ucb L059, a novel anti-convulsant drug: pharmacological profile in animais

ucb k059, a novel anti-convulsant drug: pharmacological profile in annuals

Received I4 May IYYI?.wised

MS received 2 July 1YY2.accrp~ed 1 I August IYY2

The ~inti~~~nvuls~~ntactivity of ucb LOW ((S)-n-cthyl-3-tJxtl-pyrnliidint uch LOS9 was active incorporating gcnendly

after

oral

and intrapcritoneal

features in common with scvcral diffcrcnt

within Ihe range of X0-30.0

indticcd chemically the dcvclopmcnt ~n~nti~nl~r.

administration

by pcntyicnetctra of PTZ-induced

mg/kg. kindling

types of unlicpilcptic

in inhibiting

>ic (PTZ),

picrotoxin

the actual mechanism

of action remains unknown.

and open-field

exploration,

was minimal,

and rcduccd PTZ-induced EEG

~ntic~)nvu~~nt

doses; at ph~r~~~~l~~~ic~lliy

Ncurotoxicity,

a&c

uch LOSY: Convulsions;

The term epilepsy refers to a wide range of neurological disorders characteriscd by an abnormal discharge of cerebral neuroncs. The prevafencc of epilepsy is estimated at between 3 and 6 per 1~~~ (Griffin and Wyfcs, 1991; Sander and Shnrvon, 1987). Thcrc is a gcneraf consensus for the need for new, improved drugs to treat epifepsies (Liischer and Schmidt, 1988; Porter, 19X6). Although epifcpsy is adequately controlled in the majority of patients, there remains a significant number of sufferers who arc untreated or respond only partially to drug treatment. In addition, existing drugs, even when cffcctivc, are not free from adverse side-effects and are a continuous cause of concern to clinicians, particularly in view of the chronic nature of drug treatment (Griffin and Wyfcs, 1991: Porter, 19%). uch LO59 C(S)-n-ethyl-2-oxo-pyrrofidinc acetamide) is the S enantiomer of the ethyl anafogue of piracetam, a drug widely used on account of its purported bcncfi-

Ci,rrespondrncc to: A.J. Gowur, C’NS Department, UCB Pharmacrt~tic;il Sector. Chcmin du Foricst, 1410 Braine I’Alleud. Belgium.

of action

appeared

(NMDA). SW:.

uch LWiY rctardcd

discharge

in rats. The

of action of the mdccule obscrvntion tat.

even at doses SO-100

calm hut slightly more a&c.

Petit-m&

R

although

the rotarod

lest

timcs higher than tl-c ucb LO5Y thl;;

agent. with potential anticpilcpmgenic and anti-abscncc

Epilcptogcncsis:

1. Introduction

spike

cvaluatcc! with an Irwin-type

doses. the animals anticonvulsant

profile

induced convulsil~ns and c(~nvu~si~~n~

the stcrcosp~cificity

with only mild scdaticm being ohscrvcd.

prcscnts as an orally active. safe, broad-spectrum

a unique

drugs. The compound was active. with ED,,, values

and N-methyl-D-asp,rrafc

ucb L&50, had low intrinsic ~n~ic~~nvuls~nl activity, showing

in a range of animal mod&.

rats and mice, with

~udi~~g~nic seizures. c!crtrical!y

hicucullinc.

in mice

~c~~~rnid~) was cvaluatcd in both

actions.

Ep2cpsy

cial effects on cognition in the elderly. Routine scrccning of ucb LOS9 in the audiogenic seizure-prone mouse showed potent anticonvulsant activity. The effects of ucb LOS9 were consequently determined in a wide range of anticonvulsant tests in rats and mice. The tests, selected with reference to r~c~~rnn~ended programmes for evaluating potential antiepileptic drugs (Fisher, 1989; Kupferberg, 1989: Liischer and Schmidt, 1988; Mcfdrum, 19861, included genetic animal models, models involving electrical and chemical seizure induction, as well as chemical kindling and chemically induced EEG spike-and-wave discharge as a model of absence cpifepsy. Reference anticonvulsant compounds, including clinically used drugs. were tested in parallel for comparative purposes. In addition. the effects of the R enantiomcr, ucb LOhO,wcrc cvafuatcd in a Iimitcd range of tests. Finally, the neurotoxic effects of ucb LO.59were assessed on the basis of direct observation, using an Irwin-type evaluation, locomotor activity in an open-field test and rotarod performance. The results confirm that ucb LO59 has a broad spectrum of potent anticonvulsant activity with a very wide safety margin between pharmacofogicalfy active doses and those causing neurotoxicity or adverse side-effects.

The experiments described below involved both mice and rats. Two stmins of mice were used. hoth bred in the Animal Husbandry Unit at UCB. Belgium. Female DB&derivcd mice aged -1-S weeks. weighing 14-22 g and genetically sound-sensitive were used for the audioeenic seizure tests. NMRI mice, either male or female depending on the test. aged 5-6 weeks with a bode weight c of 22-23 g were used for ail other mouse studies. Male Sprague-Dawley rats. bought from IFFA-CREDO. Belgium. aged 6-7 weeks with a body iieight of 190 & 20 g were used for the bicucuiiinc and picrotoxin tests and r&s aged 3 months with a body weight of 270-300 g at the time of implantation of EEG electrodes were used for the pentyienetretrazoie (PTZ)/EEG test. Prior to testing. the animals were housed in animal hoIding rooms ~intained at 20-21 “C. under a 12-h light-dark qeie. with lights on at 6:ott h, and allowed ad lib access to standard cube diet and water. The mice were housed in groups of 20 per cage (38 x 26 x 14 cm) containing a bedding layer of sawdust. Rats were housed in groups of four animals in wire cages (21 x 20 X 4-t cm) except for the rats implanted with EEG clcctrodes, which were housed individually in wire cages (20 X I9 X 55 cm). To reduce possible problems caused by prolonged isolation, the EEG rats were regrot.t_red at least once a week for approximately 31 min. Apart from the EEG rats. ail animals were used once only.

