mGluR5 Antagonist Induced Psychoactive Properties: MTEP ...jpet.aspetjournals.org/.../28/jpet.113.211185.full.pdf · antagonists MPEP and SIB-1893 can inhibit NMDA receptor mediated

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mGluR5 Antagonist Induced Psychoactive Properties: MTEP Drug

Discrimination, a Pharmacologically Selective Non-NMDA Effect

with Apparent Lack of Reinforcing Properties

Authors:

M. D. B. Swedberg, M. Ellgren and P. Raboisson

Department of Safety Pharmacology (MDBS, ME), Disease Biology (PR),

AstraZeneca R&D Södertälje, Sweden.

Current affiliations

Independent Consultant, Drug Discovery Pharmacology, Trosa, Sweden (MDBS)

Neuropharmacology, Addiction & Behaviour, Department of Pharmaceutical

Biosciences, Uppsala University, Uppsala, Sweden (ME)

Galderma R&D SNC, Sophia Antipolis, France (PR)

JPET Fast Forward. Published on January 28, 2014 as DOI:10.1124/jpet.113.211185

Copyright 2014 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on January 28, 2014 as DOI: 10.1124/jpet.113.211185

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Running title: Psychoactive and reinforcing effects of mGluR5 antagonists

Michael D B Swedberg, Independent Consultant, Drug Discovery Pharmacology,

Sockengatan 6, S61933 Trosa, Sweden

Phone: +46733542230

e-mail: [email protected]

Text pages: 39

Tables: 3

Figures: 3

References: 53

Abstract: 246 words

Introduction: 750 words

Discussion: 1486 words

Abbreviations:

THC, tetrahydrocannabinol

mGluR, metabotropic glutamate receptor

iGluR, ionotropic glutamate receptors

EAA, Excitatory amino acid receptors

Fenobam, McN-3377, ([N-(3-chlorophenyl)-N’-(4,5-dihydro-1-methyl-4-oxo-1H-

imidazole-2-yl)urea]

CNS, central nervous system

NMDA, N-methyl-D-aspartate

AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

MPEP, (2-methyl-6-(phenylethynyl)pyridine

MTEP, (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine)

PCP, phencyclidine


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NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline

(+)-MK-801, dizocilpine

LSD, lysergic acid diethylamide

Recommended section: Behavioral Pharmacology


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Abstract

Fenobam, a potent mGluR5 receptor antagonist, reported to have analgesic effects

in animals and anxiolytic effects in humans, also caused adverse events including

psychostimulant-type effects and ”derealization phenomena". Recent

electrophysiological, pharmacological and anatomical data show that the mGluR5

antagonists MPEP and SIB-1893 can inhibit NMDA receptor mediated activity, and

that mGluR5 receptors are highly expressed in limbic and forebrain regions. The

present studies evaluated the potential of mGluR5 receptor antagonists to cause

PCP-like psychoactive effects in a rat drug discrimination procedure, and, secondly

explored and characterized the selective mGluR5 antagonist MTEP as a

discriminative stimulus and comparing MTEP with other drugs known to be

psychoactive in humans. Additionally, the reinforcing potential of MPEP and MTEP

was compared to PCP in a rat intravenous self administration procedure. (+)-MK-

801and ketamine caused full PCP-appropriate responding. Memantine and the

mGluR5 antagonists caused no or weak partial PCP-appropriate responding. In

MTEP trained rats, MTEP, MPEP and fenobam caused full and equipotent MTEP-

appropriate responding. (+)-MK-801 and memantine caused below 70%, whereas

PCP, chlordiazepoxide and LSD caused below 50% MTEP appropriate responding.

δ-9-THC, yohimbine, arecoline and pentylenetetrazole all caused MTEP appropriate

responding below 20%. Rats self-administered PCP but not MPEP or MTEP,

indicating a lack of reinforcing effects of the mGluR5 antagonists. These data

suggest that the mGluR5 antagonists appear not to have reinforcing properties, that

discriminative effects of mGluR5 antagonists and PCP are dissimilar, and that

mGluR5 antagonists may produce psychoactive effects different from NMDA-

antagonists and other drugs with known psychotomimetic properties.


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Introduction

Excitatory amino acid (EAA) receptors play a major role in mediation of synaptic

excitation in the central nervous system (CNS) and glutamate is the most abundant

EAA in the CNS (Monaghan et at., 1989). Glutamatergic transmission is mediated via

three ligand-gated ionotropic receptors, iGluR's: N-methyl-D-aspartate (NMDA), a-

amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate (Monaghan

et at., 1989), and a G-protein coupled metabotropic receptor (mGluR; Conn and Pin,

1997). Attenuation of glutamate transmission may be beneficial for CNS disorders,

including cerebral ischemia, epilepsy, anxiety, pain and depression.

For example, anticonvulsant effects were demonstrated with competitive (Chapman

et at., 1991a) and uncompetitive NMDA antagonists like phencyclidine (PCP; Hayes

and Ba!ster, 1985; Chapman and Meldrum, 1989), and the AMPA antagonist NBQX

(Chapman et al., 1991b; Swedberg et al., 1995). NMDA receptor blockade can

cause hallucinations and other vivid psychoactive effects in humans (Aniline and

Pitts, 1982; Bey et al., 2007), mediates PCP-like discriminative effects in primates

(Nicholson et al., 2007) and rats (Koek et al., 1990; Wiletts and Balster, 1988;

Swedberg et al., 1995), and maintains self-administration in animals (Nicholson et al.,

2007) , whereas the AMPA antagonist NBQX caused no PCP-like discriminative

effects (Swedberg et al., 1995).

Recent discoveries of several mGluR receptor subtypes increases the number of

potential targets for drug discovery (Conn and Pin, 1997) and mGluR5 receptors are

implicated in several CNS functions (Schoepp, 2001) and disorders including anxiety,

depression, pain and Parkinsons' disease (Spooren et al., 2001) and

gastroesophageal reflux disease (Keywood et al., 2009), and are localized in

forebrain and limbic regions in the rat (Spooren et al., 2001) and primate (Muly et al.,

2003).

