e a n i n c r e a s e d e f f e c t of b e m e g ride, pentylenetetrazol and dioxone remains obs c u r e . If the m e t a b o l i s m of t h e c o n v u l s a n t s were accelerated by phenobarbital pretreatment, a d e c r e a s e i n s t e a d of a n i n c r e a s e in c o n v u l s a n t e f f e c t s w o u l d b e o b s e r v e d . M o r e o v e r , i t is q u e s t i o n a b l e w h e t h e r a n e f f e c t o n t h e r a t e of m e t a b o l i s m of the c o n v u l s a n t s w o u l d m a n i f e s t i t s e l f in the short period (10-120 sec) between the admini s t r a t i o n of t h e d r u g s a n d t h e m o m e n t w h e n t h e animals show their first convulsion. Another imp o r t a n t p o i n t of i n t e r e s t in t h i s c o n n e c t i o n i s t h a t p h e n y t o i n (50 m g / k g s . c . ) s h o r t e n s the hexobarb i t a l s l e e p i n g t i m e , w h e n t e s t e d two o r t h r e e d a y s a f t e r i t s a d m i n i s t r a t i o n , b u t not a f t e r f o u r d a y s ( R i i m k e , 1963). H o w e v e r , t h e p r o c o n v u l s a n t e f f e c t of p h e n y t o i n a p p e a r e d o n l y a f t e r 4 d a y s in these experiments. A c e n t r a l o r i g i n of t h e i n c r e a s e d s e n s i t i v i t y to b e m e g r i d e , p e n t y l e n e t e t r a z o l a n d d i o x o n e i s q u e s t i o n a b l e a s t h e p r o c o n v u l s a n t e f f e c t of t h e a n t i e p i l e p t i c s w a s o b s e r v e d only in c h e m o s h o c k t e s t s a n d not in t h e e l e c t r o s h o c k - t e s t u s e d . The question arises, whether the observed p r o c o n v u l s a n t e f f e c t of t h e s e a n t i e p i l e p t i c s is c o n n e c t e d w i t h t h e p r o b l e m of t h e c o n v u l s i o n s , s o m e t i m e s o b s e r v e d i n e p i l e p t i c p a t i e n t s one o r m o r e d a y s a f t e r a b r u p t t e r m i n a t i o n of a t r e a t ment with an antiepileptic drug.
ACKNOWLEDGEMENT The author is much indebted to Miss H . A . K u i p e r s (Department of Medical Statistics of the F r e e U n i v e r sity) for advice and cooperation in the statistical analysis of the data. and to M r . C . B r o s k y and Mr. H . V e r beek for very careful technical a s s i s t e n c e .
REFERENCES Conney. A. H. and J, J. Burns, 1960, Biochemical p h a r macological considerations of zoxazolamine and ehlorzoxazone metabolism, Ann. N. Y. Acad. Sci. 86 167. Conney, A.H., C.Davison, R . G a s t e l and J . J . Burns. 1960, Adaptive i n c r e a s e s in drug-metabolizing enzymes induced by phenobarbital and other drugs, J. P h a r m . Exp. T h e r . 130, 1. Cucinell, S.A., A.H. Conney M. Sansur and J . J . Burns, 1965~ Drug interactions in man, Clin. P h a r macol. T h e r . 6, 240. Eddy, N.B. and D. Leimbach, 1953, Synthetic analgesics. II. Dithienyl butenyl- and dithienylbutylamines. J. P h a r m . Exp. T h e r . 107, 385. Fingl. E. and D.G. McQuarrie. 1960, Evaluation of variables for the "timed intravenous infusion" p r o cedure for pentylenetetrazol s e i z u r e s in mice. Arch. Intern. Pharmacodyn. 126, 17. De Jongh, D.K.. 1957, An apparatus for the induction of s u p r a m a x i m a l electroshock s e i z u r e s in groups of ten mice, Acta Physiol. P h a r m a c o l . Neerl. 6, 511. Kato, R., 1961. Development of t o l e r a n c e to m e p r o b a mate: tentative enzymatic interpretation, Neuropharmacology 2. 57. Maffii, G. and M . G . S e r r a l u n g a , 1961, A new analeptic: 5 . 5 - d i e t h y l - l . 3 - o x a z i n - 2 ~ 4 - d i o n e (Dioxone) If. J. Pharmacy Pharmacol. 13, 309. Maffii. G.. V.M.DezulianandB. Silvestrini, 1961, A new ana]eptic 5,5-diethyl-l,3-oxazin-2.4-dione(Dioxone) I. J. Pharmacy Pharmacol. 13, 244. Maffii. G., G. Bianchi, P. Schiatti and B.Silvestrini, 1961, Action of 5,5- diethyl- 1,3- oxazin-2,4-dione (Dioxone) on respiration and circulation, Brit. J. Pharmacol. 16, 231. Nicholls. P . J . , 1961, Pharmacological properties of some ~ glutarimides, Arch. Int. Pharmacodyn. 133, 212. Riimke, Chr. L., 1959, The effect of tranquilizers on the convulsant effect of strychnine, pentylenetetrazol and aminopyrine, Acta Physiol. Pharmacol. Neerl. 8, 354, R~imke, Chr, L., 1961a, Beeinflussungder Krampfwirkung des Bemegrids, Naunyn-Schmiedebergs Archiv Exp. Path, Pharmakol, 241, 511. Riimke, Chr, L,, 1961b, The influence of drugs given 1-72 hours previously upon hexobarbital-narcosis and upon bemegride-induced convulsions in mice, Biochem. Pharmacol. 8, 48. Riimke, Chr. L., 1961c, An efficient design for comparing the effects of two treatments in: H.de Jonge, Quantitative methods in pharmacology (North-Holland Publishing Company, Amsterdam) p. 41.
DECREASED SEIZURE THRESHOLD A F T E R ANTICONVULSANTS R(imke. C h r . L., 1962. E n h a n c i n g and d e c r e a s i n g eff e c t s of d r u g s on the c o n v u l s a n t action of b e m e g r i de. Acta P h y s i o l . P h a r m a c o l . N e e r l . 10. 288. R0:mke. C h r . L., 1963, The influence of d r u g s on the d u r a t i o n of h e x o b a r b i t a l and h y d r o x y d i o n e n a r c o s i s in m i c e , N a u n y n - S c h m i e d e b e r g s A r c h i v Exp. Path. P h a r m a k o l . 244, 519.
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R[imke. C h r . L. and J. Bout. 1960, Die B e e i n f l u s s u n g d e r H e x o b a r b i t a l n a r k o s e d u r c h v o r h e r verabfolgte Pharmaka. N a u n y n - S c h m i e d e b e r g s A r c h i v Exp. Path. P h a r m a k o l . 240. 218. Swinyard. E . A . , N . R a d h a k r i s h n a n and L . S . G o o d m a n . 1962. Effect of b r i e f r e s t r a i n t on the convulsive t h r e s h o l d in m i c e . J. P h a r m . Exp, T h e r . 138. 337.
