232 (1993) 147-158
`European Journal of Pharmacology,
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`© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
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`EJP 52939
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`Profile of ucb L059, a novel anticonvulsant drug, in models of partial
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`and generalized epilepsy in mice and rats
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`Wolfgang Loscher and Dagmar Honack
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`Department of Pharmacology, Toxicology, and Pharmacy, School of Veterinary Medicine, Hannover, Germany
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`Received 5 October 1992, revised MS received 12 November 1992, accepted 8 December 1992
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`The novel anticonvulsant drug ucb L059 ((S)-a-ethyl-2-oxo-1-pyrrolidineacetamide) was evaluated in several rodent models of
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`partial and generalized seizures. Ucb L059 (27-108 mg/kg i.p.) increased the thresholds for tonic electroconvulsions and
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`myoclonic and clonic seizures induced by timed i.v. infusion of pentylenetetrazol (PTZ), but was ineffective in the traditional
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`maximal electroshock seizure and s.c. PTZ seizure tests in mice and rats in doses up to 500 mg/kg. The anticonvulsant potency
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`of ucb L059 in seizure threshold tests was similar to that of standard drugs, such as valproate. In amygdala-kindled rats, ucb L059
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`exerted potent anticonvulsant activity against both focal and secondarily generalized seizures at doses of 13-108 mg/kg. The
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`adverse effects of ucb L059 were quantitated in the open field and in standard tests for motor impairment, such as the rotarod
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`and chimney tests. Ucb L059 exerted only minimal effects on behaviour, e.g. slight hyperactivity, and did not impair muscle
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`activity in the rotarod test in doses up to 1700 mg/kg i.p. The data indicate that ucb L059 is an interesting new anticonvulsant
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`agent with a broad spectrum of activity and high therapeutic index.
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`Ucb L059; Seizures; Epilepsy; Piracetam; Valproate; Anticonvulsant drugs
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`1.Introduction
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`drugs, including piracetam have to be administered at
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`very high doses in order to achieve anticonvulsant
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`activity (Schmidt, 1990; Fischer et al., 1991; Keller,
`Nootropic drugs, such as piracetam (2-oxo-1-pyrro­
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`1991).
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`lidineacetamide), are used clinically as cognition-en­
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`hancing agents in the elderly (Rosenberg et al., 1990;
`More recently, ucb L059 ((S)-a-ethyl-2-oxo-l-pyrro­
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`lidineacetamide), an ethyl analogue of piracetam (fig.
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`Sarter, 1991). The mechanism of action of these drugs
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`1), was shown to exert potent anticonvulsant effects at
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`is unknown, but some evidence suggests that effects on
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`relatively low doses (5-30 mg/kg) in various models of
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`energy metabolism and n:euronal excitability might be
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`involved (Rosenberg et al., 1990; Sarter, 1991). More
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`generalized seizures in rats and mice (Gower et al.,
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`recently, piracetam and other nootropic drugs were
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`1992). The novel drug appeared to be more potent
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`reported to exert anticonvulsant effects in different
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`against tonic seizures than against clonic seizures, which
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`seizure models, including chemical kindling in rats
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`would be similar to standard antiepileptic drugs, such
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`(Schmidt, 1990; Fischer et al., 1991; Keller, 1991).
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`as phenytoin and carbamazepine. Models of partial
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`Piracetam has also been reported to lessen cognitive
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`epilepsy, such as amygdala-kindling (McNamara, 1984),
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`disturbances and to improve seizure protection in
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`were not included in the study of Gower et al. (1992).