Fenobam ([N-(3-chlorophenyl)-N’-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-

yl)urea]; McN-3377), recently characterized as an mGluR5 receptor antagonist with


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anxiolytic (Porter et al., 2005) and analgesic effects in rodents (Montana et al., 2009)

had shown anxiolytic effects in humans (Pecknold et al, 1980, 1982; Lapierre and

Oyewumi, 1982), and was dissimilar to the classic anxiolytic diazepam in being more

psychostimulant (Itil et al., 1978; Friedmann et al., 1980) and causing hallucinations

(Friedmann et al., 1980). The more selective mGluR5 antagonist MPEP (2-methyl-6-

(phenylethynyl)pyridine; Gasparini et al., 1999) had anxiolytic-like activity in rats

(Ballard et al., 2005) and like SIB-1893 ((E)-2-methyl-6-styryl-pyridine) inhibited

NMDA receptor activity in electrophysiological and pharmacological studies (O'Leary

et al., 2000; Movsesyan et al., 2001) and had antagonistic effects on primate

cocaine drug discrimination and self-administration (Lee at al., 2005). The more

recent mGluR5 antagonist MTEP (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine) was

more selective towards NMDA and other receptors and more potent in rat anxiety

models compared to MPEP (Busse et al., 2004; Cosford et al., 2003).

Psychoactive and hallucinatory effects of fenobam in humans (Friedmann et al.,

1980; Pecknold et al, 1982), MPEP antagonism of NMDA receptor mediated effects

(O'Leary et al., 2000; Movsesyan et al., 2001), and on cocaine self-administration

and drug discrimination (Lee et al., 2005), warrants further characterization of

behavioral effects of mGluR5 antagonists.

In contrast to hyperactivity and ataxia often observed with NMDA antagonists such

as PCP (Swedberg et al., 1995), mGluR5 antagonists in our discovery program

reduced spontaneous activity, caused flat body posture in rodents at higher doses in

the Irwin screen (Irwin, 1968) or on spontaneous locomotor activity, but caused no

other systematic behavioral effects or interacted with pentylenetetrazole convulsion

thresholds (unpublished data).

In development of CNS active drugs, regulatory and safety aspects include

investigation of potential for abuse liability, and applying a proactive abuse liability

assessment strategy (Swedberg, 2013), the present studies were designed to assess


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NMDA antagonist like discriminative effects of mGluR5 receptor antagonists in rat

PCP drug discrimination, and to investigate the feasibility of establishing MTEP drug

discrimination and assessing the selectivity of MTEP discrimination using other

mGluR5 antagonists, NMDA antagonists and other drugs with psychoactive or

hallucinogenic properties in humans. Cannabinoid (CB1) receptor agonists like δ-9-

THC are psychoactive in humans (D’Souza et al., 2004) and animals (Wiley, 1999).

LSD is a well known hallucinogenic (Passie et al., 2008) with discriminative effects in

animals (Appel et al., 2004). Arecoline, a cholinergic agonist and active ingredient in

the betel nut is a CNS stimulant in humans (Bhat et al., 2010) and psychoactive in

animals (Meltzer and Rosecrans, 1981). Yohimbine, an α2 adrenergic receptor

antagonist can cause CNS excitation and dizziness and reinstatement of drug self-

administration (Fitzgerald, 2013). Pentylenetetrazole is a nonspecific CNS stimulant

(Bloom, 1990) and chlordiazepoxide a sedative anxiolytic (Rall, 1990), both

psychostimulant and sedative / anxiolytic in humans and animals, respectively.

Additionally, MTEP was compared to MPEP and PCP in a rat intravenous self

administration procedure to evaluate the reinforcing potential of mGluR5

antagonism.


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Materials and Methods

All animal experiments were performed in accordance with the guidelines of The

Swedish National Board for Laboratory Animals under a protocol approved by the

Ethical Committee of Southern Stockholm, Sweden. Studies were carried out in

accordance with the Declaration of Helsinki and with the Guide for the Care and Use

of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of

Health.

Drug Discrimination Experiments

Subjects. Forty male Wistar rats (Scanbur BK AB, Sollentuna, Sweden and

Moellegaard, Ry, Denmark) weighing 240 to 250 g at the beginning of the

experiments were housed in pairs in a colony room with water accessible at all times

and lights on between 0600 and 1800 hr. By restricting access to food, animals were

kept at approximately 80% of free feeding weight.

Apparatus. Sixteen operant chambers equipped with two response levers, cue lights,

a house light and a food magazine were used (Med Associates Inc., St Albans, VT).

Food pellets used were 45-mg Dustless Precision Pellets (Bio-Serv, Frenchtown,

NJ). Experiments were run and data collected by a PC with DRDIWin PC Software

(Ellegaard Systems A/S, Faaborg, Denmark) and LOIS DRDI software (R&D IS,

AstraZeneca R&D Södertälje).

