Neurotoxic/neuroprotective profile of carbamazepine, oxcarbazepine and two new putative antiepileptic drugs, BIA 2-093 and BIA 2-024

Abstract

We investigated and compared the toxicity profile, as well as possible neuroprotective effects, of some antiepileptic drugs in cultured Ž . Ž . w x rat hippocampal neurons. We used two novel carbamazepine derivatives, S -y -10-acetoxy-10,11-dihydro-5H-dibenz b,f azepine-5-Ž . w x Ž . carboxamide BIA 2-093 and 10,11-dihydro-10-hydroxyimino-5H-dibenz b,f azepine-5-carboxamide BIA 2-024 , and compared their effects with the established compounds carbamazepine and oxcarbazepine. The assessment of neuronal injury was made by using the Ž . Ž . 3-4,5-dimethylthiazol-2-yl -2,5-diphenyl MTT assay, as well as by analysing morphology and nuclear chromatin condensation Ž . propidium iodide staining , after hippocampal neurons were exposed to the drugs for 24 h. The putative antiepileptic drugs, BIA 2-093 or Ž . BIA 2-024 at 300 mM , only slightly decreased MTT reduction, whereas carbamazepine or oxcarbazepine were much more toxic at lower concentrations. Treatment with the antiepileptic drugs caused nuclear chromatin condensation in some neurons, which is characteristic of apoptosis, and increased the activity of caspase-3-like enzymes, mainly in neurons treated with carbamazepine and Ž . oxcarbazepine. The toxic effect caused by carbamazepine was not mediated by N-methyl-D-aspartate NMDA or by a-amino-3-hydroxy-Ž . 5-methyl-isoxazole-4-propionate AMPA receptors. Moreover, the antiepileptic drugs failed to protect hippocampal neurons from the toxicity caused by kainate, veratridine, or ischaemia-like conditions. q

Evaluation of neurotoxic and neuroprotective pathways affected by antiepileptic drugs in cultured hippocampal neurons

“…Lower concentrations of OXC (0.01 and 0.03 mM) did not affect cell viability. The observed toxic effects caused by high concentrations of OXC on the hippocampal neurons are in agreement with previous studies (Ambrosio et al, 2000;Araujo et al, 2007).…”

“…Our group previously demonstrated that ESL is less toxic to cultured neurons than CBZ and OXC, namely at high concentrations (0.3 mM). Indeed, ESL did not induce apoptosis or caused structural damage to the neuritic network (Ambrosio et al, 2000;Araujo et al, 2004), whereas CBZ was found to induce apoptosis in cultured cerebellar granule cells (Gao et al, 1995;Nonaka et al, 1998) and OXC increased the activity of caspase-3-like enzyme (Ambrosio et al, 2000). However, the effect of ESL metabolites, S-Lic and R-Lic, on the viability of neuronal cells, has not been addressed yet, and it is of interest to determine the direct effects of S-Lic and R-Lic on the viability of cultured neurons, since in the in vitro system (cultured neurons) there is no metabolism of ESL as it occurs in vivo.…”

“…The cells were cultured in serum-free Neurobasal medium, supplemented with B27 supplement, glutamate (25 lM), gentamicin (0.12 mg/ml), as described previously (Ambrosio et al, 2000;Araujo et al, 2004;Brewer et al, 1993) and GlutaMAX™ (0.5 mM). Cultures were kept at 37°C in a humidified incubator in 5% CO 2 /95% air, for 7-8 days.…”

“…The hippocampal neurons were exposed to AEDs for 24 h, as previously described (Ambrosio et al, 2000;Araujo et al, 2004), or for other periods as indicated in the figure legends. The drugs were prepared in DMSO (except VPA, which was prepared in water), and further diluted in 200 ll aliquots of culture medium taken from each well and then added back to the correspondent well.…”

“…Cultured hippocampal neurons were exposed to the AEDs for 24 h, at concentrations of 0.01, 0.03, 0.1 and 0.3 mM (for ESL, S-Lic, R-Lic, CBZ, OXC and LTG) and of 0.01, 0.03, 0.1, 0.3, 0.5, 1 and 3 mM for VPA. The concentrations of the AEDs that were utilized in these study are within the range of those tested in functional assays using rat hippocampal neurons, namely for the evaluation of effects upon Ca 2+ and Na + channels (Ambrosio et al, 1999;Ambrosio et al, 2000;Brady et al, 2011;Hebeisen et al, 2011) and in hippocampal neural progenitor cells (Hsieh et al, 2004).…”