The day before the experiment, the mice were subjected to a preseiection test and only mice in which a tonic convulsion was provoked by an acoustic stimulus were retained: approximately 10% of the population did not meet this criterion, For drug testing, the mice were injected orally tp.0.) with uch LO59 or the rcference drugs and 60 min later placed in individual cages in a sound-attenuated cabinet. After 30 s to allow for orientation, a 911-JB. IO- to 20-Hz acoustic stimulus was delivered for 30 s via loud speakers positioned directly above each cage. During the 30-s sound deiivcry. the presence of wild running, and clonic and tonic eonvuisions was noted for each mouse. For each of these three separate parameters, the % protection afforded by each dose of drug was caicuIated using the formula cited in the Statistics section. From this data, the ED,,, value. defined as the effective dose producing 50% protection. was computed for each drug.

MI3 seizures were induced in male mice by the method of Swinyard et al. (19731. The animals were subjcctcd to a SO-mA ac current (250 cpsl for il.2 s deiivercd via cornea1 electrodes, 60 min after oral administration of ucb LOS9 or reference drugs. This electrical stimulus induced tonic convulsions in 80% or more of control mice. The number of mice exhibiting tonic convulsions was noted per group and the CTC protection per dose was calculated using the formula given in the Statistics section. From this data, the EDst, value, being the effective dose affording 50% protection. was computed for each drug.

The effects of ucb LOS9 were examined on seizure activity induced by a range of chemicals, including PTZ, bicuculiine, picrotoxin and N-methyl-D-aspartate (NMDA). Where appropriate, reference drugs were included for comparison. 2L3.I. PTZ The effects of ucb LO59 and reference drugs, administered orally 61) min prior to PTZ CiOr, mg/kg i.p.1, were determined in female mice. The dose of PTZ (l(K) mg/kgI was ascertained in preliminary experiments as being the maximal dose causing clonic convulsions in 100% and tonic convulsions in 75% or more of control mice. In a separate experiment, the ability of a range of doses of ucb LO59 p.o. to displace the dose-response curve for PTZ (60-l2tt mg/kg i.p.1 was assessed. 2.2.3.2. Bicrrcullim The protective effects of ucb LOS9 and reference drugs against convulsions induced by bicucullinc (4.0 mg/kg i.p.1 were determined in female mice. after oral administration 60 min before the test. The dose of bicueuiiine was ascertained in preliminary experiments as being one which caused clonic convulsions in 100% and tonic convulsions in 75% oi- more of n&e mice. In addition, the ability of a range of doses of ucb LO59 p-0. to displace the dose-response curve for bicuculline 12.75-4.5 mg/kg i.p.I was examined. L2.3.3. NM04 Female NMRI mice wcrc pretreated with ucb LO59 i.p. 60 min prior to intracerebral ti.c.v.1 injection into the right lateral ventricle of 4 pi (I nmoll of NMDA. The i.c.v. injections were given to non-anaesthctised mice, using a free-hand method based on that described by Clark et al. (1968), in which the head of the mouse is positioned in a specially constructed mould. The presence of tonic and clonic convulsions was noted. A similar experiment was carried out with MK-801. 2.2.3Lc. PTZ-indtrced khrdlimg itz mice Male mice were injected i.p. once daily for II consecutive days with ucb LO59 (5.4, 17.11or 54.0 mg,/kgI or saline, 60 min before the administration of PTZ f55.0 mg/kg i.p.1. Imm~djat~l~ after the PTZ injection, the mice

were placed in individual cages and observed for tonic and clonic convulsions. 2.2.1. Chemically

induced

seizures in rats

The effects of ucb LO59 were determined against bicuculline- and picrotoxin-induced seizures in rats. 2.2.4.1. Bicmrlline Rats were pretreated with ucb LO59 or selected reference antiepileptic drugs either p.o. 60 min or i.p. 30 min prior to administration of bicuculline 0.6 mg/kg injected i.v. via the tail vein. The incidences of tonic and clonic convulsions and mortality were noted. The severity of the clonic convulsions was rated subjectively as follows: 0 = absent, 1 = mild, limited to forelimbs, 2 = moderate, short-lived clonus of both fore- and hindlimbs without loss of the righting reflex and 3 = severe, prolonged clonus of both foreand hindlimbs with loss of the righting reflex. In addition, the ability of ucb LO59 and refcrcnce drugs to displace the dose-response curve for bicuculline was determined. 2.2.4.2. Picroroxirl Rats were pretreated with ucb LO59 p.o. 60 min prior to i.v. administration of picrotoxin (2.75, 3.0 or 3.25 mg/kg). The incidence of tonic and clonic convulsions and mortality was recorded. The severity of convulsions was also rated, using the scale noted above. 2.2.5. PTZ-hduced rats

spike-and-ware

discharge

(SWD)

in

Rats implanted with cortical surface EEG electrodes were used. The experiment had a cross-over design in which each rat received vehicle or ucb LO59 (5.4 or 17.0 mg/kg i.p.) at weekly intervals. The order of treatment was arranged according to a random latin-square design. Twenty minutes after i.p. injection of ucb LO59 or saline, each rat was injected with PTZ 25 mg/kg i.p. During EEG testing, the rats were placed in individual cages located in a sound-attenuated chamber. The EEG was monitored from 20 min before injection of ucb LO59 up to 2 h after injection of PTZ. The cumulative duration of SWD and number of SWD episodes per 20-min epoch of testing were determined by direct measurements from the EEG paper trace. The criteria for SWD were as follows: repetitive frequency of the spikes between 6 and II Hz, spike amplitude between 200 and 600 /.LVwith simultaneous activity on both the left and right cortical EEG. A parallel experiment was carried out with clonazepam (0.1 and 0.3 mg/kg i.p.1. 2.3. Neurotoxicity

testing

2.3.1. Nertrotoxicity testing in mice Potential neurotoxicity in mice was assessed in three different tests, each carried out independently with