Discrimination training. Training and testing procedures were similar to those

described earlier (Swedberg et at., 1995). Briefly, an autoshaping procedure was

used to train rats to approach the response levers and learn that food pellets were

available. Prior to any drug administration, rats were trained to press either response

lever (left or right) once (a FR1 schedule), in order to produce a food pellet. The

production of one food pellet constitutes the completion of one trial. After learning


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that pressing any of the two levers would produce food, a drug lever and a no drug

lever was assigned to each rat in a balanced fashion. From this point and on, each

animal received either an injection of the training drug (Drug) or no injection (No

drug), prior to training sessions, and were required to press the lever appropriate to

the pretreatment received ("correct lever") in order to produce a food pellet. Animals

were run on a single alternation schedule with an increase in the fixed ratio (FR)

response requirement every other day until an FR10 was reached. Presses on the

incorrect lever had no programmed consequences other than to reset the FR value

on the correct lever, thus requiring the rats to emit 10 correct responses

consecutively in order to obtain a food pellet. At FR10 a double-alternation schedule

was introduced so that pairs of two consecutive drug (D) and no drug (N) sessions

alternated (D, D, N, N, D, D, N, N etc). A session lasted until 50 food pellets had

been earned (equalling 50 trials) or until 15 min had elapsed, whichever occurred

first. Rats were then trained to reach a criterion of 90% correct responding with no

more than 9 responses on the incorrect lever prior to the first reinforcement in each

session, for 8 consecutive sessions. The two sessions following immediately after

the 8th criterion session were acquisition test sessions in which the training

conditions (D and N) were tested and 10 consecutive responses on either lever

would produce a food pellet (i.e. both levers were "correct"), and the same

performance criteria were applied.

During testing, animals were run according to a single-alternation schedule (D, N, D,

N etc.) and tests were interspersed between the training sessions (D, N). Formally:

D, T, N, D, T, N, T, D, N, T etc. Test sessions were identical with training sessions,

except that during tests both levers were correct and ten consecutive presses on any

of the levers would produce a food pellet. Test sessions were typically run on

Tuesdays and Fridays, provided that the animals performed according to the criterion

on the training days. If training day performance fell below criteria for any rat on a


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single training day, the upcoming test was postponed for that rat and it was tested

again only after completing two consecutive training sessions during which criteria

were met. During testing the appropriate vehicle of each drug was tested as the No

Drug condition.

Several groups (n=8/group) of male Wistar rats were trained to discriminate 2.8

mg/kg of PCP administered intraperitoneally 30 minutes prior to training, or 2 mg/kg

of MTEP administered intraperitoneally 30 minutes prior to training, from no drug.

Data analysis

Drug discrimination results are expressed as the mean (±SEM) of the individual

percentages of drug responding during drug and no drug sessions, respectively, in

rats completing at least 10 trials. Rates of responding are expressed as the mean

(±SEM) number of responses per second in all rats tested. ED50 values for the

discriminative effects were determined as the dose producing 50% training drug

appropriate responding. Response rate suppressing effects are shown as maximal

suppression as a percentage of vehicle control rates. ED50 values and 95%

confidence limits were calculated by use of analysis of variance and linear regression

techniques (GraphPad Prism v.5, GraphPad Software, Inc., La Jolla, CA, USA;

Snedecor and Cochran, 1967). Sigma Plot for Windows version 12.5 (Systat

Software Inc, Chicago, IL, USA) was used for graphics.

Drugs and doses producing less than 20% training drug appropriate responding are

considered not to produce discriminative effects similar to those of the training drug.

Drugs and doses producing between 20 and 80% training drug appropriate

responding are considered to produce discriminative effects partly similar to those of

the training drug. Drugs and doses causing 80% or more training drug appropriate

responding are considered to fully share the discriminative effects of the training

drug, and drugs producing full training drug like discriminative effects and having no


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significant effects (<50% reduction relative to vehicle alone) on response rates are

considered to produce discriminative effects identical to those of the training drug.

Drug Self-Administration Experiments

Animals

Male Wistar rats (n=24, 8 per test drug; Taconic Europe A/S, Ry, Denmark) were

housed in a temperature-controlled environment on a 12-h light/dark cycle (lights off

at 1800 hours) with ad libitum access to food and water. The rats were allowed to

acclimate in their new environment during at least one week before the start of the

experiment. The studies were carried out in accordance with the Declaration of

Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted

and promulgated by the U.S. National Institutes of Health.

Surgery

Intravenous catheters (CamCaths, Ely, UK) were implanted into the right jugular vein

under isoflurane (Forene Abbott, Solna, Sweden) anesthesia. Rats were

administered post-operative analgesia (Buprenorphine, Temgesic, Schering-Plough,

Brussels, Belgium; 0.06 mg/kg sc) and antibiotic (Amoxicillin, Bimoxyl vet., Ceva

Animal Health, Dublin, Ireland; 0.75 mg/kg sc). The catheters were flushed with a

heparin solution (50 U/ml; Heparin LEO, LEO Pharmaceuticals, Copenhagen,

Denmark) before and after every session and a heparinized glycerol lock-solution

(500 U/ml Heparin LEO, 50:50 heparin:glycerol) was used over week-ends. Catheter

patency was tested before the start of the study with an infusion of the short-acting

anesthetic propofol (Rapinovet vet. 10 mg/mL, Schering-Plough Animal Health,

Ballerup, Denmark). The patency test was also performed during the course of the

study in the event there were signs of non- satisfactory catheter function. Failure to

respond to propofol resulted in recatheterization in the left jugular vein (n=4).


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Apparatus

Eight operant chambers (Med Associates) each equipped with two retractable

response levers, two cue lights, a house light and an infusion pump (PHM-100,

3.33rpm; Med Associates) were used. Each chamber was enclosed in a sound

attenuating box, and a ventilating fan that operated throughout the sessions served

as white noise. A 10 mL plastic syringe placed in the pump was connected to the

implanted catheter through CoEx tubing (Harvard Apparatus, Kent, UK) and

protected by a flexible metal leash (CamCaths). Experiments were run and data

collected by SARAWin PC Software (Ellegaard Systems A/S).

Self-administration training

Rats were food trained on a fixed ratio (FR) 1 schedule of reinforcement. Following

catheter implantation, intravenous self-administration was initiated with cocaine (0.5

mg/kg/inf), available on an FR5 schedule during daily 3-hour sessions. Once stable

self-administration behavior was achieved (>10 delivered infusions and <15%

variation in the number of infusions per rat during three consecutive sessions),

vehicle was substituted for cocaine to cause extinction of responding (≤10 delivered

infusions for three consecutive days). Dose response curves were generated for PCP

(0.03 -1.0), MPEP (0.1-3.0) and MTEP (0.01-3.0; all doses in mg/kg/infusion) in

separate groups of animals (n=8). Each dose was given until stable self-

administration behavior or extinction was reached and was preceded by a cocaine

session to re-establish responding if the animal failed to respond on the previous

dose.

