DOI 10.1007/s00702-004-0155-6 J Neural Transm (2004) 111: 1121–1139

Effects of piracetam alone and in combination with antiepileptic drugs in rodent seizure models W. Fischer1, H. Kittner1, R. Regenthal2 , E. Russo3 , and G. De Sarro3 1 Rudolf-Boehm-Institute of Pharmacology and Toxicology, and Institute of Clinical Pharmacology, University of Leipzig, Germany 3 Department of Experimental and Clinical Medicine, Faculty of Medicine and Surgery, University of Catanzaro, Italy 2

Received December 12, 2003; accepted March 27, 2004 Published online May 14, 2004; # Springer-Verlag 2004

Summary. The nootropic drug piracetam was investigated in various experimental models of epilepsy. Generally, piracetam exhibits no or only moderate anticonvulsant properties against generalized tonic or clonic seizures. However, in many cases it did increase the anticonvulsant effectiveness of conventional antiepileptics, as shown in the maximal electroshock seizure (MES) threshold test, the traditional MES test or in DBA=2 mice. A pharmacokinetic interaction does not seem to be responsible for this effect. In lethargic mice, a model of absence seizures, piracetam significantly decreased the incidence and duration of spike-wave discharges. Furthermore, in the cobalt-induced focal epilepsy model piracetam reduced the number of spikes=min and in the hippocampal stimulation model it increased the anticonvulsant potency of phenobarbital and phenytoin after single and repeated administration. In conclusion, the well tolerated piracetam itself did not show marked anticonvulsant effects in most screening tests, however, its co-medication with antiepileptic drugs improved seizure protection in various models which may bear potential clinical significance. Keywords: Piracetam, nootropic drugs, antiepileptic drugs, anticonvulsants, seizure models. Introduction Around 0.5–1% of the human population or more than 50 million people worldwide suffer from epileptic diseases (Meinardi, 1993; Sander and Shorvon, 1996). Despite the development of various novel antiepileptic drugs in recent years, about one third of the affected patients is resistant to current pharmacotherapies (L€ oscher and Schmidt, 2002). In many of these cases, seizure

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control is inadequate or limited by side effects of the used drugs. Disturbances of learning and memory are also frequently described in epileptic patients and are a major factor influencing social life and personality (Helmstaedter and Kurthen, 2001; Rahmann et al., 2002). Besides the type, duration and frequency of seizures including the underlying neuropathological changes, the chronic treatment with antiepileptic drugs may also contribute toward symptomatology of cognitive disorders and behavioural changes (Trimble, 1987; Kwan and Brodie, 2001; Brunbech and Sabers, 2002). Surprisingly, memory disturbances tend to be regarded as unavoidable sequelae of certain forms of epilepsy and only few experimental and clinical studies have been undertaken to mitigate these symptoms (Mondadori and Schmutz, 1986). An interesting aspect to compensate intellectual dysfunctions in epileptic patients could be the additive administration of ‘‘memory-enhancing’’ agents (Mondadori et al., 1984; Schmidt, 1990; L€ oscher and H€ onack, 1993). Nootropic drugs like piracetam improve learning and memory in a variety of experimental paradigms (Nicholson, 1990; Vernon and Sorkin, 1991). Piracetam is widely used in the therapy of age-related cognitive disturbances (Sarter, 1991; Israel et al., 1994; Flicker and Grimley, 2002; Waegemans et al., 2002) including Alzheimer’s disease (Tariska and Paksy, 2000; Tsolaki et al., 2001) and in the treatment of poststroke deficits (De Deyn et al., 1997; Kessler et al., 2000; Sareen, 2002). Further clinical experience has shown that this drug is effective against some types of myoclonus (Obeso et al., 1986; Fedi et al., 2001; Uthman and Reichl, 2002). For a combination therapy with antiepileptic drugs, nootropics should be of special interest which exhibit anticonvulsant actions by themselves or increase the anticonvulsant potency of antiepileptics in co-medication. Previous experimental studies showed that most nootropics including piracetam possess no marked anticonvulsant properties (for review, see Voigt and Morgenstern, 1988) or reveal only modest effects in special tests including chemical kindling in rats at higher doses (Mare
s, 1989; Schmidt, 1990; Keller, 1991; L€ oscher and H€onack, 1993). A detailed analysis of the profile of piracetam in a broad spectrum of seizure models is, however, missing and appears to be justified since controlled clinical trials show that this drug may be useful as adjunctive medication to lessen cognitive disturbances and=or to improve seizure protection in epileptic patients receiving carbamazepine (Kunnecke and Malan, 1979; Chaudhry et al., 1992). For this purpose, we have examined the effects of piracetam in various electrically and chemically-induced seizure tests including the cobalt epilepsy and the hippocampal stimulation model as well as in two genetic models of epilepsy. Furthermore, we investigated its influence on the anticonvulsant potency of various conventional antiepileptic drugs. Materials and methods Animals The basic screening tests (maximal electroshock seizure (MES) test, pentylenetetrazol (PTZ) seizure test, see below) were carried out on male albino mice (strain 01, University Leipzig, breeding centre Leipzig-Probstheida, Germany), weighing 19 to 25 g (4 to 5 weeks old). In the

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two genetic models of epilepsy, DBA=2 mice (from Harlan, Correzzana, Milano, Italy), weighing 8–14 g (22–26 day old), and lethargic (lh=lh) mice (bred at the University of Catanzaro, Italy), weighing 30–36 g (6–8 months old), were used. After adaptation to laboratory conditions for some days, the animals were allocated to the experimental groups by means of a randomized schedule; each mouse was used for one experiment only. For determination of cobalt-induced spike activity and electrically-evoked hippocampal afterdischarges, male Wistar rats (own breeding stock, formerly ‘‘Jelei: WIST’’), weighing 250 to 300 g at the beginning of the experiments, served as subjects. The animals were kept in colony cages under standard laboratory conditions on a natural light-dark cycle (or a 12-h light=dark cycle in DBA=2 and lh=lh mice) with free access to commercial food pellets and tap water. The screening experiments were carried out between 9 and 12 h to avoid circadian influences. Animal care and handling was conducted in compliance with the German and Italian Animal Welfare Act and was approved in each case by the local governmental authority in accordance with the European Communities Council Directive of laws and policies.