NORTH-HOLLAND PUBL. COMP.. AMSTERDAM
INCREASED SUSCEPTIBILITY OF MICE TO SEIZURES AFTER SOME ANTICONVULSANT DRUGS CHR. L. R U M K E Department o f Pharmacology and Neurobiochemistry, F r e e University, A m s t e r d a m , The Netherlands Accepted 12 May 1967 Chr. L. ROMKE, Increased susceptibility of mice to s e i z u r e s a f t e r some anticonvulsant drugs, European J. P h a r m a c o l . 1 (1967) 369-377. The effects of some antiepileptic drugs given one to four days beforehand, on the convulsant effects of bemegride, pentylenetetrazol and dioxone in mice were studied. Phenobarbital, methylphenobarbital, primidone and methoin were shown to enhance the effect of these convulsants, when given two days before t h e i r administration. A single dose of phenytoin was found to have an anticonvulsant effect in the f i r s t two days; a f t e r four days the CED50 of bemegride, pentylenetetrazol and dioxone was diminished. These enhanced convulsant effects after antiepileptics could be observed in c h e m o s h o c k - t e s t s with t h r e e s t r u c t u r a l l y different convulsants; however, they were not parallelled by an increased sensitivity to a s u p r a m a x i m a l electroshock. In r a t s phenobarbital did not i n c r e a s e the convulsant effect of bemegride or pentylenetetrazol, given two or t h r e e days l a t e r . The nature of the observed effects in mice could not be elucidated.
1. I N T R O D U C T I O N M a n y d r u g s a r e k n o w n to p r o l o n g t h e d u r a t i o n of h e x o b a r b i t a l o r h y d r o x y d i o n e n a r c o s i s in m i c e or rats when given shortly before the adminis t r a t i o n of t h e s e a n e s t h e t i c s . H o w e v e r , s e v e r a l of t h e s e d r u g s s h o r t e n t h e d u r a t i o n of t h e n a r c o sis when the time interval between the adminis t r a t i o n i s a b o u t two d a y s ( s e e , a m o n g s t o t h e r s , RiJmke a n d B o u t , 1960; R i i m k e , 1963). Similar effects with other drugs have been described by various authors: Conney and Burns (1960), a n d C o n n e y e t al. (1960) s h o w e d t h a t t h e d u r a t i o n of z o x a z o l a m i n e p a r a l y s i s in r a t s i s shortened by pretreatment with phenobarbital, phenylbutazone and orphenadrine. K a t o (1961) o b s e r v e d t h a t p r e m e d i c a t i o n of r a t s w i t h p h e n o barbital or meprobamate, one or two days beforehand, decreases meprobamate paralysis t i m e . I n b o t h c a s e s t h e a c t i v i t y of t h e l i v e r m i crosomal enzyme system metabolizing the drug w a s f o u n d to b e e n h a n c e d . C h r o n i c t r e a t m e n t of experimental animals with phenobarbital was s h o w n b y C u c i n e l l e t al. ( 1 9 6 5 ) t o c a u s e a c c e l e r a t e d m e t a b o l i s m of b i s h y d r o x y c o u m a r i n , diphenylhydantoin and antipyrin resulting in dim i n i s h e d e f f e c t s of t h e s e d r u g s . In t h e w o r k d e s c r i b e d here, antiepileptic
d r u g s w e r e g i v e n to m i c e a t d i f f e r e n t t i m e s b e f o r e t h e a d m i n i s t r a t i o n of b e m e g r i d e , p e n t y l e n e tetrazol and dioxone (see Maffii, Dezulian and S i l v e s t r i n i , 1961; M a f f i i a n d S e r r a l u n g a , 1961; M a f f i i , B i a n c h i , S c h i a t t i a n d S i l v e s t r i n i , 1961) to s e e w h e t h e r t h e t i m e i n t e r v a l i n f l u e n c e d t h e a c t i o n of t h e a n t i e p i l e p t i c s o n t h e c o n v u l s a n t e f f e c t of t h e s e d r u g s . T h e s a m e a n t i e p i l e p t i c s w e r e g i v e n a t t i m e i n t e r v a l s f r o m + 1 h o u r to s e v e r a l d a y s b e f o r e h a n d to s e e w h e t h e r t h e t i m e of a d m i n i s t r a t i o n i n f l u e n c e d t h e i r e f f e c t on t h e i n c i d e n c e of m a x i m a l e l e c t r o s h o c k s e i z u r e s i n m i c e , a n d on t h e r e a c t i o n - t i m e of m i c e in t h e hot p l a t e t e s t . In a d d i t i o n s o m e e x p e r i m e n t s w e r e d o n e o n r a t s to s t u d y t h e e f f e c t of p h e n o b a r b i t a l o n t h e i r s e n s i t i v i t y to b e m e g r i d e a n d p e n t y l e n e tetrazol on the second and third day after its administration. Preliminary r e p o r t s of s o m e of these experiments have been given (Rtimke, 1961a, 1961b, 1962).
2. M A T E R I A L S AND M E T H O D S M a l e a n d f e m a l e m i c e of t h e i n b r e d C P B - N s t r a i n w e r e u s e d in a l l e x p e r i m e n t s . The anim a l s in a n y o n e e x p e r i m e n t w e r e a l w a y s of t h e same sex and their weight range did not exceed
370
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L. RIIMKE
6 g. All e x p e r i m e n t s w e r e p e r f o r m e d at 23oc. P h e n o b a r b i t a l - s o d i u m , t r i m e t h a d i o n e , phenyt o i n - s o d i u m and ethionine w e r e i n j e c t e d subcutaneously. P r i m i d o n e (Mysoline (R); I m p e r i a l C h e m i c a l I n d u s t r i e s Ltd.), m e t h y l p h e n o b a r b i t a l , me t ho i n and p h e n a c e m i d e w e r e given by g a v a g e * . The drugs to be i n j e c t e d and those to be given by mouth w e r e d i s s o l v e d o r suspended in n o r m a l s a l i n e so that each a n i m a l r e c e i v e d 0.2 m l p e r 10 g body weight. In each e x p e r i m e n t c o n t r o l s w e r e t r e a t e d s i m u l t a n e o u s l y with the s a m e v o l u m e of s al i n e, a d m i n i s t e r e d by the s a m e route as the drug to be studied. The activity of the convulsant a g e n t s a f t e r p r e m e d i c a f i o n and in c o n t r o l a n i m a l s was m e a s u r e d by d e t e r m i n i n g the CED50 and the MED. The f o r m e r value was defined in t h e s e e x p e r i m e n t s as the dose of the convulsant agent which c a u s e s at l e a s t a clonic convulsion in 50%of the a n i m a l s a f t e r an i n t r a v e n o u s injection lasting 5 se c into one of the tail veins. B e m e g r i d e (Megimide (R); A s p r o Nicholas (Ltd.), p e n t y l e n e t e t r a zol (Cardiazol (R)) and dioxone ( 5 , 5 - d i e t h y l - l , 3 o x a z i n - 2 , 4 - d i o n e ; Lepetit) w e r e diluted with s a line to give solutions containing 0.5 m g / m l b e m e g r i d e , 1.5 m g / m l p e n t y l e n e t e t r a z o l and 0.75 m g / ml dioxone. Convulsions w e r e o b s e r v e d 5-15 s e c a f t e r injecting the convulsant. The d e t e r m i n a t i o n of the CED50's in d r u g - a n d s a l ' n e p r e t r e a t e d a n i m a l s took p l a c e following a " s e m i - s e q u e n t i a l " design. A d e t a i l e d account of this method has been given e l s e w h e r e (Rtimke, 1961c). The d e sign and the s t a t i s t i c a l a n a l y s i s d e s c r i b e d in e x a m p l e 3 of the quoted publication was applied and the r a t i o of the CED50 in drug p r e t r e a t e d and in c o n t r o l a n i m a l s was calculated. An a n t i c o n v u l sant effect is r e f l e c t e d by a C E D 5 0 - r a t i o s i g n i f i cantly g r e a t e r than 1; a d e c r e a s i n g effect upon the s e i z u r e t h r e s h o l d is shown by a C E D 5 0 - r a t i o which is l e s s than 1. This d e c r e a s i n g effect is r e f e r r e d to as a ' p r o c o n v u l s a n t e f f e c t' throughout this paper. In each e x p e r i m e n t of this kind at l e a s t 20 d r u g - and 20 s a l i n e - p r e t r e a t e d a n i m a l s w e r e i n j e c t e d with one of the convulsant agents in di f f eren t doses. Most of the g i v e n C E D s 0 - r a tios have been obtained by a v e r a g i n g the r e s u l t s of two e x p e r i m e n t s , p e r f o r m e d on d if f e r e n t days. In all tables the n u m b e r of a n i m a l s (N) r e f e r s to the total n u m b e r of p r e t r e a t e d and c o n t r o l m i c e , which w e r e used in the e x p e r i m e n t s . In the s t a t i s t i c a l t e s t s 5% (two-sided) was chosen as the l e v e l of s i g n i f i c a n c e . The m i n i m a l e f f e c t i v e dose (MED) of the c o n * Thanks are due to the mentioned firms for supplying their drugs used in this experiment.