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`epileptic patients receiving carbamazepine (Chaudhry
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`In the present study, we examined the anticonvul­
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`et al., 1991). Since many epileptic patients have mem­
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`sant profile of ucb L059 in a battery of seizure models
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`ory disturbances, either due to the disease itself or to
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`previously proposed for the evaluation of new anticon­
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`the chronic treatment with standard antiepileptic drugs
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`vulsant drugs in mice and rats (Loscher and Schmidt,
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`(Trimble, 1991), an anticonvulsant drug with cognition­
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`1988). The seizure tests included models with threshold
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`enhancing activity would offer important advantages
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`as well as suprathreshold induction of seizures in order
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`for the treatment of epilepsy. However, most nootropic
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`to ensure that effects of ucb L059 against specific
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`seizure types were not missed. Furthermore, amygdala­
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`kindled rats were used as a model to evaluate drug
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`activity against complex partial seizures (Loscher and
`Correspondence to: W. Llischer, Department of Pharmacology, Tox­
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`Schmidt, 1988). The selectivity of the anticonvulsant
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`icology and Pharmacy, School of Veterinary Medicine, Biinteweg 17,
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`W-3000 Hannover 71, Germany.
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`effects produced by ucb L059 was examined by using
`ARGENTUM Exhibit 1097
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`IPR2016-00204
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`148 ~O Fig. 1. Chemical structure of ucb L059 ((S)-a-ethyl-2-oxo-l-pyrro- lidineacetamide). models for quantitative determination of 'minimal neu- rotoxicity', such as the rotarod and chimney tests. All tests were used in a standardized manner as recently described (L6scher et al., 1990, 1991a, b; L6scher and Nolting, 1991). 2. Materials and methods 2.1. Animals Male NMRI-mice were obtained from a commercial breeder (Winkelmann Versuchtstierzucht GmbH, Bor- chen, F.R.G.) at the age of 4 weeks (body weight 19-27 g) and were allowed to adapt to the laboratory for 1 week before the experiments were started. All experi- ments with drug injections were then carried out within the next week to minimize the effect of increasing age on drug susceptibility. Each mouse was used for only one experiment. Female Wistar rats (Winkelmann) were bougth at an age of 11-12 weeks (body weight 180-200 g) and were used after at least 1 week of adaptation to the laboratory. Female rats were used because they are known to eliminate drugs less rapidly than male rats, which was thought to be an advantage for the drug potency studies. Furthermore, at the age used, the female rats had almost reached their final body weight and, in contrast to adult male rats, could be kept in groups of 10 (or more) in one cage, which is an advan- tage for large scale studies. Mice and rats were kept in groups of 10 in plastic cages at controlled temperature (25°C) and humidity (about 40%) with a 12-h light cycle beginning at 7 a.m. They received standard diet (Altromin, Lage, F.R.G.) and tap water ad libitum. All drug injections were given in the forenoon at an ambient temperature of 23-25°C. 2.2. Tests used to evaluate anticonvulsant activity against generalized seizures The threshold for seizures induced by maximal (tonic hindlimb extension) electroshock in mice was deter- mined via transauricular electrodes (i.e. copper elec- trodes introduced bilaterally into the ears) by means of a stimulator (BMT Medizintechnik GmbH, Berlin, F.R.G.) which delivered a constant current (adjustable from 1 to 200 mA regardless of the impedance of the test object; self-adjusting stimulus voltage maximally 7000 V) with sinusoidal pulses (50/s) for 0.2 s. The stimulus intensity was varied by an up-and-down method in which the current was lowered or raised in 0.l-log intervals if the preceding animal did or did not show hind limb extension, respectively. The data thus generated in groups of 20 mice were used to calculate the threshold current for inducing hind limb extension in 50% of the mice (CC50 with confidence limits for 95% probability), using the method of Kimball et al. (1957). Each group of animals was used for only one threshold determination. Control groups, which re- ceived the vehicle used for drug administration, were tested together with the drug-treated animals, using the same pretreatment time at which the drug was tested. In additional experiments with ucb L059, the threshold for tonic electroconvulsions was determined with transauricular electrodes in rats (20 animals per group), using 0.6-log intervals for the up-and-down method instead of the 0.l-log intervals used for mice. The maximal (tonic hindlimb extension) elec- troshock seizure (MES) test with supramaximal stimu- lation was carried out with corneal copper electrodes and with apparatus described above, using a fixed current of 50 mA in mice and 150 mA in rats with a pulse frequency of 50/s for 0.2 s. The tonic extension of the hind limbs was used as endpoint in both species. Ten animals were used per group. In several control groups (with or without vehicle injection) of mice and rats tested prior to the drug experiments, all animals of a group exhibited tonic extension of hindlimbs so that, in contrast to the threshold test, daily control groups were not necessary for experiments with the MES test. The threshold for different types of pentylenetetra- zol (PTZ)-induced seizures was determined by infusing a 1% solution of PTZ into the tail vein of unrestrained freely moving mice at a rate of 0.3 ml/min with an infusion pump (Perfusor-E, Braun Melsungen, F.R.G.). Groups of 10-12 mice were used per threshold deter- mination. In untreated control mice, the following seizure types occurred during PTZ infusion (described in order of appearance): (1) one or more generalized myoclonic twitches of the whole body, (2) repeated clonic seizures of fore- and/or hindlimbs without loss of righting reflexes, (3) a generalized clonus with re- peated clonic seizures of fore- and hindlimbs, during which animals fell onto their side, i.e. exhibited loss of righting reflexes, (4) clonic seizures with loss of righting reflexes followed by backward extension of forelimbs (forelimb tonus), often but not always followed by (5) tonic hindlimb extension. In the experiments with ucb L059, the following endpoints were used for quantifica- tion of drug effects: (1) the initial myoclonic twitch, (2)
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`the first generalized clonus with loss of righting re- flexes, and (3) the forelimb tonus. The threshold for each endpoint was calculated as the dose (mg/kg) of PTZ inducing this endpoint during infusion. The threshold for tonic hindlimb seizures could not be quantitated in all animals because the infusion was usually stopped after 2 min and not all animals exhib- ited tonic hindlimb seizures during this period. Increas- ing the infusion time did not resolve this problem, since several mice of a group died without showing hindlimb tonus. However, all animals showed forelimb tonus within 2 min, and this seizure type proved to be a more reliable endpoint with less variation than hindlimb tonus. All drug-treated groups were compared with vehicle-treated groups on each day. For the s.c. PTZ seizure test, before evaluation of the anticonvulsant drugs, a dose-response curve for PTZ was determined in groups of 10 mice or rats. PTZ was injected s.c. in the back of the neck of the animals. The animals were then observed for 30 min after injection and the first generalized clonic seizure with loss of righting reflexes was used as the endpoint. In this way, the dose inducing this seizure type in 97% of the animals (CD97) was