Therapeutic doses of topiramate are not toxic to the developing rat brain

Antiepileptic drugs (AEDs) used to treat seizures in pregnant women, infants, and young children may cause cognitive impairment. One of the implicated mechanisms is enhancement of apoptotic neuronal death, which occurs physiologically in the developing brain. We investigated whether topiramate, one of the newer antiepileptic drugs, has neurotoxic properties in the developing rat brain. Topiramate slightly but significantly enhanced apoptotic neuronal death in the 7-day-old rat brain at doses of 50 mg/kg and above. These doses are several folds higher than reported ED(50) doses in infant rodent seizure models that respond to topiramate. Electron microscopy confirmed that dying neurons following topiramate treatment displayed the same morphological features as neurons undergoing physiological cell death during development. When compared to the neurotoxicity profile of phenytoin, valproate, and phenobarbital, the separation between the effective anticonvulsant dose and the neurotoxic dose was greater for topiramate and the neurotoxic effect was lower.     

Is the persistent sodium current a specific target of anti-absence drugs?

The persistent Na+ current (INaP) has been proposed as the putative target of the anti-absence antiepileptic drugs. Accordingly, the effect of reference anti-absence drugs ethosuximide (ESM) and valproate (VPA), and of the new antiepileptic drug levetiracetam (LEV), on INaP have been tested in CA1 hippocampal neurons and compared to the classic anticonvulsant phenytoin (PHT) and the neuroprotective agent riluzole (RIL). Whole-cell patch-clamp recordings of the slowly inactivating current, fully characterized as INaP, were performed with a standard voltage-step protocol on thin hippocampal slices prepared from rat brain. Both PHT (100 microM) and RIL (10 microM) strongly depressed INaP, whereas ESM (1 mM) induced a slight decrease of INaP and VPA (1 mM) had no effect. Likewise, 60-min perfusion with relevant concentrations of LEV (10, 32 or 100 microM) did not modify INaP. In conclusion, these data question the impact of INaP depression as an anti-absence mechanism, and also disclaim the involvement of INaP in the antiepileptic mechanism of LEV.     

抗癫痫药物四种对中青年绝经前女性骨代谢的影响

目的研究不同的常用抗癫痫药物对中青年女性骨代谢的影响。方法通过测量各组受试者的血清钙、25-羟基维生素D、碱性磷酸酶、甲状旁腺激素、胰岛素生长因子-1、胰岛素结合蛋白-3、骨密度等,分析长期单药服用不同抗癫痫药物如苯妥英钠(PHT)、卡马西平(CBZ)、丙戊酸钠(VPA)、左乙拉西坦(LEV)等对受试者骨代谢的影响。结果服用CBZ、PHT和VPA的受试者血清钙浓度低于服用LEV的受试者(P=0.002);服用PHT的受试者的ALP浓度显著高于服用CBZ、LEV和VPA的受试者(P=0.000);与LEV组相比,PHT组血清IGF-1水平降低(P=0.03);与LEV组相比,PHT组血清的IGFBP-3浓度显著降低(P=0.000)。结论抗癫痫药物会影响骨代谢,导致患者血清钙下降、骨量减低。LEV较其他抗癫痫药物对骨代谢的影响较小,但仍能造成患者碱性磷酸酶含量下降等。抗癫痫药物对中青年女性的骨代谢会产生一定影响,在临床用药过程中需监测相关指标。     

The antiepileptic drug levetiracetam stabilizes the human epileptic GABAA receptors upon repetitive activation

