Novel Systemically Active Antagonists of the Glycine Site ofthe N-Methyl-D-aspartate Receptor: Electrophysiological,Biochemical and Behavioral Characterization
CHRIS G. PARSONS, WOJCIECH DANYSZ, GUNTER QUACK, SABINE HARTMANN, BIANKE LORENZ,CHRISTINE WOLLENBURG, LEOKADIA BARAN, EDMUND PRZEGALINSKI, WOJCIECH KOSTOWSKI, PAWEL KRZASCIK,BORIS CHIZH and P. MAX. HEADLEY
Department of Pharmacology, Merz and Co., D-60318 Frankfurt am Main, Germany (C.G.P., W.D., G.Q., S.H., B.L., C.W.), Institute ofPharmacology PAN, Smetna 12, Cracow, Poland (L.B., E.P.), Department of Pharmacology, Institute of Psychiatry and Neurology, Sobieskiego1–9, Warsaw, Poland (W.K., P.K.) and Department of Physiology, School of Medical Sciences, University Walk, Bristol BS8 1TD, England (B.C.,P.M.H.)
Accepted for publication July 30, 1997
ABSTRACTA series of novel tricyclic pyrido-phthalazine-dione derivativeswas tested for antagonistic effects at the strychnine-insensitivemodulatory site of the N-methyl-D-aspartate (NMDA) receptor(glycineB). All compounds displaced [3H]MDL-105,519 bindingto rat cortical membranes with IC50 values of between 90 nMand 3.6 mM. In patch-clamp experiments, steady-state inwardcurrent responses of cultured hippocampal neurons to NMDA(200 mM, glycine 1 mM) were antagonized by these same com-pounds with IC50 values of 0.14 to 13.8 mM. The antagonismobserved was typical for glycineB antagonists, i.e., they in-duced desensitization and their effects were not use or voltagedependent. Moreover, increasing concentrations of glycinewere able to decrease their apparent potency. Much higher
concentrations (.100 mM) were required to antagonize a-ami-no-3-hydroxy-5-methyl-4-isoxazolepropionic acid-inducedcurrents. They were potent, systemically active NMDA receptorantagonists in vivo against responses of single neurons in therat spinal cord to microelectrophoretic application of NMDAwith ID50 values in the low milligram per kilogram i.v. range.They also inhibited pentylenetetrazol-, NMDA- and maximalelectroshock-induced convulsions in mice with ED50 valuesranging from 8 to 100 mg/kg i.p. The duration of anticonvulsiveaction was rather short but was prolonged by the organic acidtransport inhibitor probenecid (200 mg/kg). The agents testedrepresent a novel class of systemically active glycineB antago-nists with greatly improved bioavailability.
Glutamate is probably the major excitatory transmitter inthe CNS but is also likely to be involved in numerous patho-logical and excitotoxic processes (see Danysz et al., 1995). Itis therefore not surprising that there is a great deal of inter-est in the development of glutamate antagonists for thera-peutic use (see Meldrum, 1985; Lipton and Rosenberg, 1994Muir and Lees, 1995; Danysz et al., 1995). Glutamate acti-vates three major types of ionotropic receptor, namelyAMPA, kainate and NMDA and several types of metabo-tropic receptors.
Antagonism of NMDA receptors may potentially have awide range of therapeutic applications ranging from acuteneurodegeneration (e.g., stroke), chronic neurodegeneration(e.g., Parkinson’s disease, Alzheimer’s disease, Huntington’sdisease) to symptomatic treatment (e.g., Parkinson’s disease,
drug dependence, depression, anxiety, chronic pain, etc.).Functional modulation of NMDA receptors can be achievedthrough actions at different recognition sites such as: theprimary transmitter site (competitive), the phencyclidine sitelocated inside the cation channel (uncompetitive), the poly-amine modulatory site and the strychnine-insensitive, co-agonistic glycine site (glycineB, Danysz et al., 1987; Johnsonand Ascher, 1987; Wong et al., 1987; Kleckner and Dingle-dine, 1988; Fadda et al., 1988).
Although several uncompetitive and competitive NMDAreceptor antagonists are already used clinically or are at anadvanced stage of development (Turski, 1990; Lipton andRosenberg, 1994; Danysz et al., 1995) less is known about thetherapeutic potential of full antagonists acting at the gly-cineB site (Carter, 1992; Kemp and Leeson, 1993; Leeson andIverson, 1994; Kulagowski and Leeson, 1995). Initial preclin-ical evidence, which suggested that a different, perhaps moreReceived for publication April 8, 1997.
ABBREVIATIONS: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; CNS, central nervous system; DH, dorsal horn; GABA,g-aminobutyric acid; GlycineB, strychnine-insensitive, co-agonistic glycine site of the NMDA receptor; MES, maximal electroshock; NMDA,N-methyl-D-aspartate; PTZ, pentylenetetrazol; TI, therapeutic index; 5,7-DCKA, 5,7-dichlorokynurenic acid; HEPES, N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid; ACPC, 1-aminocyclopropanecarboxylic acid.
promising therapeutic profile might be expected from gly-cineB antagonists, was obtained either with local intracere-broventricular administration of full glycineB antagonistswith poor pharmacokinetic properties or systemic adminis-tration of partial agonists (e.g., Moroni et al., 1992; Bubser etal., 1992; Baran et al., 1994; Pellegrini-Giampietro et al.,1994; Salituro et al., 1994). In such studies glycineB antago-nists have lacked many of the side effects classically associ-ated with NMDA receptor blockade, such as: 1) lack of neu-rodegenerative changes in the cingulate/retrosplenial cortexeven after high doses (Chen et al., 1993; Haggerty et al., 1993;Hargreaves et al., 1993; Berger et al., 1994); 2) lack of psy-chotomimetic-like effects (Koek and Colpaert, 1990; Danyszet al., 1994; Loscher et al., 1994; Tortella and Hill, 1996); 3)lack of learning-impairing effects at anticonvulsive doses(Chiamulera et al., 1990; Murata and Kawasaki, 1993;Faiman et al., 1994).
However, some full glycineB antagonists with improved,but by no means optimal pharmacokinetic properties (Baronet al., 1992; Carling et al., 1993; Rowley et al., 1993; Wood-ward et al., 1995) were also reported to have good therapeuticindices after systemic administration in models of hyperal-gesia (Vaccarino et al., 1993; Millan and Seguin, 1994; Lairdet al., 1996), as anxiolytics (Kehne et al., 1995), as possibleanti-psychotomimetics (Bristow et al., 1995, 1996), as neuro-protective agents in models of focal ischemia (Warner et al.,1995) and trauma (Tsuchida and Bullock, 1995) as antiepi-leptics, even in models of partial complex seizures (McCabe etal., 1993; Chapman et al., 1995; Smith et al., 1994) and inblocking spreading depression (Obrenovitch and Zilka, 1996).These studies seem to confirm the promising therapeuticprofile of systemically active glycineB antagonists.
Merz has developed a series of tricyclic “pyrido-phthala-zine-diones” which are also moderately potent glycineB an-tagonists in vitro but show a much better in vivo systemicavailability and/or penetration of the blood-brain barrierthan most glycineB receptor antagonists reported to date.
MethodsIn vitro Receptor Binding Studies
Membrane preparation and protein determination. Tissuepreparation was performed according to Foster and Wong (1987).Male Sprague-Dawley rats (200–250 g) were decapitated and theirbrains were removed rapidly. The cortex was dissected and homog-enized in 20 volumes of ice-cold 0.32 M sucrose with a glass-Teflonhomogenizer. The homogenate was centrifuged at 1000 3 g for 10min. The pellet was discarded and the supernatant was centrifugedat 20,000 3 g for 20 min. The resulting pellet was resuspended in 20volumes of distilled water and centrifuged for 20 min at 8000 3 g.Then the supernatant and the buffy coat were centrifuged threetimes (48,000 3 g for 20 min) in the presence of 50 mM Tris-HCl, pH8.0. All centrifugation steps were carried out at 4°C. After resuspen-sion in 5 volumes of 50 mM Tris-HCl, pH 8.0, the membrane sus-pension was frozen rapidly at 280°C. On the day of assay themembranes were thawed and washed four times by resuspension in50 mM Tris-HCl, pH 8.0, and centrifugation at 48,000 3 g for 20 min.The final pellet was suspended in assay buffer. The amount ofprotein in the final membrane preparation was determined accord-ing to the method of Lowry et al. (1951) with some modifications(Hartfree, 1971). The final protein concentration used for our studieswas between 250 and 500 mg/ml.
In initial experiments, stock solutions (10 mM) were made indistilled water or dimethyl sulfoxide. In later experiments with
[3H]5,7-DCKA and [3H]MDL-105,519 most stock solutions weremade in 10% saturated Tris buffer (pH 9.5) to improve solubilityfurther. Serial dilutions of these stock solutions were then made indistilled water before addition of 50 ml aliquots to the assay tubes(total volume, 500 ml).
