19. Glutamatergic compounds - a perspective

Editors: Spanagel, Rainer; Mann, Karl F.

Title: Drugs for Relapse Prevention of Alcoholism, 1st Edition

Copyright 2005 Springer

> Table of Contents > Glutamatergic compounds: a perspective

Glutamatergic compounds: a perspective

Daniel Bachteler

Rainer Spanagel

Central Institute of Mental Health, Department of Psychopharmacology, University of Heidelberg, (CIMH), J5, 68159 Mannheim, Germany

Introduction

It is well known that ethanol alters the synaptic action of glutamate - the major excitatory neurotransmitter in our brain. Thus, high intoxicating doses of ethanol decrease glutamate levels and reduce the activity of glutamate receptors, especially the NMDA receptors. As a consequence of chronic alcohol intake, adaptive changes in the glutamate system are observed, which result in a hyper-glutamatergic state. It is suggested that a hyper-glutamatergic state is a trigger for alcoholic patients to further drink alcohol during withdrawal or to relapse after a period of abstinence. This causal relationship of chronic alcohol intake and long-lasting adaptive changes within the glutamatergic system and its involvement in relapse mechanisms provides the basis for the glutamatergic theory of alcoholism [1, 2].

Glutamate's postsynaptic actions are mediated either through the interaction with ionotropic glutamate receptor channels (iGluR), including NMDA, AMPA and Kainate receptors, or by G protein coupled metabotropic glutamate receptors (mGluR). Among these, the NMDAR complex represents one of the highest affinity targets for ethanol in the brain [3]. Its composition offers several potential sites to interfere with alcohol-related actions. These include, for example, blockade of the receptor channel, selective antagonism of subunits or special binding sites, or functional antagonism. Selective pharmacological manipulation of glutamate receptors has been shown to decrease alcohol intake and preference as well as relapse behavior, including the severity of withdrawal symptoms in various animal models. Moreover, current research demonstrates a functional interplay not only within, but also among receptor types, as for example NMDA and mGluR5. In addition to pharmacological targeting of glutamate receptors, interventions in glutamate release mechanisms as well as re-uptake processes could also represent a promising approach in interfering with relapse mechanisms.

In the following sections, we will review different glutamatergic compounds that have been tested preclinically in different animal models of alcoholism, including basic home cage drinking models such as the alcohol deprivation effect model and more sophisticated operant conditioning paradigms

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such as cue-induced reinstatement of ethanol-seeking behavior (for detailed description of these paradigms see Chapter by Spanagel).

Substances acting at ionotropic glutamate receptors (iGluR)

NMDA receptors

NMDAR is a heteromeric subunit complex, composed of NR1 and NR2 subunits (NR2A-D), each of which exists in several splice variants. At least six pharmacologically distinct binding sites have been recognized until the present, through which compounds may alter the receptor's activity. Functional receptors in the adult mammalian central nervous system (CNS) are formed by combinations of NR1 and NR2 subunits, which express the glycine and glutamate binding sites, respectively. Glutamate acts as an agonist and glycine as a co-agonist at the NMDAR. The endogenous polyamines spermine and spermidine also influence the activity of the NMDAR, in particular at the NR2B subunit-containing receptors [4]. Yet another important binding site lies within the NMDAR complex channel, where un-/non-competitive antagonists block the receptor in a use-dependent manner, i.e., the channel must first be opened by an agonist for the antagonist to bind [5].

Depending on the NMDA receptor subunit composition, acute alcohol inhibits NMDA receptor activation at behaviorally relevant concentrations [6]. For example, Lovinger and colleagues [7] showed in voltage-clamped hippocampal neurons that the ion current induced by the glutamate receptor agonist NMDA was concentration-dependent inhibited by ethanol. At an intoxicating concentration of 50 mM ethanol, the NMDA-activated current was inhibited by more than 60%. Despite intensive research, the site of action of ethanol on the NMDA receptor still remains unknown. However, recently Ren et al. [8] identified a residue in the fourth membrane-associated domain in the NR2A subunit that is suggested to interact with or forms part of a site of ethanol action.