Maximal electroshock seizure threshold (MES-T) test (mice) The threshold for maximal electroshock seizures was determined via ear-clip electrodes by a constant current stimulator (rodent-shocker type 221; Hugo Sachs Elektronik, March-Hugstetten, Germany) which delivered a constant current (sinusoidal pulses adjustable between 1–150 mA, 50=s, 0.2 s duration). The endpoint was the tonic extension of the hindlimbs. The stimulus intensity was varied by an ‘‘up-and-down’’ method whereby the current was raised or lowered in 1-mA steps if the preceding animal did not or did show tonic hindlimb extension, respectively (for further methodical details, see Fischer et al., 2001). Current intensity-effect curves were constructed on the basis of the percentage of animals responding with the endpoint at the corresponding current value. The calculation of CC50-values (current intensity in mA, necessary to induce tonic hindlimb extension in 50% of the mice tested) and the statistical comparisons were performed using a computer-supported probit analysis according to the method of Litchfield and Wilcoxon (1949). In the case of multiple comparisons between different drug-treated and the corresponding control group, a-correction was performed.

Maximal electroshock seizure (MES) test (mice) Maximal electroshock seizure was induced in mice via ear-clip electrodes by a constant suprathreshold current (rectangular 20-ms impulses, 50 mA, 35=s, 0.4 s duration) following the method of Swinyard et al. (1952). The prevention of the hindlimb tonic extensor component was regarded as the endpoint of protection. The dose-response curves were estimated by testing 4–5 doses and 8 (sometimes 16) animals per dose. The calculation of ED50 values (dose that protects 50% of the animals against MES-induced tonic hindlimb extension) including the 95% confidence limits and the statistical analysis were performed according to the traditional method of Litchfield and Wilcoxon (1949).

Pentylenetetrazol seizure threshold (PTZ-T) test (mice) Clonic convulsions were induced by administering of the chemoconvulsant PTZ (85 mg=kg s.c.) in the neck of unrestrained mice at the time of peak effect for the respective test drug (i.p. or vehicle i.p.). The appearance of the first generalized clonus (repeated clonic seizures of the foreand hindlimbs lasting
5 s with an accompanying loss of righting reflex) was recorded during individual observation for 30 min (see Krall et al., 1978). The number of animals in the group (n ¼ 9 or 12) with clonic seizures and the latency time were analysed for statistical significance using Fisher’s exact probability test and the Logrank test, respectively.

Audiogenic seizures (DBA=2 mice) Each DBA=2 mouse was placed under a hemispheric perspex dome and 1 min was allowed for habituation and assessment of locomotor activity. Auditory stimulation (12–16 kHz, 109 dB) was

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applied for 1 min or until tonic extension occurred. The seizure response was classified behaviourally using the following scale: 0 ¼ no response, 1 ¼ wild running, 2 ¼ clonus, 3 ¼ tonus, 4 ¼ respiratory arrest (see De Sarro et al., 1988). Behavioural changes were observed during the period between drug administration and auditory testing. Significant differences between groups of combined treatment (piracetam plus vehicle and piracetam plus antiepileptic drugs) were estimated using Fisher’s exact probability test (incidence of seizure phases). The percentage of mice showing 50% of clonic or tonic phase ED50 values (95% confidence limits) were calculated using a computer-supported probit analysis according to the method of Litchfield and Wilcoxon (1949). The relative anticonvulsant activities were determined by comparison of respective ED50 values (95% confidence limits).

Absence epileptic seizures in lethargic (lh=lh) mice All mice were chronically implanted with five electrodes under halothane anaesthesia. At least 1 week after surgery, each mouse underwent five daily electroencephalogram (EEG) recordings without drug administration to observe the EEG pattern. Then, during the 5-h recording session, mice received i.p. either vehicle or test drug at 60 min after each baseline recording (at least 6 mice per dose). The identification of absence seizures was based on the expression of bilaterally synchronous electroencephalographic bursts of 5–6 Hz spike-and-wave discharges (SWDs), as previously described by Hosford et al. (1992) (i.e., amplitude not less than 60 mV and frequency range of 5–6 Hz; seizures must have a duration no shorter than 0.6 s). The quantification of absence seizures was based on the number and duration of spike-and-wave discharges (SWDs) or poly-spikes, as previously described (Russo et al., 2003). The data were statistically analysed using one-way repeated analysis of variance (ANOVA) followed by Student-Newman-Keuls test for multiple comparisons.

