peripherally acting nmda receptor/glycineb site receptor

Neuropharmacology 48 (2005) 360–371

www.elsevier.com/locate/neuropharm

Peripherally acting NMDA receptor/glycineB site receptorantagonists inhibit morphine tolerance*

Wojciech Danysza, Ewa Kozelab, Chris G. Parsonsa, Meik Sladeka,Tanja Bauera, Piotr Popikb,)

aMerz Pharmaceuticals GmbH, Eckenheimer Landstrasse 100, 60318 Frankfurt/Main, GermanybInstitute of Pharmacology PAN, Smetna 12, 31-343 Krakow, Poland

Received 17 June 2004; received in revised form 29 October 2004; accepted 27 November 2004

Abstract

The present study focused on the role of peripheral ionotropic N-methyl-D-aspartate (NMDA) receptors in the development oftolerance to morphine-induced antinociception. An initial experiment revealed that NMDA channel blocker memantine, and

NMDA receptor/glycineB site antagonist MRZ 2/576 inhibited maximal electroshock-induced convulsions (MES) in female NMRmice with respective potency of 5.93 and 20.8 mg/kg, while other NMDA receptor/glycineB site antagonists MRZ 2/596 and MDL105,519 were ineffective, supporting lack of CNS activity of the latter two agents. This observation was also supported by blood–brain barrier experiments in vitro. In male Swiss mice, morphine (10 mg/kg) given for 6 days twice a day (b.i.d.) produced tolerance

to its antinociceptive effects in the tail-flick test. The NMDA receptor/glycineB site antagonists, MRZ 2/576 at 0.03, 0.1, 0.3 mg/kgand MRZ 2/596 at 0.1, 0.3, 3 and 10 mg/kg attenuated the development of morphine tolerance. Similarly, in male C57/Bl mice,morphine (10 mg/kg) given for 6 days b.i.d. produced tolerance to its antinociceptive effects in the tail-flick test. Like in Swiss mice,

in C57/Bl mice morphine tolerance was attenuated by both MRZ 2/576 and MRZ 2/596. Another NMDA receptor/glycineB sitereceptor antagonist, MDL 105,519 (that very weakly penetrates to the central nervous system) also inhibited morphine tolerance atthe dose of 1 but not 0.1 mg/kg. Moreover, both naloxone hydrochloride (5 and 50 mg/kg) and centrally inactive naloxone

methiodide (50 mg/kg) inhibited morphine tolerance suggesting the involvement of peripheral opioid receptors in this phenomenon.The present data suggest that blockade of NMDA receptor/glycineB sites in the periphery may attenuate tolerance to theantinociceptive effects of morphine.� 2004 Elsevier Ltd. All rights reserved.

Keywords: Antinociception; Pain; Tolerance; Glutamate; NMDA receptor/glycineB site antagonist; Blood–brain barrier

* The referees of the present paper suggested to assess brain

penetration of MRZ 2/596 using brain microdialysis. It was not

possible at the time of manuscript preparation but such opportunity

appeared after manuscript acceptance. The study revealed, that in rats

after application of MRZ 2/596 at the dose of 30 mg/kg ip maximal

concentrations in brain reach c.a. 100 nM (NZ3) concentrations

corrected for in vitro recovery. Thus, it is unlikely that dose used in the

present study (0.05–1 mg/kg) resulted in brain levels of MRZ 2/596

that would affect NMDA receptors assuming lack of differences in

blood brain barrier between rat and mice.) Corresponding author. Institute of Pharmacology, Polish Acad-

emy of Sciences, 12 Smetna Street, 31-343 Krakow, Poland. Tel.: C48

12 6623375; fax: C48 12 6374500.

E-mail address: [email protected] (P. Popik).

