Peripherally acting NMDA receptor/glycineB site receptor antagonists inhibit morphine tolerance

The present study focused on the role of peripheral ionotropic N-methyl-D-aspartate (NMDA) receptors in the development of tolerance to morphine-induced antinociception. An initial experiment revealed that NMDA channel blocker memantine, and NMDA receptor/glycine(B) site antagonist MRZ 2/576 inhibited maximal electroshock-induced convulsions (MES) in female NMR mice with respective potency of 5.93 and 20.8 mg/kg, while other NMDA receptor/glycine(B) site antagonists MRZ 2/596 and MDL 105,519 were ineffective, supporting lack of CNS activity of the latter two agents. This observation was also supported by blood-brain barrier experiments in vitro. In male Swiss mice, morphine (10 mg/kg) given for 6 days twice a day (b.i.d.) produced tolerance to its antinociceptive effects in the tail-flick test. The NMDA receptor/glycine(B) site antagonists, MRZ 2/576 at 0.03, 0.1, 0.3 mg/kg and MRZ 2/596 at 0.1, 0.3, 3 and 10 mg/kg attenuated the development of morphine tolerance. Similarly, in male C57/Bl mice, morphine (10 mg/kg) given for 6 days b.i.d. produced tolerance to its antinociceptive effects in the tail-flick test. Like in Swiss mice, in C57/Bl mice morphine tolerance was attenuated by both MRZ 2/576 and MRZ 2/596. Another NMDA receptor/glycine(B) site receptor antagonist, MDL 105,519 (that very weakly penetrates to the central nervous system) also inhibited morphine tolerance at the dose of 1 but not 0.1 mg/kg. Moreover, both naloxone hydrochloride (5 and 50 mg/kg) and centrally inactive naloxone methiodide (50mg/kg) inhibited morphine tolerance suggesting the involvement of peripheral opioid receptors in this phenomenon. The present data suggest that blockade of NMDA receptor/glycine(B) sites in the periphery may attenuate tolerance to the antinociceptive effects of morphine.     

Antinociceptive activity of combination of morphine and NMDA receptor antagonists depends on the inter-injection interval

The actual time-course of morphine antinociception is shorter than what would be predicted from its elimination kinetics, suggesting the presence of an acute tolerance phenomenon. Since antagonists acting at NMDA subtype of glutamate receptors were repeatedly shown to prolong acute morphine antinociception, acute tolerance may be attributed to hyperactivity of NMDA receptors. The ability of various site-selective NMDA receptor antagonists to affect morphine antinociception (tail-flick test) was assessed in mice 30 and 120 min after acute morphine challenge. Competitive NMDA receptor antagonist 3-(2-carboxypiperazin-4-yl)-1-propenyl-1-phosphonic acid (D-CPPene) (SDZ EAA 494; 0.1-1 mg/kg), low-affinity channel blockers 1-amino-3,5-dimethyl adamantane (memantine) (1-10 mg/kg) and 1-amino-1,3,3,5,5-pentamethyl-cyclohexan hydrochloride (MRZ 2/579) (1-10 mg/kg), glycine site antagonists 5-nitro-6,7-dichloro-1, 4-dihydro-2,3-quinoxalinedione (ACEA-1021) (5 or 10 mg/kg) and 8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridaliono(4, 5-b)quinoline-5-oxide choline salt (MRZ 2/576) (1-10 mg/kg) were administered intraperitoneally (i.p.) 15 or 30 min prior to the tail-flick test (i.e., interval between injections of morphine and NMDA receptor antagonist was either 0-15 or 90-105 min). ACEA-1021, MRZ 2/576 and to the lesser extent, memantine and MRZ 2/579 enhanced morphine antinociception when tests were conducted 120 but not 30 min post-morphine. D-CPPene potentiated morphine antinociception irrespective of the interval between morphine administration and the tail-flick test. The results suggest that NMDA receptor antagonists may restore analgesic activity of morphine in acutely tolerant mice.     

Uncompetitive NMDA receptor antagonists potentiate morphine antinociception recorded from the tail but not from the hind paw in rats.