Cobalt-induced focal epileptiform activity (rats) Cobalt-induced focal epilepsy was experimentally elicited in rats according to previous methods (Fischer et al., 1967; K€astner et al., 1972). Briefly, the dura of anaesthetized rats (ketamine-HCl 100 mg=kg i.p. plus xylazine-HCl 15 mg=kg i.p.) was incised and a cobalt-agar-pellet (about 1 mm in length and 0.8 mm in diameter; 0.5–1 mg metallic cobalt) was inserted directly into the right sensorimotor cortex. For EEG-recording six stainless-steel electrodes were positioned epidurally above the bulbus olfactorius, the sinus sagittalis superior (reference electrode; 2 mm frontomedially to the bregma), the sensorimotor and the visual cortex. The electrodes were connected to a miniature socket which was embedded on the surface of the skull with dental cement. Single spikes or polyspike discharges were found as first signs of pathological events within the first two weeks after cobalt implantation. The epileptiform activity then gradually declined and disappeared after some weeks. Seven days after surgery, first control EEG-recordings were performed in the state of passive wakefulness. The following parameters were used for quantification: number of spikes and spike-wave groups (‘‘cobalt spindles’’) per min and the number of spikes per group. Only animals with a stable spike activity registered over the sensorimotor cortex were used for drug testing. Statistical differences between the control recordings before and after drug administration (intra-individual comparisons) were evaluated by the Wilcoxon signed rank test.

Electrically-evoked hippocampal afterdischarges (rats) Under ketamine=xylazine anaesthesia (see above), the rats were fixed to a stereotaxic apparatus (TSE, Bad Homburg, Germany) and bipolar deep electrodes were implanted into the right dorsal hippocampus (AP 2.5 to 3.0 posterior to bregma, L ¼ 1.8 mm lateral to the midline and V ¼ 2.7– 3.0 mm ventral to the skull surface; stereotaxic coordinates according to Fifkova´ and Mar
sala, 1967). For EEG-recording six stainless-steel electrodes were positioned epidurally above the bulbus olfactorius, the sinus sagittalis superior, the sensorimotor and visual cortex (for further details, see Fischer et al., 2001). Two weeks after surgery, the rats were habituated to the recording setup and EEG-recordings (Bioscript BST 1, Zw€onitz, Germany) were performed.

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Constant current stimulations were delivered to the deep electrodes in the hippocampus, when a stable EEG response had been established from the freely behaving animals. The stimulus train (rectangular 1-ms current impulses, 60–300 mA, 50=s, 5 s duration) was applied from a HSEstimulator (type 215=1, HSE, March-Hugstetten, Germany) coupled to a stimulus isolation unit and a constant current unit. The individual stimulation threshold for hippocampal rhythmic spike activity (‘‘afterdischarges’’) was estimated with a series of stimulations, commencing with 60 mA and increasing in 20 mA steps every 2 min until an afterdischarge (amplitude at least twice the voltage of the prestimulus EEG signal, frequency
1=s) was elicited. To ensure a stable response (
20 s), this threshold value was increased by a factor of 1.2 and the response was retested every three days. Drugs were studied when the duration and pattern of the hippocampal afterdischarges remained almost constant over 3–4 control tests. Localization of the deep electrodes was verified histologically (cresyl violet-stained frontal sections). Statistical differences (intra-individual comparisons) were established by the paired Student’s t-test.

Plasma levels of phenobarbital, valproate as well as piracetam (rats) The two antiepileptics, phenobarbital (20 mg=kg i.p., 60 min before blood drawing) and valproate (150 mg=kg i.p., 30 min before) were injected in combination with vehicle (controls) or piracetam (500 mg=kg i.p., 60 min before) in four groups of 6–7 rats. Individual blood samples of 300– 500 ml, taken from the retro-orbital venous plexus under short diethylether anaesthesia, were collected into Eppendorf tubes and centrifuged at 10,800 rev=min for about 1 min. Subsequently, 100 ml of the supernatant were pipetted into Abbott system cartridges and the total plasma level of phenobarbital or valproate was determined by an Abbott TDx analyzer (Abbott, Irving, TX, USA), which is based on a fluorescence polarization immunoassay (FPIA) technique. For comparison of the plasma levels of phenobarbital and valproate in vehicle- or piracetam-treated rats, Student’s t-test was used. In addition, the plasma levels of piracetam were determined in the same piracetam-phenobarbital and piracetam-valproate-treated groups by liquid–liquid sample extraction and reversed phase liquid chromatography and UV-detection at 206 nm according to Louchahi et al. (1995). The plasma levels were expressed as means  SEM in mM or mM (6–7 rats for each group).

Drugs and solutions The drugs used were: carbamazepine, clonazepam, ethosuximide, phenobarbital-Na, valproate-Ca (Arzneimittelwerk Dresden, Germany), lamotrigine (Glaxo Wellcome, Verona, Italy), piracetam (Isis-Chemie, Zwickau, Germany), pentylenetetrazol (Knoll, Ludwigshafen, Germany), phenytoin-Na (G€ odecke, Freiburg, Germany), valproate-Na (Sigma-Tau, Pomezia, Italy, as indicated in DBA=2 and lh=lh mice). All doses refer to the salts. The drugs were dissolved as follows: pentylenetetrazol in 0.9% NaCl-solution, phenobarbital-Na, phenytoin-Na (with 1–2 drops of 1 N NaOH) in distilled water; piracetam, carbamazepine, clonazepam, ethosuximide and valproateCa were given in a 2% suspension of hydroxyethylcellulose. Animals in the control groups received equivalent volumes of the vehicle (10 ml=kg in mice; 2 ml=kg in rats) and were always tested together with the respective experimental group.

