l:11mp<'clll
`Joumal
`of l'hamwmlog_1·.
`222 ( 1992)
`19J-20J
`' 1992 Ebevicr
`Science
`Puhlishers
`RV. All rights
`re�er'l:d
`0014-2999/lJ2/$05Jl0
`
`ucb L059, a novel anti-convulsant
`drug:
`pharmacol
`ogical
`profile
`in animals
`
`Alma J. Gower, Michel
`Jean Gobert and Ernst Wulfcrt
`Noyer,
`Rene Vcrloes.
`UCB Plumnan·wirnl
`St'cTor.
`Cht:111i11
`du Foriest. /.110 Braim· I Allcml,
`Belgium
`
`Rccdved
`14 May 1992,
`revised
`MS received
`2 July 1992.
`accepted 11 August
`1992
`
`The anticonvulsant
`activity
`of ucb U159 ((S)-a-ethyl-2-oxo-pyrrolidine
`acetamidc)
`was evaluated
`in a range of animal
`model�.
`uch Lll59 was active
`after oral and intraperitoneal
`adminisiration
`in both rats and mice, with a unique
`profile
`of action
`incorporating
`features in common
`with several
`different
`types
`of antiepilcptic drugs.
`The compound
`was actiw,
`with ED,,. values
`generally with:n
`the range of 5.0-30.0 mg/kg,
`in inhibiting
`audiogenic seizures. electrically
`induced
`convulsions
`and convulsions
`induced
`chemically
`by pcntylcnetetra
`lie (PTZ),
`hicucullinc,
`picrotoxin
`and N-methyl-D-asp
`... rtatc
`(NMDA).
`uch L059 retarded
`the development
`of PTZ-indueed kindling
`in mice and reduced
`PTZ-induced
`EEG spike w1v·. discharge
`in rats.
`The R
`enantiomer,
`ucb L060, had low intrinsic
`anticonvulsant
`activity,
`showing
`the stcreospecificity
`of action
`of the molecule
`although
`the actual
`mechanism
`of action
`remains
`unknown.
`Ncurotoxicity,
`evaluated
`with an Irwin-type
`qbservation
`t:st.
`the rotarod
`test
`and open-field
`exploration,
`was minimal, with
`only mild sedation
`heing ohscrvcd,
`even at doses 50-100
`times higher
`than tl-e
`anticonvulsanl
`doses;
`at pharmacologically
`active doses,
`the animals
`appeared
`calm hut sliglitly
`more active.
`ucb L059 thi;�
`prcscnh
`as an ornlly active,
`safe, broad-spectrum
`anticonvu
`lsant
`agent.
`with potential anticpilcplogenic
`and anti-absence
`actions.
`
`ucb L059: Convulsions:
`Epilcptogcncsis:
`Petit-mat:
`Ep;lepsy
`
`I.Introduction
`
`cial effects
`on cognition
`in the elderly.
`Routine screen­
`ing of ucb L059 in the audiogcnic
`seizure-pr
`one mouse
`showed potent
`anticonvulsant
`activity. The
`effects
`of
`ucb L059 were consequently
`determined
`in a wide
`The term epilepsy refers
`to a wide range of neuro­
`range of anticonvulsant
`tests in rats and mice. The
`disorders
`characterised
`by an abnormal
`dis­
`logical
`tests,
`selected
`with reference
`to recommended
`pro­
`charge
`of cerebral
`neurones.
`The prevalence
`of epilepsy
`grammes for evaluating potential
`antiepilcptic
`drugs
`3 and 6 per IOOO (Griffin
`is estimated
`at between
`and
`(Fisher,
`1989; Kupferberg,
`1989; Losehcr
`and Schmidt,
`1991; Sander and Shnrvon,
`1987).
`There is a
`Wyles,
`1988; Meldrum, 1986).