[3H]5,7-DCKA binding assay. Incubations were performed ac-cording to the methods modified from previous groups (Yoneda et al.,1993). Membranes were suspended and incubated in 50 mM Tris-HCl, pH 8.0, for 45 min at 4°C with a fixed [3H]5,7-DCKA concen-tration of 10 nM. Nonspecific binding was defined by the addition ofunlabeled glycine at 1 mM.
[3H]MDL-105,519 binding assay. MDL-105,519 is a novel, se-lective high-affinity antagonist at the glycineB site and was recentlyintroduced as a commercially available radioligand for binding stud-ies at this site (Baron et al., 1996, 1997; Hofner and Wanner, 1997).Experiments with [3H]MDL-105,519 were performed as with[3H]5,7-DCKA, except that a fixed [3H]MDL-105,519 concentrationof 2 nM was used.
[3H]Glycine binding assay. [3H]Glycine-binding assays wereperformed according to Kessler et al. (1989). Membranes were sus-pended and incubated in 50 mM Tris-acetate, pH 7.4. Compoundswere incubated with 20 nM [3H]glycine for 30 min at 4°C in thepresence of 100 mM strychnine. Nonspecific binding was defined bythe addition of unlabeled 5,7-DCKA (10 mM).
Incubations were terminated using a Millipore filter system. Thesamples, all in duplicate, were rinsed three times with 2.5 ml ice-coldassay buffer over glass-fiber filters obtained from Schleicher andSchuell (Keene, NH) under a constant vacuum. For the [3H]glycineassay, 10 mM MgSO4 was added to the stop buffer. Filtration wasperformed as rapidly as possible. After separation and rinse thefilters were placed into scintillation liquid (5 ml; Ultima Gold) andradioactivity retained on the filters was determined with a conven-tional liquid scintillation counter (Liquid Scintillation Analyser,Hewlett Packard, Palo Alto, CA).
Patch Clamp
Hippocampi were obtained from rat embryos (E20 to E21) andwere then transferred to calcium- and magnesium-free Hanks’ buff-ered salt solution (Gibco, Eggenstein, Germany) on ice. Cells weremechanically dissociated in 0.05% DNase/0.3% ovomucoid (Sigma,Deisenhofen, Germany) after an 8-min preincubation with 0.66%trypsin/0.1% DNase (Sigma, Deisenhofen, Germany). The dissoci-ated cells were then centrifuged at 18 3 g for 10 min, resuspended inminimum essential medium (Gibco, Eggenstein, Germany) andplated at a density of 150,000 cells cm22 onto poly-DL-ornithine(Sigma)/laminin (Gibco) poly-L-lysine (Sigma)-precoated plastic Petridishes (Falcon, Heidelberg, Germany). The cells were nourished withNaHCO3/HEPES-buffered minimum essential medium supple-mented with 5% fetal calf serum and 5% horse serum (Gibco, Egg-enstein, Germany) and incubated at 37°C with 5% CO2 at 95%humidity. The medium was exchanged completely after inhibition offurther glial mitosis with cytosine-b-D-arabinofuranoside (5 mMSigma) after about 5 days in vitro. Thereafter the medium wasexchanged partially twice weekly.
Patch-clamp recordings were made from these cultured neurons,after 12 to 15 days in vitro, with polished glass electrodes (3–5megohm) in the whole-cell mode at room temperature (20–22°C)with the aid of an EPC-7 amplifier (List). Test substances wereapplied by switching channels of a custom-made fast superfusionsystem with a common outflow (15- to 20-ms exchange times). Thecontents of the intracellular solution were as follows (mM): CsCl,120; triethanolamine-Cl, 20; ethyleneglycol-bis(b-aminoethyl ether)-N,N,N9,N9-tetraacetic acid, 10; MgCl2, 1; CaCl2, 0.2; glucose, 10;ATP, 2; cAMP, 0.25; pH was adjusted to 7.3 with CsOH or HCl. Theextracellular solutions had the following basic composition (mM):NaCl, 140; KCl, 3; CaCl2, 0.2; glucose, 10; HEPES, 10; sucrose, 4.5;tetrodotoxin (3 3 1024). For most experiments glycine (1 mM) waspresent in all solutions, a concentration sufficient to cause approxi-
1997 Systemically Active GlycineB Antagonists 1265
mately 80% activation of glycineB receptors. Experiments to test theglycine dependence of the Merz glycineB antagonists were performedin the continuous presence of increasing concentrations of glycine(1–10 mM). Only results from stable cells were accepted for inclusionin the final analysis, i.e., after recovery of responses to NMDA by atleast 75% of their depression by the antagonists tested.
Microelectrophoretic Application of NMDA and AMPA toSpinal Neurons in Vivo
Details of the experimental procedure have been described else-where (Cumberbatch et al., 1995). Male Wistar rats (290–350 g) wereanesthetized with halothane, and tracheal, carotid and jugular can-nulae were inserted. A laminectomy (T10-L2) was performed, andthe spinal cord was cut at T10-T11. Anesthesia was switched to andmaintained with a-chloralose (50 mg/kg i.v., then 10–15 mg/kg/hr).Arterial blood pressure was monitored continuously and systolicpressure remained above 100 mm Hg. Core temperature was main-tained close to 37°C.
Extracellular recordings of single DH neuron action potentialswere made by multibarrel glass electrodes containing 3.5 M NaCl(recording barrel), sodium salts of NMDA (100 mM in 100 mM NaCl)and AMPA (10 mM in 200 mM NaCl), both at pH 7.5 to 8.0, and 150mM NaCl for automated current balancing. Amino acids were ejectedin regular cycles (40-s ejection period, 30–40 s interstimulus inter-val) with 60-s intervals between cycles. Drugs were dissolved in anaqueous solvent (0.606 g Tris; 5.0 g glucose; 0.5 g Tween 80; 95 mlwater) at pH 8.5 to 9.0 (MRZ 2/502 and 2/516) or in 0.9% saline (MRZ2/570, 2/571, 2/576 and 2/577). Typical drug concentrations were 3 to5 mg/ml. Drugs were given intravenously in a cumulative dose-doubling regime when the variability of the control amino acid re-sponses did not exceed 10%. Drug effects were expressed as percent-ages of the mean of last three predrug responses. Spike counts werecompensated for ongoing activity. Calculations of ID50 values wereperformed on individual cells with commercially available software(GraFit, Erithacus Software Ltd, Staines, Middlesex, England). Sta-tistical analysis was by the Mann-Whitney test on original spikecount data.
Anticonvulsive Activity
Male albino Swiss mice (19–21 g) housed 10 to 15 per cage wereused for the NMDA lethality test (Leander et al., 1988). For PTZ-induced convulsions male albino Swiss mice (25–34 g) housed 40 percage were used, whereas in the MES and motor impairment testsNMR female mice (18–28 g) housed 5 per cage were used. All ani-mals were kept with water and food ad libitum under a 12-hrlight-dark cycle (light on at 6 A.M.) and at a controlled temperature(20 6 0.5°C). All experiments were performed between 10 A.M. and 5P.M. Tested agents were usually injected i.p. 15 min before theinduction of convulsions. MRZ 2/502 was dissolved in saline withNaOH added. The choline salts of Merz glycineB antagonists weredissolved in distilled water. Most other agents were dissolved in thefollowing solution: 0.606 g Tris; 5.0 g glucose; 0.5 g Tween 80; 95 mlwater. All experiments were performed strictly according to theanimal rights commission allowance F 77–51 (Hessen).
In the test of NMDA-induced convulsions in mice, a dose-responserelationship for NMDA was first performed to determine the ED97
dose which was then used for testing antagonistic properties. Afterinjection of the ED97 dose of NMDA the animals were placed in asmall cage and observed for 20 min. Death preceded by clonic con-vulsions and tonic seizures was the pharmacological endpoint.
PTZ was injected at a dose of 90 mg/kg (i.p). The presence ofgeneral tonic convulsions was then scored for 30 min because thisparameter is more sensitive to NMDA receptor antagonists thanclonic convulsions. The pharmacological endpoint was taken as thepresence of tonus in the hind limbs with stretching.
The MES test was performed together with tests for myorelaxantaction (traction reflex) and motor coordination (rotarod). For the
traction reflex test mice were placed with their forepaws on a hori-zontal rod and were required to place all four paws on the wirewithin 10 sec. To test ataxia (motor coordination) mice were placedon rotarod (5 rpm) and were required to remain on the rod for 1 min.Only mice not achieving the criteria in all three repetitions of eachtest were considered to exhibit myorelaxation or ataxia, respectively.These tests were followed by MES (100 Hz, 0.5-s shock duration,50-mA shock intensity, 0.9-ms impulse duration, Ugo Basile, Com-erio, Italy) applied through corneal electrodes. The presence of tonicconvulsions was scored (tonic extension of hind paws with minimumangle to the body of 90°). The aim was to obtain ED50 values for allparameters scored (anticonvulsive activity and motor side effects)with use of the Litchfield Wilcoxon test for quantal dose responses.Division of the ED50 for side effects (ataxia or myorelaxation) by theED50 for antagonism of electroshock convulsions was used as a TI.