PURPOSE: GABAA receptors from the brain of patients afflicted with mesial temporal lobe epilepsy (MTLE) become less efficient (run-down) when repetitively activated by GABA. Experiments were designed to investigate whether the antiepileptic drug, levetiracetam (LEV), which is used as an adjunctive treatment for medically intractable MTLE, counteracts the GABAA receptor run-down. METHODS: GABAA receptors were microtransplanted from the brains of patients afflicted with MTLE into Xenopus oocytes. The GABA-current run-down, caused by repetitive applications of GABA, was investigated using the standard two-microelectrode voltage-clamp technique. Additionally, the GABA-current run-down was investigated directly on pyramidal neurons in human MTLE cortical slices. RESULTS: It was found that, in oocytes injected with membranes isolated from the MTLE neocortex, the GABA-current run-down was inhibited by a 3-h pretreatment with 0.5-100 microM LEV. Moreover, the GABAA receptors of pyramidal neurons in human neocortical slices exhibited a current run-down that was significantly reduced by 1 microM LEV. Interestingly, the run-down in oocytes injected with membranes isolated from the MTLE hippocampal subiculum was not affected by LEV. CONCLUSIONS: We report that the antiepileptic LEV strengthens GABA inhibition of neuronal circuits by blocking the receptor run-down in the cortex whilst leaving the run-down of GABAA receptors in the hippocampal subiculum unaltered. These findings point to the GABAA receptor run-down as an important event in epileptogenesis and as a possible target for testing and screening antiepileptic drugs.     

Short-term use of antiepileptic drugs is neurotoxic to the immature brain

Previous studies have shown that the long-term use of antiepileptic drugs can cause nervous system damage. However, short-term antiepileptic drug treatment is frequently given to infants, especially neonates, to control seizure. Whether the short-term use of antiepileptic drugs is neurotoxic remains unclear. In the present study, immature rats, 3–21 days of age, were intraperitoneally injected with phenobarbital and/or topiramate for 3 consecutive days. Hematoxylin-eosin and immunohistochemical staining revealed that phenobarbital and topiramate, individually or in combination, were cytotoxic to hippocampal CA1 neurons and inhibited the expression of Glu R1 and NR2 B, excitatory glutamate receptor subunits. Furthermore, the combination of the two drugs caused greater damage than either drug alone. The results demonstrate that the short-term use of antiepileptic drugs damages neurons in the immature brain and that the combined use of antiepileptic drugs exacerbates damage. Our findings suggest that clinicians should consider the potential neurotoxic risk associated with the combined use of antiepileptic drugs in the treatment of seizure.     

Levetiracetam exhibits protective properties on rat Schwann cells in vitro

Oxidative stress and inflammation represent pathways causing substantial damage to the peripheral nervous system. Levetiracetam (LEV) is a commonly used antiepileptic drug targeting high-voltage activated N-type calcium channels. Recent evidence suggests that LEV may also act as a histone deacetylase inhibitor, suggesting that this drug exhibits both anti-inflammatory and anti-oxidative effects, and as such may represent an interesting candidate for treating inflammatory diseases affecting the peripheral nerve. Therefore, we analysed the influence of LEV ex vivo on purified Schwann cells from neonatal P3 rats as well as on dorsal root ganglia prepared from E15 rat embryos. LEV diminished a lipopolysaccharide (LPS)-induced increase of the pro-inflammatory signature molecules tumour necrosis factor alpha, matrix metalloproteinase 9 (MMP-9), and caspase 6. Furthermore, LEV decreased LPS-induced cell death and protected cells against oxidative stress in a glutamate-based oxidative stress model. MMP-2 activity, usually elevated during myelination and repair, was also found to be up-regulated following LEV, while LEV exhibited no negative effects on myelination. Intracellular sodium or calcium concentrations were unaltered by LEV. Thus, LEV may be a promising, well-tolerated drug that - besides its antiepileptic potential - mediates anti-inflammatory, anti-oxidative, and anti-apoptotic properties that may potentially be useful in treating diseases of the peripheral nerve.     

Pattern of antiepileptic drug-induced cell death in limbic regions of the neonatal rat brain