calculated in mice (80 mg/kg) and rats (90 mg/kg) by the method of Litchfield and Wilcoxon (1949). After administration of these doses, the following seizure types were observed in untreated mice and rats (described in the order of appearance): (1) one or more generalized myoclonic twitches of the whole body, (2) repeated clonic seizures of fore- and/or hindlimbs without loss of righting reflexes (correspond- ing to the 'threshold seizure' proposed by Swinyard (1969) for anticonvulsant drug evaluation in the s.c. PTZ test), (3) a generalized clonic seizure of fore- and hindlimbs, during which animals fell onto their side, i.e. exhibited loss of righting reflexes, (4) loss of right- ing followed by tonic forelimb seizure, and/or (5) loss of righting with tonic fore- and hindlimb seizure. In the controls (with or without vehicle injection), endpoints 1, 2 and 3 were observed in 9-10 animals of a group. In contrast, especially in mice, the number of animals that also exhibited generalized tonic seizures (extension of hind- and/or forelimbs) after these doses varied. For drug potency studies, PTZ was injected s.c. in groups of 10 animals at the time of the maximal anticonvulsant effect (see below) and the animals were observed for 30 min for the occurrence of seizures. None of the vehicles used during these experiments affected PTZ-induced seizures. For the threshold models, the significance of differ- ences between individual (vehicle-treated) control groups and drug-treated groups was calculated by Stu- dent's t-test. In case of significant drug effects, the doses increasing the MES or PTZ threshold by 20% (TID20) were determined by log-linear regression anal- ysis from dose response curves, using at least three 149 doses per drug (for more details see l_~scher et al., 1991a, b). For comparison with the anticonvulsant po- tency of ucb L059, TID20 values of seven standard antiepileptic drugs were determined in the seizure threshold models. Swinyard et al. (1952) originally pro- posed the use of TID20 values to compare the potency of anticonvulsant drugs, and TID20 values have been used to compare GABA mimetic drugs in seizure threshold models (Lfscher, 1982, 1985). In this respect, it is important to not that the TIDz0 values of drugs such as vigabatrin are much closer to the doses effec- tive in epileptic patients than the more commonly used TIDs0 values (L6scher, 1982, 1985). Thus, in the case of drugs with relatively low potency, the concept of TID20 measurements, as originally proposed by Swin- yard et al. (1952), might yield predictive data in terms of clinical effectiveness. In all seizure tests, anticonvulsant potency was quantified at the time of the maximal anticonvulsant effect (I_dSscher et al., 1991a, b). For ucb L059, the time of the peak effect was determined for the electrocon- vulsive threshold in mice and for kindled seizures (see below) in rats. 2.3. Amygdala kindling For implantation of stimulation and recording elec- trodes, rats were anaesthesized with chloral hydrate (360 mg/kg i.p.) and a bipolar electrode was stereo- taxically implanted in the right basolateral amygdala (according to the surgery methods described in the atlas of Paxinos and Watson (1986)). The coordinates for electrode implantation were AP--2.2, L-4.8, V-8.5, measured from the bregma. Skull screws served as the indifferent reference electrode. The electrode assem- bly was attached to the skull with dental acrylic ce- ment. After a postoperative period of 2 weeks, con- stant current stimulations (500/xA, 1 ms, monophasic square-wave pulses, 50/s for 1 s) were delivered to the amygdala at intervals of 1 day until 10 stage 5 seizures were elicited. The electrical susceptibility of the stimu- lated region (threshold for induction of afterdis- charges) was recorded on the first day of the experi- ment (initial afterdischarge threshold) as well as after kindling (with an interval of at least 4 days after the 10th stage 5 seizure), using an ascending stairstep procedure (Freeman and Jarvis, 1981). The initial cur- rent intensity was 10/zA, and the current intensity was increased in steps of about 20% of the previous current at intervals of 1 min until an afterdischarge lasting at least 3 s was elicited. Since almost all fully kindled animals exhibited generalized seizures (stage 4-5) at the afterdischarge threshold current, it was not neces- sary to determine the threshold for generalized seizures separately. In fully kindled rats, the afterdischarge threshold determined with interstimulation intervals of