Chemicals
The following agents were used: ACPC, S-AMPA, 7-chlorothio-kynurenic acid, 5,7-dichlorothiokynurenic acid, D-cycloserine, 5,7-DCKA, 3-amino-1-hydroxypyrrolidin-2-one [(1R)-HA-966],kynurenic acid, trans-2-carboxy-5,7-dichloro-4-phenyl-aminocarbon-ylamino-1,2,3,4-tetradroquinoline (L-689,560), 7-chloro-3-(cylopro-pylcarbonyl)-4-hydroxy-2(1H)-quinoline (L-701,252), 3-(-3-hy-droxyphenyl)prop-2-ynyl-7-chloro-4-hydroxy-2-(1H)-quinolone-3-carboxylate (L-701,273), 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinoline (L-701,324), D-serine, L-serine (all Tocris CooksonLtd, Bristol, U.K.); NMDA, glycine, tetrodotoxin, PTZ, warfarin,probenecid, salts and buffers (all Sigma); all pyrido-phthalazine-dione derivatives (Institute for Organic Synthesis, Riga, Latvia). Thebasic structure of these derivatives is given in figure 1. The followingradioligands were used: [3H]5,7-DCKA (56.6 Ci/mmol) and [3H]gly-cine (48.4 Ci/mmol) (Dupont NEN, Cologne, Germany); [3H]MDL-105,519 {[3H](Z)-2-(phenyl)-3-(4,6-dichloroindol-3-yl-2-carboxylicacid) propenoic acid, 74.0 Ci/mmol} (Amersham, Braunschweig, Ger-many).
ResultsIn Vitro Receptor Binding Studies
The tricyclic pyrido-phthalazine-diones tested displaced[3H]MDL-105,519 binding to rat cortical membranes withIC50 values of between 90 nM and 3.9 mM (see table 1).Standard glycineB antagonists, partial agonists and full ago-nists also showed their expected potencies in this assay (seetable 1). Nonspecific binding determined with 1 mM glycinewas only 10 to 15% and all compounds tested displacedbinding to nonspecific levels with Hill coefficients very closeto unity (fig. 2).
In contrast, although potencies were roughly similar in the[3H]5,7-DCKA and [3H]glycine assays, the maximal displace-ment by antagonists and agonists/partial agonists wasgreater than nonspecific binding determined with 1 mM gly-cine and 10 mM 5,7-DCKA, respectively. The reason for this
Fig. 1. Basic structure of the pyrido-phthalazine-diones.
1266 Parsons et al. Vol. 283
remains unclear and confounded the interpretation of thedata and resulted, in many cases, in Hill coefficients as low as0.5 and associated high variability in the data (see also Baronet al., 1991; Yoneda et al., 1993). As such the results are notpresented. [3H]MDL-105,519 binding therefore seems to bethe more suitable, commercially available radioligand fordetermining binding to the glycineB site.
Patch Clamp
Steady-state inward current responses of cultured hip-pocampal neurons to NMDA (200 mM with 1 mM glycine)were antagonized by these tricyclic pyrido-phthalazine-dio-nes with IC50 values of 0.14 to 13.8 mM. The relative poten-cies of standard glycineB antagonists, partial agonists andfull agonists were similar to those reported in the literatureand correlated well with [3H]MDL-105,519 binding (Pearson
Product Moment Correlation Coefficient 5 0.941, P , .0001).The antagonism observed with most compounds was typicalfor glycineB full antagonists, i.e., they induced glycine-sensi-tive desensitization (fig. 3). The degree of this desensitiza-tion, however, seemed to depend largely on the potency ofantagonist tested: 5,7-DCKA and 7-chlorokynurenic acidwere 10 times more potent against steady-state currentsthan against peak currents, whereas L-689,560 andL-701,324 were almost equieffective against these two com-ponents (table 2). In agreement with previous reports, partialagonists were equipotent against both components despiteshowing relatively low potency (Kemp and Priestley, 1991).The NMDA receptor antagonism by Merz tricyclic pyrido-phthalazine-diones was mediated at the glycineB site as ev-idenced by the parallel shift in the concentration-responsecurves in the presence of increasing glycine concentrations
TABLE 1Concentration-dependent displacement of [3H]MDL 105,519 binding to cortical membranes by glycineB antagonists, partial agonistsand agonistsa
a Estimation of IC50 values and curve fitting were made according to the four-parameter logistic equation (Excel, Microsoft Software). Data are presented asmeans 6 S.E. from separate experiments.
b 5,7-Dichlorokynurenic acid.c 1-Aminocyclopropanecarboxylic acid.
Fig. 2. Representative experiment showing the concentra-tion-dependent displacement of [3H]MDL 105,519 binding tocortical membranes by glycineB antagonists. Membranes wereincubated with [3H]MDL 105,519 (2 nM) in 50 mM Tris-HCl, pH8.0, for 45 min at 4°C. Nonspecific binding (13.2 6 0.2%) wasdefined by the addition of unlabeled glycine (1 mM). The ef-fects of each concentration of displacer were assessed induplicate and plotted as % specific binding against concen-tration. Estimation of IC50 values and curve fitting were madeaccording to the four-parameter logistic equation (GraFit,Erithacus Software).
1997 Systemically Active GlycineB Antagonists 1267
(fig. 4). Thus the Kb values of MRZ 2/502 as assessed accord-ing to the Cheng-Prussoff relationship were similar in 1, 3and 10 mM glycine (79.9 6 18.3, 124.4 6 29.3 and 118.2 624.8 nM corresponding to IC50 values of 0.28 6 0.03, 1.06 60.09 and 3.08 6 0.14 mM, respectively). Furthermore, the
effects of MRZ 2/502 were not voltage-dependent (fig. 4).Choline derivatives had potencies similar to the free acids invitro (table 2).
In contrast, the three of these glycineB antagonists testedwere only very weak antagonists of inward currents to AMPA
Fig. 3. Concentration dependence of the blockade of NMDA receptors by MRZ 2/502. (A) Peak and plateau (steady state) NMDA currentresponses of cultured hippocampal neurons were normalized to control levels and plotted as means (6 S.E.) against MRZ 2/502 concentration(0.1 mM, n 5 6; 0.3 mM, n 5 6; 1.0 mM, n 5 6; 3.0 mM, n 5 5; 10.0 mM, n 5 5; 30.0 mM, n 5 4). Estimation of IC50 values and curve fitting weremade according to the four-parameter logistic equation. (Peak IC50 5 0.80 6 0.12 mM, Hill coefficient 5 1.06 6 0.07; steady-state IC50 5 0.28 60.03 mM, Hill coefficient 5 0.96 6 0.05). (B) Concentration-dependence of the blockade of NMDA receptors by MRZ 2/502 on a single culturedhippocampal neuron. NMDA (200 mM) was applied for 2.5 s every 30 s in the continuous presence of glycine (1 mM) at a constant membranepotential of 270 mV. MRZ 2/502 (0.1, 0.3 and 1.0 mM) was continuously present for a least 1.5 min at each concentration. Equilibrium responsetraces have been superimposed to allow better resolution of differences in the kinetics of desensitization of individual responses.
TABLE 2Antagonism of NMDA- and AMPA-induced inward currents in cultured hippocampal neuronsIC50 values (6S.E.) against peak and steady-state components of these responses were determined from data from at least three concentrations producing between15% and 85% inhibition in the presence of 1 mM glycine and at least four cells per concentration. EC50 values (6S.E.) of partial agonists and glycine were determinedin the nominal absence of glycine. Intrinsic activity of partial agonists and agonists was based on the maximal inhibition of steady-state currents in the presence of1 mM glycine and maximal potentiation in the absence of glycine and normalized to 82.5% activation of the glycineB site by 1 mM glycine.
(100 mM). MRZ 2/502, 2/514 and 2/516 had IC50 valuesagainst peak AMPA-induced currents of 25.1 6 2.0, 72.7 64.7 and 17.6 6 1.5 mM, respectively, but were essentiallyinactive against steady-state currents that had IC50 values .
100 mM (fig. 5). This profile of action, although very weak, istypical for competitive AMPA receptor antagonists whichpreferentially block the peak nondesensitized, low-affinitystate of the AMPA receptor (see Parsons et al., 1994).