We investigated the effects of pretreatment with low-affinity, uncompetitive NMDA receptor antagonists on morphine-induced antinociception in rats using the same intensity of thermal stimulus applied to the tail and the paws. Similar baseline responses to thermal stimuli of the same intensity were recorded from tails and hind paws. However, morphine produced equal antinociception from the tail and hind paw when used at doses of 2.5 and 6 mg/kg, respectively. These doses were used in further experiments. Thirty minutes before morphine, rats were administered the NMDA receptor antagonists dextromethorphan (2.5--30 mg/kg), memantine (2.5--15 mg/kg) and MRZ 2/579 (1-amino-1,3,3,5,5-pentamethyl-cyclohexane HCl) (1.25--10 mg/kg). All three compounds significantly and dose-dependently potentiated morphine-induced antinociception recorded from the tail. However, none of these NMDA receptor antagonists affected morphine antinociception recorded from the paw. These findings suggest that low-affinity NMDA receptor antagonists modulate differently morphine antinociceptive activity recorded from the tail and hind paws.     

Morphine tolerance and dependence in mice with history of repeated exposures to NMDA receptor channel blockers.

Mice were subjected to two successive treatment protocols: first with NMDA receptor channel blockers (14 days, once a day) and second with morphine (5 mg/kg, 8 days, once a day). Treatment with the higher doses of dizocilpine (1 mg/kg), memantine (30 mg/kg), and MRZ 2/576 (30 mg/kg) upon discontinuation revealed only minor behavioral abnormalities attributable to the state of withdrawal. Following repeated administration of low-dose morphine, tolerance to morphine analgesia developed in mice preexposed to dizocilpine (1 mg/kg but not 0.3 mg/kg) but not memantine (10 and 30 mg/kg), MRZ 2/579 (10 and 30 mg/kg), or saline. There were no signs of morphine dependence in any treatment group. Overall, the present study found only minor effects of the subchronic administration of high doses of NMDA receptor channel blockers, suggesting that clinical use of NMDA receptor channel blockers such as memantine will not be accompanied by increased propensity to induction of morphine tolerance and dependence.     

N-Methyl-D-aspartate receptor modulators block hyperalgesia induced by acute low-dose morphine

1. Following opioid-induced antinociception in mice, hyperalgesic responses may be observed. The present study was designed to evaluate the effect of different N-methyl-d-aspartate (NMDA) receptor modulators (magnesium, dextromethorphan, d-serine) on the development of morphine-induced hyperalgesia in mice. The tail-flick test was used to assess the effects of morphine alone and in combination with the NMDA receptor modulators. 2. Administration of a single low dose (2 mg/kg) of morphine to mice produced antinociception that was followed by hyperalgesia. 3. Administration of magnesium sulphate (5 mg/kg) and d-serine (10 mg/kg) alone produced a transient antinociceptive response, whereas dextromethorphan (10 mg/kg) alone produced a prolonged antinociceptive response that had a relatively delayed onset after 4 h. 4. When coadministered with morphine, the NMDA receptor blockers magnesium (2 mg/kg) and dextromethorphan (2 and 5 mg/kg) and the NMDA receptor agonist d-serine (2, 5 and 10 mg/kg), maintained the duration of the antinociceptive response to morphine and inhibited the development of the hyperalgesic response. Coadministration of dextromethorphan (10 mg/kg) with morphine produced antinociception at 30-120 min and at 4-24 h. 5. The results of the present study suggest that coadministration of low-dose NMDA receptor antagonists, as well as the NMDA receptor agonist d-serine, with morphine can inhibit morphine-induced hyperalgesia.     

Interaction of memantine and ketamine in morphine- and pentazocine-induced antinociception in mice

The interaction between uncompetitive NMDA receptor antagonists (memantine and ketamine), and morphine (mu-opioid receptor agonist) and pentazocine (kappa-opioid receptor agonist) was studied in the writhing test in mice. Memantine and ketamine, administered at subthreshold doses, potentiated antinociceptive effect of the threshold (1 mg/kg) dose of morphine. The effects of the threshold (6 mg/kg) dose of pentazocine were not significantly changed by ketamine, and were significantly enhanced by the higher dose of memantine (15 mg/kg). Simultaneously performed experiments in the chimney test have shown that combination of morphine or pentazocine with an NMDA receptor antagonist did not induce significant alterations in the motor coordination of mice. The obtained results have shown that NMDA receptor antagonists (ketamine, memantine) are able to enhance the antinociceptive activity of opioids (morphine, pentazocine). It is necessary to underline that this effect was more apparent for morphine (mu-opioid receptor agonist) + NMDA antagonists than for pentazocine (kappa-opioid receptor agonist). These results may have some importance for clinical practice.     