Numerous glutamatergic compounds interfering with the different binding and modulatory sites of the NMDAR complex have been tested in animal models of alcoholism. Initially, some studies produced conflicting results: Rassnick et al. [9] described the attenuation of ethanol self-administration in a free-choice operant task by intra-nucleus accumbens microinjection of the competitive NMDAR antagonist AP-5 (2-amino-5-phosphopentanoic acid) without affecting water consumption. This selectivity was questioned by Shelton and Balster [10], who demonstrated that the competitive NMDA antagonist CPPene, as well as the non-competitive antagonist phencyclidine (PCP) decreased both ethanol and saccharin self-administration. No effects were reported by Danysz et al. [11] using a two-bottle free-choice ethanoldrinking paradigm with the non-competitive NMDAR antagonist MK-801 (dizocilpine). Only recently have competitive and uncompetitive antagonists

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been studied in either the alcohol deprivation effect model or the cue-induced reinstatement paradigm of ethanol-seeking behavior. In the following sections, two promising compounds, memantine and neramexane - both uncompetitive NMDAR antagonists - will be described in more detail.

Firstly, evidence of the alcohol anti-relapse properties of the low affinity, uncompetitive NMDAR antagonist memantine, originally developed for the treatment of moderate to severe Alzheimer's dementia [12], was published by H lter et al. [13]. In a long-term drinking paradigm, rats had voluntary access to different ethanol solutions and water in their home cages. After months of continuous access to ethanol, a deprivation phase was introduced with water as the only available liquid. Re-presentation of alcohol after 3 to 14 days led to a temporary increase in ethanol consumption and preference, known as the alcohol deprivation effect. Implanted with osmotic mini-pumps delivering 5.0 l/h of memantine, rats no longer showed an alcohol deprivation effect after having regained access to alcohol, whereas saline control animals showed the characteristic increase in consumption (Fig. 1). A lack of sedation or behavioral impairment further demonstrated the potential usefulness of this compound. A similar effect of memantine on alcohol intake was described by Piasecki et al. [14] in a free-choice, limited access procedure. Administration of 4.5-24 mg/kg significantly, but not dose-dependently, affected alcohol intake in rats; however, only at 6 mg/kg did it selectively decrease consumption. Rats, operantly trained to lever press for ethanol, showed reduced responding at the 9 mg/kg dose in an extinction procedure. This effect, however, was not selective for ethanol, since water-reinforced responses diminished as well [14].

Initial characterization of the Alzheimer drug and amino-alkyl-cyclohexane neramexane (MRZ 2/579), which displays a pharmacokinetic and pharmacodynamic profile very similar to memantine, provided support for the assumption that neramexane could be useful in the treatment of alcohol abuse [15]. Thus, this compound produced selective effects on ethanol-seeking behavior [14], suppressed ethanol withdrawal seizures in a dose-dependent manner [16], and prevented the acquisition of ethanol-induced conditioned place preference (Kotlinska, pers. comm.). Further, neramexane's effects on ethanol intake have been employed in operant and non-operant animal models. Using an extinction procedure, Bienkowski et al. [17] reported a selective and dose-dependent suppression of operant ethanol self-administration at 2.5-7.5 mg/kg without alterations in locomotor activity, given alone or in combination with ethanol. Voluntarily consuming ethanol experienced rats no longer showed an alcohol deprivation effect, when chronically administered with neramexane (9.6 mg/day) via osmotic minipumps [18]. The same rats were tested for a second alcohol deprivation effect three weeks later in the absence of the drug. The animals exhibited an alcohol deprivation effect to the same extent as control rats, showing that the effect of neramexane on relapse drinking behavior requires the presence of the drug and that a short-term treatment might only be of limited benefit. Long-term alcohol-experienced rats also underwent an operant