Fig. 4. (A) Glycine counteracts the antagonistic effects of MRZ 2/502 on NMDA-induced currents. NMDA (200 mM) was applied for 2.5 s every 30 s at270 mV in the continuous presence of glycine (1, 3 and 10 mM). Pooled steady-state responses were quantified as in figure 3 and plotted againstantagonist concentration (4–8 per concentration). The four-parameter logistic equation was used to fit the data (solid curves) and to calculate the IC50values for MRZ 2/502 (1 mM glycine, 0.28 6 0.03 mM, Hill coefficient 0.96 6 0.05; 3 mM glycine, 1.06 6 0.09 mM, Hill coefficient 1.09 6 0.06; 10 mMglycine, 3.08 6 0.14 mM, Hill coefficient 1.23 6 0.05). The Kb values of MRZ 2/502 as assessed according to the Cheng-Prussoff relationship were similarin 1, 3 and 10 mM glycine (79.9 6 18.3, 124.4 6 29.3 and 118.2 6 24.8 nM, respectively). (B) Voltage independence of the blockade of NMDA receptorsby MRZ 2/502. NMDA (200 mM) was applied for 2.5 s every 30 s in the continuous presence of glycine (1 mM) at various membrane potentials. Peakand plateau (steady-state) NMDA current responses in the absence and presence of MRZ 2/502 have been plotted as means against membranepotential (n 5 2). The upper left insert shows original data for the i.v. curve in the presence of MRZ 2/502 (1 mM).
Fig. 5. Higher concentrations of MRZ 2/502 also antagonize AMPA-induced inward currents in cultured hippocampal neurons. (A) AMPA (100 mM)was applied for 1 s every 15 s at 270 mV. The left and right profiles show control and recovery responses, respectively. The middle three profilesshow equilibrium responses in the continuous presence of 10, 30 and 100 mM MRZ 2/502, respectively. (B) Pooled responses were quantified aspeak and steady-state (plateau) currents after subtraction of any leak current and plotted, after normalization to control, as means 6 S.E. againstMRZ 2/502 concentration. At least six cells were tested at each concentration. The four-parameter logistic equation was used to fit the data (solidcurves) and to calculate the IC50 values of MRZ 2/502: Peak IC50 5 25.1 6 2.0 mM (Hill coefficient 5 0.72 6 0.04); steady-state IC50 5 149.6 621.4 mM (Hill coefficient 5 1.52 6 0.15).
1997 Systemically Active GlycineB Antagonists 1269
Microelectrophoretic Application of NMDA and AMPA toSpinal Neurons in Vivo
The ability of these glycineB antagonists to act as NMDAreceptor antagonists in vivo was assessed by intravenousadministration against responses of single neurons in the ratspinal cord to local microelectrophoretic application of AMPAand NMDA. The data shown are from 22 rats. The com-pounds were tested in a log 2, cumulative dose-progression(typically 0.5–16 mg/kg). Unfortunately, limited solubility insaline, characteristic for most compounds tested, made itdifficult to test higher i.v. doses because large volumes (.1ml) were required. More recent data indicate that solubilityof choline derivatives for i.v. injection can be increased fur-ther by substituting 5.5% fructose for 0.9% NaCl.
The choline derivative of the mother compound MRZ 2/577was tested over a range of i.v. doses (0.5–16 mg/kg), and onlyat the top doses (4–16 mg/kg) produced slight inhibition ofresponses of DH neurons to iontophoretic NMDA. This effectwas dose-dependent (mean ID50, 33.7 6 7.2 mg/kg; slope,1.3 6 0.3, n 5 5) and statistically significant at 16 mg/kg (to67 6 5% control, P , .05, n 5 5). No significant reduction ofAMPA responses was observed at the doses tested (90 6 3%control at 16 mg/kg, n 5 5).
MRZ 2/502 was tested over a range of i.v. doses (0.5–8mg/kg) and dose-dependently reduced responses of spinal DHneurons to iontophoretic NMDA (mean ID50, 1.6 6 0.3 mg/kg,n 5 6, fig. 6, table 3). Higher doses were required to reduceresponses to iontophoretic AMPA (mean ID50, 5.5 6 1.8 mg/kg, n 5 6). The effects on both NMDA and AMPA developedwithin 20 to 30 s after injection, and the responses recovered
quickly with a t1/2 of 8 to 15 min. Similar results wereobtained with its water-soluble choline salt, MRZ 2/576 (figs.6 and 7, table 3). This compound appeared to be somewhatless potent than MRZ 2/502 in reducing responses to NMDA(ID50, 2.8 6 0.7 mg/kg) but was more selective (AMPA ID50 .16 mg/kg). Thus, at top doses tested on each cell (2–8 mg/kg;mean, 4 mg/kg), NMDA responses were reduced to 30 6 7%control, whereas responses to AMPA were reduced to 88 66% control (n 5 7). The true difference in potency of MRZ2/502 and 2/576 is less than it appears because the cholinederivative has a larger molecular weight. Thus, on a molarbasis the potencies of MRZ 2/502 and 2/576 were not signif-icantly different (6.1 6 1.1 mmol/kg vs. 7.6 6 1.9 mmol/kg,respectively: Mann-Whitney P . .05).
MRZ 2/514 was not tested because of solubility problems.However, its choline salt MRZ 2/570 was tested and produceda selective and dose-dependent inhibition of responses toNMDA of spinal DH neurons (fig. 7, table 3) with an ID50 of4.5 6 0.7 mg/kg. No reduction of the AMPA response wasobserved even at the top doses tested. (At 8–16 mg/kg; mean,9 mg/kg, AMPA responses were 100 6 9% control, cf. reduc-tion of NMDA to 29 6 4% control.)
Both MRZ 2/516 (0.5–8 mg/kg) and its water-soluble cho-line salt, MRZ 2/571 (0.5–16 mg/kg) dose-dependently antag-onized responses of DH cells to exogenous NMDA (table 3).Again, the choline salt appeared to be less potent than thefree acid (ID50 of MRZ 2/571 for the NMDA response was4.7 6 0.5 mg/kg, n 5 6; for MRZ 2/516 it was 2.0 6 0.3 mg/kg,n 5 7). Both compounds substantially attenuated AMPAresponses. Thus, at the top doses tested on each cell (mean, 4;range, 2–8 mg/kg), MRZ 2/516 reduced NMDA responses to11 6 2% control; responses of these cells to iontophoreticAMPA were 42 6 10% control (n 5 7). It was possible withthese two compounds to calculate ID50 values for the AMPAresponse; they were 9.2 6 1.1 mg/kg for MRZ 2/571 and 4.1 61.4 mg/kg for MRZ 2/516.
Anticonvulsive Activity
The ability of Merz glycineB antagonists to act as NMDAreceptor antagonists in vivo was confirmed by use of threeconvulsion models in rodents. All Merz glycineB antagonistsinhibited MES- and PTZ-induced convulsions in mice withED50 values ranging from 8 to 100 mg/kg i.p. (table 4). MostMerz compounds were also active against NMDA-inducedconvulsions although they appeared to be considerably lesspotent in this model. In the MES model, choline salts seemedto have a somewhat longer duration of action (fig. 8). Theiranticonvulsive potency was increased after i.v. administra-tion (table 5). MRZ 2/570 was somewhat less potent after s.c.administration, and was also active, although considerablyless potent, after oral administration (table 5). At doseswithin the anticonvulsive range, myorelaxation (tractiontest) and ataxia (rotarod test) were observed. However, noneof the Merz glycineB antagonists showed any serious acutetoxicity, i.e., minimal lethal doses were all greater than 100mg/kg (table 4). In contrast, of the standard compoundstested and despite their low nanomolar in vitro affinity forthe glycineB site, only L-701,324 was systemically active andwas not much more potent than the Merz compounds. More-over, the TI of L-701,324 was actually worse than for thecholine salts of the Merz compounds (see table 4).
Fig. 6. Effects of intravenous MRZ 2/502 and MRZ 2/576 on re-sponses to iontophoretic NMDA and AMPA in the spinal DH of a-chloralose-anesthetized rats. (A) Activity of a DH WDR neuron (depth,800 mm from the dorsal pial surface) was evoked by cycles of ionto-phoretic applications of NMDA (N, 10 nA, 40 s) and AMPA (A, 5 nA,40 s). (B) Activity of a DH WDR neuron (920 mm depth) responses toiontophoretic NMDA (13 nA, 40 s) and AMPA (9 nA, 40 s). MRZ 2/502and MRZ 2/506 were given i.v. in a dose-doubling regime at 6-min (twocycles) intervals between the injections. The left-hand profiles repre-sent three cycles of the control responses. The calibration bar relates toboth panels A and B.
1270 Parsons et al. Vol. 283
The anticonvulsive effect of MRZ 2/570 (30 mg/kg) in theMES test was attenuated by very high doses of systemicglycine with an ED50 5 688 mg/kg and by lower doses ofD-cycloserine with an ED50 5 8.8 mg/kg, full and partialagonist of the glycineB site, respectively, which indicates thattheir in vivo effects are also mediated at the glycineB site.