v u l san t agents was defined in t h e s e e x p e r i m e n t s as the m i n i m a l dose which c a u s e d at l e a s t a clonic convulsion with continuous i n t r a v e n o u s inj e c t i o n into one of the t a i l v e i n s at a constant r a t e of 0.30 ml p e r min f r o m a 1%o solution of b e m e g r i d e , a 2.5%o solution of p e n t y l e n e t e t r a z o l , or a 0.75%0 solution of dioxone. It was m e a s u r e d with a t u b e r c u l i n - s y r i n g e a c c u r a t e to 0.01 ml. In each e x p e r i m e n t the MED was obtained in 10 d r u g - and in 10 s a l i n e - p r e t r e a t e d a n i m a l s . Most of these e x p e r i m e n t s w e r e r e p e a t e d once o r twice on d i f f e r e n t days. Analysis of v a r i a n c e ( r a n d o m i z e d blocks) t e c h n i q u e s w e r e applied in the s t a t i s t i c a l a n a l y sis of the r e s u l t s of the M E D - e x p e r i m e n t s . The s t a t i s t i c a l a n a l y s i s was always b a s e d upon the l o g a r i t h m s of the o b s e r v a t i o n s . The r e s u l t s of the M E D - e x p e r i m e n t s have finally been e x p r e s s ed in the M E D - r a t i o (and its 95% confidence l i m its), which is the r a t i o of the ( g e o m e t r i c ) m e a n MED in p r e t r e a t e d a n i m a l s and the ( g e o m e t r i c ) m e a n MED in the c o n t r o l s (cf. Fingl and Mc Q u a r r i e , 1960; Swinyard et al., 1962). An an t i convulsant effect is sh o w n i n t h e s e t e s t s when the M E D - r a t i o e x c e e d s 1; a ratio, which is s m a l l e r than 1 i n d i cat es an e n h a n c e m e n t of c o n v u l s a n t ef f e c t (a p r o c o n v u l s a n t effect). An M E D - r a f i o is concluded to differ s i g n i f i c a n t l y f r o m 1 if its 95% confidence i n t e r v a l does not contain 1. The influence of d i f f e r e n t p r e t r e a t m e n t on the i n ci d en ce of s u p r a m a x i m a l e l e c t r o s h o c k s e i z u r e s was t e s t e d with an ap p ar at u s d e s c r i b e d by De Jongh (1957) which allows 10 m i c e to be t r e a t e d s i multaneously. An e l e c t r i c a l s t i m u l u s of 0.2 sec duration was given. The c u r r e n t was such that about 50% of the c o n t r o l a n i m a l s showed the e x t e n s o r component of a s e i z u r e . E a c h e x p e r i m e n t was p e r f o r m e d upon 4 groups of 5 + 5 d r u g - and s a l i n e - p r e t r e a t e d animals. Most experiments w e r e r e p e a t e d once with d i f f e r e n t a n i m a l s on a d i f f er en t day. The p e r c e n t a g e s g i v en in t a b l e s 6 and 7 r e f e r to the d i f f e r e n c e b et w een the p e r centage of all p r e t r e a t e d a n i m a l s r e a c t i n g with an e x t e n s o r s e i z u r e and the p e r c e n t a g e of r e acting c o n t r o l s . S t a t i s t i c a l s i g n i f i c a n c e of the d i f f e r e n c e between t h ese two p e r c e n t a g e s was d e t e r m i n e d in a c o m b i n e d 2 × 2 - t a b l e t e s t on the r e s u l t s f r o m all s e p a r a t e g r o u p s of 5 + 5 m i ce. A slight m o d i f i c a t i o n of E d d y ' s hot plate method (Eddy and L e i m b a c h , 1953) was u sed , to d e t e r m i n e the r e a c t i o n t i m e s in d r u g - and s a l i n e - p r e t r e a t e d animals. Most hot p l at e e x p e r i m en t s w e r e p e r f o r m e d twice on d i f f e r e n t days upon groups of m i c e which s h o r t l y a f t e r w a r d s w e r e to be t r e a t e d with a c h e m o - o r an e l e c t r o shock. The t e m p e r a t u r e of the plate was 56oc.