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`150 1 day was not different from the afterdischarge thresh- old values determined with interstimulation intervals of 1 min, thus demonstrating that the short interstimula- tion interval did not bias the afterdischarge threshold determinations. In addition to afterdischarge thresh- old, the following parameters of kindled seizures were measured in fully kindled rats after stimulation with either the afterdischarge threshold current or supra- threshold stimulation with 500/~A: seizure severity was classified according to Racine (1972): 1, immobility, eye closure, twitching of vibrissae, sniffing, facial clonus; 2, head nodding associated with more severe facial clonus; 3, clonus of one forelimb; 4, rearing, often accompa- nied by bilateral forelimb clonus; 5, rearing with loss of balance and falling accompanied by generalized clonic seizures. Seizure duration was the duration of limbic (stage 1-2) and/or motor seizures (stage 3-5); limbic seizure activity sometimes occurred after termination of stage 3-5 seizures but was not included in the seizure duration. Afterdischarge duration was the total time that spikes with a frequency of at least 1/s were present in the EEG, recorded from the site of stimula- tion. The anticonvulsant effects of ucb L059 in fully kin- dled rats were determined in a group of eight rats with reproducible stage 5 seizures in two ways: (1) kindled seizures were evoked by suprathreshold stimulation with 500 /zA after i.p. drug injection; (2) the focal seizure threshold (afterdischarge threshold) was deter- mined after i.p. drug injection. The drug was injected 0.5 h, 1 or 2 h prior to stimulation. In the experiments with suprathreshold stimulation, control responses were determined 2-3 days before and after each drug ad- ministration, and the next drug experiment was only undertaken if all rats showed reproducible stage 5 seizures. Similarly, in the experiments for the determi- nation of the afterdischarge threshold, the control fo- cal seizure threshold was determined 2-3 days before and after drug treatment. For control determinations, rats received an i.p. injection of vehicle (saline) with the pretreatment time of the respective drug experi- ment (see below). For all drug experiments, at least 4 days were interposed between two drug injections in order to avoid changes in drug potency due to drug accumulation or tolerance. The anticonvulsant activity of piracetam was evalu- ated in additional experiments with another group of fully kindled rats. Experiments were done at 500/zA as described above. The significance of differences between pre-drug control responses and responses after drug injection was calculated by the Wilcoxon signed rank test for paired replicates. The EDs0 for suppression of secon- darily generalized (stage 4-5) seizures was calculated by the method of Litchfield and Wilcoxon (1949) as described previously (L6scher et al., 1986). 2.4. Tests used for quantification of adverse effects Groups of 10 mice or rats were used per dose of ucb L059. The animals were observed at various times up to 192 h after injection for adverse effects as described below. Groups of 10 mice or rats injected i.p. with vehicle (saline) were used as controls. Minimal neurological deficit, such as sedation and impaired motor function, was quantitated with the rotarod test and the chimney test. In the chimney test, the animals had to climb backwards in a plastic tube of 25 cm length and 3 cm inner diameter in the case of mice and 50 cm length and 5.5 cm inner diameter in the case of rats. Neurological deficit was indicated by the inability of the animals to climb backwards up the tube within 30 s. Whereas untreated control mice were able to perform the test without previous training (normal mice climb backwards through the tube within less than 5-10 s), rats had to be trained (and data were then used as individual control) before drug adminis- tration. The rotarod test was carried out with rods of differ- ent diameters, which were produced by inserting a metal rod into a rubber (mice) or foam rubber (rats) tube in order to