Data analysis

Self-administration data from FR5 sessions are presented as the mean (±SEM) of

the number of delivered infusions/3 hours and of intake (mg/kg)/3 hours, respectively.


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Statistical analysis on group values was performed using a one-way ANOVA followed

by Dunn’s multiple comparisons test (GraphPAD Prism, GraphPAD Software Inc.,

CA, USA) when appropriate.

Drugs

MTEP (3-((2-methyl-1,3-thiazol-4-yl)ethynyl)-pyridine) was dissolved in 20 %

hydroxypropyl-β-cyclodextrin (HPβCD; AstraZeneca R&D Mölndal, Sweden) and 80

% water, MPEP (2-methyl-6-(phenylethynyl)-pyridine; AstraZeneca R&D Mölndal,

Sweden) was dissolved in 5 % HPβCD and 95% water, and fenobam [3-((2-methyl-

1,3-thiazol-4-yl)ethynyl)-pyridine; AstraZeneca R&D Mölndal, Sweden] was dissolved

in 40% HPβCD and 60% sterile water. (-)-δ-9-THC (100 mg/ml in ethanol; Lipomed,

Arlesheim, Switzerland) was pipetted into vials and exposed to a slow flow of

nitrogen gas to evaporate the ethanol. A mixture of PEG and Tween 80 was added to

the THC such that in the planned final solution the concentration of PEG and Tween

80 reached 3 to 5 %. (+)-MK-801 hydrogen maleate ((Research Biochemicals, Inc.,

(RBI), Natick, MA, USA(), phencyclidine (PCP; Lipomed), arecoline HBr (Sigma-

Aldrich, St- Louis, Mo, USA), pentylenetetrazol (PTZ; Sigma-Aldrich),

chlordiazepoxide HCl (Sigma-Aldrich), ketamine HCl (Sigma-Aldrich) and memantine

HCl (RBI) were dissolved in saline. Yohimbine HCl (Sigma-Aldrich) was dissolved in

sterile water. Lysergic acid diethylamide (LSD; Lipomed) was dissolved in 5% acetic

acid and 5% mannitol, and pH was adjusted to pH~5 by adding NaOH). MTEP, was

administered in volumes of 10 ml/kg. MPEP was administered at 4ml/kg (PCP study)

or 10 ml/kg (MTEP study, fenobam was administered at 5 ml/kg (PCP study) or 10

ml/kg (MTEP study), and all other drugs were given at 2 ml/kg. Doses given refer to

the weight of the base. The following routes of administration and pretreatment times

were used: PCP (i.p., 15 min), (+)-MK-801 (i.p., 15 min), ketamine (i.p., 15 min),

fenobam (p.o., 30 min), MPEP (p.o., 15 min), memantine (i.p., 15 min), MTEP (p.o.,

15 min, PCP study), MTEP (i.p., 30 min, MTEP study), LSD (s.c., 30 min and i.p., 15


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min),chlordiazepoxide (i.p., 30 min), yohimbine (s.c., 30 min), arecoline (s.c., 30 min),

PTZ (i.p., 30 min) and δ-9-THC (i.p., 20 min). Drug discrimination and self-

administration studies are key pharmacological tools to determine psychoactive and

reinforcing effects of drugs as part of abuse liability assessment, requiring exposures

including and exceeding estimated therapeutic levels in order to cover non-intended

use, typically self-medication beyond recommended doses (EMA 2006; FDA 2010;

ICH 2009).


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Results

Acquisition of drug discrimination. Rats were successfully trained to discriminate PCP

at 2.8 mg/kg i.p., 15 min or, MTEP at 2 mg/kg, i.p., 30 min, from no drug. The number

of sessions required to reach the criterion for testing (see Methods), including

shaping sessions (FR1 to FR7) and training sessions (FR10) for two representative

groups (n=8/group) of rats were 43.38 (range: 29 to 59) for PCP, and 55.63 (range:

37 to 71) for MTEP (table 1). Further scrutiny of the results show that the difference

between the two groups lies in the FR10 phase in which the MTEP group requires 12

more sessions to reach the testing criteria (table 1).

PCP caused a dose dependent increase in PCP appropriate responding with an ED50

of 1.55 mg/kg in rats trained to discriminate 2.8 mg/kg of PCP, i.p., 15 min, from no

drug (fig. 1, A/C , table 2). Rates of responding showed a small increase with dose

(fig. 1, B/D, table 2). (+)-MK-801caused a full and dose dependent increase in PCP

appropriate responding with an ED50 of 0.18 mg/kg, and a decrease at the highest

dose (fig.1, A, table 2), at which dose rates of responding had decreased to 45% of

vehicle alone rates (fig. 1, B, table 2). Ketamine caused a full and dose dependent

increase in PCP-appropriate responding with an ED50 of 10.1 mg/kg (fig. 1, A, table

2) and a reduction in rates of responding to 68% of vehicle alone, at the highest

dose (fig. 1, B, table 2).

Memantine caused a partial and dose dependent increase in PCP appropriate

responding with a maximal effect of 36.37% at 12.08 mg/kg and at the highest dose,

21.58 mg/kg, only one rat produced more than 9 food pellets and 88.5 % of the

responses were emitted on the PCP-appropriate lever (fig. 1, C, table 2). Rates of

responding after memantine decreased dose dependently causing a maximal

decrease to 8% of vehicle alone (fig. 1, D, table 2).

Fenobam caused partial PCP-appropriate responding (31.52%; fig. 1, C, table 2) at a

dose that reduced rates of responding to 71% of vehicle alone rates (fig. 1, D, table


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2), whereas at the next higher dose, 2.69 mg/kg, PCP appropriate responding was

reduced to 0.45% (fig. 1, C, table 2) and response rates were reduced to 46% of

vehicle alone rates (fig. 1, D, table 2).

