A novel class of amino-alkylcyclohexanes as uncompetitive, fast, voltage-dependent, N-methyl-D-aspartate (NMDA) receptor antagonists--in vitro characterization.

The fact that potent NMDA receptor channel blockers produce phencyclidine-like psychotropic symptoms in man and rodents implies that uncompetitive antagonism of NMDA receptors may not be a promising therapeutic approach. However, recent data indicate that agents with moderate affinity such as memantine and neramexane (MRZ 2/579) are useful therapeutics due to their strong voltage-dependency and rapid unblocking kinetics. Merz has developed a series of novel uncompetitive NMDA receptor antagonists based on an amino-alkylcyclohexane structure. These compounds displaced [(3)H]-MK-801 binding to rat cortical membranes with K(i) values between 1 and 100 microM and inward current responses of cultured hippocampal neurons to NMDA were antagonized in a strongly voltage-dependent manner with rapid blocking/unblocking kinetics. Three of these compounds, with similar biophysical properties to memantine, were chosen for development. MRZ 2/759 (1-ethenyl-3,3,5,5-tetramethyl-cyclohexylamine), 2/1010 (1,3,3,5-tetramethyl-6-azabicyclo[3.2.1]octane) and 2/1013 (8,8,10,10-tetramethyl-1-azaspiro[5.5] undecane) displaced [(3)H]-MK-801 binding with K(i) values of 1.18, 2.59 and 3.64 microM, respectively. They were similarly potent against NMDA-induced currents in hippocampal neurons - IC(50) values of 1.51, 3.06 and 2.20 microM, respectively. In line with their moderate affinity, all were voltage-dependent (delta = 0.86, 0.96 and 0.89, respectively) and fast, open-channel blockers (k(on) 7.90, 1.70 and 2.60 x 10(4) M(-1) sec(-1), k(off) 0.13, 0.12 and 0.24 sec(-1), respectively). These compounds are also NMDA receptor antagonists in the CNS following systemic administration and have good therapeutic indices in a variety of in vivo behavioural models where glutamate is known to play a pivotal role. In view of their relatively low affinity and associated rapid kinetics, they should prove to be useful therapeutics in a wide range of CNS disorders.

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.

Modulation of NMDA receptors by glycine--introduction to some basic aspects and recent developments.

Glycine is a co-agonist at NMDA receptors and it's presence is a prerequisite for channel activation by glutamate or NMDA. Physiological concentrations reduce one form of NMDA receptor-desensitization. Interactions between the glycineB site and other domains of the NMDA receptor are complex and include the glutamate, Mg2+ and polyamines sites. Glycine shows different affinities at various NMDA receptor subtypes probably via to allosteric interactions between NMDA2 subunits and the glycine recognition site on the NMDAR1 subunit. There is still some debate whether the glycineB site is saturated in vivo but it seems likely that this depends on regional differences in receptor subtype expression, local glycine or D-serine concentrations and the expression of specific glycine transporters. GlycineB antagonists and partial agonists have been reported to have good therapeutic indices as neuroprotective agents against focal ischaemia and trauma, anti-epileptics, anxiolytics, anti-psychotomimetics and in models of chronic pain. They clearly lack two potentially serious side effects classically associated with NMDA receptor blockade, namely neurodegenerative changes in the cingulate/retrosplenial cortex and psychotomimetic-like effects. This improved therapeutic profile may be partially due to the ability of full glycineB antagonists to reveal glycine-sensitive desensitization and possibly also via functional and/or regional NMDA receptor subtype selectivity.

Novel systemically active antagonists of the glycine site of the N-methyl-D-aspartate receptor: electrophysiological, biochemical and behavioral characterization.

A series of novel tricyclic pyrido-phthalazine-dione derivatives was tested for antagonistic effects at the strychnine-insensitive modulatory site of the N-methyl-D-aspartate (NMDA) receptor (glycineB). All compounds displaced [3H]MDL-105,519 binding to rat cortical membranes with IC50 values of between 90 nM and 3.6 microM. In patch-clamp experiments, steady-state inward current responses of cultured hippocampal neurons to NMDA (200 microM, glycine 1 microM) were antagonized by these same compounds with IC50 values of 0.14 to 13.8 microM. The antagonism observed was typical for glycineB antagonists, i.e., they induced desensitization and their effects were not use or voltage dependent. Moreover, increasing concentrations of glycine were able to decrease their apparent potency. Much higher concentrations (>100 microM) were required to antagonize alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced currents. They were potent, systemically active NMDA receptor antagonists in vivo against responses of single neurons in the rat spinal cord to microelectrophoretic application of NMDA with ID50 values in the low milligram per kilogram i.v. range. They also inhibited pentylenetetrazol-, NMDA- and maximal electroshock-induced convulsions in mice with ED50 values ranging from 8 to 100 mg/kg i.p. The duration of anticonvulsive action was rather short but was prolonged by the organic acid transport inhibitor probenecid (200 mg/kg). The agents tested represent a novel class of systemically active glycineB antagonists with greatly improved bioavailability.