provide traction. For mice, the rod had a diameter of 2.5 cm and rotated at 6 r.p.m., whereas for rats the rod had a diameter of 6 cm and rotated at 8 r.p.m. Neurological deficit was indicated by the in- ability of the animals to maintain their equilibrium for at least 1 rain on the rotating rod. Untreated mice were able to remain on the rod for several min, whereas rats had to be trained before drug experiments. After drug treatment, mice or rats that were not able to maintain their equilibrium on the rod for 1 min were put on the rod twice. Only animals that were not able to remain on the rod at three subsequent l-rain attempts were considered to exhibit neurological deficit. TDs0 was determined in the chimney test at the time of the maximum effect of ucb L059. TDs0 values with confidence limits for 95% probability were calcu- lated by the method of Litchfield and Wilcoxon (1949), using a computer program (Pharm/PCS). For compari- son with the TDs0 values of ucb L059, TDs0 values were determined in the same tests with seven standard antiepileptic drugs as described elsewhere (L6scher and Nolting, 1991). In addition to these quantitative estimations of neu- rological deficit, behavioural alterations caused by ucb L059 were recorded in the animals' home cages and in an open field (90-100 cm in diameter). Muscle tone was estimated by palpation of the abdomen. The extent of sedation, ataxia and muscle relaxation after adminis- tration of ucb L059 was determined with a rating system as described previously (L6scher et al., 1987). In short, animals were taken out of cage, placed in an open field and observed for about 1 min; sedation and
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`ataxia were rated separately (abdominal muscle tone was evaluated by palpation at the end of the observa- tion period). Sedation: 1, slightly reduced forward loco- motion; 2, reduced locomotion with rest periods in between (partly with dosed eyes); 3, reduced locomo- tion with more frequent rest periods; 4, no forward locomotion, animal sits quietly with closed eyes. Ataxia: 1, slight ataxia in hindlegs (tottering of the hind quar- ters); 2, more pronounced ataxia with dragging of hind legs; 3, further increase in ataxia and more pronounced dragging of hind legs; 4, marked ataxia, animals lose balance during forward locomotion; 5, very marked ataxia with frequent loss of balance during forward locomotion. The same protocol for recording adverse effects was also used in kindled rats. Rectal body temperature was measured with an electronic ther- mometer. The weight of the animals was recorded daily, before and after drug injection. The significance of differences in body weight or body temperature between pre-drug and post-drug data determined in the same group of animals was calculated by using the Wilcoxon signed rank test for paired replicates. 2.5. Drugs The drugs used in this study were kindly provided by the following companies: ucb L059 by UCB Pharma- ceutical Sector (Braine l'Alleud, Belgium); phenobarbi- tal (as sodium salt), valproate (as sodium salt), carba- mazepine, phenytoin, and ethosuximide by Desitin 151 Arzneimittel GmbH (Hamburg, F.R.G.); diazepam and donazepam by Hoffmann-La Roche (Basle, Switzer- land). PTZ was purchased from Caesar and Loretz (Hilden, F.R.G.) and piracetam from Sigma (Munich, F.R.G.). Ucb L059 was dissolved in saline and injected i.p. at an injection volume of 10 ml/kg in mice and 2-4 ml/kg in rats. Piracetam, phenobarbital (as sodium salt), valproate (as sodium salt), phenytoin (by means of dilute NaOH), diazepam (by means of dilute HC1) and ethosuximide were freshly dissolved in saline and injected i.p. at a volume of 10 ml/kg in mice and 2-3 ml/kg in rats. Drugs, i.e. carbamazepine and clon- azepam, which were insoluble in aqueous vehicles were dissolved by means of lipophilic vehicles. We used either warmed glycofurol (tetraglycol; tetrahydrofur- furylalcohol polyethylene glycol ether) or warmed polyethylene glycol 400 (PEG 400) to dissolve the drug and warmed saline was then added under continuous stirring. In order to avoid the possible effects of vehicle alone, maximum concentrations of 30% PEG 400 or 10% glycofurol were used; at these concentrations, the solvents do not alter the seizure thresholds or the behaviour of animals in the chimney and rotarod tests (Lrscher et al., 1990). The injection volume was 10 ml/kg in mice and 2-10 ml/kg in rats. All doses and concentrations of drugs given in this study refer to the free acid or base of the respective drug. TABLE 1 Effect of ucb L059 on the threshold for tonic (hindlimb extension) electroshock seizures in mice and rats. The seizure threshold (stimulation via ear electrodes) was determined as CCs0 (with confidence limits for 95% probability) in groups of 20 animals per dose. For ucb L059, the effect of three different doses on the threshold is shown. Control groups (40 mice and 60 rats) received i.p. vehicle injection. Significant differences between controls and drug-treated groups are indicated (ap < 0.001). The dose which increased the threshold by 20% (TID20) was calculated from the dose response curves for ucb L059 in mice and rats. For comparison, TID2o values for standard antiepileptic drugs determined in the same model in mice are shown. All determinations were done at the times of the peak drug effects, i.e. 60 min (ucb L059 in mice and rats), 5 min (valproate), 15 min (diazepam, clonazepam), 30 min (phenobarbital, ethosuximide), and 120 min (phenytoin), respectively. Not effective is indicated by n.e., not determined by n.d. Drug Dose Mice Rats (mg/kg i.p.) Threshold TID20 Threshold TID20 (CCs0 (mg/kg (CCs0 (mg/kg in mA) i.p.) in mA) i.p.) Ucb L059 Ucb L059 Phenobarbital Carbamazepine Phenytoin Valproate Ethosuximide Diazepam Clonazepam 0 7.4 (6.8- 8.0) 18.4 (17.4-20.0) 27 8.3 (7.7- 9.0) a 22.9 (20.8 - 25.2) a 54 9.6 (8.9 -- 10.3) a 22.4 (20.8 -- 24.1) a 108 9.6 (8.9-- 10.3) a 24.6 (22.1 --27.2) a 40 2.9 1.2 4.9 50 n.e. 1.6 0.37 29 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
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`152 CCso [mA} 0.5 I 2 3 Hours after administration Fig. 2. Effect of ucb L059 (54 mg/kg i.p.) on the threshold for tonic (hindlimb extension) electroconvulsions in mice at different times after administration. Data are means (and upper part of confidence limits for 95% probability) from groups of 20 mice. Controls (white bars) received an i.p. injection of saline and were tested at the same time after injection as the drug-treated groups (black bars). The electroconvulsive threshold is shown as CCs0 in mA. Significant differences between individual control and drug treated groups are indicated by asterisks (P < 0.001). 3. Results 3.1. Effect of ucb L059 on thresholds for generalized and clonic seizures As shown in fig. 2, ucb L059 caused a long-lasting increase in the threshold for tonic (hindlimb extension) electroconvulsive seizures in mice. After i.p. injection of 54 mg/kg, maximal effects were observed between 0.5 and 2 h. On the basis of these data, all subsequent anticonvulsant tests with mice were done 1 h after injection of ucb L059. As shown in table 1, ucb L059 dose dependently increased the electroconvulsive threshold at 27 and 54 mg/kg, but a further increase in dose did not result in more pronounced effects on seizure threshold, possibly indicating slow absorption of the drug after high doses (see below). The potency of ucb L059 to increase the electroconvulsive threshold was similar to that of val- proate. A comparable anticonvulsant activity was demonstrated in rats for the electroconvulsive thresh- old (table 1). The anticonvulsant activity of ucb L059 against dif- ferent types of generalized seizures was determined after timed i.v. infusions of pentylenetetrazol (PTZ). The drug exerted significant effects against myoclonic and clonic seizures, whereas the threshold for forelimb tonus was not altered significantly (table 2). The anti- convulsant potency of ucb L059 against myoclonic seizures was about twice as high as that of valproate or ethosuximide (table 2). 