The induction of neuronal apoptosis throughout many regions of the developing rat brain by phenobarbital and phenytoin, two drugs commonly used for the treatment of neonatal seizures, has been well documented. However, several limbic regions have not been included in previous analyses. Because drug-induced damage to limbic brain regions in infancy could contribute to emotional and psychiatric sequelae, it is critical to determine the extent to which these regions are vulnerable to developmental neurotoxicity. To evaluate the impact of antiepileptic drug (AED) exposure on limbic nuclei, we treated postnatal day 7 rat pups with phenobarbital, phenytoin, carbamazepine, or vehicle, and examined nucleus accumbens, septum, amygdala, piriform cortex, and frontal cortex for cell death. Histologic sections were processed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay to label apoptotic cells. Nucleus accumbens displayed the highest level of baseline cell death (vehicle group), as well as the greatest net increase in cell death following phenobarbital or phenytoin. Phenobarbital exposure resulted in a significant increase in cell death in all brain regions, whereas phenytoin exposure increased cell death only in the nucleus accumbens. Carbamazepine was without effect on cell death in any brain region analyzed, suggesting that the neurotoxicity observed is not an inherent feature of AED action. Our findings demonstrate pronounced cell death in several important regions of the rat limbic system following neonatal administration of phenobarbital, the first-line treatment for neonatal seizures in humans. These findings raise the possibility that AED exposure in infancy may contribute to adverse neuropsychiatric outcomes later in life.     

Levetiracetam has an activity-dependent effect on inhibitory transmission

Purpose: Previous work has shown that levetiracetam (LEV) binds the vesicular protein SV2A and reduces excitatory neurotransmitter release during trains of high‐frequency activity, most likely by accessing its binding site through vesicular endocytosis into excitatory synaptic terminals. Because there are differences in excitatory and inhibitory transmitter release mechanisms, and there are suggestions that neurons differ in their SV2A expression, we were curious whether LEV also reduces inhibitory transmission. Methods: We used patch‐clamp recording from CA1 neurons in rat brain slices to quantify the effects of LEV on inhibitory postsynaptic currents (IPSCs). We were able to elicit pure IPSCs by stimulating inhibitory terminals close to neuronal soma and blocking excitatory postsynaptic currents with specific antagonists. Key Findings: We found that LEV reduces inhibitory currents in a frequency‐dependent manner, with the largest relative effect on the later IPSCs in the highest frequency trains. However, in contrast to excitatory postsynaptic currents (EPSCs), LEV reduced IPSC trains after a briefer, 30 min incubation. When spontaneous activity during incubation was blocked with antagonists of excitatory transmission, LEV no longer reduced IPSCs. If slices were returned to LEV‐free artificial cerebrospinal fluid (ACSF) after LEV incubation, but prior to recording, the IPSC reduction failed to appear. However, if synaptic activity was limited by treating with excitatory transmitter antagonists, after the initial LEV exposure, LEV still diminished trains of IPSC. The concentration required to diminish IPSC trains was lower than for EPSCs. Significance: LEV exerts a qualitatively similar, frequency‐dependent effect on both IPSCs and EPSCs. The much shorter latency for IPSC reduction is consistent with the greater levels of spontaneous inhibition in brain slices, supporting the hypothesis that vesicular uptake is necessary for the entry of LEVs into terminals. The vesicular entry of LEV resembles the cell entry pathways for tetanus and botulinum neurotoxins, but is unique for small, neuroactive drugs. Although the reduction of IPSC trains by LEV initially seems counterintuitive for an antiepileptic drug, there are multiple reasons that disruption of γ‐aminobutyric acid (GABA) release could ultimately attenuate pathologic discharges.     

Inhibition of multidrug transporters by verapamil or probenecid does not alter blood-brain barrier penetration of levetiracetam in rats

Overexpression of multidrug efflux transporters such as P-glycoprotein (Pgp; ABCB1) or multidrug resistance proteins (MRPs; ABCC) in the blood-brain barrier has recently been suggested to explain, at least in part, pharmacoresistance in epilepsy, which affects about 30% of all patients with this common brain disorder. The novel antiepileptic drug (AED) levetiracetam (LEV) is an effective and well tolerated drug in many patients with otherwise AED-refractory epilepsy. One explanation for the favorable efficacy of LEV in pharmacoresistant patients would be that LEV is not a substrate for Pgp or MRPs in the BBB. In the present study, we used in vivo microdialysis in rats to study whether the concentration of LEV in the extracellular fluid of the cerebral cortex can be modulated by inhibition of Pgp or MRPs, using the Pgp inhibitor verapamil and the MRP1/2 inhibitor probenecid. Local perfusion with verapamil or probenecid via the microdialysis probe did not increase the extracellular brain concentration of LEV, which is in contrast to various other AEDs which have been studied previously by the same experimental protocol in this model. The data indicate that brain uptake of LEV is not affected by Pgp or MRP1/2 which may be an important reason for its antiepileptic efficacy in patients whose seizures are poorly controlled by other AEDs.     