Results Effect of piracetam alone and in combination with valproate on the threshold for electroshock-induced tonic seizures Single administration of piracetam (100–500 mg=kg i.p., given 60 min before testing), or repeated administration (500 mg=kg i.p., once daily for 6 days) did not influence the threshold for electrically-induced tonic (hindlimb extension) seizures in mice. On the other hand, the standard antiepileptic drug valproate (150 mg=kg i.p., 30 min before) as reference substance significantly increased the electroconvulsive threshold by about 40% (Fig. 1). In co-medication,

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Fig. 1. Effect of piracetam (PIR, 500 mg=kg) alone and in combination with the antiepileptic drug valproate (VP, 150 mg=kg) on the threshold for tonic (hindlimb extension) electroshock seizures (MES-T) in mice. In one series of experiments (‘‘Acute’’), mice were given PIR and VP alone or in combination 60 and 30 min before threshold determination. In the second series (‘‘6d Vehicle’’ and ‘‘6d PIR’’), mice were injected with vehicle or PIR once daily for 6 days and on the 7th day received PIR or VP (‘‘6d Vehicle’’) and PIR alone or in combination with VP (‘‘6d PIR’’), respectively. The columns represent the CC50-values (with confidence limits for 95% probability) of drugs or drug combinations (16–20 animals per dose), expressed in percent of the control MES thresholds which were determined in parallel (C ¼ 100%, vehicle=vehicle 10 ml=kg i.p.). Means of the control MES thresholds were 5.9 (5.6–6.2) mA (‘‘Acute’’) and 6.0 (5.6–6.4) mA (‘‘6d Vehicle’’), respectively. Doses of drugs (in mg=kg i.p.) are indicated below the columns. ? P < 0.05 (Probit analysis)

piracetam tended to increase the anticonvulsant effectiveness of valproate after acute administration and significantly increased the potency of valproate after subchronic application. Effect of piracetam on maximal electroshock seizures Piracetam (100–1000 mg=kg i.p., 60 min before MES) exhibited no protective effects in the traditional MES test in mice. When piracetam (300, 1000 mg=kg) was given as co-medication with phenobarbital, phenytoin or carbamazepine, the ED50 values of the antiepileptic drugs were generally reduced by 20–40%, combinations with phenobarbital revealed statistical significance (Fig. 2). Influence of piracetam on pentylenetetrazol-induced clonic seizures In the PTZ seizure threshold test in mice with clonic convulsions as endpoint, piracetam (100–1000 mg=kg, i.p., 60 min before PTZ) revealed no seizure protective effects. Only at the highest dose the latency time to the first generalized clonus was significantly increased (Table 1). On the other hand, the two reference antiepileptic drugs ethosuximide (150 mg=kg i.p.) and valproate (200 mg=kg i.p.) significantly protected mice against clonic seizures (10 of 12 animals each)

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Fig. 2. Influence of piracetam (PIR) on the anticonvulsant effectiveness of phenobarbital (PB), phenytoin (PHT) and carbamazepine (CBZ) in the maximal electroshock seizure (MES) test in mice. The columns represent the ED50 values (with confidence limits for 95% probability) of drug combinations, expressed as percent of the parallel determined ED50 values of the antiepileptic drug alone (controls ¼ 100%, injection of vehicle 10 ml=kg i.p.). Doses of PIR (in mg=kg i.p.) are indicated below the columns. The average control ED50 values were: PB 14.0 (60 min before MES), PHT 7.6 (90 min before) and CBZ 9.3 mg=kg i.p. (60 min before), respectively. ? P < 0.05 (Litchfield and Wilcoxon, 1949) Table 1. Effects of piracetam (PIR) alone and in co-medication with ethosuximide (ETHO) and valproate (VP) on pentylenetetrazol (PTZ)-induced clonic seizures in mice Substance

Dose (mg=kg i.p.)

Time of application before PTZ

PTZ seizure threshold test (85 mg=kg s.c.) Number of mice=with seizures

(min) Vehicle PIR Vehicle PIR Vehicle ETHO þ Vehicle ETHO þ PIR Vehicle VP þ Vehicle VP þ PIR

– 100 300 – 500 1000 – – 50 – 50 þ 500 – – 50 – 50 þ 500

60 60 60 60 60 60 30 þ 60 30 þ 60 30 þ 60 30 þ 60 30 þ 60 30 þ 60

12=12 12=12 12=12 9=9 9=9 9=9 12=12 12=11 12=11 9=9 9=9 9=9

Latency to the first generalized clonus (min)

(% Control)

7.76  0.90 5.86  0.67 6.33  1.30 7.54  0.91 7.63  0.83 11.25  0.89 6.95  0.79 9.33  1.06 10.09  1.13 6.83  0.85 8.69  0.37 10.42  1.3

100  11.6 75.7  8.6 81.6  16.7 100  12.1 101.2  11.0 149.2  11.8 100  11.4 134.3  15.3 145.2  16.2 100  12.4 125.9  5.4 152.6  19.6

All calculated data are given as means  SEM. On the left, doses of piracetam and antiepileptic drugs, respectively (control groups were treated with vehicle). In the middle, number of animals within the group vs. animals with generalized clonic seizures. On the right: latency time to the first generalized clonus. Significance level:  P< 0.05 (Logrank test)

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and increased the latency time (164.3  35.4 and 216.5  49.8%, respectively; P<0.05 each). The co-medication of a low dose of ethosuximide (50 mg=kg i.p.) with piracetam (500 mg=kg i.p.) did not increase the effectiveness of the former, but significantly increased the latency time (Table 1). Very similar results were obtained when a low dose of valproate (50 mg=kg i.p.) was given in combination with piracetam. Anticonvulsant activity of piracetam in audiogenic seizure prone DBA=2 mice Piracetam (administered i.p., 60 min before auditory stimulation) exerted dosedependent anticonvulsant effects against the tonic, clonic and wild running phase of audiogenic seizures in DBA=2 mice. The respective ED50 values were 103 (86–122), 268 (203–354) and 493 (359–674) mg=kg, respectively. Low doses of piracetam (25 and 50 mg=kg i.p.) did not influence the incidence and the severity of audiogenic seizures, whilst piracetam (100 mg=kg i.p.) tended to Table 2. ED50 values (95% confidence limits) of antiepileptic drugs alone or in co-medication with piracetam (50 and 100 mg=kg i.p.) against the audiogenic seizure phases in DBA=2 mice Seizure phase