`included
`genetic
`animal models,
`general
`consensus
`for the need for new, improved
`models involving electrical
`and chemical
`seizure
`induc­
`drugs to treat epilepsies
`(Loscher
`and Schmidt,
`1988:
`tion, as well as chemical
`kindling
`and chemically
`in­
`Porter,
`1986).
`Although
`epilepsy
`is adequately
`con­
`duced EEG spike-and-wave
`discharge
`as a model of
`trolled
`in the majority
`of patients,
`there remains
`a
`absence
`epilepsy. Reference
`anticonvulsant
`com­
`significant
`number of sufferers
`who arc untreated
`or
`pounds,
`including clinically
`used drugs.
`were tested
`in
`respond
`only partially
`to drug treatment.
`In addition,
`parallel
`for comparative
`purposes.
`In addition.
`the
`existing
`drugs,
`even when effective,
`arc not free from
`effects
`of the R enantiomcr,
`uch L060, were evaluated
`adverse
`side-effects
`and are a continuous
`cause of
`in a limited
`range of tests.
`Finally,
`the ncurotoxic
`concern to clinicians,
`particularly
`in view of the chronic
`effects of
`uch L059 were
`assessed
`on the basis of direct
`nature of drug treatment
`(Griffin
`and Wyles, 1991;
`observation,
`using an Irwin-type
`evaluation,
`locomotor
`Porter,
`1986).
`activit
`y in an open-field
`test and rotarod
`performance.
`ucb L059 ((S)-a-ethyl-2-oxo-pyrrolidinc
`acetamide)
`The results
`confirm
`that uch L059 has a broad spec­
`is the S enantiomer
`of the ethyl analogue
`of piracetam,
`trum of potent
`anticonvulsant
`activity
`with a very wide
`a drug widely
`used on account
`of its purported
`henefi-
`safety
`margin between
`pharmacologically
`active dosl'S
`Correspondence
`to: A.J. Gower,
`CNS Department,
`UCB Pharma­
`ceutical
`Sector,
`Chemin
`du Foriest,
`1420 Brainc
`l'Allcud.
`Belgium.
`and those causing
`neurotoxicity
`or adverse
`side-effects.
`ARGENTUM Exhibit 1098
` Argentum Pharmaceuticals LLC v. Research Corporation Technologies, Inc.
`IPR2016-00204
`
`Page 00001
`
`

`
`in
`
`mice
`
`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 au- dioeenic seizure tests. NMRI mice, either male or female depending on the test. aged 5-6 weeks with a bode weight of 22-23 g were used for ail other mouse c 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
`
`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 sub- jected 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 rcfer- ence 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 deiiv- cry. 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 caicu- Iated 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
`the Statistics section. From this data, the EDst, value, being the effective dose affording 50% protec- tion. 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-re- sponse curve for bicuculline 12.75-4.5 mg/kg i.p.I was examined. L2.3.3. NM04 Female NMRI mice wcrc pre- treated 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 car- ried 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, 17.11 or 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
`
`Page 00002
`
`

`
`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 mortal- ity 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 fore- and hindlimbs with loss of the righting reflex. In addi- tion, 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 picro- toxin (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 spike-and-ware discharge (SWD) in rats Rats implanted with cortical surface EEG elec- trodes 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-at- tenuated 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 /.LV with 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 spec- trum of behavioural and CNS parameters, by direct observation of spontaneous behaviour or after manipu- lation 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 re- tained. 