Effects of levetiracetam on axon excitability and synaptic transmission at the crayfish neuromuscular junction

Levetiracetam (LEV) is a widely prescribed antiepileptic drug, but its actions on neuronal function are not fully characterized. Since this drug is believed to enter neurons by binding to a vesicular protein during endocytosis, we used motor axons of the crayfish opener neuromuscular junction to examine potential impacts of LEV on axon excitability. Two electrode current clamp from the inhibitory axon of the opener showed that LEV reduced action potential (AP) amplitude (AP_amp) and suppressed synaptic transmission, although the latter occurred with a longer delay than the reduction in AP_amp. Comparison of antidromic and orthodromic conducting APs in LEV suggested that this drug preferentially reduced excitability of the proximal axon, despite the expectation that it entered the axon at the terminals and should affect the distal branches first. Results presented here suggest that LEV modulates axonal excitability, which may in turn contribute to its antiepileptic effects.     

Levetiracetam compared to phenytoin for the prevention of postoperative seizures after craniotomy for intracranial tumours in patients without epilepsy

Anticonvulsant drugs are frequently given after craniotomy. Phenytoin (PHT) is the most commonly used agent; levetiracetam (LEV) is a new anticonvulsant drug with fewer side effects. To compare the incidence of seizures in patients receiving either prophylactic PHT or LEV perioperatively, 971 patients undergoing a craniotomy were analysed retrospectively during a 2-year period. PHT was used routinely and LEV was administered when PHT was contraindicated. Seizures documented during the first 7 days after craniotomy were considered. A total of 235 patients were treated with an antiepileptic drug: 81 patients received LEV, and 154 patients, PHT. Two patients receiving LEV (2.5%) and seven receiving PHT (4.5%) had a seizure despite this treatment. No patient had a documented side effect or drug interaction. The data show that LEV may be an alternative option in patients with contraindications to PHT.     

Anfälle in der Neugeborenenperiode

In the USA 1.5–3.0/1,000 neonates suffer an epileptic seizure in the postnatal period while the rate is even higher in preterm babies. In neonates and preterm babies it is often difficult to clinically recognize a seizure. Seizure semiology greatly differs from what is seen at a later age and the correlation between clinical seizures and electroencephalographic (EEG) patterns is weak. The introduction of amplitude-integrated EEGs as bedside monitoring in neonatal intensive care units has had a great impact on the recognition and treatment of seizures. The prognosis of children is determined primarily by the underlying disease, such as hypoxic ischemic encephalopathy (HIE), infarct/bleeding and central nervous system (CNS) infections. However, in recent papers it was demonstrated that a seizures can itself provoke morphological changes and inhibit brain development, which is even more pronounced in cases with preexisting CNS lesions (e.g. HIE). An increase of apoptotic necrosis of neuronal cells was found in neonatal rats after various antiepileptic (AE) drugs, e.g. phenobarbital (PB), valproate (VPA), phenytoin (PHT), carbamazepine (CBZ) and lamotrigine (LTG), but not after levetiracetam (LEV) or topiramate (TPM). The discussion that PB might hamper and inhibit brain development triggered the use of LEV and TPM in newborns. At least in some small cohorts the results are indicative for the efficacy of LEV in preterm and term babies to control seizures: therefore, LEV is used nowadays in some hospitals as the first drug in neonatal seizures.     

Comparison of reproductive effects of levetiracetam and valproate studied in prepubertal porcine ovarian follicular cells