MPEP caused increased PCP responding with a maximal effect of 33.1% at a dose

of 22.97 mg/kg (fig. 1, C, table 2) reducing rates of responding to 51% of vehicle

alone rates (fig. 1, D, table 2). MTEP caused no PCP-appropriate responding (<1%

at all doses; fig. 1, C, table 2) up to a dose reducing rates of responding to 43% of

vehicle alone rates (fig. 1, D, table 2).

MTEP caused a dose dependent increase in MTEP appropriate responding with an

ED50 of 0.42 mg/kg and a maximal effect of 99.01% at 2.0 mg/kg (fig. 2, A, table 3),

while rates of responding were not affected (fig. 2, B, table 3).

MPEP caused a dose dependent increase in MTEP-appropriate responding with an

ED50 of 0.62 mg/kg and a maximal effect of 99.97% at 6.89 mg/kg (fig. 2, A, table 3),

and a maximal response rate decrease to 91% of vehicle alone (fig. 2, B, table 3).

Fenobam caused a dose dependent increase in MTEP-appropriate responding with

an ED50 of 0.96 mg/kg and a maximal effect of 100% at 8.06 mg/kg (fig. 2, A, table

3), and a maximal response rate decrease to 87% of vehicle alone (fig. 2, B, table

3).

(+)-MK-801 caused maximal MTEP appropriate responding of 67.3% at 0.19 mg/kg

(fig. 2, A, table 3), with an ED50 of 0.15 mg/kg, and a maximal response rate

decrease to 74% of vehicle alone rates (fig. 2, B, table 3). (+)-MK-801 at 0.1 mg/kg

caused 25.58% MTEP appropriate responding (fig. 2, A) and rates of responding

were not affected (fig. 2, B).

Memantine caused a dose dependent partial increase in MTEP appropriate

responding with an ED50 of 11.42 mg/kg and a maximal effect of 54.25% at 12.95

mg/kg (fig. 2, A, table 3), and a maximal response rate decrease to 47% of vehicle


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alone (fig.2, B, table 3). At 6.47 mg/kg of memantine, MTEP responding was 33.3 %

(fig. 2, A) and rates of responding were not affected (fig. 2, B).

PCP caused maximal MTEP appropriate responding of 27.55% at 2.8 mg/kg (fig. 2,

C, table 3) and rates of responding at this dose was 90% of vehicle alone rates (fig.

2, D, table 3).

LSD after s.c. administration caused maximal MTEP appropriate responding of

47.3% at 0.3 mg/kg (fig. 2, C, table 3) and rates of responding at this dose were 33%

of vehicle alone rates (fig. 2, D, table 3), whereas LSD at 0.1 mg/kg caused 6.98%

MTEP responding (fig. 2, C) and rates of responding were not affected (fig. 2, D).

LSD after i.p. administration caused maximal MTEP appropriate responding of

35.44% at 0.3 mg/kg (fig. 2, C, table 3) and rates of responding at this dose were

46% of vehicle alone rates (fig. 2, D, table 3), whereas LSD at 0.1 mg/kg after i.p.

administration, caused 24.59% MTEP responding (fig. 2, C) and rates of responding

were not affected (fig. 2, D).

Chlordiazepoxide caused a dose dependent increase in MTEP appropriate

responding with a maximal effect of 40.04% at the highest dose, 18.8 mg/kg (fig. 2,

C, table 3), and rates of responding at this dose were reduced to 49% of vehicle

alone rates (fig. 2, D, table 3).

δ-9-THC caused maximal MTEP appropriate responding of 16.67% at 0.09 mg/kg

(fig. 2, E, table 3) and rates of responding were reduced to 38% of vehicle alone

rates at 0.94 mg/kg (fig. 2, F, table 3). Yohimbine caused MTEP appropriate

responding of <9% at any dose (fig. 2, E, table 3) and a maximal reduction in rates of

responding to 91% of vehicle alone occurred at 3 mg/kg (fig. 2, F, table 3). Arecoline

at 5.6 mg/kg caused maximal MTEP appropriate responding of 19.74% (fig. 2, E,

table 3) and a response rate reduction to 41% of vehicle alone (fig. 2, F, table 3), and

at 10 mg/kg of arecoline none of the rats was able to respond.


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PTZ caused MTEP appropriate responding of <1% at any dose (fig. 2, E, table 3),

and a maximal reduction in rates of responding to 71% of vehicle alone rates

occurred at 17.5 mg/kg (fig. 2, F, table 3).

When cocaine 0.5 mg/kg/infusion was available, rats self-administered a total mean

(±SEM) of 46.6(7.8), 35.9 (1.6) and 34.8 (1.1) infusions in the PCP, MPEP and

MTEP groups, respectively (fig. 3, A, C, E), whereas the same groups self-

administered saline at a mean (±SEM) of 5.2 (0.5), 4.4 (0.6) and 5.8 (0.9) infusions

respectively. PCP (0.03 to 1.0 mg/kg/infusion) maintained self-administration and the

number of infusions over the 3-h session generated yielded an inverted U shaped

dose response curve with a maximal mean (±SEM) intake of 31.0(6.5) infusions at

0.1 mg/kg/infusion (statistically significant difference from vehicle, p=0.01; fig. 3, A).

The total amount of PCP self-administered increased with dose (fig 3, B) reaching a

maximal mean (±SEM) total intake of 10.6 (2.7) mg/kg over the 3-h session . MPEP

(0.1 to 3 mg/kg/infusion; fig. 3, C) and MTEP (0.01 to 3 mg/kg/infusion; fig. 3,E) did

not maintain self-administration as the infusion rates were less than 7.5 and 6.5

respectively over the 3-h session. The MPEP and MTEP groups reached a maximal

mean (±SEM) total intakes of 13.9 (1.6) and 6.0 (1.7) mg/kg respectively over the 3-h

session at the highest doses (fig. 3, D, F).