Probenecid (200 mg/kg) 30 min before administration ofthe tested agents prolonged considerably the duration ofanticonvulsive action in the MES test. For example the half-lives of 2/514 and 2/570 30 mg/kg were about 40 and 80 min,
respectively, in the absence of probenecid. In the presence ofprobenecid the half-lives were prolonged to about 180 and210 min, respectively (fig. 8). Probenecid alone at the doseused (200 mg/kg) had no effect on MES-induced convulsionsper se. Warfarin (50 mg/kg i.p.) was also able to slightlyincrease the potency of Merz glycineB antagonists in the MEStest (table 6). However, it is not clear whether this reflectsmoderate binding to plasma albumins of the Merz glycineB
antagonists because higher doses of warfarin (100 mg/kg)alone showed some anticonvulsive actions.
Fig. 7. Pooled data on the effects of i.v. MRZ 2/576(0.5–4 mg/kg, n 5 6), MRZ 2/570 (1–8 mg/kg, n 5 5) andMRZ 2/571 (1–8 mg/kg, n 5 6) on responses to ionto-phoretic NMDA and AMPA in the spinal DH of a-chlora-lose-anesthetized rats. Examples of experimental proto-col are shown in figure 6. Data are presented as meanpercentages of control values 6 S.E. Significant differ-ence from control is shown as *P , .05; **P , .01,Mann-Whitney test.
TABLE 4Effect of Merz glycineB antagonists and selected reference agents on convulsions induced by MES, PTZ and NMDAED50 values are in milligrams per kilograms (95% confidence limits are shown in parentheses). The therapeutic index (TI) was also calculated as the ED50 for inhibitionof traction reflex (Tract.) impairment or rotarod failure (Rot.) divided by the ED50 for MES-induced seizures. None of the Merz glycineB antagonists showed any acutetoxicity, i.e., minimal (Min) lethal doses were all more than 100 mg/kg.
Formulation MES ED50 TI Tract. TI Rot. PTZ ED50 NMDA ED50 Min Lethal
TABLE 3Effects of intravenous glycineB antagonists on responses to iontophoretic NMDA and AMPA in the spinal DH of a-chloralose-anesthetized ratsID50 values (6S.E.) against responses of spinal DH neurons to iontophoretic NMDA and AMPA were determined from data from at least three doses producing between15% and 85% inhibition and at least five cells per dose. NT 5 not tested.
1997 Systemically Active GlycineB Antagonists 1271
DiscussionThe present data illustrate that the tricyclic pyrido-
phthalazine-dione derivatives studied are moderately potent
and selective glycineB antagonists in vitro. Most compoundsdisplaced [3H]DCKA, [3H]MDL-105,519 and [3H]glycinebinding to rat cortical membranes with high nanomolar af-finity and antagonized steady-state inward current re-sponses of cultured hippocampal neurons to NMDA withhigh nanomolar to low micromolar affinity. The NMDA re-ceptor antagonism observed was typical for glycineB antago-nists, i.e., showed competition with glycine, revealed glycine-dependent desensitization (Mayer et al., 1989a,b; Lerma etal., 1990, Vyklicky et al., 1990; Parsons et al., 1993) and wasnot use- or voltage-dependent. Much higher concentrationswere required to antagonize steady-state inward current re-sponses to AMPA and, in this case, the antagonism observedwas reminiscent of a competitive interaction (Parsons et al.,1994). These effects on AMPA receptors agree closely withprovisional unpublished data which indicate a least a 100-fold lower affinity in displacing [3H]AMPA binding (the Ki
values of MRZ 2/502, 2/514 and 2/516 were 24 6 7 mM, 33 69 mM and 26 6 5 mM, respectively), but no activity at up to 10mM on many other, well characterized CNS receptors (otherionotropic glutamate receptor recognition sites and severalsubtypes of adenosine, adrenergic, dopamine, GABA, seroto-nin, opioid, histamine, cholinergic, purinergic and sigma re-ceptors and voltage-activated channels; Pan Labs, Bothell,WA). These Merz glycineB antagonists were also testedagainst glutamate (100 mM)-induced neurotoxicity in cul-tured cortical neurons and provided near complete protectionat 30 mM (data not shown).
The ability of these glycineB antagonists to act as NMDAreceptor antagonists in vivo was assessed by i.v. administra-tion against responses of single neurons in the rat spinal cordto microelectrophoretic application of AMPA and NMDA.They were potent NMDA receptor antagonists in vivo withID50 values in the low milligram per kilogram range. On amolar basis these in vivo doses are about 20-fold higher thanthose required for NMDA receptor antagonism in vitro,which indicates relatively good penetration to the CNS.Somewhat higher doses of the free acids also antagonizedresponses to AMPA. However, two of the choline derivativeswere more selective; this apparent lack of selectivity con-trasts with the in vitro assays. As such, it seems that at leastsome of the observed effects on AMPA may be secondary todepression of NMDA receptor-mediated background activity(Chizh et al., 1996).
Merz glycineB antagonists also inhibited PTZ-, NMDA-and MES-induced convulsions in mice after i.p. administra-tion. They were most potent against MES- and PTZ-inducedconvulsions but less potent against NMDA-induced convul-sions. The reason for these differences remains unclear. Wehave extensive data that the MES test is a good index forNMDA receptor antagonism in vivo (Parsons et al., 1995),and this test was therefore used for further characterizationof the in vivo activity of Merz glycineB antagonists.
As exemplified by MRZ 2/570, Merz glycineB antagonistswere found to be most potent against MES-induced convul-sions after i.v. administration, somewhat less active after i.p.and s.c. administration and least potent after p.o. adminis-tration. Their anticonvulsive actions were reversed by bothsystemic glycine and D-cycloserine administration, which in-dicates that their in vivo effects are also mediated at theglycineB site. The duration of anticonvulsive action after i.p.administration was rather short (30–40 min) but was pro-
Fig. 8. Probenecid prolongs the anticonvulsive action of MRZ 2/514and MRZ 2/570 in the MES model in mice. Probenecid (200 mg/kg) 30min before administration of the tested agents prolonged considerablythe duration of anticonvulsive action, which indicates that organic acidtransport in the choroid plexus out of the brain plays an important rolein the short duration of action of the compounds tested. Probenecidalone at the dose used (200 mg/kg) has no effect on MES-inducedconvulsions per se.
TABLE 5MRZ 2/570 was more potent against MES-induced seizures afteri.v. administration but was less potent after s.c. or p.o.administration
a The therapeutic index (TI) was calculated as the ED50 for inhibition of tractionreflex (Tract.) impairment or rotarod failure (Rot.) divided by the ED50 for MES-induced seizures. NT 5 not tested.
TABLE 6Warfarin was also able to increase the potency of Merz glycineBantagonists which suggests moderate binding to plasmaalbuminsWarfarin (50 mg/kg i.p.) 30 min before administration of the tested agents in-creased their potency against MES-induced seizures.
longed by probenecid, which indicates rapid transport out ofthe brain by the organic acid transporter in the choroidplexus (Moroni et al., 1988; Leeson and Iverson, 1994; San-tamaria et al., 1996). Warfarin was able to slightly increasetheir potency, which suggests only moderate binding toplasma albumins. Moreover, higher doses of warfarin alonealso had anticonvulsive activity, which indicates that thispotentiation may actually have been caused by synergisticinteractions. These compounds had a similar in vivo potencyto the standard glycineB antagonist L-701,324 despite beingat least 20-fold less potent as NMDA receptor antagonists invitro. Taken together with the relatively modest effects ofwarfarin, this would tend to indicate that plasma albuminbinding of these Merz glycineB antagonists is much less thanseen previously with other glycineB antagonists (T. Priestley,personal communication). This fact might partially accountfor their much improved pharmacokinetics, i.e., penetrationto the CNS.
Initial results from microdialysis studies indicate thatMRZ 2/570 reaches peak extracellular concentrations in thebrain of about 1.7 mM at 20 min after i.p. administration of 30mg/kg in rats. The half-life of MRZ 2/570 in the rat brainreflects that seen for anticonvulsive activity in mice and canalso be prolonged by probenecid (200 mg/kg i.p.) (peak con-centrations of 2.6 mM with a half-life of about 90 min; M.Hesselink, unpublished data).