DECREASED SEIZURE THRESHOLD AFTER ANTICONVULSANTS T h e m e a s u r e m e n t s in d r u g - a n d s a l i n e - p r e t r e a t e d a n i m a l s w e r e m a d e in r a n d o m o r d e r . T h e o b s e r v e r d i d not k n o w w h e t h e r a n a n i m a l h a d b e e n given a drug or a saline premedication. The m e a s u r e d r e a c t i o n t i m e is the t i m e e l a p s i n g b e tween the f i r s t contact with the plate and the f i r s t l i c k i n g r e s p o n s e . In c o n t r o l s t h e a v e r a g e r e a c t i o n t i m e w a s a p p r o x i m a t e l y 10 s e c at t h e m o s t . T h e r e s u l t s of hot p l a t e r e a c t i o n t i m e d e t e r m i n a t i o n s in s e c f r o m 44 e x p e r i m e n t s w i t h 20 m i c e e a c h s h o w e d t h a t the d i s t r i b u t i o n of t h e r e a c t i o n t i m e s i s not n o r m a l . H o w e v e r , t h e d i s t r i b u t i o n of t h e l o g a r i t h m s of t h e s e r e a c t i o n t i m e s a p p r o x i m a t e s the n o r m a l d i s t r i b u t i o n v e r y w e l l . T h e r e f o r e t h e s t a t i s t i c a l a n a l y s i s of t h e r e s u l t s of t h e s e e x p e r i m e n t s w a s a l w a y s b a s e d on t h e l o g a r i t h m s of t h e r e a c t i o n t i m e s . T h e r e a c t i o n t i m e r a t i o i s t h e r a t i o of t h e ( g e o m e t r i c ) m e a n r e a c t i o n t i m e in p r e t r e a t e d a n i m a l s a n d t h e ( g e o m e t r i c ) m e a n r e a c t i o n t i m e in t h e c o n t r o l s . T h e d r u g s t u d i e d i s c o n c l u d e d to p r o l o n g t h e r e a c t i o n t i m e s i g n i f i c a n t l y if t h e r e a c t i o n t i m e r a t i o e x c e e d s 1, a n d if i t s 95% c o n f i d e n c e i n t e r v a l d o e s not c o n t a i n 1. T h e M E D - e x p e r i m e n t s in r a t s w e r e c a r r i e d out on m a l e a n d f e m a l e a n i m a l s of t h e i n b r e d TNO-WU-strain, weighing 150-200 grams. Bem e g r i d e a n d p e n t y l e n e t e t r a z o l w e r e g i v e n in a c o n t i n u o u s i n t r a v e n o u s i n f u s i o n into one of the t a i l v e i n s at a c o n s t a n t r a t e of 1 m l p e r m i n f r o m a 2.5 m g / m l , a n d a 7.5 m g / m l o r 3.75 m g / m l solution, respectively. T h e a n a l y s i s of v a r i a n c e of the r e s u l t s of t h e M E D - a n d hot p l a t e e x p e r i m e n t s w a s d o n e w i t h t h e h e l p of o n e of t h e E l e c t r o l o g i c a E L - X 1 o r E L - X 8 c o m p u t o r s of the M a t h e m a t i c a l C e n t r e a t Amsterdam.
Table 1 The influence of drugs given ½ to 120 hours ,beforehand on the CED50 of bemegride in mice. premedieation phenobarbital
primidone
100 100 100 100
methoin
s.c. s.c. s.c. s.c.
s.c. s.c. s.c. s.c. s.c. s.c. or. or. or. I or.
60 or. 60 or. 60 or. 60 or.
interval CED50 sig~ifin h r s ratio icance a~ ½ 1 2 12 24 48 72 96 120
0.98 0.86 1.10 1.30 4.20 1.48 0.71 0.77 0.94 0.97
1 24 48 72
1.57 1.02 0.89 1.00
+++
1½ 24 48 72
1.57 1.14 0.85 1.02
++~
+*+ +
trimethadione
500 500 500 500
s.c. s.c. s.c. s.c.
1½ 24 48 72
>3.00 1.15 1.06 1.32
phenytoin
50 50 50 100 50 100 50 50
s.c. s.c. s.c. s.c. s.c. s.c. s.c. s.c.
1½ 24 48 48 72 72 96 120
1.01 1.46 1.00 1.16 1.09 0.94 0.87
1 24 48 48
0.95 1.16
phenaeemide
125 or. 125 or. 125 or. 500 or. +
++ ++÷
3.1. CED50-experirnents
do se mg/kg 0.01 0.1 1 10 100 100 100 100 100 100
sl nificanee: 3. E X P E R I M E N T S AND R E S U L T S
371
-, +~ +~
+ E+ + +
*
++
++ + +
1.02 1.80
1.05
+++
N 80 80 80 80 80 80 80 80 80 80 80 80 80 40 40 80 80 80 40 40 80 8o 80 80 40 4o 4o 4o 80 4o 4o 4o 4o 8o
P > 0.05 0.05 >~P > 0.01 0.01 >/P > 0.001 P ~< 0.001
on the convulsant a c -
tion o f bemegride T h e r e s u l t s of C E D 5 0 - e x p e r i m e n t s on t h e e f f e c t s of s e v e r a l d r u g s on t h e c o n v u l s a n t a c t i v i t y of b e m e g r i d e a r e s h o w n in t a b l e 1. In t h e s e e x p e r i m e n t s the t i m e interval b e t w e e n p r e m e d i c a tion and intravenous bemegride administration r a n g e d f r o m ½ to 120 h o u r s . P h e n o b a r b i t a l (10 m g / k g ) , p r i m i d o n e , m e t h oin, t r i m e t h a d i o n e and p h e n a c e m i d e had an a n t i 1 1 c o n v u l s a n t e f f e c t , if g i v e n 5 t o 15 h o u r s b e f o r e b e m e g r i d e . Of t h e s e d r u g s p h e n o b a r b i t a l (100 mg/kg), primidone and methoin had a proconvulsant effect when the i n t e r v a l b e t w e e n p r e t r e a t m e n t a n d b e m e g r i d e a d m i n i s t r a t i o n w a s 48
h r s . T h r e e d a y s a f t e r a s i n g l e d o s e of p h e n o b a r b i t a l (100 m g / k g ) t h e c o n v u l s a n t e f f e c t of b e m e gride was still enhanced. No e f f e c t u p o n t h e CED50 of b e m e g r i d e w a s o b s e r v e d w h e n p h e n y t o i n (50 m g / k g ) w a s g i v e n 1½ h r s b e f o r e h a n d . H o w e v e r , one d a y a f t e r a s i n g l e p h e n y t o i n i n j e c t i o n of 50 m g / k g a n d e v e n t w o d a y s a f t e r a s i n g l e i n j e c t i o n of 100 m g / k g t h e CED50 of b e m e g r i d e w a s i n c r e a s e d . A s i g n i f i c a n t d e c r e a s e of t h e C E D 5 0 (a p r o c o n v u l s a n t e f f e c t ) w a s f o u n d a f t e r a 96 h r s i n t e r v a l b e t w e e n p h e n y t o i n a d m i n i s t r a t i o n (50 m g / k g ) and i n j e c t i o n of b e m e g r i d e . A r e l a t i v e l y s m a l l d o s e of p h e n o b a r b i t o n e (0.1
372
CHR. L. RUMKE
m g / k g ) i n c r e a s e d t h e c o n v u l s a n t e f f e c t of b e m e g r i d e g i v e n 30 m i n l a t e r .
on the convulsant action of pentylenetetrazol
Table 3 The influence of drugs given ½ to 96 hours beforehand on the CED50 of dioxone in mice.
3.2. CED50-experiments
T h e r e s u l t s of C E D s 0 - e x p e r i m e n t s on t h e i n f l u e n c e of p r e t r e a t m e n t w i t h s e v e r a l d r u g s on t h e c o n v u l s a n t e f f e c t of p e n t y l e n e t e t r a z o l a r e s h o w n in t a b l e 2. Table 2 The influence of drugs given ½ to 120 hours beforehand on the CED50 of pentylenetetrazol in mice. Jremedication phenobarbital
!primidone methoin phenytoin
dose mg/kg 0.1 10 100 100 100 100
interval CED50 signifratio icance in h r s
s.c. s.c. s.c. s.c. s.c. s.c.
½ 1 24 48 72 96
1.12 2.06 2.29 0.85 0.89
N
1.00
-
80 80 40 80 80 80
~++ + ~+ -
100 or. 100 or.
1 48
1.37 0.91
+ +
40 80
60 or. 60 or.