Drug þ saline

Drug þ piracetam (50 mg=kg)

Drug þ piracetam (100 mg=kg)

Wild running Carbamazepine Diazepam Ethosuximide Lamotrigine Phenobarbital Phenytoin Valproate-Na

10.6 (8.1–13.8) 0.49 (0.34–0.71) 290 (207–406) 6.1 (4.6–8.1) 7.1 (5.6–9) 4.3 (3.1–6) 84 (63–114)

7.6 (5.4–10.7) 0.25 (0.16–0.39) 262 (183–375) 4.5 (3.4–5.9) 3.9 (3.1–4.9) 2.9 (2.1–4.0) 46 (29–73)

5.9 (4.2–8.3) 0.21 (0.14–0.31) 186 (139–219) 3.9 (2.8–5.4) 3.4 (2.5–4.6) 2.6(1.8–3.8) 38 (27–53.5)

Clonus Carbamazepine Diazepam Ethosuximide Lamotrigine Phenobarbital Phenytoin Valproate-Na

4.4 (3.6–5.4) 0.28 (0.2–0.39) 138 (96–198) 3.5 (2.4–5.1) 3.4 (2.3–5) 2.5 (1.8–3.5) 43 (33–56)

3.0 (2.6–4.1) 0.14 (0.10–0.2) 106 (78–144) 2.3 (1.6–3.3) 1.5 (0.9–2.5) 1.4 (0.9–2.2) 21.2 (16.1–27.9)

2.6 (1.8–3.7) 0.12 (0.09–0.16) 80 (49–131) 2.0 (1.4–2.9) 1.3 (0.8–2.1) 1.2 (0.8–1.8) 19.5 (15.1–25.2)

Tonus Carbamazepine Diazepam Ethosuximide Lamotrigine Phenobarbital Phenytoin Valproate-Na

3.0 (2.6–3.8) 0.24 (0.15–0.39) 90 (70–116) 1.1 (0.7–1.8) 2.4 (1.7–3.4) 2.0 (1.6–2.5) 31 (22–43)

1.8 (1.2–2.7) 0.11 (0.07–0.17) 70 (52–94) 0.7 (0.5–1.0) 1.2 (0.8–1.8) 0.9 (0.6–1.35) 15.2 (10.4–22.2)

1.6 (1.1–2.3) 0.10 (0.06–0.17) 63 (47–84) 0.6 (0.4–0.9) 1.1 (0.7–1.73) 0.69 (0.42–0.91) 13.4 (9.5–18.9)

Significant differences in the ED50 values among concurrent groups of antiepileptic drug þ saline- or antiepileptic drug þ piracetam-treated groups are denoted by  P < 0.05 and  P < 0.01 (method of Litchfield and Wilcoxon, 1949)

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reduce the incidence of clonic and tonic phases (not shown). However, piracetam (50 or 100 mg=kg i.p.) administered simultaneously with conventional antiepileptic drugs, markedly reduced their ED50 values (Table 2). Effects of piracetam on the number and duration of SWDs in lh=lh mice Lower doses of piracetam (50, 100 and 200 mg=kg) were not able to induce a significant reduction in the number of SWDs in comparison with a group of mice receiving only vehicle. At a higher dose, piracetam (400 mg=kg) showed a clear reduction in the incidence of SWDs and this action was found most evident during the 90–120 min period post injection (Fig. 3A, Table 3). The

Fig. 3. Anti-absence effects of piracetam and valproate-Na, given alone and in co-medication, in lethargic (lh=lh) mice. A Time- and dose-dependent reduction of the number of epileptic spike-and-wave discharges (nSWDs) by piracetam (100, 200 and 400 mg=kg, n ¼ 6 mice for each dose); B Effects of piracetam on the cumulative duration of SWDs (dSWDs). C Time- and dose-dependent reduction of the number of nSWDs by valproate-Na (100 mg=kg) alone and by two combinations of valproate-Na (100 and 200 mg=kg) with piracetam (100 mg=kg each). D Effects of valproate-Na and the two combinations with piracetam on the cumulative duration of SWDs (dSWDs). All values are expressed as means  SEM and represent each 30-min period of recording. ? P < 0.05, ?? P < 0.01, compared to the vehicle controls (one-way repeated ANOVA)

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Table 3. Number of episodes and duration of spike-and-wave discharges in lethargic (lh=lh) mice following treatment with piracetam and antiepileptic drugs alone or in co-medication Substance

Dose (mg=kg i.p.)

Number of episodes of spike-and-wave discharges (n=period 90–120 min)

Duration of spike-and-wave discharges (s=period 90–120 min)

Vehicle Piracetam

– 50 100 200 400 – 100 200 100 þ 100 200 þ 100 – 100 200 100 þ 100 200 þ 100 – 100 300 100 þ 100 300 þ 100

19.6  2.1 18.2  2.1 17.9  1.8 17.3  1.6 13.1  1.5 19.8  2.2 15.3  2.1 15.2  2.0 12.7  1.6 7.8  1.0 19.7  2.1 23.2  2.3 23.4  2.3 17.1  2.1 17.1  2.2 19.6  2.0 16.3  1.8 14.6  1.7 12.6  1.6 9.6  1.6

321  18 277  15 242  15 219  14 183  13 322  18 228  14 179  13 202  14 167  12 314  16 338  17 379  18 290  15 291  15 321  18 212  14 175  13 174  12 162  12