separate groups of mice. The lcsts were the Irwin test. measurement of locomotor activity alid measurement of rotarod performance. The Irwin test, based on that described by Irwin (1968). relies on the subjective scoring of a wide spectrum of behavioural and CNS parameters, by direct observation of spontaneous behaviour or after manipulation according to a standard protocol. Locomotor activity was measured in an open field, 31-cm square with 15-cm high walls, equipped with infrared photocells located in the walls I.5 cm above a metal grid floor. The photocells were spaced 3 cm apart, measured from centre to centre. in triads, with 4.5 cm between the triads. Activity was expressed as the distance moved, calculated on the basis of the number of interruptions of the photobeams. during a 20-min period. Rotarod performance was assessed in terms of the number of mice per dose-group able to remain for at least 60 s on a 3-cm diameter rod rotating at a constant speed of 6 rpm. The mice were pretrained the day before drug-testing and only mice able to reach the 60 s criterion within three consecutive trials were retained. 2.3.2. Nearotoxicity

testing in rats

Both the Irwin test and measurement of locomotor activity were used to assess the potential neurotoxicity of ucb LO59 in rats. Each test was carried out separatcly, using different groups of rats. The Irwin test was similar to that used for mice. but adapted where necessary to be applicable to rats. As with mice locomotor activity was measured in an open field. The open field consisted of a l-m square arena, with 40-cm high walls surrounding a grid floor. Two horizontal rows of infrared photocells, spaced 6 cm apart, were located in the walls, one row at 2 cm and the other row at 10 cm above the grid floor. Locomotor activity was recorded automatically in terms of distance, calculated on the basis of the number of photobeams interrupted. Rearing was also counted, in terms of the interruptions of the upper row of photobeams. 2.4, Drugs and injections ucb LO59 ((SI-cY-ethyl-2-oxo-pyrrolidine acctamide) and the R enantiomer, ucb LO60, were synthesised in the research chemical laboratories of UCB. Each compound is a white crystalline powder which dissolves readily in saline or water giving solutions of about pH 6.0. The reference drugs included phenytoin (Vcl, Bclgium); carbamazepine (Sigma); clonazepam and diazepam (Hoffman - La Roche, Switzerland), sodium valproate (Sigma; obtained both as the sodium salt and as valproic acid) and MK-801 hydrogen maleate (( + )-

The ~~vu~s~~nt agents used were pentylenetetrazol (PTZ; ~~~rdi~~z~~~~ Janssen Chimica): hicucuilinc and picrotosin (Fluka) and NMDA (N-methyl-D-aspartate: Sigma?. Bicuculline was dissolved in a minimal quantity of glacial acetic acid f& administration to mice, and in a ~~~irna~ quantity of hydrochloric acid for administration to rats: in both cases the pH was adjusted to 5-6 with NaOH and the soIutions were diluted to volume with saline. PTZ. picrotoxin and NMDA were dissohed in saline. For i.p. and p.o. administration, a dose-volume of 10 m!/kg body weight was l.scd for mice and 5 ml/kg body weight was used for rats. A dose-volume of 1 t~~~jiig body weight was used for i-v. administration to rats,

In all experiments except the SWD/EEG study. the number of animals responding in each group was de-

termined for each parameter measured. The statistical significances of differences from the control group were c&ufated using the F&her test. The number of mice responding peg group was tiqed to calculate the % protection tP%;) for each parameter. by applying the following formula:

where nt = the number of animals responding

in the

test group, with Nt = the number of animals tested and nc = the number of animals responding in the control group. with Nc = the number of animals tested. The effective dose of drug affording 50% protection tEDSI,) was then computed using a probit analysis (Finney, 1971). or. in the case of the bicucilline rat test, the method of Berkson (1953). In the SWD/EEG study, as each rat served as its own control, the control and test data were compared using a Wilcoxon signed ranks matched pairs test (Siegel and Castcllan, 198zO.

3. Results

The ED,,, values for uch LO59 and the reference drugs, administered p.0.. are given in table I. ucb LO59 dose dependently protected against audiogenic seizures elicited in mice, being approximately equiactivc against tonic and clonic convulsions (ED,,, values 7.0 and 9.7 mg/kg, respectively) but requiring 3-4 times higher doses to prevent wild ~nni~~g. This proPtIe of ucb LOS9 resembled that of clonazepam and sodium valproate, which were both equiactive against tonic and clonic seizures but less effective against wild running. In contrast, phenytoin and carbamazepinc were both preferentially active against tonic convulsions, with approximately 10 times higher doses required to block clonic than tonic convulsions. in terms of the EDSir values for protection against tonic convulsions; the order of potency of the drugs tested was as follows: clonazcpam -%=. phenytoin = ucb LO59 = carbamazepine X- sodium valproate. When ucb LO59 was administered i.p., the EDs,, value with 95% confidence limits for protection against tonic seizures was 8.6 (6.2- I 1.2) mg/kg.

Inhihilion h? uch LtKY and reference antiepiteplic drugs of convulsions induced in mice. WR = uild running: CC = clonic convulsion: TC = tonic convulsion. C1mvulLmt

test

Audiogrnic ~rizure~

Mazimal electroshock Bicuculline -t mg/kg i.p. Pentylenetrtrazoie 100 mg/k_e i.p.

PXlmrter WR CC TC TC CC TC CC l-C

ED,,, mg/kg p.~. (Y5G confidence limits) uch LCiS4 ) 3 I .7 IX-44.5 4.7f 7.5-11.91 7.0 ( 4.4- 9.1) 23.5 f IO. I -Sk-l) > 170 ‘9.5 (21 .Y-42.6) 1 700 :’ 5.-b I700 2%3W inhib.

Phenytrtin

Cnrllamazepinr

Clcmazepam

Na Valproate

a X0.6 3xf-YK!u S-53.2) 4.7 ( 4.2- 5.31 2.4 ( 1.9- 7.81

2 75.5 70.0 ” 7.0 ( 6.3- 7.9) 4.1 ( 2.7- 5.‘) 137 . “..7 iI 19.3 (16.3-21.7) 75 5 iI 17:5 t 14-Y-2&l?)