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free-choice ethanol self-administration paradigm, in which they received acute treatment of neramexane (2 and 4 mg/kg). Herein, neramexane had a short-lasting reductive, selective effect on lever pressing for ethanol during the alcohol-deprivation effect, but not for water. More recently, Bienkowski et al. [16] tested the same compound in both an operant and non-operant two-bottle choice setup. Non-operant ethanol intake was not affected by a chronic 6-day infusion of neramexane via osmotic minipumps (9.6 mg/day). Only intermittent injections (5 mg/kg) resulted in a significant, progressive decrease in operant responding. This finding is in line with a recent study that showed a dose-dependent reduction of the alcohol deprivation effect following intermittent injections of neramexane (4 and 6 mg/kg; Fig. 1) [19]. Bachteler et al. [20] evaluated whether neramexane would also reduce craving for alcohol in the reinstatement paradigm, applying different doses (1, 2, 4 mg/kg) to rats. Animals were trained to lever press for ethanol in the presence of a distinct set of cues. After extinction, the animals were exposed to the respective cues that initiated reinstatement of responding. However, intraperitoneal administration of neramexane did not result in a significant reduction of lever presses on the ethanol lever at the used doses, but led to a suppression of lever presses on both levers at the highest dose, thereby displaying unspecific response behavior (Fig. 2). This finding is in line with a recent study using the high affinity non-competitive NMDA receptor antagonist MK-801 in the reinstatement paradigm. Thus doses up to 0.15 mg/kg of MK-801 did not reduce the number of lever presses on the ethanol lever following cue presentation. MK-801 rather had a tendency to increase responding, but this effect did not produce significance [21]. The latter results may suggest that neramexane and other un- or non-competitive NMDA receptor antagonists might be more useful in the treatment of relapse behavior than in craving (however, it is important to note that relapse often occurs in the context of craving) and further shows that the neurochemical substrates underlying the expression of an alcohol deprivation effect and reinstatement behavior are different.

Figure 1. Alcohol deprivation effect. Total ethanol intake (g/kg/day) in rats before and after an alcohol deprivation period (AD) of two weeks. The average of three days measurements as a baseline drinking (-1) is shown. To test for an effect on the ADE, different compounds acting at the glutamatergic system were i.p.-administered. Animals were treated with A the uncompetitive NMDAR antagonist memantine (4.8 mg/day); B the competitive NMDAR antagonist CGP 37849 (2, 8 mg/kg); C the NMDA/glycine binding site antagonist L-701,324 (2, 5 mg/kg); D the NMDAR 2B subunit selective antagonists ifenprodil (5, 10 mg/kg); E the NMDA channel blocker neramexane (4, 6 mg/kg); F ethanol (0.8, 1.6 g/kg) as a functional NMDAR antagonist; G the AMPA receptor antagonist GYKI 52466 (4, 10 mg/kg); F the mGlu5 receptor antagonist MPEP (3, 10 mg/kg). Controls received saline. Data was adapted after [13; A], [19; B-F], [31, G], [42; H].

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In line with several drug discrimination experiments that show a substitution to the ethanol cue following the application of different uncompetitive NMDA receptor antagonists [22, 23], memantine and neramexane also exhibited a dose-dependent substitution to the ethanol cue [18, 22], suggesting that these compounds exert their effects on ethanol consumption, at least in part, by generalizing for some of the stimulus properties of ethanol. In other words

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uncompetitive NMDAR antagonists can be viewed as substitution drugs for the treatment of alcoholism. Based on this new concept, a multicenter, randomized, double-blind, placebo-controlled study with neramexane was launched in 2002. Surprisingly, neramexane treatment had no significant effect on either relapse rates or total number of abstinence days. However, compliance problems and the use of a very low dose of neramexane might have confounded the outcome of this study.

Figure 2. Cue-induced reinstatement in rats. Modulation of ethanol-responding under stimulus conditions associated with ethanol (active lever) and water (inactive lever) during a 30-min reinstatement session. Animals were pretreated with A the uncompetitive NMDAR antagonist MK-801 (0, 0.05, 0.15 mg/kg); B the competitive NMDAR antagonist CGP 37849 (0, 5, 10 mg/kg); C the NMDA channel blocker neramexane (0, 1, 2, 4 mg/kg); D the NMDA/glycine binding site antagonist L-701,324 (0, 2, 4 mg/kg); E the AMPA/Kainate receptor antagonist CQNX (0, 0.5, 1.5 mg/kg); F the AMPA receptor antagonist GYKI 52466 (0, 4, 10 mg/kg); G the mGlu5 receptor antagonist MPEP (0, 1, 3, 10 mg/kg). Data was adapted after [20; C], [21; A, B, D, E], [31; F], [42; G].