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 sepa- ratcly, 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 photo- beams. 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 com- pound 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, Bcl- gium); carbamazepine (Sigma); clonazepam and di- azepam (Hoffman - La Roche, Switzerland), sodium valproate (Sigma; obtained both as the sodium salt and as valproic acid) and MK-801 hydrogen maleate (( + )-
`
`Page 00003
`
`

`
`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 administra- tion 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 dis- sohed 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 ap- proximately 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 PXl- ED,,, mg/kg p.~. (Y5G confidence limits) mrter Audiogrnic ~rizure~ WR CC TC Mazimal electroshock TC Bicuculline CC -t mg/kg i.p. TC Pentylenetrtrazoie CC 100 mg/k_e i.p. l-C uch LCiS4 Phenytrtin Cnrllamazepinr Clcmazepam Na Valproate 3 I .7 IX-44.5 ) a X0.6 2 75.5 U.O.SX h > 2SY 4.7f 7.5-11.91 3xf-Y K!u S-53.2) 70.0 ” D.03X * >217 (1513 -270 ) 7.0 ( 4.4- 9.1) 4.7 ( 4.2- 5.31 7.0 ( 6.3- 7.9) 0.029 (0.024-0.03h) 177 (IS7 -226 ) 23.5 f IO. I -Sk-l) 2.4 ( 1.9- 7.81 4.1 ( 2.7- 5.‘) 0.3 (0.15 -0.5’) ) 73.x ( 2x.3- I20.4) > 170 X0.h :’ 137 7 iI . “.. 0.05 * 33Y.Ot254 -h26 ) ‘9.5 (21 .Y-42.6) 14.n(11.5-1~.4) 19.3 (16.3-21.7) 0.029 (tMll7-0.12 ) 222.0 (15X -494 ) 1 700 :’ 75 7 il 75 5 iI __ ._ 5.-b I700 7.7( 6.1- 9.w 17:5 t 14-Y-2&l?) 0.07x W.Oh -0. I1 J 250 (221 -2x7 ) 0.037 (0.020-0.05 1) ifdl (124 -22Y) 2%3W inhib. 6 Inactive up to dose given: h limits could not he caIcula~ed.
`
`Page 00004
`
`

`
`197
`
`TABLE 2 Inhibition by ucb LO5Y or MK-WI i.p. of convulsions induced tr) NMDA (I nmol i.c.v.) in mice N = number of mice per drug and control group. %ug treatmect N Percentage uf mice responding (i.p. mg/kgf Tonic convulsion Clonic convulsion Control Drug (‘ontrol Drug u& 1.0% 5.4 20 41i.0 15.0 75.0 55.0 17.0 30 32.5 h.7 h 13.3 53.3 54.1’ 50 52.0 ‘2.0 ;1 78.0 70.0 170.0 JO 55.0 0 h 77.5 32.5 h MK-HOI 0.034 10 60.0 60.0 70.0 70.0 0. IOK 20 60.0 15.0 “ 75.0 50.0 0.340 20 60.0 0 h 75.0 0 h ” P = O.Ot; h P = WlOi: Fisher lest vrrsus control. ucb LO59 p.o. produced dose-dependent protection against tonic convulsions induced by maximal elec- troshock 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. 3.3. Clrernically induced conwisions in nzice 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 depen- dently protected against tonic convulsions although only the latter two were effective against clonic convui- sions. 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.59 for inhibition of tonic convul- sions 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 bicucuf- line-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
`Pentylenetetrazole (mg/kg i.p.1 Fig. 1. Effect of uch LO.59 on tonic convulsions induced by penlylenete~r~z~~le in 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) diffcr- ence from the corresponding 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 pro- duced 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-
`
`treatment Fig. 2. Effect of ucb LO59 on the development of pentylen~tetrazole- induced kindling in mice. Groups of 20 mice were injacted once daily with either vehicle control (:.I. ucb LOSY 5.4 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.