U.O.SXh D.03X *

> 2SY >217 (1513 -270 ) 177 (IS7 -226 ) 73.x ( 2x.3- I20.4) 33Y.Ot254 -h26 ) 222.0 (15X -494 ) 250 (221 -2x7 ) ifdl (124 -22Y)

X0.h :’ 14.n(11.5-1~.4) 75 ._ 7 il __ 7.7( 6.1- 9.w

6 Inactive up to dose given: h limits could not he caIcula~ed.

0.029 (0.024-0.03h) 0.3 (0.15 -0.5’) ) 0.05 * 0.029 (tMll7-0.12 ) 0.07x W.Oh -0. I1 J 0.037 (0.020-0.05 1)

197

TABLE

2

Inhibition by ucb LO5Y or MK-WI NMDA (I nmol i.c.v.) in mice

ucb LO59 p.o. produced dose-dependent protection against tonic convulsions induced by maximal electroshock in mice, with an EDS,, value of 23.5 mg/kg (table 1). This value was approximately 5-10 times higher than the corresponding values for phenytc+ and carbamazepine. Neither ucb LO59 nor any of tt,e reference drugs protected against cionic=convulsions.

%ug treatmect (i.p. mg/kgf

3.3. Clrernically induced conwisions in nzice

5.4 17.0 54.1’ 170.0 MK-HOI 0.034 0. IOK 0.340

ucb LO59 p.o. at doses of 5.4 mg/kg and higher inhibited tonic convulsions induced by a maximal dose of PTZ (100 mg/kg) by 25-30% (fig 1, table 1). This effect was not dose-dependent and no inhibition of cionic convulsions was obtained. The reference drugs, phenytoin, carbamazepine, clonazepam and sodium valproate, all dose dependently protected against tonic convulsions although only the latter two were effective against clonic convuisions. In contrast, at submaximal doses of PTZ (fig. 11, ucb LO59 was able to antagonise tonic convulsions in a dose-dependent manner. According to this data, the EDS,, values of ucb LO.59for inhibition of tonic convulsions were 68 mg/kg against PTZ 90 mg/kg and 28 mg/kg against PTZ 80 mg/kg. ucb LO59 p.o. dose dependently reduced bicucufline-induced tonic convulsions in mice (table 1). The resulting EDS,, value, 29.5 mg/kg, approached that of phenytoin and of carbamazepine. ucb LOS9 (17-170 mg/kg) afso reduced clonic convulsions but the effect was not dose-dependent and varied between 30-60%; an EDa, vlaue could not be determined.

- .

6-o

70

80

Pentylenetetrazole

90

100

(mg/kg i.p.1

Fig. 1. Effect of uch LO.59 on tonic convulsions induced by penlylenete~r~z~~lein mice. Exh value given is the percentage (%I of mice responding per group of 20. The mice received either vehicle (:.). ucb LO59 17 mg/kg (0 ), ucb LOS9 54 mg/kg ( A ) or uch LO59 170 mg/kg (0) p-o. 60 min before pentylenetetr;tzole was injected i.p. Solid symbols indicate a s~~~~is~ically si~nifi~un~ (P < 0.05) diffcrence from the corresponding control.

i.p. of convulsions induced tr)

N = number of mice per drug and control group. N

Percentage uf mice responding Tonic convulsion

Clonic convulsion

Control

Drug

(‘ontrol

Drug

20 30 50 JO

41i.0 32.5 52.0 55.0

15.0 h.7 h ‘2.0 ;1 0 h

75.0 13.3 78.0 77.5

55.0 53.3 70.0 32.5 h

10 20 20

60.0 60.0 60.0

60.0 15.0 “ 0 h

70.0 75.0 75.0

70.0 50.0 0 h

u& 1.0%

” P = O.Ot; h P = WlOi: Fisher lest vrrsus control.

NMDA (1 nmol i.c.v.) causeti tonic convulsions in up to 60% and clonic con~lsions in up to 80% of the control mice. Tonic convulsions were reduced by ucb LO59 (5.4-170 mg/kg i.p.), although clonic convulsions were significantly reduced only at 170 mg/kg. The effects were not dose-related, unlike those of OK-801 (0.034-0.34 mg/kg) (table 2). 3.4. PTZ-induced kindling in nzice Single daily injections of PTZ (55 mg/kg i.p.1 produced a progressive increase in the number of control mice with clonic convulsions, increasing from less than 25% on day 1 to 90% on day 11. Pretreatment with ucb LO59 (5.4-54.0 mg/kg i.p.) caused dose-dependent re-

2

4

5

Qays

of treatment

6

7

8

9

10

11

Fig. 2. Effect of ucb LO59 on the development of pentylen~tetrazoleinduced kindling in mice. Groups of 20 mice were injacted once daily with either vehicle control (:.I. ucb LOSY5.4 mg/kg (c’t. uch LIHY 17 mg/kg (u) or ucb LOSY S4 ml/kg (a) i.p. 60 min before pentyleneletrazole was administered (55 mg/kg i.p.). Each value is the percentage (Q) of mice per group with clonic convulsions. Solid symbols indicate in s~~tisti~lly significant (P i O.ftS)diffcrencc from the control value.

A!

CONVULSIONS

TONIC

0.4

BI MORTALITY

I

1

0.6

0.6

J ,

I 0.6

0.4

Bicuculllne (mg/kg I.v.1

8icuculllne (mg/kg I.v.) Fig. 5. Effccr of uch LtW t 1. uzb LtW

I .i

admrnizterd

Each \aluc

mg/Lp

on t.,\b tonic convulGcms and (B) mortahty t T b. uch LCW 5.1 mgjkg i> the pcrccntagr

induced in rats hy bicucullinc.

(~1 or uch LO59 Ii me,

of ra

1 0.6

responding.

kp ( u 1p.o. 60 min

Solid symbols indicdtc

Groups

of ‘0 rats received vehicle control

hrfnre

hicuculline

(0.4, 0.h or 0.X mg/kg

;I statistically

significant

(P < MS)

differrncc

i.v.) was from the

control.

tardation of this phenomenon (fig. 2). The intensity of rhc effect of uch LO59 was such that after 54 mg/kg. the incidence of clonic convulsions remained 5% over the first 6 days and never exceeded 20% even after days of kindling.

11

elicited

by bicuculline

ues obtained

(0.6 mg/kg

i.v.). The EDS,, val-

for both p.o. and i.p. administration

are

given in tahlc 3, from which it can be seen that the potency of ucb LOS9 was similar to that of carbamazepine.