Inhibitory effects of MPEP,an mGluR5 antagonist,and memantine,an <Emphasis Type="Italic">N</Emphasis>-methyl-<Emphasis Type="SmallCaps">D</Emphasis>-aspartate receptor antagonist,on morphine antinociceptive tolerance in mice

Abstract Rationale. Inhibition of N-methyl-D-aspartate (NMDA) receptors by memantine, an NMDA-receptor antagonist, and other antagonists of ionotropic receptors for glutamate inhibit the development of opiate antinociceptive tolerance. The role of metabotropic receptors for glutamate (mGluR) in opiate tolerance is less known. Objective. In the present study, we examined the effect of 2-methyl-6-(phenylethynyl)-pyridine (MPEP), the mGluR type-I (subtype mGluR5) antagonist, as well as the effect of co-administration of low doses of memantine and MPEP on morphine antinociceptive tolerance in mice. Methods. Morphine antinociceptive activity was tested twice, before and after chronic morphine administration, in the tail-flick test using a cumulative dose–response protocol. Tolerance was induced by six consecutive days of b.i.d. administration of morphine (10 mg/kg, s.c.). Saline, memantine (7.5 mg/kg and 2.5 mg/kg, s.c.), MPEP (30 mg/kg and 10 mg/kg, i.p.) and the combination of both antagonists at low doses was given 30 min prior to each morphine injection during its chronic administration. A separate experiment assessed the effects of memantine, MPEP and their combination on acute morphine antinociception using a tail-flick test. Results. MPEP (30 mg/kg but not 10 mg/kg) as well as memantine (7.5 mg/kg but not 2.5 mg/kg) attenuated the development of tolerance to morphine-induced antinociception. When given together, the low doses of MPEP (10 mg/kg) and memantine (2.5 mg/kg) also significantly attenuated opiate tolerance. None of the treatments with glutamate antagonists produced antinociceptive effects or significantly affected morphine-induced antinociception. Conclusions. The data suggest that both mGluR5 and NMDA receptors may be involved in the development of morphine antinociceptive tolerance. Electronic Publication     

Drug discrimination analysis of NMDA receptor channel blockers as nicotinic receptor antagonists in rats

Rationale Antagonists acting at the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors inhibit various phenomena associated with exposures to nicotine (e.g., tolerance, sensitization, dependence, and intravenous self-administration). These effects are often discussed in terms of nicotine-induced glutamate release with subsequent glutamate-dependent stimulation of dopamine metabolism and neuronal plasticity in brain areas critically involved in drug-addiction mechanisms. However, it is also well established that certain types of NMDA receptor antagonists (channel blockers) potently bind to nicotinic receptors and may act as nicotinic receptor antagonists.Objective The present study aimed to evaluate the discriminative-stimulus effects of the NMDA receptor channel blockers (+)MK-801, dextromethorphan, and memantine in rats trained to discriminate nicotine from its vehicle.Methods Adult male Wistar rats were trained to discriminate 0.6 mg/kg nicotine from saline under a two-lever, fixed-ratio 10 schedule of food reinforcement. During test sessions, injections of (+)MK-801 (0.03–0.3 mg/kg, i.p.), dextromethorphan (30 mg/kg, s.c.), or memantine (1–10 mg/kg, i.p.) were co-administered with s.c. nicotine (0.075–0.6 mg/kg; interaction tests) or saline (generalization tests). Additional interaction and generalization tests were conducted with the selective nicotinic receptor antagonists mecamylamine (0.1–3 mg/kg, s.c.) and MRZ 2/621 (0.3–10 mg/kg, i.p.), and the mGlu5 receptor antagonist MPEP (3–10 mg/kg, i.p.).Results In generalization tests, none of the compounds produced any appreciable levels of substitution for nicotine. The nicotine discriminative-stimulus control was dose dependently attenuated by mecamylamine (ED₅₀=0.67 mg/kg) and MRZ 2/621 (ED₅₀=9.7 mg/kg). Both agents produced a marked downward shift in the nicotine dose–response curve. Memantine and MPEP slightly attenuated nicotine discriminative-stimulus effects, while (+)MK-801 and dextromethorphan did not affect the nicotine-appropriate responding.Conclusions NMDA receptor channel blockers, such as (+)MK-801, dextromethorphan, and memantine, have minimal interactions with the discriminative-stimulus effects of nicotine.     