`
`80
`
`90
`
`100
`
`2
`
`4
`
`5
`
`Qays of
`
`6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`Page 00005
`
`

`
`CONVULSIONS
`
`BI MORTALITY
`
`I 1 J , I 1 0.4 0.6 0.6 0.4 0.6 0.6 8icuculllne (mg/kg I.v.) Bicuculllne (mg/kg I.v.1 Fig. 5. Effccr of uch LtW on t.,\b tonic convulGcms and (B) mortahty induced in rats hy bicucullinc. Groups of ‘0 rats received vehicle control t 1. uzb LtW I .i mg/Lp t T b. uch LCW 5.1 mgjkg (~1 or uch LO59 Ii me, kp ( u 1 p.o. 60 min hrfnre hicuculline (0.4, 0.h or 0.X mg/kg i.v.) was admrnizterd Each \aluc i> the pcrccntagr of ra responding. Solid symbols indicdtc ;I statistically significant (P < MS) differrncc 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 11
`
`days
`
`of kindling. ucb LO59, like the reference drugs tested, dose dependently reduced the incidence of tonic convulsions
`
`Al TONIC CONVULSIONS
`
`B) MORTALITY
`
`100 -
`
`lmg/kg I.v.) elicited by bicuculline (0.6 mg/kg i.v.). The EDS,, val- ues obtained 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 carba- mazepine. Neither ucb LO59 nor phenytoin, carba- mazepine or sodium valproate were able to alter the incidence of clonic convulsions: however, all of these drugs reduced the severity of convulsions. Clonazepam was exceptional in reducing the incidcncc of clonic convulsions.
`
`O-
`
`I, I 1 2.75 3.0 3.25
`
`Picrotoxrn
`
`J , I 1 2.15
`3.0
`
`Picrotoxln
`
`(mg/kg
`
`I.v.1
`
`Fig. 4. Effect of uch Ltl% on (,\I tcmic convulsi;rns and (B) mortality induced in rats hy picrotcwin. Groups of N rats received vehicle control ( ‘. 1. uch IMY 5.1 ms /kg ( ) or uch LtW I7 mg/kg f
`q
`1 p.o. 60 min hefore picrotoxin (1.7.5. 3.0 or 3.?i mg/kg i.v.) was administered. Each value is the wrscntage of ras responding. Solid symholh indizlte a st:ltistic;dly significant (P < 0.05) difference from the control.
`
`Page 00006
`
`A! TONIC
`3.25
`

`
`TABLE 3 Effrct of uch LOSY and reference drugs on tonic co~;*~ulsions induced by hicucullme (0.h mg/kg i.v.) in rats. Values given are doses affording 5115 protection (ED,,, mg/kg). with confidcnee limits in parentheses. .--.- __._..^ __ Test drug ED,,, (me/kg) P.o. (10 min 1.p. 30 min before i&u...;iinl: before: IbiLu ~2r.c ucb LOSY 7.7( 5.0 - Ii.‘) ) 5.1 f 3.1 - 8.6) Ph~nyti~in > lb0 iY.7 f 10.3 - 37.X) 41G inhib. at 160 Carbamazepine Y.7( 7.3 - Il.4 ) 15.6 I 13.0 - 1X.X) Sodium
`
`valproate
`
`329
`
`(302
`
`injection
`
`(mini after PTZ
`
`mg/kg. At 170 mg/kg, ucb LOS? caused slight effects, consisting of reduced activity. reduced body muscle 01 4 I 4 8 0 20 40 60 89 100 120
`-357 ) 10x (100 -117 1 Clomrzepam 0.09 ( o.os- 0.14) 0.10 t 0.07- 0.12) In common with all of the reference antiepileptic drugs tested, except clonazepam, ucb LO59 was consid- erably 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 exempli- fied 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 paral- lel 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 convul- sions (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. 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 im- mobile. After p.o. administration, no gross effects were ob- served in mice in the Irwin test at