PURPOSE: Long-term valproate (VPA) treatment has been associated with reproductive endocrine disorders characterized by hyperandrogenism and polycystic changes in the ovaries in women with epilepsy. Levetiracetam (LEV) is a promising, new antiepileptic drug that may represent an alternative to VPA for many patients. Here the effect of LEV and VPA on basal and gonadotropin-stimulated steroid secretion from prepubertal porcine ovarian follicular cells was compared and the conversion of testosterone to estradiol is measured. METHODS: Ovarian follicles were obtained from prepubertal pigs. Follicular theca and granulosa cells were cocultured and different concentrations of LEV or VPA added to the control or gonadotropin-stimulated cultures. RESULTS: VPA, but not LEV, caused a significant increase of LH-stimulated testosterone secretion and decreased FSH-stimulated estradiol secretion. VPA decreased conversion of testosterone to estradiol in both basal and FSH-stimulated cultures, while LEV only decreased testosterone to estradiol conversion after FSH stimulation and only at the highest, nontherapeutic drug concentration. Both drugs increased basal testosterone secretion at therapeutic drug levels. VPA also reduced basal estradiol secretion, while LEV decreased basal estradiol secretion only at nontherapeutic drug levels. CONCLUSION: Both LEV and VPA affect endocrine function in the prepubertal ovary. But while VPA alters both basal and gonadotropin-stimulated testosterone and estradiol secretion at therapeutic drug concentrations, LEV only affects basal hormone secretion at this concentration level. The possibility that LEV could be an alternative treatment to VPA if reproductive endocrine problems emerge in adult women, is discussed. However, extrapolation to the clinical situation is problematic and particular emphasis is placed on the need for further studies.     

Hyperoxic exposure leads to cell death in the developing brain

Premature infants have high incidence of motor and cognitive impairment in later life. Supraphysiological oxygen concentrations are routinely used in neonatal intensive care units and elicit injury to premature lungs and retina. Since the effects of hyperoxia on the developing brain are scarce, we studied the effects of high oxygen on this tissue. Wistar rat pups were exposed from birth until day 5 to 21% or 80% oxygen. The neuronal density and apoptosis in CA1 and dentate gyrus of hippocampus, prefrontal cortex, parietal cortex, subiculum, and retrosplenial cortex were assessed by immunohistochemistry and ELISA cell death assay. Neuronal density of the investigated brain areas were significantly decreased in the hyperoxia group. Furthermore, using ELISA cell death and TUNEL assays, we observed increased cell death in the developing brain. Our results show that hyperoxia induces cell death in the developing rat brain. This may be one of the important mechanisms that cause motor and cognitive impairment in later life of premature infants.     

The effect of levetiracetam on status epilepticus-induced neuronal death in the rat hippocampus

PurposeLevetiracetam has been reported to be well tolerated and effective in status epilepticus (SE) refractory to benzodiazepine. Because of little preclinical or clinical data concerning the outcomes of LEV in SE-induced neuronal death and vasogenic edema, we investigated the effect of LEV on SE-induced injury in comparison to diazepam (DZP), and valproate (VPA).MethodsTwo hours after pilocarpine-induced SE, rats were given one of the following drugs; (1) DZP, (2) LEV, (3) VPA, (4) DZP + LEV, (5) DZP + VPA, and (6) DZP + oxiracetam. Three–four days after SE, neuronal damage and vasogenic edema were evaluated by Fluoro-Jade B (FJB) staining and serum-protein extravasation, respectively.ResultsLEV (≥50 mg/kg) was effective to protect neuronal damage from SE in comparison to DZP and VPA. LEV as an add-on drug with DZP could not alleviate neuronal damage as compared to LEV alone. VPA (≥100 mg/kg) was effective to protect neuronal damage from SE, as compared to DZP. VPA as an add-on drug with DZP reduced neuronal damage, as compared to DZP alone.ConclusionThese findings suggest that LEV may negatively interact with DZP, and be more effective to prevent SE-induced neuronal death as a first line drug than as a second line therapy after BDZ treatment.     

Urethane anesthesia produces selective damage in the piriform cortex of the developing brain.

The potential induction of neuronal death by neuroactive drugs at specific stages of embryonic or postnatal development is a serious concern in treating brain disease. Recent evidence indicates that NMDA antagonists, GABA agonists, ethanol and some anesthetics can all produce massive neuronal cell loss at critical times during development. We show here that the anesthetic urethane, once used clinically, produces a selective lesion of the piriform cortex, a region not previously implicated in such toxicity, in the developing brain. Young rats were injected with urethane at 1, 2, 3, and 4 weeks of age and brain damage was measured 1-4 days later. We found that urethane produces a large lesion in subfields of the piriform cortex and that the damage is most severe in 2 week-old animals. These data, together with other recent reports, show that there are multiple neuronal death-inducing pathways in the developing nervous system. It will be important to determine if anesthetics used in pregnant women and young children may have similar effects.     