The fact that most of the very high-affinity, standard gly-cineB receptor antagonists tested in the present study wereinactive in vivo indicates that attempts to improve systemicactivity solely by increasing the in vitro potency of glycineB
antagonists with poor pharmacodynamic properties may bethe wrong approach. Moreover, the ability of some full an-tagonists of the glycineB site to unmask NMDA receptorglycine-sensitive desensitization may underlie their promis-ing therapeutic profile (Parsons et al., 1993) and seems, inpart, to be inversely related to affinity. The present datashow a trend toward a greater antagonism of steady-statethan peak inward current responses to NMDA by lower af-finity glycineB full antagonists, i.e., the lower affinity com-pounds induced a greater degree of glycine-sensitive desen-sitization (Johnson and Ascher, 1987; Kleckner andDingledine, 1988; Mayer et al., 1989a,b; Lerma et al., 1990,Vyklincky et al., 1990; Parsons et al., 1993). This finding is inline with a recent report by Molnar and Erdo (1996). Incontrast, the partial agonists (1R)-HA-966 and D-cycloserineshowed no differentiation between peak and steady-statecomponents despite relatively low affinity. ACPC behavedessentially like a full agonist, with higher concentrationscausing antagonism, probably via effects at a different rec-ognition site. Similar differences in the profiles of some par-tial agonists and full antagonists have also been reportedpreviously and attributed to differential effects on allostericinteractions between the agonist and glycineB sites (Kempand Priestley, 1991; Grimwood et al., 1995).
Receptor desensitization may represent a physiologicalprocess serving as an endogenous control mechanism to pre-vent long-term neurotoxic activation of glutamate receptorsbut allow their transient physiological activation (Parsons etal., 1993). Ischemia increases not only the concentration ofextracellular glutamate but also that of glycine, and althoughthis later effect is less pronounced, it actually persists formuch longer (Globus et al., 1991). Hence, some full glycineB
antagonists could restore normal synaptic transmission un-der excitotoxic conditions by increasing NMDA receptor de-sensitization to its physiological level (Parsons et al., 1993).Indeed, our own provisional data indicate these Merz gly-cineB antagonists are neuroprotective against NMDA-in-duced lesion of the nucleus basalis fo Meynert in rats as wellas in a global ischemia model in gerbils (Danysz et al., 1996).
Doses within the anticonvulsive range also produced my-orelaxation and ataxia. It should be stressed, however, thatthe MES test was used purely as an index of NMDA receptorantagonism in vivo and not as a preclinical model for thepossible utility of Merz glycineB antagonists against gener-alized seizures. The relatively poor therapeutic indices in theMES test do not necessarily contradict the hypothesis dis-cussed above. Although the biophysical properties of someglycineB full antagonists may allow therapeutically relevantconcentrations to block chronic, low-level pathological acti-vation of NMDA receptors while leaving their synaptic acti-vation intact, precisely these properties may also underliethe poor therapeutic indices relative to antiepileptic activityseen in the present study because of the synaptic nature ofboth seizures and normal glutamatergic transmission. Thisin turn does not mean that poor therapeutic indices can beexpected in all models of disturbed glutamatergic neuro-transmission. The literature indicates that some glycineB
antagonists have much improved therapeutic indices thanothers belonging to a similar class. For example, MDL100,458 and MDL 102,288 are equipotent as glycineB antag-onists in vitro but exhibit strikingly different in vivo profilesin audiogenic seizures in DBA/2 mice and in separation-induced ultrasonic vocalizations in rat pups, a model of anx-iolytic activity (Kehne et al., 1995). The reason for thesedifferences is not clear, although the compounds may possi-bly act preferentially at different NMDA receptor subtypes(Danysz et al., 1995).
Our own data also indicate that these Merz glycineB an-tagonists may have improved potency and therapeutic indi-ces in some indications and show a very different behavioralprofile to competitive and uncompetitive NMDA receptor an-tagonists. For example, in rats in the open-field test theyactually attenuate the hyperlocomotion induced by both PCPand amphetamine at nonataxic doses and show no disruptionof prepulse inhibition (Danysz et al., 1996; see also Bristow etal., 1995, 1996). These data indicate a lack of psychotomi-metic potential and possibly even antipsychotic activity ofthese tricyclic pyrido-phthalazine-diones. Furthermore, theyantagonize morphine place preference in rats at low, nona-taxic doses, which suggests therapeutic potential in thetreatment of opioid abuse (Danysz et al., 1996). MRZ 2/570(5–10 mg/kg i.p.) has no negative effects on working or ref-erence memory in the radial maze (W. Danysz, unpublisheddata). Finally, even very high doses of up to 100 mg/kg i.p.MRZ 2/576 does not cause any neurodegenerative changes inthe cingulate or retrosplenial cortex of female or male rats (A.Schwaier, unpublished data).
We have now initiated extensive testing of these glycineB
antagonists in preclinical models of various disorders thathave been associated with disturbances of the glutamatergicsystem. These compounds could be useful in the treatment ofthe following disorders: 1) acute excitotoxicity such as is-chemia during stroke, trauma, hypoxia, hypoglycemia andhepatic encephalopathy; 2) chronic neurodegenerative dis-
1997 Systemically Active GlycineB Antagonists 1273
eases such as Alzheimer’s disease, vascular dementia, Par-kinson’s disease, Huntington’s disease, multiple sclerosis,amyotrophic lateral sclerosis, AIDS-neurodegeneration,olivopontocerebellar atrophy, Tourette’s syndrome, motorneuron disease, mitochondrial dysfunction, Korsakoff syn-drome, Creutzfeldt-Jakob disease; 3) other disorders relatedto long-term plastic changes in the CNS such as chronic pain,drug tolerance, dependence and addiction (e.g., opioids, co-caine, benzodiazepines and alcohol); 4) epilepsy (partial com-plex seizures), tardive dyskinesia, schizophrenia, anxiety,depression, visceral pain, spasticity and tinnitus.
In conclusion, these tricyclic pyrido-phthalazine-dionesrepresent a novel class of systemically active glycineB antag-onists with much improved penetration to the CNS. Theyshould prove to be useful tools to elucidate the therapeuticpotential of this class of NMDA receptor antagonists in var-ious disorders that involve disturbances of glutamatergictransmission.
References
BARAN, H., LOSCHER, W. AND MEVISSEN, M.: The glycine/NMDA receptor partialagonist D-cycloserine blocks kainate-induced seizures in rats. comparisonwith MK-801 and diazepam. Brain Res. 652: 195–200, 1994.
BARON, B. M., SIEGEL, B. W., SLONE, A. L., HARRISON, B. L., PALFREYMAN, M. G.AND HURT, S. D.: [3H]-5,7-Dichlorokynurenic acid, a novel radioligand labelsNMDA receptor-associated glycine binding sites. Eur. J. Pharmacol. Mol.Pharmacol. 206: 149–154. 1991.
BARON, B. M., HARRISON, B. L., MCDONALD, I. A., MELDRUM, B. S., PALFREYMAN,M. G., SALITURO, F. G., SIEGEL, B. W., SLONE, A. L., TURNER, J. P. AND WHITE,H. S.: Potent indoleline and quinoline-containing N-methyl-D-aspartate an-tagonists acting at the strychnine-insensitive glycine binding site. J. Phar-macol. Exp. Ther. 262: 947–956, 1992.
BARON, B. M., HARRISON, B. L., KEHNE, J. H., SCHMIDT, C. J., VANGIERSBERGEN,P. L. M., WHITE, H. S., SIEGEL, B. W., SENYAH, Y., MCCLOSKEY, T. C., FADAYEL,G. M., TAYLOR, V. L., MURAWSKY, M. K., NYCE, P. AND SALITURO, F. G.:Pharmacological characterization of MDL 105,519, an NMDA receptor gly-cine site antagonist. Eur. J. Pharmacol. 323: 181–192, 1997.
BARON, B. M., SIEGEL, B. W., HARRISON, B. L., GROSS, R. S., HAWES, C. AND
TOWERS, P.: [3H]-MDL 105,519, a high affinity radioligand for the NMDAreceptor-associated glycine recognition site. J. Pharmacol. Exp. Ther. 262:947–956, 1996.
BERGER, P., FARREL, D., SHARP, F. AND SKOLNICK, P.: Drugs acting at the strych-nine-insensitive glycine receptor do not induce HSP-70 protein in the cin-gulate cortex. Neurosci. Lett. 168: 147–150, 1994.
BRISTOW, L. J., LANDON, L., SAYWELL, K. L. AND TRICKLEBANK, M. D.: The glycine/NMDA receptor antagonist, L-701,324 reverses isolation-induced deficits inprepulse inhibition in the rat. Psychopharmacology 118: 230–232, 1995.
BRISTOW, L. J., FLATMAN, K. L., HUTSON, P. H., KULAGOWSKI, J. J., LEESON, P. D.,YOUNG, L. AND TRICKLEBANK, M. D.: The atypical neuroleptic profile of theglycine/N-methyl-D-aspartate receptor antagonist, L-701,324, in rodents.J. Pharmacol. Exp. Ther. 277: 578–585, 1996.
BUBSER, M., KESEBERG, U., NOTZ, P. K. AND SCHMIDT, W. J.: Differential behav-ioural and neurochemical effects of competitive and non-competitive NMDAreceptor antagonists in rats. Eur. J. Pharmacol. 229: 75–82, 1992.