Vehicle Ethosuximide Ethosuximide þ Piracetam Vehicle Carbamazepine Carbamazepine þ Piracetam Vehicle Valproate-Na Valproate-Na þ Piracetam

Values are given as mean duration of spike-and-wave discharges (SWD)  SEM (groups of 6–8 lethargic mice), determined in the testing period from 90–120 min. Reference period (period 30 min before drug injection).  P < 0.05,  P < 0.01, compared to vehicle; one way repeated ANOVA followed by Student-Newman-Keuls test

mean total duration of SWDs was not affected by 50 mg=kg piracetam, 100 mg=kg piracetam tended to decrease and the two higher doses (200 and 400 mg=kg) significantly decreased the duration of SWDs (Fig. 3B, Table 3). Again, the effects were most pronounced in the time period from 90–120 min after injection. The antiepileptic drug valproate-Na (100 mg=kg i.p.) when given alone slightly reduced the number of SWDs (Fig. 3C). The effectiveness of valproate was further increased by piracetam (100 mg=kg i.p.) which at this dose had no effect by itself. When the duration of SWDs was considered, the effects of valproate alone were very marked, but the co-medication of piracetam further decrease the duration of SWDs (Fig. 3D). Qualitatively similar results can be observed with ethosuximide (100 and 200 mg=kg i.p.) alone and in combination with piracetam, whereas carbamazepine (100 and 200 mg=kg i.p.) alone or in combination with piracetam did not significantly affect the incidence of SWDs. Table 3 summarizes the anti-absence effects of piracetam and the three antiepileptics alone or in co-medication in the time period from 90–120 min.

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Effects of piracetam upon cobalt-induced focal epileptiform activity The application of metallic cobalt into the cerebral cortex of rats results in the development of a chronic epileptiform discharging focus accompanied by myoclonic twitches of body musculature. The brain pathology manifests as single spike-wave activity or trains of spike discharges (‘‘cobalt spindles’’) and can be observed especially in the state of passive wakefulness. Beside the primary focus around the cobalt implant, often a mirror focus was present on the contralateral side at 2–4 weeks. Administration of piracetam (100 and 300 mg=kg i.p.) significantly reduced the number of spikes=min to 81.6  14.3 and 77.2  14.9% (n ¼ 8 animals; P<0.05) and tended to reduce the number of spike groups=min to 88.1  12.9 and 88.2  12.5%, respectively, but not the number of spikes=group 30 min after injection (Fig. 4). On the other hand, the antiepileptics ethosuximide (200 mg=kg i.p.) and clonazepam (0.5 mg=kg i.p.) significantly reduced the number of spikes=min as well as the spike groups=min within 30 and 60 min after administration (Fig. 4). Effects of piracetam upon electrically-evoked hippocampal afterdischarges In unrestrained rats stimulated by chronically implanted hippocampal electrodes, piracetam alone (500 mg=kg i.p.) tended to reduce the duration of hippocampal afterdischarges but did not influence the focal stimulation threshold to induce spike activity (Fig. 5). The two antiepileptics, phenobarbital and

Fig. 4. Effect of piracetam (PIR, 300 mg=kg) in comparison with ethosuximide (ES, 200 mg=kg i.p.) and clonazepam (CAZ, 0.5 mg=kg i.p.) on the cobalt-induced focal epileptiform activity in rats. The columns represent the means  SEM (n ¼ 6–8 animals). The rats were tested before (controls ¼ 100%) and 30 and 60 min after drug administration (intra-individual comparison) in the state of passive (relaxed) wakefulness. The time of EEG recording and the doses of drugs (in mg=kg i.p.) are given below the columns. Control values were 49.6  9.1 (spikes=min), 5.4  1.6 (groups=min) and 4.7  0.4 (spikes=group); ? P < 0.05 (Wilcoxon signed rank test). (&) ¼ spikes=min, ( ) ¼ spike groups=min, (&) ¼ spikes=group

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Fig. 5. Effect of piracetam (PIR, 500 mg=kg) and phenobarbital (PB, 20 mg=kg) or phenytoin (PHT, 20 mg=kg) alone and in combination after single co-administration (‘‘ac’’ ¼ acute) as well as after 6 daily injections (‘‘6d’’) of piracetam (tests on the 7th day after PIR þ PB or PIR þ PHT co-administration) on the duration of electrically-evoked hippocampal afterdischarges (top) and the stimulation threshold for hippocampal spike activity (bottom). The rats were tested three days before (controls ¼ 100%) and 55–60 min (PIR), 55–60 min (PB and PIR þ PB) and 80–90 min (PHT and PHT þ PIR), respectively, after drug application (intraindividual comparison). Each rat underwent four drug tests, separated by one (or two) control trials. The columns represent the means  SEM of 6 animals each (at the base of the columns in the lower diagram: number of animals with increased threshold=number of animals per group). The doses of drugs (in mg=kg i.p.) are given below the columns. The individual control values (duration of initial spike phase) were 24 to 42 s, the control thresholds (increased by a factor of 1.2) were between 160 and 240 mA. ? P< 0.05, ?? P < 0.01 (paired t-test)

phenytoin (20 mg=kg i.p. each) alone reduced the duration of hippocampal afterdischarges but did not significantly elevate the focal stimulation threshold at this dose. The co-medication of piracetam, first given as single combination with phenobarbital and phenytoin, respectively, showed a marked increase in the effectiveness of both antiepileptics including a significant elevation of the stimulation threshold. After subchronic pre-medication of piracetam (500 mg=kg i.p. once daily for 6 days), in both cases a complete suppression of the