Selective agonist of group II glutamate metabotropic receptors, LY354740, inhibits tolerance to analgesic effects of morphine in mice

1. Antagonists of glutamate N-methyl-D-aspartate (NMDA) subtype receptor inhibit the development of tolerance to the antinociceptive effects of opioids. Another way to inhibit the function of glutamate receptors is the stimulation of presynaptic metabotropic group II (mGluRII) receptors. Because LY354740 ((+)-2-aminobicyclo [3,1,0] hexane-2,6-dicarboxylic acid) is the first systemically active agonist of group II mGlu receptors, we investigated if this compound might inhibit the development of tolerance to antinociceptive effects of morphine and fentanyl. 2. As assessed by cumulative dose-response approach in the tail-flick test, administration of 10 mg kg(-1) morphine bid s.c. to male Albino Swiss mice for 6 days, right-shifted morphine dose-response curve by approximately 4 fold. In a separate group of mice, 12 injections of 0.04 mg kg(-1) of fentanyl over 3 days, right-shifted fentanyl dose-response curve by approximately 3.3 fold. 3. In experiment 1, LY354740 (1 and 10, but not 0.1 mg kg(-1)) as well as the reference compound, an uncompetitive NMDA receptor antagonist memantine (7.5 mg kg(-1)) inhibited the development of morphine tolerance. Neither LY354740 (10 mg kg(-1)) nor memantine (7.5 mg kg(-1)) affected the development of tolerance to fentanyl. In experiment 2, neither LY354740 (1 and 10 mg kg(-1)) nor memantine (7.5 mg kg(-1)) affected the tail-flick antinociceptive response, or the acute antinociceptive effect of morphine. 4. The present results are the first to suggest that the development of antinociceptive morphine tolerance may be inhibited by metabotropic group II glutamate agonist.     

Effects of short-acting NMDA receptor antagonist MRZ 2/576 on morphine tolerance development in mice

The present study sought to evaluate the ability of a short-acting glycineB site NMDA receptor antagonist, MRZ 2/576, to affect morphine tolerance development in mice. It was found that MRZ 2/576 (10 mg/kg, i.p.) significantly retarded development of morphine analgesic tolerance (20 mg/kg, s.c., 8 days, once a day; tail-flick test) when administered 120 min or 150 min after each daily morphine injection. MRZ 2/576 did not affect the development of morphine tolerance when administered immediately, 15, 30, 60, 90, 180, 240, 300 or 360 min after the daily morphine injections. Thus, short-acting NMDA receptor antagonists may be useful in exploring the temporal characteristics of opioid tolerance (i.e., periods after morphine injection that are critical for tolerance induction) and the present study suggests that after morphine administration there is a period of NMDA receptors activation crucial for the development of tolerance.     

Effects of the NMDA receptor channel blockers memantine and MRZ 2/579 on morphine withdrawal-facilitated aggression in mice

Rationale: Opioid withdrawal is known to facilitate aggressive behavior in laboratory rodents. Aggression develops as the somatic signs disappear and thus may reflect protracted withdrawal-related behavioral alterations. Antagonists acting at the NMDA receptor are known to attenuate the expression of morphine withdrawal syndrome in laboratory animals. Objective: The present study aimed to evaluate the effects of low-affinity NMDA receptor channel blockers (memantine and MRZ 2/579) on aggression facilitated by morphine withdrawal in mice. Methods: Significant increases in aggressive behavior were observed 48 h after repeated morphine administration (8 days, b.i.d., 10–80 mg/kg, SC) was discontinued. Separate groups of mice were treated intraperitoneally with vehicles or different doses of memantine (1, 3, 10 or 30 mg/kg) or MRZ 2/579 (1, 3 or 10 mg/kg) 48 h after the last morphine injection. Results: Both compounds dose-dependently reduced the expression of aggressive behavior while having no significant effect upon the intensity of non-aggressive social contacts. Memantine significantly diminished the occurrence of all recorded components of aggressive behavior (attacks/bites, threats, tail rattling) while MRZ 2/579 affected mainly the appetitive events of aggressive bursts (threats, tail rattling). For both compounds, anti-aggressive effects occurred at dose levels that did not produce motor impairment in the Rotarod test. Conclusions: Taken together with the evidence on the lack of selective anti-aggressive effects of these drugs in morphine-naive mice, attenuation of the aggression observed in the present study may be due to specific interaction with morphine withdrawal-triggered processes. Received: 2 May 1999 / Final version: 17 November 1999     