Neither

ucb

LO59

nor

mazepine

or sodium valproate

incidence

of clonic convulsions:

phenytoin,

carba-

were able to alter the however,

all of these

drugs reduced the severity of convulsions. Clonazepam ucb

LO59, like

dependently

the

reference

drugs

tested,

dose

reduced the incidence of tonic convulsions Al TONIC

was exceptional convulsions.

in reducing

CONVULSIONS

the incidcncc

of clonic

B) MORTALITY 100

-

O-

I,

3.0

Picrotoxrn Fig. 4. Effect of uch Ltl% on (,\I

3.25

tcmic convulsi;rns and (B)

,

1

I

3.0

2.15

Picrotoxln

lmg/kg I.v.)

uch IMY 5.1 ms /kg ( ) or uch LtW I7 mg/kg the wrscntage

J

1

I

2.75

3.25

(mg/kg

I.v.1

mortality induced in rats hy picrotcwin. Groups of N rats received vehicle control (

f q 1 p.o. 60 min hefore picrotoxin

of ras responding. Solid symholh indizlte

(1.7.5. 3.0 or 3.?i mg/kg

i.v.) was administered.

a st:ltistic;dly significant (P < 0.05) difference

from the control.

‘.1.

Each value is

TABLE Effrct

3 drugs on tonic co~;*~ulsions induced

of uch LOSY and reference

by hicucullme (0.h mg/kg

i.v.) in rats.

Values given are doses affording confidcnee

5115 protection

(ED,,,

mg/kg).

with

limits in parentheses. .--.- __._..^__

Test drug

ucb LOSY Ph~nyti~in

ED,,,

(me/kg)

P.o. (10 min

1.p. 30 min

before i&u...;iinl:

before: IbiLu ~2r.c

7.7( 41G

Carbamazepine

5.0 -

Ii.‘)

)

> lb0 Y.7(

3.1 -

8.6)

iY.7

5.1

f

f 10.3 -

37.X)

15.6

I 13.0 -

1X.X)

inhib. at 160 7.3 -

Il.4

)

01 0

Sodium valproate

Clomrzepam

329

(302

-357

0.09 ( o.os-

) 0.14)

10x

(100

0.10 t

0.07-

-117

20

Time

1

0.12)

Fig. 5. Effect produced

In common with all of the reference antiepileptic drugs tested, except clonazepam, ucb LO59 was considerably more effective against submaximal doses of bicuculline, ie. 0.6 mg/kg and lower. than against a maximal dose, i.e. 0.8 mg/kg. This finding is exemplified in fig. 3 for p.o. injection although simiiar results were obtained after i.p. administration. Thus, apart from clonazepam, none of the drugs produced a parallel displacement of the bicuculline dose-response curve. ucb L059, tested at 17 and 54 mg/kg p.o. only, dose dependently reduced picrotoxin-induced tonic convulsions (fig. 4). This effect reached statistical significance at 54.0 mg/kg against all three doses of picrotoxin (2.75, 3.0 and 3.25 mg/kg i.v.) , although statistically significant effects after 17 mg/kg were only obtained against the lowest dose of picrotoxin, i.e. 2.75 mg/kg i.v.

4

I

4

8

40

60

89

100

(mini after

LO5Y 17 mg/kg administered in s/min.

f

(. f. ucb

120

injection

of uch LO59 on the spike-and-wave

in rats by pentyi~n~t~tr~zo~e

received either vehicle control

indicate

PTZ

PTZt. A

discharge (SWD) group of eight rats

L%Y 5.4 mg/kg

(17 1 i.p. 20 min hefore

PTZ

fc. ! or ucb

(25 mg/kg

i-p.) was

in a cross-over design. The duration of SWD. expressed

was measured direcrly from the EEC

trace. Solid symbols

a statistically significant (P < O.OS) difference

from the con-

tfui.

tone arId increased response to touch. With increasing doses up to 1700 mg/kg, more widespread signs of sedation occurred but were rated moderate rather than severe, even at the highest dose. In the open field, ucb LO59 i.p. slightly increased locomotor activity in mice; this effect reached a maximal and significant level of 14% at the dose of 54 mg/kg only (data not shown). In the rotarod test in mice, p.o. administered ucb LO59 had little effect up to 1700 mg/kg. At higher doses (2213-4255 mg/kg) performance was significantly impaired but the effect was not dose-dependent

3.6. PTZ-induced SWD in rats ucb LO59 (5.4 and 17.0 mg/kg, i.p.) reduced the mean cumulative duration of PTZ-induced SWD (fig. 5). The effect at 17 mg/kg was maintained throughout the 2 h testing period. The reduction was due to a decrease in the number of episodes of SWD rather than to a decrease in the duration of each episode. Clonazepam (0.1 and 0.3 mg/kg i.p.) also reduced PTZ-induced SWD. However, ucb LO59 produced no change in the baseline EEG whereas after clonazepam there was an increased proportion of lower-frequency, higher-amplitude EEG activity. Both ucb LO59 and clonazepam reduced the proportion of time spent immobile.

TABLE

4

Effect of ucb LMY compared irwin and Rotarod MAD

= minimal

ED5,, = effective

to reference

antiepileptic

drugs in the

tests. active

dose

causing

dose at which

50%

overt

hehavioural

changes.

of the mice were unahlr

to

remain on the rod, tested 60 min after dosing. Drug

MAD

ucb LO54 Phenytoin

(mg/kg

p.o.)

Mouse

Rat

1711

17011

‘5 ._’ _.

Necro-

Rotarod

Irwin test

251

ED,,, (mg/kg

toxicity pat

index ’

Mouse

Mttlise

> 17t111

> 243

93.6

I Y.Y

(no limits) Carbamezepine

70.X

‘3.h

YO

17.‘)

txo- 100) Clonazepam

0.03

KY4

0.053

17.7

(t).37-t).~Y) Sodium valproate

After p.o. administration, no gross effects were observed in mice in the Irwin test at doses below 170 mg/kg. At 170 mg/kg, ucb LOS? caused slight effects, consisting of reduced activity. reduced body muscle

144.2

‘XX.4

507

3 .I!