1½ 48
1.46 0.88
+ ++
40 80
1½ 1½
1.08 0.93 1.21 1.34 0.95 0.90 0.68 1.00
÷ ÷+÷ +~ -
40 80 40 40 80 40 80 40
50 100 50 100 50 50 50 50
s.c. s.c. s.c. s.c. s.c. s.c. s.c. s.c.
24 24 48 72 96 120
Phenobarbital (10 m g / k g ) , p r i m i d o n e a n d m e t h o i n i n c r e a s e d t h e CED50 of p e n t y l e n e t e t r a z o l , w h e n g i v e n 1-1½ h r s b e f o r e h a n d . W h e n p h e n y t o i n w a s a d m i n i s t e r e d 1½ h o u r b e f o r e p e n t y l e n e t e t r a z o l , no s u c h e f f e c t w a s s e e n , b u t o n e day a f t e r a p h e n y t o i n i n j e c t i o n of 50 m g / k g o r 100 m g / k g t h e CED50 w a s i n c r e a s e d . When the interval b e t w e e n a single p h e n o b a r b i t a l (100 m g / k g ) , p r i m i d o n e o r m e t h o i n p r e m e d i c a t i o n w a s two d a y s , the C E D 5 0 ' s of p e n t y l e n e t e t r a z o l w e r e d e c r e a s e d . The s a m e enhanced convulsant effect was observed with p h e n y t o i n , ff the i n t e r v a l w a s 96 h r s . P h e n o b a r b i t o n e (0.1 m g / k g ) d i d n o t i n c r e a s e t h e c o n v u l s a n t e f f e c t w h e n g i v e n 30 m i n b e f o r e pentylenetetrazol. 3.3. CED50-experiments
on the convulsant ac-
tion of dioxone T a b l e 3 s u m m a r i z e s t h e r e s u l t s of C E D 5 0 e x p e r i m e n t s on t h e i n f l u e n c e of s e v e r a l d r u g s on t h e c o n v u l s a n t a c t i o n of d i o x o n e . T h e C E D 5 0 ' s of
premedication
dose mg/kg
phenobarbital
0.1 10 100 100 100 100
primidone
s.c. s.c. s.c. s.c. s.c. s.c.
interval CED50 signifin h r s ratio
N
21 1 24 48 72 96
1.00 1.29 1.74 0.85 0.81 0.98
÷+ +++ 4+ +++ -
40 40 40 80 80 40
100 or. 100 or.
1 48
1.26 0.75
++ +
40 40
methoin
60 or. 60 or.
1 48
1.81 0.95
~+ +
40 80
phenytoin
50 s.c. 50 s.c.
24 96
1.70 0.86
+++ +
40 80
dioxone w e r e i n c r e a s e d by p r e t r e a t m e n t with p h e n o b a r b i t o n e (10 m g / k g ) , p r i m i d o n e a n d m e t h oin, g i v e n 1-1½ h r s e a r l i e r . When the interval between premedication with one d o s e of p h e n o b a r b i t o n e (100 m g / k g ) , p r i m i d o n e o r m e t h o i n w a s two d a y s , t h e C E D 5 0 ' s w e r e decreased. O n e d o s e of p h e n y t o i n , g i v e n o n e day e a r l i e r , had a m a r k e d anticonvulsant effect. However, four days after such a single dose the convulsant a c t i o n of d i o x o n e w a s e n h a n c e d . 3.4. E x p e r i m e n t s of the minimal effective dose (MED) of bemegride T h e r e s u l t s of t h e M E D - e x p e r i m e n t s on t h e i n f l u e n c e of s e v e r a l d r u g s on t h e c o n v u l s a n t e f f e c t of b e m e g r i d e a r e s h o w n in t a b l e 4. P h e n o b a r b i t a l (10 m g / k g ) , m e t h y l p h e n o b a r b i t a l (20 m g / k g ) , p r i m i d o n e , m e t h o i n a n d t r i m e t h a d i o n e i n c r e a s e d t h e M E D - r a f i o of b e m e g r i d e i.e. d e c r e a s e d i t s c o n v u l s a n t e f f e c t w h e n g i v e n 1 l ~ - 1 ~ h r s b e f o r e it. A s m a l l d o s e of p h e n o b a r b i t a l (0.1 m g / k g ) , a n d 20 m g / k g p h e n y t o i n d e c r e a s e d t h e M E D - r a tio significantly when given ½ hour b e f o r e b e m e g r i d e a d m i n i s t r a t i o n . In t h e s e d o s e s , t h e s e d r u g s m a y b e c o n c l u d e d to e n h a n c e t h e c o n v u l s a n t e f fect. T w o d a y s a f t e r a d m i n i s t r a t i o n of one l a r g e d o s e of p h e n o b a r b i t a l , m e t h y l p h e n o b a r b i t a l a n d p r i m i d o n e t h e c o n v u l s a n t e f f e c t of b e m e g r i d e w a s i n c r e a s e d . O n e day a f t e r a s i n g l e d o s e of 50 m g / k g p h e n y t o i n , a n d one a n d t w o d a y s a f t e r a s i n g l e d o s e of 100 m g / k g p h e n y t o i n t h e c o n v u l s a n t e f f e c t of b e m e g r i d e w a s s t i l l d e c r e a s e d . Two d a y s a f t e r a s i n g l e m e t h o i n , t r i m e t h a d i o n e or phenacemide pretreatment or four days after
D E C R E A S E D S E I Z U R E T H R E S H O L D A F T E R ANTICONVULSANTS Table 4 T h e i n f l u e n c e of d r u g s on t h e MED of b e m e g l : i d e in m i c e . MED-ratio with 95% interval
Table 5 The i n f l u e n c e of d r u g s on the MED of p e n t y l e n e t e t r a z o l and d i o x o n e in m i c e .
dose mg/kg
interval in h r s
0.1 s . c . 10 s . c . 100 s . c .
½ ½ 48
0.88(0.78-0.997) 40 1.37(1.19-1.58) 20 0.83(0.72-0.96) 40
methylphenobarbital
20 or. 200 or.
1 48
1.37(1.14-1.61) 0.83(0.70-0.97)
20 40
primidone
100 o r . 100 or.
1 48
1.37(1.12-1.68) 0.86(0.75-0.95)
20 60
60 or. 60 or.
11 48
1.78(1.43-2.22) 1.10(0.98-1.25)
20 60
s.c. s.c. s.c. s.c. s.c. s.c. s.c.
~1 24 24 48 48 96
0.85(0.77-0.93) 0.90(0.75-1.07) 1.28(1.14-1.44) 1.16(1.03-1.31) 1.02(0.87-1.18) 1.25(1.02-1.55) 0.99(0.85-1.15)
60 40 60 60 40 40 60
trimethadione
500 s . c . 500 s . c .
1½ 48
2.08{1.90-2.28) 1.00(0.76-1.31)
20 20
phenacemide
500 or.
48
0.88(0.76-1.02)
40
premedication phenobarbital
methoin phenytoin
20 50 50 100 50 100 50
phenytoin administration no significant t h e M E D of b e m e g r i d e w a s o b s e r v e d . 3.5.