Clinically available NMDA antagonist, memantine, attenuates tolerance to analgesic effects of morphine in a mouse tail flick test

Converging lines of evidence indicate that N-methyl-D-aspartate (NMDA) receptor antagonists attenuate the development of morphine tolerance tested in antinociception assays in rodents. The present study extends these findings to the effects of clinically available NMDA receptor antagonist, memantine. Male Albino Swiss mice were tested for analgesia using the tail-flick apparatus. Preliminary experiment was designed to find out the optimal dose of morphine and the number of injections that would produce tolerance to its analgesic effects. In the main experiment, during the development of tolerance period (6 days), mice received 10 mg/kg sc b.i.d. morphine injections in the animal room (non-associative tolerance). This treatment resulted in 5.8 fold rightward shift of morphine cumulative dose-response effect from 3.39 mg/kg on day 1 to 16.19 mg/kg on day 8 of the experiment. Memantine pretreatment (5 and 10 mg/kg, but not 2.5 mg/kg), given 30 min prior to each morphine dose during the development of tolerance period, inhibited the rightward shift of morphine cumulative dose-response curve. Thus, pretreatment with memantine at doses of 2.5, 5 and 10 mg/kg resulted in ED50 values of 12.13, 4.74 and 1.95 mg/kg, respectively, corresponding to 3.35, 1.02 and 0.94 fold changes. These data indicate that low affinity, clinically available NMDA receptor antagonist, memantine, may be used to inhibit the development of morphine tolerance.     

Effects of a novel uncompetitive NMDA receptor antagonist, MRZ 2/579 on ethanol self-administration and ethanol withdrawal seizures in the rat

It has been repeatedly reported that NMDA receptors may contribute to ethanol-induced discriminative stimulus effects and withdrawal syndrome. However, the role of NMDA receptors in the reinforcing properties of ethanol remains unclear. The aim of the present study was to evaluate effects of the novel low-affinity, uncompetitive NMDA receptor antagonist, 1-amino-1,3,3,5,5-pentamethyl-cyclohexane hydrochloride (MRZ 2/579), on ethanol self-administration and ethanol withdrawal-associated seizures in rats. Both an operant (lever pressing for ethanol) and non-operant two-bottle choice setups were employed to initiate ethanol self-administration. In another procedure, forced treatment with high doses (9--15 g/kg/day) was used to induce physical dependence on ethanol. MRZ 2/579 delivered chronically by osmotic minipumps (9.6 mg/day, s.c.) did not alter either operant or non-operant ethanol drinking behaviour in a maintenance phase of ethanol self-administration. In contrast, repeated daily injections of the drug (5 mg/kg, i.p.) led to a progressive decrease in operant responding for ethanol. MRZ 2/579 (0.5--7.5 mg/kg, i.p.) and another low-affinity NMDA receptor antagonist, memantine (1--10 mg/kg, i.p.) dose-dependently suppressed ethanol withdrawal seizures with efficacies comparable with that of a standard benzodiazepine derivative, diazepam. The results of the present study indicate that: (i) intermittent administration of MRZ 2/579 may lead to a gradual decrease of operant responding for ethanol; and (ii) the group of low-affinity uncompetitive NMDA receptor antagonists may be an interesting alternative to benzodiazepines in the treatment of alcohol withdrawal.     

Effects of nicotinic and NMDA receptor channel blockers on intravenous cocaine and nicotine self-administration in mice.