(372-h37) ” Neurotoxicity index is the ratio between the EDq,, value for inhihition of tonic convulsionselicited in audiogenic mice (see tahlc I) and the ED5,, value obtained

in the rotarod test.

;md did not permit the dctcrmination

of an ED,,,

value

f tshk Il. Ncurotosicity indices n-xc dctcmiincd for ucb LOSS) and the rcfcrcncc drugs in mice. in terms of the ratio between

the ED,,,

value for

impairment

of rotarod

p~rf~~~ance and the ED,,, value for inhibition of audiogenic scizurcs. These values are shown in table 4. From this data. it can be seen that the neurotoxicity indcs of ucb LO59 was at least 10 times higher than

effects occurring from 5.4-170

mg/kg i.p. and 54-540

mg/kg p.o. (table 5). The increases never exceeded X)-30% of control values. Minimal sedation, apparent as a reduction

in rearing coupled with a tendency to

reduce locomotor activity, was observed at 1700 mg/kg i.p. but not 1700 mg/kg p.o. 3.8. liffkts of the R mantiorner. ucb LO60, in aruiconwlsant tests in rodimts

at of phenytoin. carbamarcpine or clonazepam and almost 100 times higher than that of sodium valproatc.

ucb LO60 was active only at very high doses in protecting against convulsant activity induced in mice

Zn the rat. the only ohservablc changes produced by ucb LO551 p.o. occurred at the highest dose tested ( 1700 mg/kgl. At this dose. minimal signs of sedation ap-

and rats (table 6). ED5(, values of 1064 mg/kg and 1400 tally induced convulsions

peared. clmsisting of slight decreases in both spontaneous activity and reactivity.

other tests, namely, convulsions induced by MES, PTZ and bicucullinc in mice, and by bicuculline in rats, ucb

In the open-field

test.

uch LO59 slightly

~~~orn~~t~~ractivity in rats with

TABLE

Efkrt \vR

statistically

increased significant

p.o. were obtained against tonic and clonic audiogeniin mice, respectively. In the

LO60 was inactive or produced only slight inhibition the highest doses tested.

h of uch LthO in anticonvulsant

IC’SISin rodents.

= urlil running: CT‘ = chmic c~mvulsion: TC = tonic convulsion.

~~~lnvul\ant tr‘\t

Specicx

Mouse

Route of administration Prctreatmenl time

Paramrter

Resuh 35% inhihition at 1700 mg/kg ED5,, = IhOO mg/kg ”

P.O.

WR

h0 min

CC TC

ED,,,

P.O.

TC

31% inhibition

at 540 mg/kg

TC

Wi

at 1700 mg/kg

‘I’C

Y3 mg/kg

TC

540 mp/kg

= IOh

(77Y-l4S3)

mg/kg

60 min PSI.

inhihition

60 min P.O.

’

60 min

Lp. 30 min a hia~tiW

Up to dose

given: h limits could not he calculated.

*’

at

4. Discussion The results show that uch LO59

i:; an anticonvuisant

compound, cffcctivc in a wide range of anima! models Wed in epilepsy research. -*iii5 ED,,, vahrcb gGiXil!~j within the range of 5-?O mg/kg: Thus, ucb LO59 prevcntcd audiogenically elicited seizures in genetically susceptible

mice

rc4ured

sions and protected

electrically

variety of chcmoconvulsants, jected

PTZ,

induced

convul-

against the convulsant effects of a

bicucullinc

including systcmicalfy in-

and picrotoxin

and centrally

injected NMDA. ucb LOS9 also rctardcd the dcvclopment of kindling induced in mice by subthreshold ucb LOS9 was able to reduce the SWD observed on the EEG after injection of PTZ (25 mg/kgk The compound was equally active after oral and i-p. administration in both rats and mice. Moreover, although not systematically investigated, activity was similar in both male and female animals, indicating no sex-related differences. The range of tests was selected to give a general overview of the activity profile of ucb LOS9. The choice of tests was guided by recommended approaches to the evaluation of potential, novel antiepileptic drugs (Kupferberg, 1989; Lijscher and Schmidt, 1988; Meldrum, 1986). The audiogenic mouse model is used widely for routine screening. In this test, ucb LOS9 was equally effcctivc against clonic as against tonic convulsions, although higher doses were required to block wild running. This profile was also obtained with clonazepam and sodium valproate, whereas phenytoin and carbamazepine were preferentially active against tonic convulsions. Although all commonly used antiepileptic drugs are active, the audiogenic mouse is not representative of a particular type of epilepsy in man and the model does not differentiate different classes of epileptic drugs. Moreover, audiogenic seizures can be attenuated by drugs other than ccnventional anticpileptics (Chapman et al., 1984; Seyfried, 1979). The ability of drugs to prevent MES-induced seizures in animals is considered predictive of a potential clinical effect against generalized seizures of the grand mal type. Thus, phenytoin and earbamazepine are active, sodium valproate and hcnzodiazcpincs arc only active at high doses, and ethosuximidc is inactive (Chapman et al., 1984; Fisher, 1989; Liischer and Schmidt, 19%: Swinyard et al., 1988). Apart from sodium vaIproatc, our findings with the rcfcrencc drugs arc in agreement with these published observations. The potency of uCb LO59 in this test was similar to its potency in the other acute tests. In view of its activity in tile MES test, it can be predicted that ucb LO59 will be effective in grand mal epilepsy. Testing a novel compound against acutely administcrcd chem[)convul~nts is useful to confirm the antidoses of PTZ. In addition,

convulsant activity of a novel comp~)und and to diffcrcntiatc its profile. The agents used in the present stud! can bc divided into those that interact with the Y_ aminobutyric acid (GABA)-bcnzodi;izcpinc-chloride ionophorc to impair GABAcrgic ncur~)transmissj~~n. namely PTZ, hicuculhnc and picrotoxin (Parrant and Wcbstcr, I989; Meldrum, 19x4: Woodbury. 1~~0). and NMDA,