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Very recently, several compounds acting at different sites of the NMDA receptor were comparatively tested in the alcohol deprivation effect model by Vengeliene et al. [19], including the competitive antagonist CGP 37849, the glycine binding site antagonist L-701.324, and ifenprodil. Repeated administration of these agents produced a significant dose-dependent reduction of alcohol intake in long-term drinking rats during the alcohol deprivation effect to a similar extent (Fig. 1). However, the effect of administration of ifenprodil on the alcohol deprivation effect was weaker but still significant. In all experiments, water intake was not affected.

Besides selective channel blockade of the NMDAR, the competitive and glycine binding sites are therefore interesting for drug development in alcohol dependence. However, it should be emphasized that in recent studies on cue-induced reinstatement of ethanol-seeking behavior, competitive and the already aforementioned uncompetitive NMDAR antagonists were not effective (Fig. 2) [20, 21], whereas L-701,324 dose-dependently reduced ethanol-seeking behavior (Fig. 2) [21]. Furthermore, since NMDARs are involved in almost all of the functions of the CNS, therapeutic intervention may potentially be associated with side-effects (e.g., sedation and cognitive disturbances).

AMPA/kaniate receptors

Synaptic plasticity, underlying associative (i.e., Pavlovian conditioning) and non-associative learning, depends on changes in the glutamatergic system, and more specifically to the AMPA glutamate receptor subtype [24, 25]. The ionotropic AMPA receptor is a heterodimer which may be formed by four different protein subunits, named A, B, C and D, and synaptic plasticity associated to this receptor is clearly dependent on its subunit composition [26]. Interestingly, neuroplastic changes associated to AMPA subunit composition have been proposed to underlie main features of addictive behavior, including drug seeking and relapse behavior. Thus, it has been shown that abstinence or

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extinction after cocaine self-administration is accompanied with an up-regulation of the GluR-A and B/C sub-units within the nucleus accumbens [27, 28].

Interestingly, following chronic alcohol treatment, GluR-C, but not GluR-A or GluR-B, seems to be upregulated in several brain regions [29]. This observation is in accordance with studies in GluR-A and GluR-C knockout mice. GluR-A knockout mice did not differ from wild-type animals in ethanol intake and preference, nor in the alcohol deprivation effect [30]. However, in GluR-C knockout mice a blunted cue-induced reinstatement response and a lack of the alcohol deprivation effect were observed, when compared to wild-type controls [31]. In contrast, under operant and home cage drinking conditions, no difference in genotype could be observed, showing that the GluR-C subunit is not involved in acquisition and maintenance of alcohol reinforcement. For further pharmacological validation of this genetic model, the AMPA antagonist GYKI 52466 was tested on alcohol relapse behavior by Sanchis-Segura et al. [31]. This compound dose-dependently reduced cue-induced reinstatement and the alcohol deprivation effect (Figs 1, 2). Similar to the observations in GluR-A and GluR-C knockouts, GYKI 52466 did almost not affect baseline alcohol consumption in the home cage nor did it disrupt operant self-administration of ethanol in rats [32]. In conclusion, the GluR-C subunit seems to be involved in the neuroplastic changes underlying the ability of cues to promote alcohol-seeking behavior and therefore GluR-C containing AMPA receptors could be a suitable therapeutic target to prevent relapse.

Another antagonist of the AMPA/Kainate receptor, NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline), was only effective in reducing operant responding for ethanol or sucrose at doses (3 and 6 mg/kg) which also led to a significant disruption of the animals' locomotor activity [32]. However, the AMPA/kainite antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione disodium) attenuated reinstatement of cue-induced ethanol-seeking behavior significantly ([21]; Fig. 2) supporting the role of AMPA/kainite receptor in alcohol craving and relapse.