0028-3908/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuropharm.2004.11.005

1. Introduction

Over the last decade research has provided compel-ling evidence that glutamate receptors are cruciallyinvolved in phenomena related to opioid tolerance (seeMao, 1999; Price et al., 2000 for reviews). Antagonists ofthe ionotropic N-methyl-D-aspartate (NMDA) receptorcomplex, including memantine, the moderate affinityand highly voltage-dependent clinically used NMDAchannel blocker (Parsons et al., 1999) inhibit thedevelopment of morphine tolerance (Trujillo and Akil,

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361W. Danysz et al. / Neuropharmacology 48 (2005) 360–371

1991; Marek et al., 1991; Popik et al., 2000a) (seeBespalov and Trujillo, 2002 for the recent review).

One obstacle to the introduction of NMDA receptorantagonists into clinical practice is undesired ‘‘phency-clidine side-effects’’ profile, that is centrally mediated(Parsons et al., 1999). In turn, NMDA receptorantagonists which weakly penetrate to the brain mighthave a more favorable profile. However, such com-pounds may not be applicable to the inhibition oftolerance to morphine-induced antinociception which isbelieved to be primarily of central origin (McNally andWestbrook, 1998; McNally, 1999; Ueda and Inoue,1999). On the other hand, recent data reported byKolesnikov and colleagues demonstrated that local(topical) application of uncompetitive NMDA receptorantagonists, (C)MK-801 or ketamine, inhibited toler-ance to topically applied morphine (Kolesnikov et al.,1996; Kolesnikov and Pasternak, 1999b; Kolesnikovand Pasternak, 1999a), suggesting a peripheral compo-nent of antinociceptive morphine tolerance. Sincesystemic rather than local administration of compoundsis more favorable from the therapeutic perspective, theaim of the present study was to investigate whetherantagonism of NMDA receptors in the peripheralnervous system (PNS) would inhibit tolerance to theantinociceptive effects of systemically administeredmorphine. As pharmacological tools we used recentlydeveloped NMDA receptor antagonists acting at theNMDA receptor/glycineB site. These compounds reachrelatively low brain levels after systemic application ascompared to plasma values (MRZ 2/576: w2%(Hesselink et al., 1999b), MDL 105,519: 0.01–0.08%(Opackajuffry et al., 1998)) or seem to be lacking CNSactivity as was the case with MRZ 2/596 (see Sections3.1.3 and 4). In contrast, the free brain levels ofmemantine are over 50% of plasma concentration(Hesselink et al., 1999a).

2. Methods

2.1. In vitro

2.1.1. Receptor bindingTissue preparation was performed according to

Foster and Wong (1987) with some modifications. MaleSprague-Dawley rats (200–250 g, Janvier, Le Genest-Isle, France) were decapitated and their brains wereremoved rapidly. Tissue was then processed as describedpreviously (Parsons et al., 1997). The amount of proteinin the final membrane preparation was determinedaccording to Hartfree (1971) and adjusted to 250–500 mg/ml.

Membranes were suspended and incubated in 50 mMTris–HCl, pH 8.0 for 45 min at 4 �C with a fixed[3H]MDL-105,519 concentration of 2 nM. MDL-105,519

is a selective high affinity antagonist at the NMDAreceptor/glycineB site and has recently been introduced asa commercially available radioligand (Amersham Bio-sciences, Freiburg, Germany) (Baron et al., 1996;Baron et al., 1997). Non-specific binding was definedby the addition of unlabeled glycine at 100 mM.Incubations were terminated using a Millipore filtersystem (Millipore, Schwalbach Germany). The samples,all in duplicate, were rinsed three times with 2.5 ml ice-cold assay buffer over glass fibre filters (Schleicher andSchuell, Dassel, Germany) under a constant vacuum.Filtration was performed as rapidly as possible (max2 s). Following separation and rinse, the filters wereplaced into scintillation liquid (5 ml; Ultima Gold)and radioactivity was determined with a liquid scintil-lation counter (both Packard BioScience, Dreieich,Germany).

2.1.2. Patch clampPatch clamp recordings were made from rat hippo-

campal neurons, after 12–15 days in vitro, with polishedglass electrodes (3–5 mU) in the whole cell mode at roomtemperature (20–22 �C) with the aid of an EPC-7amplifier (HEKA, Lambrecht, Germany) – detailedmethods described in Parsons et al. (1999).