which

is an cxciratorq

amino

acid receptor

(EAA; McGcer and McGccr, 198% Watkins ct af.. 1990). ucb ‘LO59 was active against aif of thcsc chemoconvulsants with an overall profile of activity distinct from that of any of the rcferencc drugs used. uch Lo.59 was very potent when tested against submaximal doses of PTZ and bicuculline, but was less active against maximal convulsant doses. In mice. this difference was particularly striking for PTZ-induced convulsions but less so for bicucufline-induced convulsions. Unlike the reference drugs, ucb LOS9 was only weakly active against convulsions induced by PTZ ( 100 mg/kg). In contrast, !owering the dose of PTZ slightly. to 90 mg/kg, revealed potent activity. predominantly againsl tonic convulsions. Both phenytoin and carbamazcpine also preferentially inhibit tonic rather than clonic convulsions induced by PTZ. A parallel study of PTZ in the rat was not undertaken. However. in the rat agonist

bicuculline

model,

ucb

LO59 was selectively

active

against submaximal bicuculline doses and against tonic convulsions and mortality. This profile is common to carbamazep~ne and sodium valproate. It is interesting to note that although neither ucb LOS9 nor carbamazepinc or sodium valproate affected the incidoncc of clonic convulsions, ail of them reduced their severity. Clonazepam was clearly different from the other drugs. since not only did it consistently inhibit both types of convulsions but it was also equally effective against all doses of bicucuifine, causing a parallel shift of the bicuculline dose-response curve. The introduction of testing against submaximal

convul~nts grammes,

is an extension which

tend tu ~nfine

doses of such agents. The LO59, showing

doses of the chcmo-

of rcc~~mmended protesting to maximal

results obtained

with ucb

activity, ikIStratc the hIitatiOnS of Standard Screening methods and support the CCMX~ Of fiScher and Schmidt (198X) that it is important not to evaluate novel compounds cxclusivcly against [email protected]

maximal doses of convulsants so as not to miss it'ItCrCSting new drugs. A prediction of what preferential activity against submaximal rather than maximal convufsant doses means in terms of the clinical profile of the drug is difficult to make at this stage. ucb LO59 was also cffcctivc in antagonising convdsions induced by i.c.v. injcctcd

NMDA. in contrast. a

single study showed that ucb LOS9 was unable to antaganise convulsions induced by NMDA administered i.p. at 300 mg/kg (data not shown). According to Morcau

ncui-otcl\icit! c;~ii hc attributed

10 poor absorption

of

1111’C~~lllp~N.l~lct.

At

\\itt;

appcarcd cafm. dcspitc the dctcction of

UC+

\fight

WCC,

hut

~Il~tiLY~ll~~UlSilIlt JOSCS.

c(~ns~stcn~ incrcascs

ration. particularly ity with uch L.03

As

in open-ficfd

anticpifcptic

and drowsiness

(Griffin

tIXXllCd

cxpfo-

in rats. The abscncc of ncurotoxicindicates a potcntiaf important ad-

vantage over csisting scdatinn

iIllilllillS

and Wyfcs.

drugs.

arc often

1001: Porter.

for

unwanted

which effects

IYXh).

noted in the Introduction.

ucb LOS)

is the S

cr~~l~t~~~n~cr of ii-ctllyf-‘-coxes-pyrmllidinc acctamide, Ihc cthyf 3~illO~UC of pitXctiHl1. Piracctitrtt itscff is not Nt .~~t;..l~n‘.1l!~~:::t! :igytt in 4!:anrf:lrd :Inim;tf mo(jcfs h\lt is .L ,,,..I I.(. cffcctivc [Brown tcstcd

as

adjunct

ct al,. IYYl). against

scizurcs vulsions

therapy The

in

human

myoclonia

K enanti~~mcr. ucb LOf%. was

audiogcnic-.

MES-

and PTZ-induced

in mice and against bicucuffine-induced conin both rats and mice. It was found to have

activity only at very high doses or to have only slight non-significant cffccts. The finding that the anticonvulsant activity is intrinsic to one cnantiomer and not the other is important,

since it provides cvidcncc that the

mofcculc interacts

with

conformationaf

a specific site with

stringent

requirements.

As yet. this

site has not been idcntificd

and the

mechanism of action of ucb LO59 remains the subject of investigation. In this context, the existence of an inactive optical isomer could be useful. The finding of activity against both GABAcrgic-

and EAA-mediated

convulsions is common to cfjnicafly avajfabfe anticpifcptic drugs hut suggests potential actions to he cxpforcd.

The

submaximal

scfcctivc action

doses of PTZ

of ucb LOS9

against

or bicucuffinc supports

the

notion that ucb LO59 does not interact directly with the GABA-benzodiazepine-chloride ionophore. and the preferentiaf action of i.& LOS3 against i.c.v. than i.p. administered NMDA argues against direct antagonism of N~DA receptors. A lack of undc~~anding of how an epileptic

drug

works

does not

detract

from

its

thcrapcutic vafuc and. in fact, the mechanisms of action of several classical anticpifeptic drugs are stiff not established. The data presented in this paper convincingly show. that ucb LOSY is an anticonvulsant merits

further

investigation.

feast two species. in a wide of action is unique.

compound which

It is active orally,

in at

range of modcfs. Its profile

having features

in common with

scvcraf diffcrcnt types of anticpilcptic drugs, thus supporting its potcntiaf as a broad-spectrum anticonvulsant agent. Its par~icufar activity against epifeptogenesis and against SWD

suggests specific actions typical of

cfonazepam and sodium vafproate. Finally, its low neurotoxicity indicates a pcwerfuf advantage over existing therapy.

Acknowledgements Thr

ituIh<#\

Miss Manoa

gratcf’ufly

Bcnscler. Mr.

and Doru-Gcorg

Margincanu

and thanh

Mrs.

Jo+anc

W;trlu\

for

typing the mambcript.

References

Mcldrum.

B.S..

proachch Bcrkson. J.. 1‘63. A st;lti~tj~~ill~ prczise and rclativcly ximpfc mrtfmd of estimating

the hic),gw:1y wii:t quant;rf response

logistic limction. Brown.

P.. M.J.

Waegcmans

Stcigcr.

J.C‘. Ro~hwcll. Marhdcn.