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Discussion

In-house observations suggest contrasting in-vivo effects of NMDA- and mGluR5-

antagonists (unpubl). Whereas uncompetitive NMDA antagonists typically cause

dose dependent symptoms such as hyperlocomotion and ataxia, mGluR5

antagonists at higher doses typically cause sedative effects including reduced

locomotor activity and flat body posture, consistent with other observations

(Pietraszek et al., 2005). Other data show mGluR5 antagonist-enhanced NMDA

antagonist effects on prepulse inhibition (Pietraszek et al., 2005). Clinical findings of

hallucinations caused by fenobam (Friedmann et al., 1980; Pecknold et al., 1982)

and in vitro electrophysiology data showing modulation of functional effects of NMDA

in rat neuronal cells by MPEP (Movsesyan et al., 2001) suggested the need to

characterize the psychoactive effects of mGluR5 antagonists.

Since PCP causes psychotomimetic effects in man and is readily discriminated by

animals and man, PCP drug discrimination procedures are used to predict

psychotomimetic effects of novel glutamate antagonists (e.g. Koek et al., 1990;

Swedberg et al., 1995). Koek (1999) concluded that NMDA antagonists producing

intermediate or full PCP-like discriminative effects in animals exert PCP-like effects in

humans, while NMDA antagonists lacking PCP-like discriminative effects in animals

appear not to produce PCP-like effects in man. Our data on NMDA antagonists in

PCP trained rats are consistent with those and other findings, as is the lack of PCP-

like discriminative effects of the AMPA antagonist NBQX (Swedberg et al., 1995).

PCP in the present study yielded an ED50 of 1.55 mg/kg, and a response rate

increase to 127% of vehicle alone rates at the lowest dose causing 90% or greater

PCP appropriate responding (2.8 mg/kg), in agreement with Swedberg et al. (1995)

reporting a PCP ED50 of 1.75 mg/kg and a response rate increase to 118% of vehicle

alone rates at the lowest dose causing 90% or greater PCP appropriate responding.

Additionally, in the present study, (+)-MK-801 yielded an ED50 of 0.18 mg/kg and


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maximal PCP appropriate responding of 85% at 0.25 mg/kg with a response rate of

97% of vehicle alone, as compared to an ED50 of 0.15 mg/kg and a maximal PCP

appropriate responding of 99% at 0.2 mg/kg with a response rate of 64% of vehicle

alone (Swedberg et al., 1995). These comparisons demonstrate the robustness and

reproducibility of the assay with minimal methodological differences: Swedberg et al.

(1995) used a PCP training dose of 3 mg/kg while currently it was 2.8 mg/kg.

Ketamine, like PCP and (+)-MK-801, a drug with known NMDA antagonist action

(Ellison, 1995; Koek et al. 1990; Swedberg et al., 1995) caused a dose dependent

increase and full PCP-appropriate responding at a dose causing no significant

response rate reduction compared to vehicle alone levels, consistent with Swedberg

et al. (1995). Memantine, an uncompetitive and low to moderate affinity NMDA

antagonist caused severe disruption in responding such that only one of eight rats

responded at the highest dose (21.58 mg/kg), consistent with previous findings in

rats trained to discriminate (+)-MK-801 from no drug (Grant et al., 1997) and in rats

trained to discriminate PCP from no drug (Nicholson et al., 1998), whereas in

monkeys memantine caused full PCP-appropriate responding without severe

response rate suppression (Nicholson et al, 1998). At the next lower dose of

memantine (12.08 mg/kg) in the present study seven of eight rats responded and the

mean PCP-appropriate responding was 36%.

The mGluR5 antagonists fenobam and MPEP caused non-dose dependent partial

PCP-appropriate responding with maximal effects below 34% up to doses causing

significant response rate reduction, whereas MTEP caused no PCP-appropriate

responding up to a dose causing significant response rate reduction. These findings

suggest that the partial PCP-like effects of fenobam and MPEP could be attributable

to their lower selectivity with regards to NMDA antagonist activity (O'Leary et al.,

2000; Movsesyan et al., 2001), and the lack of PCP-like effects of MTEP due to its

increased selectivity (Cosford et al., 2003).


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O'Leary et al. (2000) reported MPEP concentration dependent decrease of NMDA-

induced LDH release, observing statistically significant effects of MPEP at 20 µM

(~70% reduction) and at 200 µM (~90% reduction). Extrapolating from available

observations on dose to brain exposure relations, 5 mg/kg and 0.15 µM (Nagel et al

2007) and 3 mg/kg and 0.83 µM (Cosford et al 2003), brain exposure levels at the

high dose of MPEP in this study (22.97 mg/kg) would be predicted to 0.7 to 6.4 µM,

30- to 3- fold below the active NMDA antagonist concentration (20µM) reported by

O'Leary et al (2000). In vitro profiling (Cosford et al., 2003) shows MPEP (18µM)

bound more potently than MTEP (>300 µM) to NR2B receptors, which have been

reported to mediate PCP-like discriminative effects in rats (Nicholson et al., 2007).

These findings would not be inconsistent with NMDA and/or NR2B antagonism

mediating the partial PCP-like discriminative effects of MPEP in this study. However,

it should be emphasized that the MPEP dose (22.97 mg/kg) causing partial (33.12%)

PCP-like effects is a third of the dose (6.98 mg/kg) causing full (99.97%) MTEP-like

effects, suggesting that psychoactive effects in humans would reflect mGluR5

antagonist mechanisms before and if any NMDA antagonist effects develop.

The co occurrence of partial or no PCP-appropriate responding with severe

suppression of response rates, clearly demonstrate that drug formulation or

pharmacokinetic factors such as absorption, distribution, metabolism or excretion are

not attributable for the lack of consistent PCP-like discriminative effects of mGluR5

antagonists.