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afterdischarge activity could be registered and a tendency for a further increase of the stimulation threshold. Effects of piracetam on the plasma level of phenobarbital and valproate Piracetam (500 mg=kg i.p.) did not alter the total plasma level both of phenobarbital (20 mg=kg i.p.) [alone 86.6  3.8 vs. 90.4  4.1 mM (each n ¼ 6 rats)] or valproate (150 mg=kg i.p.) [alone 1.76  0.06 vs. 1.78  0.08 mM (each n ¼ 6 rats)]. These findings suggest that pharmacokinetic interactions between piracetam and the two tested antiepileptics, in terms of total plasma levels, are not likely. The plasma level of piracetam (500 mg=kg i.p., 60 min post injection) was 4.73  0.13 mM (n ¼ 11). Discussion Additive administration of nootropic drugs might be of clinical value in human epileptic disorders and, for this reason, the objective of the present study was (i) to examine the effects of the prototype nootropic drug piracetam in a series of seizure models and (ii) to determine its influence on the anticonvulsant potency of conventional antiepileptics. Our study focused on standard screening tests such as the MES, MES-T and s.c.-PTZ seizure threshold test in mice (L€ oscher and Schmidt, 1988), two genetic animal models, the lethargic mouse and the audiogenic seizure susceptible DBA=2 mouse as well as two rat models, the cortical cobalt epilepsy model and the hippocampal afterdischarge model. First, piracetam exhibits no marked anticonvulsant properties in most screening models for generalized tonic-clonic seizures including electroshock or various chemically-induced seizures (Moyersoons et al., 1969; Dlaba
c et al., 1981; Morgenstern et al., 1989; Fischer et al., 1991; present study). This also holds true for MES threshold tests (see Dlaba
c et al., 1981; present study). However, in audiogenic seizure models a marked effect of piracetam was obtained. In the present study, piracetam exerted anticonvulsant activity against the tonic, clonic and wild running phase in the susceptible DBA=2 mouse, a genetic animal model for generalized tonic-clonic seizures (Kellogg, 1976). In previous studies, piracetam had revealed protective effects against audiogenic seizures in rats (Moyersoons et al., 1969; Bene
sova´, 1980). In the PTZ-kindling, a model for primary generalized seizures (Corda et al., 1991), piracetam was even more potent than in the models considered in the present study. Thus, in fully kindled rats, a dose-dependent anticonvulsant activity for piracetam was described (Schmidt, 1990; Keller, 1991; Genkova-Papasova and LazarovaBakarova, 1992). Piracetam also significantly suppressed the PTZ-kindling development (Schmidt, 1987). Next, the effects of piracetam were studied in models predictive for myoclonic and=or absence seizures. In the s.c.-PTZ seizure threshold test, the latency time to the first generalized clonus was not affected at low doses but significantly increased at high doses whereas the clonic convulsions were not suppressed (present study). When given in combination with low doses of valproate and ethosuximide, piracetam further increased their effect on the

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latency time to the first generalized clonus. In the i.v.-PTZ seizure threshold test, piracetam was found to produce a slight increase in the seizure threshold but had no effects on the anticonvulsant activity of valproate-Na (Morgenstern et al., 1989). In the lethargic (lh=lh) mouse, a genetic animal model of absence seizures (Hosford et al., 1992), piracetam showed a slight reduction in the incidence and duration of SWDs. Furthermore, piracetam increased the efficacy of low doses of ethosuximide and valproate on SWDs. As expected, carbamazepine, which is not suited for the treatment of absences, was devoid of an effect in the lh=lh mouse model, both in the absence and presence of piracetam. A third series of experiments focused on two rat models that are predictive for partial or complex partial seizures, the cobalt epilepsy model (K€astner et al., 1972) and the hippocampal afterdischarge model (Fischer et al., 1993, 2001). The results show that piracetam significantly reduced the number of Cospikes=min and tended to reduce the duration of electrically-induced spikedischarges, respectively. For the hippocampal afterdischarge model, also the effect of combined administration with two standard antiepileptics was determined. Especially after repeated administration of piracetam (once daily for 6 days) before the co-medication, the anticonvulsant potency of phenobarbital and phenytoin can be markedly increased. Furthermore, in the amygdala-kindling, an other model for complex partial seizures, piracetam significantly decreased the seizure severity (Schmidt, 1990; see also L€ oscher and H€ onack, 1993). We also studied the influence of piracetam on the anticonvulsant effectiveness of standard antiepileptics both in the MES-T and MES test as well as in DBA=2 mice. Piracetam tended to increase the effect of valproate on the electroconvulsive threshold when given acutely; this effect became significant upon administration of piracetam over a time period of 7 days. In the MES test in mice, piracetam increased the anticonvulsant activity of phenobarbital but only tended to increase the effectiveness of the two other tested antiepileptics, carbamazepine and phenytoin. It should be mentioned that piracetam is well tolerated and neither produced sedation nor impaired motor coordination, as revealed behaviourally and by the rotarod test (Fischer et al., 1991). In DBA=2 mice, low doses of piracetam increased the protective effects of all tested antiepileptic drugs. In previous studies in models for generalized tonic-clonic seizures (MES; rats), piracetam increased the anticonvulsant activity of carbamazepine (Mondadori et al., 1984) but failed to increase the effects of valproate, phenytoin and phenobarbital (Mondadori and Schmutz, 1986). Morgenstern et al. (1989) found no influence of piracetam on the anticonvulsant activity of phenytoin in the MES test (mice). Finally, in a special model of focal (tetanus toxin-induced) limbic epilepsy, piracetam was able to increase the anticonvulsant activity of carbamazepine (Hawkins and Mellanby, 1986). The possibility that the additive effects of piracetam in combination with standard antiepileptics are related to pharmacokinetic interactions seems to be not likely. Thus, the present experiments show no significant modulation of the total plasma levels of phenobarbital and valproate by piracetam. Previous experiments from our laboratory revealed that the levels of phenobarbital, carbamazepine and ethosuximide in rat brain cortex were not altered by piracetam (Scheibler and Kittner, 1996).