B.L.

etam in cortical myoclonus. J. Movement Chapman. of

M.J.

A.G..

Cmuchrr

anticonvulsant

drugs

seizures. Arzneim. Clark.

W.G.,

Forsch. Drug

Salnmu.

T.

of Pu-;ac-

fin press).

with

M. Orlandi.

Giorgio.

furlction

Porter.

G. Carhtrni.

V. Frau

kindling

and 0.

Bchav.

Wchster.

108% GABA

antagonists:

and mechanisms of action. in: Nruromcthods in N~ur~~tr~nsrnitt~r and A.V. Finney.

Rtzsearch.

Juorio (fiu~n~

D.J..

eds. A.A.

R.S.. 19x0. Animal

Baker

A Statistical

mod&

Trratmrni

University

of the epilepsies.

of that

J. and M. Wyles.

of Health Irwin.

S..

Economics.

RL’s. Rev.

1968,

systematic

IYYI. Epilepsy Towards Tomorrow ohservationaf

assessmenl:

la.

A

cooperative

1089. Antiepileptic

effort

of Government

(SuppI. II, 551. Liischrr. W. and D. Scflmidt.

drug development and

t..

industry.

Epilepsia

a 30

IYXX. Which animal models should he drugs? A proposal based

and clinical considerations,

G. Michcletti,

program:

M. Vergnes.

Epilepsy Rcs. 2. 145.

A. Depaulis,

L. Rumhach

R.J.

Porter

(John

induced h!

in mice: effectx of NMDA

antaptr

and anticonvulsants.

drugs: efficacy and inadequaq.

and R.J. Portrr

and SD.

Shorvon.

in:

(John Lihhey. tondon!

p. 3.

19X7. Incidencta and prcvnlencr proh1cm.s: a r&cu.

Psych&t. SO. X3X.

1070. Audiogmic

Siegel. S. and N.J. Cast&m. Brhavittur;tf


in micr.

Swinyard.

19xX. Nonparametric

Sciences: 2nd edn. IMrGraw-Hill.

c‘. Shin and J.O. McN~lrn~r~.

cffccts of conventionaf Ann. Nrurol. EA...

anticonvufsants

Fed. Proc. 3X.

f9QI.

and H.H.

Wolf. 2. 95.

P. Krogsgnard-Larsen relationships

acid receptor

and T. Honore.

eds. D.

Lodge

of

19%). Struc-

of excitatory amino

antagonists. in: The Phar-

Amino Acids: A Trends Pharmacol. and G. C[)lfin~rid~~

Sci.

(Efwvicr.

p. 4.

Wilson. W.A.. S. Stasheff. S. Swartzwelder. ;md D. Lewis,

1980. The NMDA

Receptor.

Prow, Oxford) W‘u)dhury. D.M., Anticpifeptic

198X. Mechanism>

in the development

agonists and competitive

&port,

Anti~pif~ptI~~~i~~~

29. 3.56.

H.S. White

J.C.K..

Slatistics for the Ncrw York).

in the kindling model of

~Intic~~nvuls~nt drugs. Ph~~uc~~Jl?~

NMDA

used in thr hearch for new antiepileptic on cxpcrimentul Marescau..

T.N..

Cambridge) H.J..

and

23’)‘).

Speci;tf

13 . “7 ..“W.

~1: ntncl

Vol. -1: Ncu. Anti-

YX. ll)SO.

macology of Excitatory

far assrssing thtr hrhavioural

and p~ysi~~l~~~icstate nf the mouse, Psych~jph~~rmuct~l~~~ic~(Berlin) Kupfcrherg,

tar r\;rluatittn

minor tranquilizer\

Neurtlsurg.

ture-activity

pmcrdure

Mcldrum

NMDA

I%%. Antirpileptic

J. Neurtti.

Watkins.

(office

London).

Comprehensive

quantitative

ap-

{SUSHI.).

Problems in Epifcpsy. Vol. 4. New Anti~‘~nvul~~nt Drug%.

rpilepq.

Press. London). Brain

14. 245. Griffin,

Current

Silver. J.M..

Press. New Jcrsryf p. Ihf.

1471, Prohit Analysis:

Response Curve, 3rd cdn. (Cambridge Fisher.

thtrir USC

G.B.

trst sy\tcm\

studies in epilepsy and their methodological

40.

12: Drugs us Tools Bnuttcm.

cd>. B.S.

administered

R.J..

Scyfricd. M. and R.A.

3

J.. L. Pirri and 8. Prud.h~lm. IYXO. Cimvul\ion*

Sander. J.W.A.S.

in rats: effect

inl~ihitf~rs, Ph~~rrn~~[)l. B&hem.

and nL\l

f’.plfq\t;f

1p. ZI.

eds. B.S. Meldrum D. Lccca.

320. Farrant.

Drug\.

London

Br. J. Pharmacol. frcrhand

of the conscious mouse.

lg91. Pentylonetetrazol-induced

of GABA

L&hey.

nists. hrnzodiazepinrs.

Physiof. 25. 319.

M.G.,

action.

in: C‘urrent Problems in Epifcp\!.

convulsant

crntrally

sound-induced

fo(,X. Accurate

drug

B.S.. IYXh. Preclinical

Morcau.

IYXJ. Evrtluation

Res. 34. 1261.

C.A. Vivtmi;~ and C.F. Baxter.

J. Appl.

M.

Disorders

mice

injcctittn into the la~crcrl tlrvin vcntriclc Corda,

Dity.

and B.S. M&drum. in DBA/Z

M&drum.

compounds.

IYY2. The eltectlvcne~s

acid ncurotr,ln\mltterr

5 I-w.

h;tscd on the

Am. %a~. Assac. J. Sept. 565.

and C.D.

I
to aniiconvuf~ant

eds. J.C. Watkins

p, 107. 19X0. Convulsant Woodhmy

in rpifrpsy.

in: The

and G.L. Coflin~rid%

(JRL

drugs: mechrtnisms of action. in:

Drugs: Mechanisms

J.K. Penry and D.M.

S. Clark. W.W. Anderson

recrptor

of Action.

(Raven

eds. G.H.

Press. Nrw York)

Glazer. p. 749.