Rats readily learned to discriminate MTEP from no drug within 55.63 sessions

compared to 44.38 sessions for PCP with overlapping ranges showing a comparable

speed of acquisition.

Fenobam, MPEP and METP had similar potencies and efficacies both in terms of

MTEP-appropriate responding and effects on response rates, fenobam being

somewhat less potent than MPEP and MTEP, consistent with [3H]MPEP and


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[3H]fenobam binding data (Porter et al. 2005). MPEP, MTEP and fenobam inhibited

[3H]MPEP binding to rat mGluR5 receptor transfected HEK-293 cell membranes with

mean Ki values (nM) of 3.1, 5.4 and 50.8, and inhibited [3H]fenobam binding in the

same assay with mean Ki values (nM) of 8.7, 17.8 and 56.3 (Porter et al., 2005). The

corresponding in vitro and in vivo potencies and efficacies strengthen the conclusion

that the MTEP-like discriminative effects of these compounds are attributable to

mGluR5 receptor antagonism.

PCP caused approximately 27% METP-appropriate responding at the highest dose,

2.8 mg/kg, but not at lower doses, while (+)-MK-801 caused a partial, dose related

increase with a maximum of 67% at the highest dose tested (0.19 mg/kg) and

reduced response rates to 74% of vehicle alone. Similarly, memantine caused a

partial, dose related increase with a maximum of 54% MTEP-appropriate responding

at a dose (12.95 mg/kg) causing response rate reduction to 47% of vehicle alone

rates. These findings suggest that (+)-MK-801 and memantine and to some degree

PCP in addition to their NMDA antagonist properties may to a lesser degree share

some similar downstream functional effects consistent with notions that subtle

differences in NMDA antagonist mechanisms may strongly impact neuronal effects

(Johnson and Kotermanski, 2006).

Chlordiazepoxide caused a partial, dose related increase in MTEP-appropriate

responding peaking at 40% at a dose (56 mg/kg) lowering response rates to 49% of

vehicle rates, and, although inconclusive, not inconsistent with observations of

anxiolytic activity of MTEP.

LSD caused non dose related partial MTEP-like discriminative effects peaking at 35

to 47% depending on route of administration, but at a dose (0.3 mg/kg) that reduced

response rates to 33 and 46 % of vehicle alone. Speculatively, the LSD findings may

relate to the prominent and robust discriminative effects caused by MTEP and

suggest some commonalities in the psychoactive properties, but further studies

based on these findings need to be undertaken for any conclusions to be made.


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Yohimbine, arecoline, δ-9-THC and PTZ caused less than 20% MTEP appropriate

responding up to doses causing substantial lowering of response rates, further

demonstrating the selectivity of the MTEP drug discrimination assay.

It is concluded, that mGluR5 antagonist discriminative effects are selective and

clearly dissimilar to other classes of psychoactive drugs even though in some cases

weak partial non dose dependent effects occurred, and it is reemphasized that all

non-mGluR5 antagonists causing MTEP-appropriate responding, did so at doses

substantially suppressing response rates.

PCP but not MPEP or MTEP maintained self-administration suggesting different

reinforcing properties of mGluR5- and NMDA- antagonists. Results suggest that

while mGluR5 antagonists produce distinct psychoactive effects, mGluR5

antagonism may not cause abuse as reflected in lack of self-administration.

However, it is suggested per a proactive strategy for abuse liability assessment

(Swedberg, 2013) that mGluR5 antagonists also be tested in primates (Weerts et al.,

2007) to further explore and characterize these compounds.

In conclusion, the present data show that mGluR5 receptor antagonists and NMDA

receptor antagonists produce distinctly different discriminative effects demonstrating

that mGluR5-antagonism may produce psychoactive and psychotomimetic effects

different from those caused by NMDA antagonism and other known mechanisms

mediating psychotomimetic effects. It is concluded that the reported psychostimulant

and hallucinogenic effects of mGluR5 antagonists in humans are most likely not

caused by NMDA antagonism, but by a specific antagonism of mGluR5 receptors.


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Acknowledgements

The technical assistance of Maria Ståhlberg, Charlotte Velasquez and Pernilla

Hammar is greatly appreciated.

The support of Dr Samantha Budd in the publication of these data is greatly

appreciated.


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Authorship contributions

Participated in research design: Swedberg MDB, Ellgren M and Raboisson R.

Conducted experiments: Swedberg MDB and Ellgren M

Performed data analysis: Swedberg MDB and Ellgren M

Wrote or contributed to the writing of the manuscript: Swedberg MDB and Ellgren M


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Footnotes

All authors were employees of AstraZeneca at the time of the studies.

Corresponding author: Michael D B Swedberg, Ph.D., Independent Consultant, Drug

Discovery Pharmacology, Sockengatan 6, S-61933 Trosa, Sweden.

Portions of these data were presented to the College of Problems of Drug

Dependence (CPDD), Scottsdale, AZ, June 12-17, 2010, at the 10th Annual Meeting

of the Safety Pharmacology Society, Boston, MA, Sep. 15-18, 2010 (Ellgren et al.,

2011; Swedberg et al., 2011), and at the 11th Annual Meeting of the Safety

Pharmacology Society, Innsbruck, Austria, Sep. 19-22, 2011 (Swedberg et al., 2012).


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Figure Legends

Figure 1. Percent PCP-appropriate responding (mean ±SEM, panels A and C) and

responses per second (mean ±SEM, panels B and D) in male Wistar rats trained to

discriminate PCP (2.8 mg/kg, 15 min after i.p. administration) from no drug.

A, B: PCP (i.p., 15 min, circles), , (+)-MK-801 (i.p., 15min, squares) and ketamine

(i.p., 15min, triangles). C, D: PCP (i.p., 15 min, filled circles), fenobam (p.o., 30 min,

diamonds), MPEP (p.o., 15 min, open circles), MTEP (p.o., 15 min, triangles) and

memantine (i.p., 15 min, squares). Note: at the highest dose of memantine only one

of eight rats produced nine or more reinforcements.