Previous studies have indicated that blockade of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors prevents acquisition of instrumental behaviors reinforced by food and drugs such as morphine and cocaine. The present study aimed to extend this evidence by testing whether NMDA receptor channel blocker, memantine, would exert similar effects on acquisition of cocaine and nicotine self-administration in mice. Inasmuch as memantine also acts as nicotinic receptor channel blocker, this study assessed the effects of mecamylamine and MRZ 2/621 that are more selective nicotinic blockers. Adult male Swiss mice were allowed to self-administer cocaine (0.8-2.4 microg/infusion) or nicotine (0.08-0.32 microg/infusion) during the 30-min test. Pretreatment with memantine (0.1-10 mg/kg) prevented acquisition of nicotine but not cocaine self-administration. Pretreatment with mecamylamine (0.3-3 mg/kg) and MRZ 2/621 (0.3-10 mg/kg) produced dose-dependent suppression of both cocaine and nicotine self-administration. Taken together with the previous reports, these results indicate that nicotinic receptor blockers antagonize acute reinforcing effects of cocaine while NMDA receptor blockade may have limited effectiveness.     

Short-acting NMDA receptor antagonist MRZ 2/576 produces prolonged suppression of morphine withdrawal in mice

The present study sought to evaluate the time-course of the effects of a short-acting glycine site NMDA receptor antagonist, MRZ 2/576 (half-life of about 20 min), on the expression of morphine withdrawal syndrome in mice. Morphine-naive and morphine-dependent mice (10-100 mg/kg, b.i.d., s.c., 9 days) were injected with a combination of naltrexone (vehicle or 1 mg/kg, s.c.) and MRZ 2/576 (vehicle, 0.3-10 mg/kg, i.p.) 24 h after the last morphine injection. MRZ 2/576 suppressed expression of several signs of morphine withdrawal (jumping, shaking, forelimb tremor). Effects of MRZ 2/576 were equally expressed throughout 1-h observation test of both spontaneous and naltrexone-facilitated withdrawal. These results suggest that despite its short half-life, MRZ 2/576 produces prolonged suppression of morphine withdrawal syndrome and this effect cannot be attributed to repeated morphine-induced increase in sensitivity to naltrexone.     

Brain distribution of an uncompetitive NMDA receptor antagonist; comparison to its in vitro potency in electrophysiological studies

Although the concentration of drugs in brain homogenates is relatively easy to determine, such data are sometimes misleading due to accumulation in intracellular compartments. This is apparent for uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists where concentrations assessed in this manner are much higher than those sufficient to block the NMDA channel from the extracellular space. The aim of the present study was to determine whether free brain concentrations (extracellular fluid - ECF) of a new uncompetitive NMDA receptor antagonist MRZ 2/579 (1-amino-1,3,3,5,5-pentamethyl-cyclohexane hydrochloride) following administration of doses effective in animal models are sufficient to block NMDA receptors based on its potency in vitro. This issue was addressed using brain microdialysis corrected for in vivo recovery and patch clamp experiments.MRZ 2/579 blocked steady-state inward current responses of cultured hippocampal neurones to NMDA with an IC50 of 1.11 microM at -70 mV. Much higher concentrations of MRZ 2/579 blocked voltage-activated Ca2+ channels with an IC50 of 340 microM. MRZ 2/579 (10 microM) reduced peak inward current responses of neuronal nicotinic receptors only to 72.3% of control. MRZ 2/579 (10-100 microM) had little or no effect at AMPA and GABA(A) receptors. Following chronic s.c. infusion of MRZ 2/579 (40 mg/kg day for 7 days) brain ECF (2.15 microM) and cerebro-spinal fluid (CSF) levels (2.16 microM) were twofold lower than free plasma levels (4.3 microM). MRZ 2/579 showed pronounced accumulation in brain tissue compared to free plasma (28-fold) and ECF (58-fold). After acute i.p. administration (5, 10 and 20 mg/kg) peak concentrations in ECF were 0.70, 0.96 and 2.53 microM, respectively. In conclusion, MRZ 2/579 is indeed strongly accumulated in brain tissue compared to brain ECF, CSF and plasma. However, the brain ECF levels attained following administration of behaviourally effective doses are sufficient for selective NMDA receptor blockade.     

Morphine tolerance and reward but not expression of morphine dependence are inhibited by the selective glutamate carboxypeptidase II (GCP II, NAALADase) inhibitor, 2-PMPA.