3.2. Effect of ucb L059 in the MES and s.c. PTZ tests Ucb L059 was not effective in the traditional MES test with suprathreshold stimulation (50 mA in mice and 150 mA in rats) via eye electrodes. In mice, the effect of 20 mg/kg ucb L059 was tested after 1 h, and the effect of 100 or 500 mg/kg was tested after 2 h; none of the animals were protected against tonic hindlimb extension. Similarly, only 1 of 10 rats showed protection against hindlimb extension 2 h after i.p. injection of 500 mg/kg ucb L059. TABLE 2 Effect of ucb L059 on the threshold for different seizure types induced by i.v. infusion of pentylenetetrazol (PTZ) in mice. The seizure threshold was calculated as the dose of PTZ (+ S.D.) inducing the respective seizure types in all animals of a group, using 9-10 mice per group. The effect of three different doses of ucb L059 on the threshold is shown. Controls (n = 10) received i.p. vehicle injection. Significant differences between controls and drug-treated groups are indicated (a p < 0.05; b p < 0.01; c p < 0.001)). The dose which increased the threshold for the initial myoclonic twitch by 20% (TID20) was calculated from the dose response curves for ucb I2)59. For comparison, TID20 valves for standard ant±epileptic drugs determined in the same model in mice are shown. All determinations were done at the times of the peak drug effects, i.e. 60 min (ucb L059), 5 rain (valproate), 15 min (diazepam, clonazepam), 30 min (phenobarbital, ethosuximide), and 120 rain (phenytoin), respectively. Not effective is indicated by n.e. Drug Dose PTZ threshold (mg/kg) TID20 (mg/kg) Initial Generalized Forelimb against initial i.p.) myoclonic clonus with tonus myoclonic twitch twitch loss of righting (mg/kg i.p.) reflexes Ucb L059 Ucb L059 Phenobarbital Carbamazepine Phenytoin Valproate Ethosuximide Diazepam Clonazepam 0 38.4±5.3 45.7±6.7 82.7±13.8 27 44.5±5.0 a 52.2±4.3 88.1±12.4 54 47.8±5.2 c 58.1±7.3 c 88.9±17.1 108 49.3±6.7 c 58.2±9.4 b 92.9±23.0 40 7 n.e. n.e. 85 89 0.1 0.015
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`153 TABLE 3 Effect of ucb L059 on kindled seizure parameters in fully amygdala-kindled rats with reproducible stage 5 seizures. For comparison, some experiments were done with piracetam. All amygdala stimulations were carried out with a suprathreshold current of 500/zA. Control responses were measured 2-3 days prior to drug administration in the same group of animals, which received i.p. injection of vehicle instead of drug. All data (shown as means 4-S.D.) were determined in the same two groups of 8-10 animals. Absence of S.D. indicates that all rats had the same responses, Pretreatment times for drug or vehicle are indicated in the table. Significance of differences to individual control responses is indicated (a p < 0.05; b p < 0.01). Drug Dose Time of (mg/kg pretreat- i.p.) ment (h) Kindled seizure parameters Seizure Seizure Afterdischarge severity duration duration (score) (s) (s) Ucb L059 Piracetam 0 0.5 5 53.0 ± 11.0 88.5 ± 41.3 54 0.5 3.4 + 1.5 a 40.1 ± 19.8 65.6±37.9 0 1 5 65.9± 11.2 92.1 ± 13.8 54 1 2.6 ±1.5 b 26.4± 9.8 b 47.6±42.9 0 2 4.9 +0.4 61.8± 11.3 93.0± 16.0 54 2 3.8 ± 1.2 a 51.4 ± 25.3 64.0 ± 35.0 0 1 5 62.0± 16.1 98.0± 17.1 13 1 4.5 ±0.5 41.0±18.0 a 74.5±38.8 0 1 5 59.0± 23.2 96.1 ± 16.1 27 1 3.6 ±1.2 a 42.1±12.9 a 70.6±32.9 0 1 5 57.0±16.1 104 ±29.8 54 1 1.9 ±1.0 b 33.4±14.2 b 52.0+20.5 0 1 5 58.1± 12.1 104 :t: 18.2 108 1 3.6 ± 1.1 b 44.3_+26.6 49.4±31.9 0 2 5 55.4± 10.3 98.9± 12.7 108 2 1.5 ± 1.1 b 43.1 ± 18.5 46.3± 19.5 0 1 5 43.2± 17.1 64.7±24.0 54 1 4.9 ± 0.31 45.5 ± 16.6 79.0 ± 24.7 0 1 5 47.8 ± 14.9 80.2 ± 22.7 108 1 5 45.6 ± 14.9 65.6 ± 22.9 0 1 5 54.7+ 12.4 81.6+ 12.7 216 1 3.9 ±1.2 a 34.0±15.5 a 61.2±31.8 0 2 5 60.0 ± 36.2 81.1 ± 25.8 216 2 4.13±0.99 a 45.9:t: 14.9 a 68.1 ±36.8 0 1 5 53.8+23.5 79.0+ 16.5 432 1 4.33 ± 0.51 46.6 ± 18.0 75.9 ± 24.9 0 2 4.89 ± 0.33 48.4 ± 13.9 85.3 ± 22.7 432 2 4.0 ±1.1 a 39.1± 7.6 75.8+49.5 Ucb L059 did not exert anticonvulsant effect against clonic seizures in the traditional s.c. PTZ seizure test, in which animals were observed for the occurrence of seizures 30 min after s.c. injection of PTZ (80 mg/kg in mice, 90 mg/kg in rats). Thus, all 10 mice had myoclonic and clonic seizures when PTZ was injected 2 h after administration of ucb L059, 500 mg/kg. Simi- larly, all 10 rats displayed myoclonic and clonic seizures when PTZ was injected 1 h after administration of ucb L059, 500 mg/kg. In contrast to the inefficacy against TABLE 4 Effect of ucb L059 on the focal seizure threshold (afterdischarge threshold) in fully amygdala-kindled rats. Control threshold (after vehicle injection) was determined 2 days before drug administration in the same group of rats. Thresholds were determined 1 h after drug or vehicle injection. Afterdischarge thresholds and seizure parameters recorded after stimulation with the afterdischarge threshold current are shown as means + S.D. of eight rats. Sig