E x p e r i m e n t s on the M E D ' s trazol and dioxone
N
effect on
of p e n t y l e n e t e -
The summary of the experiments on the MED's of pentylenetetrazol a n d d i o x o n e in t a b l e 5 s h o w s t h a t a s m a l l d o s e of p h e n o b a r b i t a l (0.1 mg/kg) does not significantly influence the MED's of pentylenetetrazol or dioxone. Methylphenobarbital (20 m g / k g ) , given 1 hour earlier, markedly inhibited the convulsant effects of pentylenetetrazol and dioxone. With pentylenetetrazol and dioxone one dose of phenobarbital (100 mg/kg) or methylphenobarbital (200 mg/kg) given 48 hours beforehand had a significant proconvulsant effect. O n e a n d t w o d a y s a f t e r 50 o r 1 0 0 m g / k g p h e n ytoin the convulsant effect of pentylenetetrazol was still reduced. No marked effect of phenytoin on the convulsant effect of pentylenetetrazol was observed after a four day interval. 3.6.
373
E x p e r i m e n t s on effects on the incidence of supramaximal electroshock seizures
Table 6 gives a summary of the results of the experiments on the influence of a single dose of some known antiepileptics given ½-96 hours earlier on the incidence of seizures after a supra-
premedication
dose mg/kg
interval in h r s
MED-ratio with 95% interval
N
pentylenetetrazol phenobarbital
! 0.1 s . c . 100 s . c .
½ 48
1.06(0.89-1.26) 0.80{0.70-0.91)
40 40
methylphenobarbital
20 or. 200 o r .
1 48
1.53(1.20-1.93) 0.79(0.69-0.90)
20 40
phenytoin
20 50 100 50 100 50
s.c. s.c. s.c. s.c. s.c. s.c.
½ 24 24 48 48 96
0.93(0.78-1.10) 1.32(1.13-1.55) 1.48(1.27-1.73) 1..19(1.02-1.39) 1.26(1.07-1.48) 1.07(0.95-1.21)
40 20 40 40 40 40
phenobarbital
0.1 s . c . 100 s . c .
½ 48
1.05(0.92-1.20) 0.81(0.69-0.95)
40 40
methylphenobarbital
20 or. 200 or.
1 48
1.40(1.18-1.65) 0.77(0.66-0.89)
40 40
dioxone
Table 6 The i n f l u e n c e of d r u g s g i v e n ½-96 h o u r s e a r l i e r on the i n c i d e n c e of s e i z u r e s a f t e r a s u p r a m a x i m a l e l e c t r o s h o c k in m i c e .
premedication phenobarbital
dose mg/kg
differe n%c e
N
-
80 80
35
+
40
Z-~-~
+++
2~ ½ 1 1 24 24 48 72 96
methylphenobarbital
20 s . c . 200 s . c .
1 48
-
primidone
100 or. 100 or.
1 48
-
60 o r . 60 or. 50 s . c . 100 s . c . 50 s . c .
phenytoin
significance
+12½
s.c. s.c. s.c. s.c. s.c. s.c. s.c. s.c. s.c.
methoin
0.01 0.1 10 50 50 100 100 100 100
interval in h r s
-
-
2½
40
+
+++ -
80 40 80 80
7~
-
80
12½
-
80
-
+
80
-
80
42½
+++
80
-
10
-
80
1 48
-
35
+++
80
-
15
-
80
24 48 96
-
65
++
40
-
35 0
~ -
80 80
25
-
2½
D i f f e r e n c e ~o: The d i f f e r e n c e b e t w e e n the p e r c e n t a g e of p r e t r e a t e d and of c o n t r o l a n i m a l s r e a c t i n g with a c o n v u l s i o n . S i g n i f i c a n c e : -: P > 0.05: +: P < 0.05; + + : P < 0.01; + + + : P < 0.001.
374
CHR. L. RUMKE
m a x i m a l e l e c t r o s h o c k . In t h i s t e s t one h o u r a f t e r phenobarbital, methylphenobarbital, primidone or methoin pretreatment a significant anticonvulsant effect was observed. This effect was also s e e n 24 h o u r s a f t e r p h e n o b a r b i t a l o r p h e n y t o i n a d m i n i s t r a t i o n . N o n e of t h e d r u g s s t u d i e d w a s f o u n d to i n c r e a s e t h e n u m b e r of c o n v u l s i o n s w h e n t h e i n t e r v a l b e t w e e n the a d m i n i s t r a t i o n a n d t h e e l e c t r o s h o c k t e s t w a s two d a y s . In t a b l e 7 t h e r e s u l t s a r e g i v e n of s o m e e x p e r i m e n t s in w h i c h t h e a n t i e p i l e p t i c s w e r e g i v e n on s i x c o n s e c u t i v e d a y s . T w o d a y s a f t e r t h e l a s t administration the electroshock test was perf o r m e d . No e f f e c t on t h e s e i z u r e t h r e s h o l d w a s o b s e r v e d in t h e s e e x p e r i m e n t s w i t h any of t h e drugs tested. Table 7 The incidence of s e i z u r e s after a supramaximal e l e c troshock in mice after six days of treatment with an anticonvulsant and two days of withdrawal. pretreatment phenobarbital phenobarbital primidone trimethadione phenytoin
daily dose mg/kg 50 75 50 250 50
s.c. s.c. or. s.c. s.c.
difference % -~ 16% ~ 5% 8% + 5% 8%
significance
N 100 50 50 40 50
Legend: see table 6.
3.7. E x p e r i m e n t s on the e f fe c t s on the hot plate
reaction time In t a b l e 8 t h e r e s u l t s of e x p e r i m e n t s on t h e i n f l u e n c e of s o m e d r u g s on t h e hot p l a t e r e a c t i o n t i m e h a v e b e e n s u m m a r i z e d . P h e n o b a r b i t a l (10 m g / k g ) a n d m e t h y l p h e n o b a r b i t a l (20 m g / k g ) g i v e n o n e h o u r b e f o r e t h e t e s t w e r e f o u n d to p r o l o n g t h e r e a c t i o n t i m e . One d a y a f t e r 50 m g / k g p h e n ytoin the reaction time was enhanced. These d r u g s h a d no e f f e c t in t h i s t e s t w h e n g i v e n two or more days earlier. 3.8. E x p e r i m e n l s with ethionine T h e i n f l u e n c e of e t h i o n i n e (250 m g / k g s . c . ) on t h e p r o c o n v u l s a n t e f f e c t of p h e n o b a r b i t a l w a s s t u d i e d in two g r o u p s of e x p e r i m e n t s . P h e n o b a r b i t a l (100 m g / k g s . c . ) w a s g i v e n 30 m i n a f t e r e t h i o n i n e a d m i n i s t r a t i o n . Two d a y s l a t e r the M E D of b e m e g r i d e w a s d e t e r m i n e d in the f i r s t g r o u p of e x p e r i m e n t s ; the MED of p e n t y l e n e t e t r a z o l w a s m e a s u r e d in the s e c o n d g r o u p . The f i r s t g r o u p c o n s i s t e d of t h r e e e x p e r i m e n t s and the s e c o n d g r o u p of two e x p e r i m e n t s , in e a c h of w h i c h f o u r s u b g r o u p s of 9 m i c e w e r e t r e a t e d .