Inhibition of glutamate carboxypeptidase II (GCP II; NAALADase) produces a variety of effects on glutamatergic neurotransmission. The aim of this study was to investigate effects of GCP II inhibition with the selective inhibitor, 2-PMPA, on: (a) development of tolerance to the antinociceptive effects, (b) withdrawal, and (c) conditioned reward produced by morphine in C57/Bl mice. The degree of tolerance was assessed using the tail-flick test before and after 6 days of twice daily (b.i.d.) administration of 2-PMPA and 10 mg/kg of morphine. Opioid withdrawal was measured 3 days after twice daily morphine (30 or 10 mg/kg) administration, followed by naloxone challenge. Conditioned morphine reward was investigated using conditioned place preference with a single morphine dose (10 mg/kg). High doses of 2-PMPA inhibited the development of morphine tolerance (resembling the effect of 7.5 mg/kg of the NMDA receptor antagonist, memantine) while not affecting the severity of withdrawal. A high dose of 2-PMPA (100 mg/kg) also significantly potentiated morphine withdrawal, but inhibited both acquisition and expression of morphine-induced conditioned place preference. Memantine inhibited the intensity of morphine withdrawal as well as acquisition and expression of morphine-induced conditioned place preference. In addition, 2-PMPA did not affect learning or memory retrieval in a simple two-trial test, nor did it produce withdrawal symptoms in morphine-dependent, placebo-challenged mice. Results suggest involvement of GCP II (NAALADase) in phenomena related to opioid addiction.     

Additive effect of dextromethorphan on the inhibitory effect of anti-NT4 on morphine tolerance

It has been proposed that opioid tolerance is a model of neuronal plasticity similar to learning and memory. Recent evidence suggests that neurotrophins may be involved in synaptic development and plasticity. Observations indicate that neurotrophin 4 (NT4) is required for the synaptic plasticity mediating both tolerance and memory. Also there are lines of evidence to indicate that NMDA receptors are involved in the neural plasticity underlying the development of opiate tolerance. Neurotrophins affect central transmission postsynaptically by enhancing NMDA receptor responsiveness. So we used the clinically available NMDA receptor antagonist, dextromethorphan, and the neurotrophin 4 antibody, anti-NT4, concomitantly and alone to investigate their effects on morphine tolerance. Tolerance was induced by injecting morphine (7 and 10 mg/kg i.p.) once per day for 4 days. Anti-NT4 (1 microg/rat i.c.v.) was administered 15 min before morphine. Results showed that chronic concomitant treatment of anti-NT4 with morphine in both doses inhibited the development of morphine tolerance. Also acute treatment of anti-NT4 significantly reversed the tolerance that was induced by morphine 7 mg/kg but failed to reverse the tolerance of morphine 10 mg/kg. Dextromethorphan in both doses (10 or 30 mg/kg) has an additive effect on the inhibitory effect of anti-NT4 on the reversal of morphine tolerance (7 mg/kg). These findings provide additional support for the hypothesis that NMDA receptor and NT4 may be involved in neural plasticity underlying opiate tolerance.     

Amino-alkyl-cyclohexanes are novel uncompetitive NMDA receptor antagonists with strong voltage-dependency and fast blocking kinetics: in vitro and in vivo characterization