Substances acting at metabotropic glutamate receptors (mGluR)

mGluRs comprise several subtypes (mGluR1-8), divided into three subgroups (I-III), that are based on sequence similarities, intracellular second messengers, and agonist activities [33]. Subgroup I (mGluR1,5) stimulates phosphatidyl inositol (PI) hydrolysis/Ca²⁺ signal transduction, whereas subgroups II (mGluR2,3) and III (mGluR4,6,7,8) are negatively coupled to cyclic adenosine monophosphate formation through adenylyl cyclase.

mGluRs are known to be involved in different CNS disorders [34]. In particular, using the mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) in various studies on anxiety and depression [35, 36], fear conditioning [37], or drug self-administration and conditioned place preference [38, 39 and 40], the importance of mGluR5 in different CNS disorders including

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addiction has been demonstrated. MPEP is a highly potent, selective, non-competitive mGluR5 antagonist with minimal activity at other mGluRs or ionotropic glutamate receptors.

Sharko et al. [41] were the first to describe that MPEP interferes with alcohol self-administration during periods of peak consumption in C57BL/6J mice. MPEP dose-dependently and selectively decreased operant lever pressing for 10% ethanol, but not for water. When administered alone, the compound did not produce conditioned place preference, indicating that it does not have rewarding effects itself. In the same study administration of mGluR1 and mGluR2/3 antagonists had no effects on ethanol or water intake in these animals. As a potentially promising agent in the treatment of relapse, MPEP was further investigated by B ckstr m et al. [42] for assessing its anti-relapse/anticraving potential. In a combination of the alcohol deprivation effect model and the reinstatement model of ethanol-seeking behavior by drug-associated cues, valuable results were obtained. Subchronic treatment of Wistar rats with MPEP (1, 3, and 10 mg/kg) attenuated both relapse to ethanol consumption and stimulus-induced ethanol-seeking significantly and in a dose-related manner (Fig. 1, 2). Administration of the same doses of MPEP also influenced baseline drinking of ethanol, even though this was not as pronounced as on the ADE. This study confirmed that pharmacological targeting of mGlu5 receptors may be a promising approach for the treatment of alcoholism.

The mechanism by which mGluR5 is involved in ethanol craving and relapse behavior remains unclear. There seems to be functional coupling between the NMDA receptor complex and the mGlu5 receptors so that simultaneous activation of mGluR5 could enhance and modulate synaptic transmission at NMDA receptors [43, 44, 45 and 46]. In summary, the functional coupling of the mGluR5 and NMDA receptor suggests that the blockade of mGluR5 by MPEP reduces glutamatergic signalling through NMDA receptors and thereby interacts with ethanol-seeking and relapse behavior.

Conclusions

In summary, the glutamate synapse, including signal transduction pathways, offers a variety of targets for pharmacological intervention in influencing alcohol craving and relapse behavior. Competitive and uncompetitive NMDA receptor antagonists should be further tested in clinical trials, as they may act as substitution drugs for ethanol. However, in order to achieve substitution for some of the subjective effects of ethanol, high doses should be used which might also produce some side-effects. Convincing preclinical evidence comes from studies on mGlu5 receptors showing that mGluR5 antagonists could be promising compounds for the treatment of alcoholism. Thus, MPEP - a selective mGluR5 antagonist - proved to be effective in all standard models of preclinical alcohol research and should therefore be tested in a clinical phase I/II study.

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Preclinical studies, however, should not only focus on glutamate receptors as potential targets for pharmacological intervention. Thus, it has been shown that glutamate release mechanisms mediate several neurobehavioral effects of alcohol [22] and in a preliminary study, the glutamate release inhibitor lamotrigine was able to reduce relapse drinking behavior. Furthermore, in genetically modified mice that exhibit a down-regulation of the glutamate transporter EAAT-1, which leads to augmented glutamate levels in the brain, enhanced alcohol consumption is observed, indicating that glutamate uptake mechanisms could also present an important site of action in modulating alcohol consumption and subsequently addictive behavior [47]. Furthermore, the NO/cGMP signalling pathway that is coupled to NMDA receptors is also critically involved in alcohol drinking behavior [48, 49]. Thus a variety of genes and their protein products of the glutamatergic system are involved in the action of alcohol in the CNS and promising compounds are in the pipeline of several pharmaceutical companies, waiting to be tested on alcoholic subjects.

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