Neurochemistry International 44 (2004) 17–23 NR2B subunit selective NMDA antagonists inhibit neurotoxic effect of alcohol-withdrawal in primary cultures of rat cortical neurones József Nagy ∗ , Csilla Horváth, Sándor Farkas, Sándor Kolok, Zsolt Szombathelyi Pharmacological and Drug Safety Research, Gedeon Richter Ltd., P.O. Box 27, H-1475 Budapest 10, Hungary Received 6 January 2003; received in revised form 28 February 2003; accepted 28 February 2003 Abstract N-Methyl-d-aspartate (NMDA) receptor-mediated glutamatergic neurotransmission is thought to play a central role in the development of alcohol dependence and this alteration is supposed to be due to a differential up-regulation of the NR2B type of subunits. In this work, we examined the effect of some known (CP-101,606; CI-1041 and Co-101,244) and novel indole-2-carboxamide derivative NR2B subunit selective NMDA receptor antagonists (SSNAs) (RG-13579 and RG-1103) on the neurotoxic effect of withdrawal in ethanol pre-treated cultures of rat cortical neurones. The extent of neurotoxicity was estimated by measuring the activity of lactate dehydrogenase (LDH) that was released into the culture medium during the 24h withdrawal period. Here, we demonstrate that NR2B SSNAs given in the course of the withdrawal potently reduced the LDH release in ethanol pre-treated cultures. One of our novel compound, RG-1103, proved to be more potent than the reference NR2B SSNAs tested in this work having similar potency as the most potent but non-subunit selective NMDA receptor antagonist dizocilpine (MK-801). Acamprosate, a currently used therapeutic drug for the treatment of alcoholism was also effective although only in high micromolar concentrations. According to these observations, NR2B SSNAs are potent inhibitors of ethanol-withdrawal-induced neurotoxicity and considering that these agents have acceptable side effect profiles, they could be promising therapeutic candidates in the pharmacotherapy for physical signs of acute alcohol-withdrawal and associated neuronal damage. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Primary culture of rat cortical neurones; Neurotoxicity; Alcohol-withdrawal; NMDA receptor; NR2B subunit selective antagonist; Indole-2-carboxamide derivatives 1. Introduction Alcohol dependence is a source of the major public health, social and medical problems all over the world. In the past years there has been increasing evidence that behavioural, neurophysiological and pathological effects of alcohol are mainly mediated through the glutamatergic neurotransmitter system (Tsai et al., 1995; Tsai and Coyle, 1998). Especially the N-methyl-d-aspartate (NMDA) type of glutamate re- ceptors seems to be an important site of ethanol’s action. Acutely, alcohol exerts an antagonistic effect on NMDA receptor function (Dildy and Leslie, 1989; Hoffman et al., 1989; Lovinger et al., 1989; Poelchen et al., 2001; Smothers et al., 2001), and chronic alcohol consumption leads to an increase in NMDA receptor-mediated neurotransmission (Chandler et al., 1993; Ahern et al., 1994; Rudolph et al., 1998; Nagy et al., 2001). This alteration is presumably not due to an overall increase in the NMDA receptor density, but due to differential up-regulation of different NMDA ∗ Corresponding author. Fax: +36-1-2605000. E-mail address: jozsef.nagy@richter.hu (J. Nagy). receptor subunits that could result in alterations in the com- position and functional properties of NMDA receptor com- plexes (Hoffman and Tabakoff, 1996; Rudolph et al., 1997; Darstein et al., 1998; Faingold et al., 1998). Therefore, modulators of the glutamatergic/NMDA receptor system are now believed useful pharmacotherapeutic agents in the treatment for alcohol dependence (Bisaga and Popik, 2000). Previously, alcohol induced adaptive changes were ob- served in primary cultures of rat cortical neurones in vitro af- ter 3 days of repeated ethanol treatment (Nagy et al., 2001). In these cultures, neurones became dependent on alcohol. Although light-microscopic morphology of these cultures in presence of ethanol did not differ from that of control, with- drawing ethanol resulted in neuronal cell death that could be reduced by the re-addition of ethanol. Besides ethanol, NMDA receptor antagonists like dizocilpine (MK-801) and threo-ifenprodil but not the -aminobutyric acid type A (GABA A ) receptor agonist muscimol produced a protective effect. These observations suggested that chronic ethanol treatment per se was not neurotoxic, but neuronal cell death was resulted in consequence of ethanol-withdrawal. In addi- tion, NMDA receptor function was supposed to play a role 0197-0186/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0197-0186(03)00100-1