Distinct neurobehavioral consequences of prenatal exposure to sulpiride (SUL) and risperidone (RIS) in rats

Antipsychotic treatment during pregnancy is indicated when risk of drug exposure to the fetus is outweighed by the untreated psychosis in the mother. Although increased risk of congenital malformation has not been associated with most available antipsychotic drugs, there is a paucity of knowledge on the subtle neurodevelopmental and behavioral consequences of prenatal receptor blockade by these drugs. In the present study, antipsychotic drugs, sulpiride (SUL, a selective D2 receptor antagonist) and risperidone (RIS, a D2/5HT2 receptor antagonist) were administered to pregnant Sprague-Dawley dams from gestational day 6 to 18. Both RIS and SUL prenatal exposed rats had lower birth body weights compared to controls. RIS exposure had a significant main effect to retard body weight growth in male offspring until postnatal day (PND) 60. Importantly, water maze tests revealed that SUL prenatal exposure impaired visual cue response in visual task performance (stimulus-response, S-R memory), but not place response as reflected in hidden platform task (spatial memory acquisition and retention). In addition, prenatal SUL treatment reduced spontaneous activity as measured in open field. Both behavioral deficits suggest that SUL prenatal exposure may lead to subtle disruption of striatum development and related learning and motor systems. RIS exposure failed to elicit deficits in both water maze tasks and increased rearing in open field test. These results suggest prenatal exposure to SUL and RIS may produce lasting effects on growth, locomotion and memory in rat offspring. And the differences may exist in the effects of antipsychotic drugs which selectively block dopamine D2 receptors (SUL) as compared to second generation drugs (RIS) that potently antagonize serotonin and dopamine receptors.     

Are Psychiatric Adverse Events of Antiepileptic Drugs a Unique Entity? A Study on Topiramate and Levetiracetam

PURPOSE: To investigate the hypothesis that some patients with epilepsy are generally prone to develop psychiatric adverse events (PAEs) during antiepileptic drug (AED) therapy irrespective of the mechanism of action of the drugs. METHODS: From a large case registry of patients prescribed topiramate (TPM) and levetiracetam (LEV), data of patients who had a trial with both drugs were analyzed. Demographic and clinical variables of those who developed PAEs with both drugs (group 1) were compared with those who did not (group 2). Subsequently, from the whole case registry, psychopathological features, demographic, and clinical variables of patients developing PAEs with TPM were compared with those of patients developing PAEs with LEV. RESULTS: The case registry included over 800 patients. Among 108 patients having a trial with both drugs, we identified 9 patients in group 1 and 71 in group 2. Previous psychiatric history, family psychiatric history and history of febrile convulsions showed to be significant clinical correlates. Comparing patients who developed PAEs with LEV with those who developed PAEs with TPM, there were no differences in epilepsy related variables. Well-defined DSM-IV disorders were more frequent with TPM than with LEV. Seizure freedom was associated with psychosis. Conclusions: This study suggests that a subgroup of patients is generally prone to develop PAEs during AED therapy, despite different pharmacological properties of the AEDs. A particular clinical profile and relevant variables have been identified.     

Caffeine-induced neuronal death in neonatal rat brain and cortical cell cultures

We examined the potential neurotoxicity of caffeine and. Intraperitoneal administration of caffeine (50 mg/kg, 3 times a day) produced neuronal death in various brain areas of neonatal rats 24 h later. Caffeine at doses > 300 microM was also neurotoxic in murine cortical cell cultures. Caffeine-induced neuronal death was accompanied by cell body shrinkage and attenuated by anti-apoptotic drugs including cycloheximide, high potassium, and growth factors. Two necrotic pathways, excitotoxicity and oxidative stress, did not mediate caffeine neurotoxicity. The pro-apoptotic protease caspase-3 was activated to mediate neuronal death following exposure to caffeine. The present findings suggest that caffeine may cause caspase-3-dependent neuronal cell apoptosis in neonatal rat as well as.