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An interesting question concerning the present study seems to be that for possible mechanisms by which piracetam does diminish seizure activity or increase the anticonvulsant activity of standard antiepileptics. Piracetam is a cyclic derivative of g-aminobutyric acid (GABA) that crosses the blood-brain barrier and accumulates in brain tissue (Tacconi and Wurtman, 1986; Vernon and Sorkin, 1991) but neither shows appreciable affinities for GABA receptors (Gouliaev and Senning, 1994) nor has effects on uptake, content or activity of GABA in different areas of the brain (M€ uller et al., 1999). Furthermore, this drug failed to affect Naþ currents in isolated hippocampal pyramidal neurones (Kopanitsa et al., 2000). Thus, an influence on GABA and Naþ channels, the mechanisms of action of many standard antiepileptics, can be ruled out. On the other hand, piracetam was found to weakly inhibit the binding of [3H]-glutamate at its receptor sites and to decrease the content of the excitatory transmitter glutamate in neocortex (Bering and M€ uller, 1985). Moreover, this drug attenuated the veratridine-induced elevation in intracellular Ca2þ concentration in rat hippocampal pyramidal neurones and, therefore, may reduce pathologically high Ca2þ levels (Zelles et al., 2001). Solntseva et al. (1997) demonstrated in isolated snail neurones a suppression of high-threshold Ca2þ currents by piracetam indicating possible Ca2þ channel modulating effects of this drug. Excessive Ca2þ influx into cells is assumed to play an important role in the generation of epileptiform discharges (Schwartzkroin and Wyler, 1980; Speckmann et al., 1993). Although the nature of our experimental data does not allow to speculate in too detailed terms, it is conceivable that mechanisms, modulating the intracellular Ca2þ level or influence the excitatory glutamatergic neurotransmission, may be of importance for the observed moderate anticonvulsant effects of piracetam, especially in models for partial and complex partial seizures including amygdala kindling. On the other hand, this drug failed to block Naþ channel activity, which can explain its inefficacy in electroshock-induced tonic seizure models. Because of the mostly negligible effects on GABAergic mechanisms, piracetam showed only a slight activity in the lethargic mouse absence model and in the PTZ-induced clonic seizure model.1 However, it should be mentioned that also for the clinical efficacy of piracetam in cortical myoclonic epilepsy no mechanism of action has been established as yet (Pranzatelli and Nadi, 1995). In conclusion, the present data underline that piracetam is not an anticonvulsant drug itself, showing only moderate activity in some experimental models of epilepsy. However, its adjunctive medication increased the anticonvulsant effectiveness of various antiepileptic drugs and may provide more efficient protection against seizures. In this connection, not only the ‘‘memoryenhancing’’ properties of this drug, but also the documented neuroprotective, 1

In addition, piracetam also enhances the firing rate of locus coeruleus noradrenergic neurones (Olpe and Steinmann, 1982) and increases the level of brain biogenic monoamines in old rats (Stancheva et al., 1991). It is also uncertain as to whether the recently discussed modulation of membrane fluidity or related processes (Peuvot et al., 1995; M€uller et al., 1997, 1999) play a role in suppression of epileptiform activity. Concerning the wide range of pharmacological activities of piracetam, it seems likely that a number of different mechanisms are relevant for its modest anticonvulsant effects and further special studies are needed.

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antihypoxic and antiischemic as well as hemorheologic effects may be of additional therapeutic value (Hitzenberger et al., 1998). The present results give further support to the idea of a combined use of some nootropics and antiepileptic drugs aimed to increase the efficiency of pharmacological therapy as well as to reduce the impairment of cognitive functions. The treatment of memory disorders in epileptic patients remains a topic of increasing practical importance (Helmstaedter and Kurthen, 2001). Acknowledgements The authors wish to thank Mrs. U. Kermes (deceased) for technical assistance in preparing the rats for hippocampal stimulation and Mrs. M. Klausch (Leipzig) for the determination of phenobarbital and valproate plasma levels with the Abbott TDx analyzer. We are also grateful to Prof. E. Schlicker (Bonn) for critically reviewing the manuscript.

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Tsolaki M, Pantazi T, Kazis A (2001) Efficacy of acetylcholinesterase inhibitors versus nootropics in Alzheimer’s disease: a retrospective, longitudinal study. J Int Med Res 29: 28–36 Uthman BM, Reichl A (2002) Progressive myoclonic epilepsy. Curr Treatm Opt Neurol 4: 3–17 Vernon MW, Sorkin EM (1991) Piracetam. An overview of its pharmacological properties and a review of its therapeutic use in senile cognitive disorders. Drugs & Aging 1: 17–35 Voigt J-P, Morgenstern E (1988) Nootropika und Epilepsien. Pharmazie 43: 673–676 Waegemans T, Wilsher CR, Danniau A, Ferris SH, Kurz A, Winblad B (2002) Clinical efficacy of piracetam in cognitive impairment: a meta-analysis. Dement Geriatr Cogn Disord 13: 217–224 Zelles T, Franklin L, Koncz I, Lendvai B, Zsilla G (2001) The nootropic drug vinpocetine inhibits veratridine-induced [Ca2þ ]i increase in rat hippocampal CA1 pyramidal cells. Neurochem Res 26: 1095–1100 Authors’ address: PD Dr. W. Fischer, Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, H€artelstrasse 16–18, D-04107 Leipzig, Germany, e-mail: [email protected]