`Fig. 5. Effect of uch LO59 on the spike-and-wave discharge (SWD) produced in rats by pentyi~n~t~tr~zo~e f PTZt. A group of eight rats received either vehicle control (. f. ucb L%Y 5.4 mg/kg fc. ! or ucb LO5Y 17 mg/kg (17 1 i.p. 20 min hefore PTZ (25 mg/kg i-p.) was administered in a cross-over design. The duration of SWD. expressed in s/min. was measured direcrly from the EEC trace. Solid symbols indicate 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 maxi- mal 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 signifi- cantly impaired but the effect was not dose-dependent TABLE 4 Effect of ucb LMY compared to reference antiepileptic drugs in the irwin and Rotarod tests. MAD = minimal active dose causing overt hehavioural changes. ED5,, = effective dose at which 50% of the mice were unahlr to remain on the rod, tested 60 min after dosing. Drug Irwin test Rotarod Necro- MAD (mg/kg p.o.) ED,,, toxicity Mouse Rat (mg/kg pat index ’ Mouse Mttlise ucb LO54 1711 1 7011 > 17t111 > 243 Phenytoin ‘5 ’ _. ._ 251 93.6 I Y.Y (no limits) Carbamezepine ‘3.h 70.X YO 17.‘) txo- 100) Clonazepam 0.03 KY4 0.053 17.7 (t).37-t).~Y) Sodium valproate 144.2 ‘XX.4 507 3 .I! (372-h37) ” Neurotoxicity index is the ratio between the EDq,, value for inhihi- tion of tonic convulsions elicited in audiogenic mice (see tahlc I) and the ED5,, value obtained in the rotarod test.
`
`Time
`
`doses
`
`below
`
`170
`
`Page 00007
`
`

`
`;md did not permit the dctcrmination of an ED,,, value
`
`for ucb LOSS)
`dctcmiincd
`Ncurotosicity
`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 au-
`diogenic 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
`of phenytoin. carbamarcpine or clonazepam and
`almost 100 times higher than that of sodium valproatc.
`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-
`peared. clmsisting of slight decreases in both sponta-
`neous activity and reactivity.
`In the open-field
`test. uch LO59 slightly
`~~~orn~~t~~r activity
`in rats with statistically
`
`increased
`significant
`
`i.p. and 54-540
`from 5.4-170 mg/kg
`effects occurring
`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.
`
`of the
`3.8. liffkts
`R mantiorner. ucb LO60, in aruicon- wlsant tests in rodimts
`
`in
`ucb LO60 was active only at very high doses
`protecting against convulsant activity
`induced in mice
`and rats (table 6). ED5(, values of 1064 mg/kg and 1400
`p.o. were obtained against tonic and clonic audiogeni-
`tally
`induced convulsions
`in mice, respectively.
`In the
`other tests, namely, convulsions
`induced by MES, PTZ
`and bicucullinc
`in mice, and by bicuculline
`in rats, ucb
`LO60 was inactive or produced only slight
`inhibition at
`the highest doses tested.
`
`Efkrt
`
`of uch LthO in anticonvulsant IC’SIS in rodents. \vR = urlil running: CT‘ = chmic c~mvulsion: TC = tonic convulsion. ~~~lnvul\ant tr‘\t Specicx Route of administration Paramrter Resuh Mouse Prctreatmenl time P.O. h0 min P.O. 60 min PSI. 60 min P.O. 60 min Lp. 30 min WR CC TC TC TC ‘I’C TC 35% inhihition at 1700 mg/kg ED5,, = IhOO mg/kg ” ED,,, = IOh (77Y-l4S3) mg/kg 31% inhibition at 540 mg/kg Wi inhihition at 1700 mg/kg Y3 mg/kg ’ 540 mp/kg *’ a hia~tiW
`
`Up to dose
`
`given: h limits could not he calculated.
`
`Page 00008
`
`f tshk Il.
`indices n-xc
`at
`TABLE h
`

`
`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 electrically induced convul- sions and protected against the convulsant effects of a variety of chcmoconvulsants, including systcmicalfy in- jected PTZ, bicucullinc and picrotoxin and centrally injected NMDA. ucb LOS9 also rctardcd the dcvclop- ment of kindling induced in mice by subthreshold doses of PTZ. In addition, 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, ac- tivity 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 clon- azepam 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 i