Table 8 The influence of drugs given 21 to 96 hours e a r l i e r on the reaction time of mice in the hot plate test. premeditation phenobarbital
dose mg/kg 0.01 0.1 10 100 100 100 100
s.c. s.c. s.c. s.c. s.c. s.c. s.c.
interval in hrs
ratio with 95% interval
N
½ ½ 1 24 48 72 96
1.01(0.84-1.22) 1.12(0.99-1.27) 1.28(1.13-1.45) 1.14(0.98-1.32) 0.90(0.79-1.03) 0.88(0.76-1.02) 0.97(0.86-1.10)
80 80 80 80 80 80 80
methylphenobarbital
20 or. 200 or.
1 48
1.17(1.05-1.31) 1.09(0.98-1.21)
80 80
primidone
100 or. 100 or. 100 or.
1 24 48
0.99(0.82-1.20) 1.01(0.86-1.20) 1.01(0.86-1.17)
80 80 80
60 or. 60 or.
1 48
1.09(0.93-1.28) 80 1.09(0.95-1.26) ~80
500 s . c . 500 s.c.
24 48
0.98(0.83-1.15) 0.94(0.80-1.11)
80 80
50 s.c. 50 s.c.
24 48
1.18(1.05-1.32) 1.06(1.91-1.23)
80 80
methoin trimethadione phenytoin
Ratio: The ratio of the (geometric) mean hot plate r e action time in p r e t r e a t e d animals and the (geometric) mean reaction time in controls.
S u b g r o u p s I and II r e c e i v e d a ( c o n t r o l ) s a l i n e p r e t r e a t m e n t w h e n e t h i o n i n e w a s g i v e n to s u b g r o u p s III a n d IV. T h i r t y m i n u t e s l a t e r g r o u p s I a n d I I I w e r e i n j e c t e d w i t h s a l i n e and g r o u p s I I a n d IV w e r e t r e a t e d w i t h p h e n o b a r b i t a l . Two d a y s l a t e r the MED of b e m e g r i d e and p e n t y l e n e t e t r a z o l , r e s p e c t i v e l y , w a s d e t e r m i n e d in all 4 g r o u p s of 9 m i c e . The r e s u l t s of t h e e x p e r i m e n t s a r e g i v e n in t a b l e 9. A n a l y s i s of v a r i a n c e of t h e r e s u l t s of t h e s e e x p e r i m e n t s s h o w s , that the p r o c o n v u l s a n t eff e c t of p h e n o b a r b i t a l i s a b o l i s h e d by e t h i o n i n e pretreatment. After ethionine pretreatment, p h e n o b a r b i t a l , g i v e n 48 h o u r s e a r l i e r , e x e r t s a m a r k e d a n t i c o n v u l s a n t e f f e c t . C o m p a r i s o n of s u b g r o u p s I and III in the b e m e g r i d e e x p e r i m e n t s i n d i c a t e s , t h a t e t h i o n i n e a l o n e , g i v e n two d a y s earlier e n h a n c e d the e f f e c t of t h e c o n v u l s a n t d r u g s . In o t h e r ( u n p u b l i s h e d ) e x p e r i m e n t s t h i s find has been corroborated. 3.9. E x p e r i m e n t s in rats T a b l e l 0 s u m m a r i z e s t h e r e s u l t s of s o m e e x p e r i m e n t s in r a t s on t h e i n f l u e n c e of p h e n o b a r b i t a l on t h e M E D of b e m e g r i d e a n d p e n t y l e n e t e t r a zol. ( T h e s e e x p e r i m e n t s h a v e b e e n p e r f o r m e d in c o o p e r a t i o n w i t h M r . E. S l a g e r ) . One d o s e of
DECREASED SEIZURE THRESHOLD AFTER ANTICONVULSANTS
375
Table 9 The influence of ethionine on the proconvulsant effect of phenobarbital in mice. Mean MED with 95% interval 1st p r e t r e a t m e n t I II III IV
2nd p r e t r e a t m e n t
saline saline ethionine ethionine
saline phenobarbital saline phenobarbital
Ethionine (250 m g / k g s.c.) m g / k g s.c.). The MED's of l a t e r . They a r e e x p r e s s e d lution of pentylenetetrazol,
MED-ratio with 95% interval
dose mg/kg
interval in h r s
100 s.c.s.c. 100
72 96
0.95(0.89-1.01} 40 80 1.01(0.91-1.12)
phenobarbital
100 100 s.c.
48 72
0.94(0.81-1.11) 0.97(0.84-1.12)
30 28
phenobarbital
100 s.c. 100
48 72
0.90(0.78-1.04) 0.99(0.84-1.17)
24 20
N
bemegride 10
pentylenetetrazol
41.8(38.3-49.8) 33.5(30.7-36.6) 35.9(32.9-39.2) 41.1(37.7-44.9)
45.2(41.1-49.6) 39.5(35.9-43.4) 42.2(38.4-46.3) 54.4(49.5-59.9)
was given 30 min before phenobarbital a d m i n i s t r a t i o n (100 bemegride and pentylenetetrazol were determined 48 hours in 0.01 ml of a 0.1% solution of bemegride, and a 0.25070 sorespectively.
Table 10 The influence of phenobarbital on the MED of b e m e gride and pentylenetetrazol in rats. premedieation
bemegride
,0
.11,
pentylenetet×azol
] 1) In c o n t r a s t to the other e x p e r i m e n t s , in which only one phenobarbital dose was given, phenobarbital was a d m i n i s t e r e d here on 7 consecutive days. MED's were d e t e r m i n e d 48 or 72 h r s after the end of this treatment. p h e n o b a r b i t a l (100 m g / k g s . c . ) d i d n o t d e c r e a s e t h e M E D of b e m e g r i d e s i g n i f i c a n t l y , d e t e r m i n e d 2, 3 o r 4 d a y s a f t e r i t s a d m i n i s t r a t i o n . T h e M E D of p e n t y l e n e t e t r a z o l w a s not s i g n i f i c a n t l y d e creased when given 2-3 days after a single dose of p h e n o b a r b i t a l , nor after seven consecutive d a i l y d o s e s of p h e n o b a r b i t a l .