CARLING, R. W., LEESON, P. D., MOSELEY, A. M., SMITH, J. D., SAYWELL, K.,TRICKLEBANK, M. D., KEMP, J. A., MARSHALL, G. R., FOSTER, A. C. AND GRIM-WOOD, S.: Anticonvulsant activity of glycine-site NMDA antagonists. 2. Trans2-carboxy-4-substituted tetrahydroquinolines. Bioorg. Med. Chem. Lett. 3:65–70, 1993.
CARTER, A. J.: Glycine antagonists: regulation of the NMDA receptor-channelcomplex by the strychnine-insensitive glycine site. Drugs Future 17: 595–613, 1992.
CHAPMAN, A. G., DURMULLER, N., HARRISON, B. L., BARON, B. M., PARVEZ, N. AND
MELDRUM, B. S.: Anticonvulsant activity of a novel NMDA/glycine site an-tagonist, MDL 104,653, against kindled and sound-induced seizures. Eur.J. Pharmacol. 274: 83–88, 1995.
CHEN, J., GRAHAM, S., MORONI, F. AND SIMON, R.: A study of the dose dependencyof a glycine receptor antagonist in focal ischemia. J. Pharmacol. Exp. Ther.267: 937–941, 1993.
CHIAMULERA, C., COSTA, S. AND REGGIANI, A.: Effect of NMDA-insensitive andstrychnine-insensitive glycine site antagonists on NMDA-mediated convul-sions and learning. Psychopharmacology 102: 551–553, 1990.
CHIZH, B. A., CUMBERBATCH, M. J., HERRERO, J. F. AND HEADLEY, P. M.: NMDAreceptors mediate spontaneous activity, but not phasic synaptic responses,in the spinal dorsal horn of the anaesthetized rat. J. Physiol. (Lond.) 491:113P, 1996.
CUMBERBATCH, M. J., CHIZH, B. A. AND HEADLEY, P. M.: Modulation of excitatory
amino acid responses by tachykinins and selective tachykinin receptor ago-nists in the rat spinal cord. Br. J. Pharmacol. 115: 1005–1012, 1995.
DANYSZ, W., WROBLEWSKI, J. T., BROOKER, G. AND COSTA, E.: Modulation ofexcitatory amino acid transmission by phencyclidine and glycine in the ratcerebellum in vivo. Soc. Neurosci. Abs. 13: 383, 1987.
DANYSZ, W., ESSMANN, U., BRESINK, I. AND WILKE, R.: Glutamate antagonistshave different effects on spontaneous locomotor activity in rats. Pharmacol.Biochem. Behav. 48: 111–118, 1994.
DANYSZ, W., PARSONS, C. G., BRESINK, I. AND QUACK, G.: Glutamate in CNSdisorders: A revived target for drug development? Drug News Perspect. 8:261–277, 1995.
DANYSZ, W., PARSONS, C. G., KARCZ-KUBICHA, M., GOLD, M., KALVINCH, I.,PISKUNOVA, I. AND ROZHKOV, E.: Novel systemically-active antagonists of theglycine site of the NMDA receptor: Behavioural characterisation. Soc. Neu-rosci. Abs. 22: 1530, 1996.
FADDA, E., DANYSZ, W., WROBLEWSKI, J. T. AND COSTA, E.: Glycine and D-serineincrease the affinity of the N-methyl-D-aspartate sensitive glutamate bind-ing sites in rat brain synaptic membranes. Neuropharmacology 27: 1183–1185, 1988.
FAIMAN, C. P., VIU, E., SKOLNICK, P. AND TRULLAS, R.: Differential effects ofcompounds that act at strychnine-insensitive glycine receptors in a punish-ment procedure. J. Pharmacol. Exp. Ther. 270: 528–533, 1994.
FOSTER, A. C. AND WONG, E. H. F.: The novel anticonvulsant MK-801 binds tothe activated state of the N-methyl-D-aspartate receptor in rat brain. Br. J.Pharmacol. 91: 403–409, 1987.
GLOBUS, M. Y. T., GINSBERG, M. D. AND BUSTO, R..: Excitotoxic index: A biochem-ical marker of selective vulnerability. Neurosci. Lett. 127: 39–42, 1991.
GRIMWOOD, S., KULAGOWSKI, J. J., MAWER, I. M., ROWLEY, M., LEESON, P. D. AND
FOSTER, A. C.: Allosteric modulation of the glutamate site on the NMDAreceptor by four novel glycine site antagonists. Eur. J. Pharmacol. Mol.Pharmacol. Sect. 290: 221–226, 1995.
HAGGERTY, G. C., CHARLES, V. AND LANTHORN, T. H.: Acute vacuolization study ofsubcutaneously administered D-cycloserine (DCS) in Sprague Dawley rats.Soc. Neurosci. Abs. 19: 1888, 1993.
HARGREAVES, R. J., RIGBY, M., SMITH, D. AND HILL, R. G.: Lack of effect ofl-687,414 ((1)-cis-4-methyl-HA-966), an NMDA receptor antagonist actingat the glycine site, on cerebral glucose metabolism and cortical neuronalmorphology. Br. J. Pharmacol. 110: 36–42, 1993.
HARTFREE, E. F.: Determination of protein: a modification of the Lowry methodthat gives a linear photometric response. Anal. Biochem. 48: 422–427, 1971.
HOFNER, G. AND WANNER, K. T.: Characterisation of the binding of [3H]-MDL105,519, a radiolabelled antagonist for the N-methyl-D-aspartate-associatedglycine site, to pig cortical brain membranes. Neurosci. Lett. 226: 79–82,1997.
JOHNSON, J. W. AND ASCHER, P.: Glycine potentiates the NMDA response incultured mouse brain neurones. Nature 325: 529–531, 1987.
KEHNE, J. H., BARON, B. M., HARRISON, B. L., MCCLOSKEY, T. C., PALFREYMAN,M. G., POIROT, M., SALITURO, F. G., SIEGEL, B. W., SLONE, A. L., VAN GIERS-BERGEN, P. L. M. AND WHITE, H. S.: MDL 100,458 and MDL 102,288: Twopotent and selective glycine receptor antagonists with different functionalprofiles. Eur. J. Pharmacol. 284: 109–118, 1995.
KEMP, J. A. AND LEESON, P. D.: The glycine site of the NMDA receptor: Fiveyears on. Trends Pharmacol. Sci. 14: 20–25, 1993.
KEMP, J. A. AND PRIESTLEY, T.: Effects of (1)-HA-966 and 7-chlorokynurenic acidon the kinetics of N-methyl-D-aspartate receptor agonist responses in ratcultured cortical neurons. Mol. Pharmacol. 39: 666–670, 1991.
KESSLER, M., BAUDRY, M. AND LYNCH, G.: Quinoxaline derivatives are high-affinity antagonists of the NMDA receptor-associated glycine sites. BrainRes. 489: 377–382, 1989.
KLECKNER, N. W. AND DINGLEDINE, R.: Requirement for glycine in activation ofNMDA receptors expressed in Xenopus oocytes. Science 214: 835–837, 1988.
KOEK, W. AND COLPAERT, F. C.: Selective blockade of N-methyl-D-aspartateinduced convulsions by antagonists and putative glycine antagonists rela-tionship with phencyclidine-like behavioural effects. J. Pharmacol. Exp.Ther. 252: 349–357, 1990.
KULAGOWSKI, J. J. AND LEESON, P. D.: Glycine-site NMDA receptor antagonists.Expert. Opin. Ther. Patients 5: 1061–1075, 1995.
LAIRD, J. M. A., MASON, G. S., WEBB, J., HILL, R. G. AND HARGREAVES, R. J.:Effects of a partial agonist and a full antagonist acting at the glycine site ofthe NMDA receptor on inflammation-induced mechanical hyperalgesia inrats. Br. J. Pharmacol. 117: 1487–1492, 1996.
LEANDER, J. D., LAWSON, R. R., ORNSTEIN, P. L. AND ZIMMERMAN, D. M.: N-methyl-D-aspartic acid-induced lethality in mice: Selective antagonism by phencyc-lidine-like drugs. Brain Res. 448: 115–120, 1988.
LEESON, P. D. AND IVERSEN, L. L.: The glycine site on the NMDA receptor:structure-activity relationships and therapeutic potential. J. Med. Chem. 37:4053–4067, 1994.
LERMA, J., ZUKIN, R. S. AND BENNETT, M. V. L.: Glycine decreases desensitizationof N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus oocytesand is required for NMDA responses. Proc. Natl. Acad. Sci. U.S.A. 87:2354–2358, 1990.
LIPTON, S. A. AND ROSENBERG, P. A.: Excitatory amino acids as a final commonpathway for neurologic disorders. N. Engl. J. Med. 330: 613–622, 1994.