Figure 2. Percent MTEP-appropriate responding (mean ±SEM, A, C, E) and

responses per second (mean ±SEM, B, D, F) in male Wistar rats trained to

discriminate MTEP (2.0 mg/kg, 30 min after i.p. administration) from no drug. A, B:

MTEP (i.p., 30 min, circles), MPEP (p.o., 15 min, triangles up), fenobam (p.o., 30

min, triangles down), (+)-MK-801 (i.p., 15 min, squares) and memantine (i.p., 15 min,

black diamonds), C,D: MTEP (i.p., 30 min, circles), PCP (i.p., 15 min, squares), LSD

(s.c., 30 min, triangles up), LSD (i.p., 15 min, triangles down), chlordiazepoxide (i.p.,

30 min, circles). E,F: MTEP (i.p., 30 min, circles), yohimbine (s.c., 30 min, squares),

arecoline (s.c., 30 min, open circles), PTZ (i.p., 30 min, hexagons) and δ-9-THC (i.p.,

20 min, diamonds).

Figure 3. Infusions delivered on a FR5 schedule of reinforcement (A,C,E) and total

drug intake over the 3-h session (B,D,E). A,B: PCP. C,D: MPEP, E,F: MTEP, in rats

trained to self-administer cocaine (0.5 mg/kg/infusion).


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Tables

Table 1. Sessions required to reach criteria in the different phases of the acquisition

of PCP (2.8 mg/kg) and MTEP (2.0 mg/kg) discrimination

Compound Total (FR1 to FR10) FR10 FR1 to 7

Mean Range Mean Range Mean Range

PCP 43.38 29 to 59 32.50 17 to 49 10.88 10 to 14

MTEP 55.63 37 to 71 44.75 27 to 61 10.88 10 to 12


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Table 2. PCP discrimination, maximal % PCP responding ED50 and confidence

levels, and maximal effects on response rates

Compound Max % PCP responding Max response rate effects

At dose (mg/kg)

mean SEM ED50 (95% C.L.) Rats responding (rats tested)

At dose (mg/kg)

mean SEM % of veh

Rats responding (rats tested)

PCP 2.80 99.51 0.21 1.55 (0.75 to 3.17) 8(8) 1.57 2.27 0.24 154 8(8)

(+)-MK-801 0.25 84.97 15.30 0.18 (0.06 to 0.56) 7(7) 0.34 0.65 0.24 45 7(8)

Ketamine 23.80 88.61 11.39 10.10 (7.62 to 13.39) 8(8) 23.80 1.16 0.22 68 8(8)

Memantine 21.58 88.50 0.00 N/A 1(7) 21.58 0.12 0.05 8 1(7)

Memantine 12.08 36.37 18.82 N/A 7(8) 21.58 0.12 0.05 8 1(7)

fenobam 1.50 31.52 16.49 N/A 6(6) 2.69 0.95 0.10 46 6(6)

MPEP 22.97 33.12 22.95 N/A 6(6) 6.89 1.11 0.21 47 6(6)

MTEP 0.60 0.81 0.89 N/A 6(6) 20.03 0.67 0.11 43 6(6)


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JPET #211185

39

Table 3. MTEP discrimination, maximal % MTEP responding ED50 and confidence

levels, and maximal effects on response rates

Compound Max % MTEP Responding Responses/sec

At dose (mg/kg)

mean SEM ED50 (95% C.L.) Rats responding (rats tested)

At dose (mg/kg)

mean SEM % of veh

Rats responding (rats tested)

MTEP 2.00 99.01 0.69 0.42 (0.34 to 0.53) 8(8) 2.00 1.58 0.11 97 8(8)

MPEP 6.89 99.97 0.03 0.62 (0.44 to 0.87) 7(7) 2.30 1.41 0.07 91 7(7)

fenobam 8.06 100 0 0.96 (0.69 to 1.33) 6(6) 2.69 1.37 0.12 87 6(6)

(+)-MK-801 0.19 67.3 22.92 0.15 (0.12 to 0.18) 4(6) 0.19 1.05 0.28 74 4(6)

memantine 12.95 54.25 14.39 11.42 (5.70 to 22.86) 7(7) 12.95 0.8 0.19 47 7(7)

PCP 2.80 27.55 16 N/A 7(7) 2.80 1.46 0.24 90 7(7)

LSD sc 0.30 47.3 19.75 N/A 5(7) 0.30 0.55 0.16 33 5(7)

LSD ip 0.30 35.44 19.58 N/A 6(7) 0.30 0.68 0.16 46 6(7)

CDP 18.80 40.04 19.29 N/A 6(7) 18.80 0.85 0.21 49 6(7)

THC 0.09 16.67 16.43 N/A 6(6) 0.94 0.67 0.24 38 4(6)

yohimbine 1.00 0.52 0.17 N/A 8(8) 3.00 1.94 0.17 91 8(8)

arecoline 5.60 19.74 19.69 N/A 5(7) 10.00 0 0 0 0(7)

PTZ 17.50 0.06 0.04 N/A 6(6) 17.50 1.31 0.12 71 6(6)


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Figure 1

Doses (mg/kg)

VEH .3 3 30 .1 1 10

% P

CP

Re

sp

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Figure 2

Doses (mg/kg)

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Figure 3

Doses (mg/kg/inf)

COC SAL .03 .3 .01 .1 1

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/kg

)

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Doses (mg/kg/inf)

COC SAL .03 .3 3 .01 .1 1

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Documents

mGluR5 Antagonist Induced Psychoactive Properties: MTEP ...jpet.aspetjournals.org/.../28/jpet.113.211185.full.pdf · antagonists MPEP and SIB-1893 can inhibit NMDA receptor mediated