The present study characterized the in vitro NMDA receptor antagonistic properties of novel amino-alkyl-cyclohexane derivatives and compared these effects with their ability to block excitotoxicity in vitro and MES-induced convulsions in vivo. The 36 amino-alkyl-cyclohexanes tested displaced [3H]-(+)-MK-801 binding to rat cortical membranes with K(i)s between 1.5 and 143 microM. Current responses of cultured hippocampal neurones to NMDA were antagonized by the same compounds with a wide range of potencies (IC50s of 1.3-245 microM, at -70 mV) in a use- and strongly voltage-dependent manner (delta 0.55-0.87). The offset kinetics of NMDA receptor blockade was correlated with equilibrium affinity (Corr Coeff. 0.87 P < 0.0001). As an example, MRZ 2/579 (1-amino-1,3,3,5,5-pentamethyl-cyclohexane HCl) had similar blocking kinetics to those previously reported for memantine (K(on) 10.67 +/- 0.09 x 10(4) M(-1) s(-1), K(off) 0.199 +/- 0.02 s(-1), K(d) = K(off)/K(on) = 1.87 microM c.f. IC50 of 1.29 microM). Most amino-alkyl-cyclohexanes were protective against glutamate toxicity in cultured cortical neurones (e.g. MRZ 2/579 IC50 2.16 +/- 0.03 microM). Potencies in the three in vitro assays showed a relatively strong cross correlation (all corr. coeffs. > 0.72, P < 0.0001). MRZ 2/579 was also effective in protecting hippocampal slices against 7 min. hypoxia/hypoglycaemia-induced reduction of fEPSP amplitude in CA1 with an EC50 of 7.01 +/- 0.24 microM. MRZ 2/579 showed no selectivity between NMDA receptor subtypes expressed in Xenopus oocytes but was somewhat more potent than in patch clamp experiments-IC50s of 0.49 +/- 0.11, 0.56 +/- 0.01 microM, 0.42 +/- 0.04 and 0.49 +/- 0.06 microM on NR1a/2A /2B, /2C and 2/D, respectively. In contrast, memantine and amantadine were both 3-fold more potent at NR1a/2C and NR1a/2D than NR1a/2A receptors. All Merz amino-alkyl-cyclohexane derivatives inhibited MES-induced convulsions in mice with ED50s ranging from 3.6 to 130 mg/kg i.p. The in vivo and in vitro potencies correlated indicating similar access of most compounds to the CNS. MRZ 2/579 administered at 10 mg/kg resulted in peak plasma concentrations of 5.3 and 1.4 microM following i.v. and p.o. administration respectively, which then declined with a half life of around 170-210 min. Analysis of A.U.C. concentrations indicates a p.o./i.v. bioavailability ratio for MRZ 2/579 of 60%. MRZ 2/579 injected i.p. at a dose of 5 mg/kg resulted in peak brain extracellular fluid (ECF) concentrations of 0.78 microM (brain microdialysates). Of the compounds tested MRZ 2/579, 2/615, 2/632, 2/633, 2/639 and 2/640 had affinities, kinetics and voltage-dependency most similar to those of memantine and had good therapeutic indices against MES-induced convulsions. We predict that these amino-alkyl-cyclohexanes, which all had methyl substitutions at R1, R2, and R5, at least one methyl or ethyl at R3 or R4 and a charged amino-containing substitution at R6, could be useful therapeutics in a wide range of CNS disorders proposed to involve disturbances of glutamatergic transmission.     

Effects of NMDA receptor channel blockers, dizocilpine and memantine, on the development of opiate analgesic tolerance induced by repeated morphine exposures or social defeats in mice

Development of tolerance to opiates involves various neurochemically and pharmacologically distinct processes. For instance, the diversity of opiate tolerance has been suggested by experiments comparing the establishment of diminished response to different effects of opiate agonists. Antagonists acting at N-methyl-d-aspartate (NMDA) receptors has become a very useful tool for studying opiate tolerance mechanisms since these drugs have been shown to retard the development of tolerance to analgesic properties of opiates. The present study compared the ability of two NMDA receptor channel blockers, dizocilpine and memantine, to affect the development of tolerance to morphine analgesia induced by repeated social defeat or by repeated morphine administrations. Male BALB/c mice were assessed for the tail-flick response before and after the defeat in five social confrontations, or before and after repeated morphine injections (20 mg/kg, s.c., once daily for 8 days). Repeated morphine injections were explicitly paired with environmental cues. Socially-defeated as well as morphine-treated mice developed significant tolerance to morphine analgesia. Separate groups of mice were exposed to repeated social confrontations or injections of morphine with each defeat or morphine injection followed by administration of either dizocilpine (0.03–0.3 mg/kg, i.p.) or low-affinity channel blocker memantine (3–30 mg/kg, i.p.). Both dizocilpine and memantine were effective in preventing the development of repeated morphine-induced tolerance to acute morphine analgesia. Treatments with NMDA receptor antagonists that retarded development of non-associative tolerance also suppressed the establishment of associative tolerance significantly. Social defeat-induced tolerance was prevented by dizocilpine but not by memantine. Our results suggest some degree of similarity in the mechanisms of morphine analgesic tolerance induced by pharmacological, contextual and social stimuli. Received: 13 December 1997 / Accepted: 15 April 1998