4. DISCUSSION In m i c e p h e n o b a r b i t a l , m e t h y l p h e n o b a r b i t a l , p r i m i d o n e a n d m e t h o i n h a v e b e e n s h o w n to d e crease t h e s e i z u r e t h r e s h o l d w h e n g i v e n two days before the administration of b e m e g r i d e , pentylenetetrazol o r d i o x o n e . In o u r e x p e r i m e n t s , a s i n g l e d o s e of p h e n y t o i n w a s f o u n d to h a v e a n a n t i c o n v u l s a n t e f f e c t i n t h e f i r s t two days after its administration; however, after
four days a decreasing effect upon the seizure t h r e s h o l d w a s o b s e r v e d in t h e C E D 5 0 e x p e r i ments with bemegride, pentylenetetrazol or dio x o n e , b u t n o t in t h e M E D - e x p e r i m e n t s with b e m e g r i d e o r p e n t y l e n e t e t r a z o l . An e x p l a n a t i o n f o r t h i s d i s c r e p a n c y h a s n o t b e e n found. T h e s e p r o c o n v u l s a n t e f f e c t s c o u l d b e o b s e r v e d in c h e m oshock-tests with three structurally different convulsants. The antiepileptics studied never inc r e a s e d t h e i n c i d e n c e of s e i z u r e s a f t e r a s u p r a m a x i m a l e l e c t r o s h o c k . Two d a y s a f t e r o n e d o s e of p h e n o b a r b i t a l (100 m g / k g s . c . ) t h e r a t i o of t h e L D 5 0 of b e m e g r i d e ( i . v . ) a f t e r p h e n o b a r b i t a l p r e t r e a t m e n t a n d the LD50 in c o n t r o l s w a s 0.64. T h u s , w i t h a two d a y i n t e r v a l , not o n l y t h e c o n v u l s a n t e f f e c t of b e m e g r i d e b u t a l s o i t s l e t h a l e f f e c t is e n h a n c e d ( u n p u b l i s h e d e x p e r i m e n t s ) . In r a t s p h e n o b a r b i t a l d i d not i n c r e a s e t h e c o n v u l s a n t e f f e c t of b e m e g r i d e o r p e n t y l e n e t e t r a z o l w h e n g i v e n two o r t h r e e d a y s a f t e r i t s a d m i n i s tration. The reason for this difference between t h e r e a c t i o n of r a t s a n d m i c e i s a s y e t o b s c u r e . T h e n a t u r e of t h e p r o c o n v u l s a n t e f f e c t in m i c e c a n n o t a s y e t b e e x p l a i n e d . S m a l l d o s e s of p h e n o b a r b i t a l a r e k n o w n to h a v e a s t i m u l a t o r y e f f e c t , t h e e x i s t e n c e of w h i c h c o u l d b e c o n f i r m e d in s o m e of o u r e x p e r i m e n t s . Moreover, in t h e MED-experiments, o n e d o s e of 20 m g / k g p h e n y t o i n w a s f o u n d to i n c r e a s e t h e c o n v u l s a n t a c t i o n of b e m e g r i d e . A s i m i l a r o b s e r v a t i o n w a s m a d e b y N i c h o l l s (1961). N e v e r t h e l e s s , t h e p r o c o n v u l s a n t e f f e c t s in t h e e x p e r i m e n t s d e s c r i b e d h e r e cannot be completely explained by this stimulat o r y e f f e c t of a s m a l l d o s e . One d a y a f t e r 100 mg/kg phenobarbital there is still a marked ant i c o n v u l s a n t e f f e c t . It i s u n l i k e l y t h a t two, t h r e e a n d f o u r d a y s a f t e r a d m i n i s t r a t i o n of t h i s d o s e the blood level remains between the narrow limits at which a proconvulsant (stimulatory) effect is exerted. T h e h o t p l a t e t e s t w a s p e r f o r m e d to i n v e s t i -
376
CHR. L. RiJMKE
g a t e , w h e t h e r d e c r e a s e d r e a c t i o n t i m e s , 48 h r s after giving phenobarbital and the other drugs r e f l e c t e d s o m e k i n d of g e n e r a l e x c i t a t i o n of t h e a n i m a l s . No s u c h e f f e c t w a s o b s e r v e d . Phenobarbital is known to increase the activity of s o m e l i v e r m i c r o s o m a l e n z y m e s y s t e m s i n v o l v e d in t h e m e t a b o l i s m of m a n y d r u g s , a n d e t h i o n i n e i s k n o w n to p r e v e n t t h i s i n c r e a s e d a c t i v i t y if g i v e n s h o r t l y b e f o r e p h e n o b a r b i t a l . Therefore, some experiments with ethionine w e r e d o n e to d i s c o v e r w h e t h e r t h e e n h a n c e d s e n s i t i v i t y to b e m e g r i d e a n d p e n t y l e n e t e t r a z o l , two days after phenobarbital administration, is corr e l a t e d w i t h t h e i n d u c t i o n of e n h a n c e d l i v e r m i crosomal enzyme activity. These experiments s h o w e d t h a t the p r o c o n v u l s a n t e f f e c t of p h e n o b a r b i t a l w a s a b o l i s h e d by e t h i o n i n e g i v e n 30 m i n earlier. After ethionine pretreatment phenobarbital still exerts a marked anticonvulsant effect for bemegride and pentylenetetrazol, when this i s t e s t e d two d a y s l a t e r . M o r e o v e r , two d a y s a f t e r e t h i o n i n e a l o n e t h e s e n s i t i v i t y of m i c e to t h e s e c o n v u l s a n t s i s e n h a n c e d . H o w e v e r , t h e way in w h i c h i n c r e a s e d l i v e r m i c r o s o m a l e n z y m e a c t i v i t y m i g h t c a u s e a n i n c r e a s e d e f f e c t of b e m e g ride, pentylenetetrazol and dioxone remains obs c u r e . If the m e t a b o l i s m of t h e c o n v u l s a n t s were accelerated by phenobarbital pretreatment, a d e c r e a s e i n s t e a d of a n i n c r e a s e in c o n v u l s a n t e f f e c t s w o u l d b e o b s e r v e d . M o r e o v e r , i t is q u e s t i o n a b l e w h e t h e r a n e f f e c t o n t h e r a t e of m e t a b o l i s m of the c o n v u l s a n t s w o u l d m a n i f e s t i t s e l f in the short period (10-120 sec) between the admini s t r a t i o n of t h e d r u g s a n d t h e m o m e n t w h e n t h e animals show their first convulsion. Another imp o r t a n t p o i n t of i n t e r e s t in t h i s c o n n e c t i o n i s t h a t p h e n y t o i n (50 m g / k g s . c . ) s h o r t e n s the hexobarb i t a l s l e e p i n g t i m e , w h e n t e s t e d two o r t h r e e d a y s a f t e r i t s a d m i n i s t r a t i o n , b u t not a f t e r f o u r d a y s ( R i i m k e , 1963). H o w e v e r , t h e p r o c o n v u l s a n t e f f e c t of p h e n y t o i n a p p e a r e d o n l y a f t e r 4 d a y s in these experiments. A c e n t r a l o r i g i n of t h e i n c r e a s e d s e n s i t i v i t y to b e m e g r i d e , p e n t y l e n e t e t r a z o l a n d d i o x o n e i s q u e s t i o n a b l e a s t h e p r o c o n v u l s a n t e f f e c t of t h e a n t i e p i l e p t i c s w a s o b s e r v e d only in c h e m o s h o c k t e s t s a n d not in t h e e l e c t r o s h o c k - t e s t u s e d . The question arises, whether the observed p r o c o n v u l s a n t e f f e c t of t h e s e a n t i e p i l e p t i c s is c o n n e c t e d w i t h t h e p r o b l e m of t h e c o n v u l s i o n s , s o m e t i m e s o b s e r v e d i n e p i l e p t i c p a t i e n t s one o r m o r e d a y s a f t e r a b r u p t t e r m i n a t i o n of a t r e a t ment with an antiepileptic drug.
ACKNOWLEDGEMENT The author is much indebted to Miss H . A . K u i p e r s (Department of Medical Statistics of the F r e e U n i v e r sity) for advice and cooperation in the statistical analysis of the data. and to M r . C . B r o s k y and Mr. H . V e r beek for very careful technical a s s i s t e n c e .
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