LOSCHER, W., WLAZ, P., RUNDFELDT, C., BARAN, H. AND HONACK, D.: Anticonvul-sant effects of the glycine/NMDA receptor ligands D-cycloserine and D-
1274 Parsons et al. Vol. 283
serine but not R-(1)-HA-966 in amygdala-kindled rats. Br. J. Pharmacol.112: 97–106, 1994.
LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. AND RANDELL, R. J.: Proteinmeasurement with the folin phenol reagent. J. Biol. Chem. 193: 265–275,1951.
MAYER, M. L., VYKLICKY, L. J. AND CLEMENTS, J.: Regulation of NMDA receptordesensitization in mouse hippocampal neurons by glycine. Nature 338: 425–427, 1989a.
MAYER, M. L., VYKLICKY, L. J. AND SERNAGOR, E.: A physiologist’s view of theN-methyl-D-aspartate receptor: An allosteric ion channel with multiple reg-ulatory sites. Drug Dev. Res. 17: 263–280. 1989b.
MCCABE, R. T., WASTERLAIN, C. G., KUCHARCZYK, N., SOFIA, R. D. AND VOGEL,J. R.: Evidence for anticonvulsant and neuroprotectant action of felbamatemediated by strychnine-insensitive glycine receptors. J. Pharmacol. Exp.Ther. 264: 1248–1252, 1993.
MELDRUM, B.: Possible therapeutic applications of antagonists of excitatoryamino acid neurotransmitters. Clin. Sci. 68: 113–122, 1985.
MILLAN, M. J. AND SEGUIN, L.: Chemically-diverse ligands at the glycineB sitecoupled to N-methyl-D-aspartate (NMDA) receptors selectively block thelate phase of formalin-induced pain in mice. Neurosci. Lett. 178: 139–143,1994.
MOLNAR, P. AND ERDO, S. L.: Differential effects of five glycine site antagonistson NMDA receptor desensitisation. Eur. J. Pharmacol. 311: 311–314, 1996.
MORONI, F., RUSSI, P., LOMBARDI, G., BENI, M. AND CARLA, V.: Presence ofkynurenic acid in the mammalian brain. J. Neurochem. 51: 177–180, 1988.
MORONI, F., ALESIANI, M., FACCI, L., FADDA, E., SKAPER, S. D., GALLI, A., LOM-BARDI, G., MORI, F., CIUFFI, M., NATALINI, B. AND PELLICCIARI, R.: Thiokynure-nates prevent excitotoxic neuronal death in vitro and in vivo by acting asglycine antagonists and as inhibitors of lipid peroxidation. Eur. J. Pharma-col. 218: 145–151, 1992.
MUIR, K. W. AND LEES, K. R.: Clinical experience with excitatory amino acidantagonist drugs. Stroke 26: 503–513, 1995.
MURATA, S. AND KAWASAKI, K.: Common and uncommon behavioural effects ofantagonists for different modulatory sites in the NMDA receptor/channelcomplex. Eur. J. Pharmacol. 239: 9–15, 1993.
OBRENOVITCH, T. P. AND ZILKHA, E.: Inhibition of cortical spreading depressionby L-701,324, a novel antagonist at the glycine site of the N-methyl-D-aspartate receptor complex. Br. J. Pharmacol. 117: 931–937, 1996.
PARSONS, C. G., ZONG, X. G. AND LUX, H. D.: Whole cell and single channelanalysis of the kinetics of glycine-sensitive N-methyl-D-aspartate receptordesensitization. Br. J. Pharmacol. 109: 213–221, 1993.
PARSONS, C. G., GRUNER, R. AND ROZENTAL, J.: Comparative patch clamp studieson the kinetics and selectivity of glutamate receptor antagonism by 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)-quinoxaline (NBQX) and 1-(4-amino-phenyl)-4-methyl-7,8-methylendioxyl-5h-2,3-benzodiazepine (GYKI 52466).Neuropharmacology 33: 589–604, 1994.
PARSONS, C. G., QUACK, G., BRESINK, I., BARAN, L., PRZEGALINSKI, E., KOSTOWSKI,W., KRZASCIK, P., HARTMANN, S. AND DANYSZ, W.: Comparison of the potency,kinetics and voltage-dependency of a series of uncompetitive NMDA receptorantagonists in vitro with anticonvulsive and motor impairment activity invivo. Neuropharmacology 34: 1239–1258, 1995.
PELLEGRINI-GIAMPIETRO, D. E., COZZI, A. AND MORONI, F.: The glycine antagonistand free radical scavenger 7-cl-thio-kynurenate reduces CA1 ischemic dam-age in the gerbil. Neuroscience 63: 701–709, 1994.
ROWLEY, M., LEESON, P. D., STEVENSON, G. I., MOSELEY, A. M., STANSFIELD, I.,SANDERSON, I., ROBINSON, L., BAKER, R., KEMP, J. A., MARSHALL, G. R., FOSTER,A. C., GRIMWOOD, S., TRICKLEBANK, M. D. AND SAYWELL, K. L.: 3-acyl-4-hydroxyquinolin-2(1h)-ones: Systemically active anticonvulsants acting byantagonism at the glycine site of the N-methyl-D-aspartate receptor com-plex. J. Med. Chem. 36: 3386–3396, 1993.
SALITURO, F. G., TOMLINSON, R. C., BARON, B. M., PALFREYMAN, M. G., MCDONALD,I. A., SCHMIDT, W., WU, H. Q., GUIDETTI, P. AND SCHWARCZ, R.: Enzyme-activated antagonists of the strychnine-insensitive glycine NMDA receptor.J. Med. Chem. 37: 334–336, 1994.
SANTAMARIA, A., RIOS, C., SOLIS HERNANDEZ, F., ORDAZ MORENO, J., GONZALEZ
REYNOSO, L., ALTAGRACIA, M. AND KRAVZOV, J.: Systemic DL-kynurenine andprobenecid pretreatment attenuates quinolinic acid-induced neurotoxicity inrats. Neuropharmacology 35: 23–28, 1996.
SMITH, R. D., GRZELAK, M. E. AND COFFIN, V. L.: Felbamate, a novel antiepilepticagent, does not affect cognition in rodents. Behav. Pharmacol. 5: 365–368,1994.
TORTELLA, F. C. AND HILL, R. G.: EEG seizure activity and behavioral neuro-toxicity produced by (1)-M. K.-801, but not the glycine site antagonistL-687,414, in the rat. Neuropharmacology 3: 441–448, 1996.
TSUCHIDA, E. AND BULLOCK, R.: The effect of the glycine site-specific N-methyl-D-aspartate antagonist ACEA 1021 on ischemic brain damage caused byacute subdural hematoma in the rat. J. Neurotrauma 12: 279–288, 1995.
TURSKI, L.: N-methyl-D-aspartate receptor complex. Different sites for regula-tion and clinical consequences. Arzneim. Forsch. Drug Res. 40: 511–514,1990.
VACCARINO, A. L., MAREK, P., KEST, B., WEBER, E., KEANA, J. F. W. AND LIEBE-SKIND, J. C.: NMDA receptor antagonists, MK-801 and ACEA-1011, preventthe development of tonic pain following subcutaneous formalin. Brain Res.615: 331–334, 1993.
VYKLICKY, L., JR., BENVENISTE, M. AND MAYER, M. L.: Modulation of N-methyl-D-aspartic acid receptor desensitization by glycine in mouse cultured hip-pocampal neurones. J. Physiol. (Lond.) 428: 313–331, 1990.
WARNER, D. S., MARTIN, H., LUDWIG, P., MCALLISTER, A., KEANA, J. F. W. AND
WEBER, E.: In vivo models of cerebral ischemia: Effects of parenterallyadministered NMDA receptor glycine site antagonists. J. Cereb. Blood FlowMetab. 15: 188–196, 1995.
WONG, E. H. F., KNIGHT, A. R. AND RANSOM, R.: Glycine modulates MK-801binding to the NMDA receptor in the rat brain. Eur. J. Pharmacol. 142:487–488, 1987.
WOODWARD, R. M., HUETTNER, J. E., TRAN, M., GUASTELLA, J., KEANA, J. F. W. AND
WEBER, E.: Pharmacology of 5-chloro-7-trifluoromethyl-1,4-dihydro-2,3-quinoxalinedione: A novel systemically active ionotropic glutamate receptorantagonist. J. Pharmacol. Exp. Ther. 275: 1209–1218, 1995.
YONEDA, Y., SUZUKI, T., OGITA, K. AND HAN, D. K.: Support for radiolabeling of aglycine recognition domain on the N-methyl-D-aspartate receptor ionophorecomplex by 5,7-,h-3.dichlorokynurenate in rat brain. J. Neurochem. 60:634–645, 1993.
Send reprint requests to: Dr. Chris. G. Parsons, Dept. of Pharmacology,Merz & Co., Eckenheimer Landstrasse 100–104, D-60318 Frankfurt am Main,Germany.
1997 Systemically Active GlycineB Antagonists 1275