2.1.3. BBB permeability studiesAn in vitro model of the BBB has been established as

a first screen for BBB permeability.

2.1.3.1. Preparation and cultivation of BBCEC. Bovinebrain capillary endothelial cells (BBCEC) were isolatedfrom brains, purified, and cultured according to Meresseet al. (1989). Briefly, after mechanical homogenizationmicrovessels were seeded onto dishes coated with anextracellular matrix secreted by bovine corneal endo-thelial cells (Gospodarowicz et al., 1976). Pure coloniesof endothelial cells were seeded onto gelatin-coateddishes (Corning Costar, Bodenheim, Germany) in thepresence of Dulbecco’s Modified Eagle’s Medium(DMEM; Gibco Invitrogen GmbH, Karlsruhe,Germany) supplemented with 20% calf serum (Hyclone,Utah, USA), 2 mM glutamine, 50 mg/ml of gentamycin(Biochrom, Berlin, Germany), and bovine fibroblastgrowth factor (bFGF; Roche, Mannheim, Germany;1 ng/ml added every other day). BBCEC in passage 4–6were used for co-cultivation/transport studies.

2.1.3.2. Preparation and cultivation of rat corticalastrocytes. Astrocytes were prepared mechanically fromcortices of newborn rats as described by Booher andSensenbrenner (1972). The cell suspension was seededonto 12-well plates (Corning, Wiesbaden, Germany) andcultivated in Dulbecco’s Modified Eagle’s Medium(DMEM, Gibco Invitrogen GmbH, Karlsruhe,Germany) containing 10% FCS (HyClone, Utah,

362 W. Danysz et al. / Neuropharmacology 48 (2005) 360–371

USA), 2 mM glutamine and 50 mg/ml gentamycin(Biochrom, Berlin, Germany).

2.1.3.3. Co-cultivation of BBCEC with rat corticalastrocytes. For co-cultivation the BBCEC were seededonto a collagen-coated filter (Transwell Col; Corning,Wiesbaden, Germany) and placed into 12-well plates(Corning, Wiesbaden, Germany) containing differenti-ated astrocytes in BBCEC culture medium.

2.1.3.4. Transport studies. The transport studies wereperformed between 8 and 12 days of co-cultivation inKrebs Ringer buffer. The amount of substance passingthrough the endothelial cell layers after 10, 20, and30 min was quantified in samples taken from theabluminal (brain) side of the filter (nZ 3).

As a control, collagen coated filters without cells wereused.

Transport studies were performed with the followingcompounds: pyrido-phthalazine-diones – MRZ 2/501,its choline salt MRZ 2/596, MRZ 2/502 and its cholinesalt MRZ 2/576, all at a concentration of 10 mM, andcompared to other NMDA receptor/glycineB siteantagonist [3H]-MDL 105,519 (0.02 mM) (known todiffuse very slowly under physiological conditions intothe brain) and NMDA receptor channel blockermemantine (10 mM) representing a drug which showsa good penetration through the BBB. For testing theBBB integrity, the permeability of 0.1 mM [14C]-sucrose,a functional blood–brain barrier-impermeant marker,was determined every day before the actual experiments.

The permeability calculations were based on theclearance principle published by Siflinger-Birnboimet al. (1987).

2.1.3.5. BBB integrity study. Co-cultures of BBCEC andastrocytes were incubated with morphine sulphate atconcentrations of 3, 6 and 10 mg/ml for 1 h, 6 h, and 24 h(on the luminal side only) and the permeability of [14C]-sucrose across the BBB in vitro was tested afterwards inthe presence of morphine.

Drug concentrations were analyzed via HPLC incase of NMDA receptor/glycineB site antagonists (seeHesselink et al., 1999b), GC/MSD in case of memantine(Hesselink et al., 1999a), or liquid scintillation counting incase of radiolabelled substances having approximatedetection limits of 100 nM,10 nMand2 nM, respectively.

2.2. In vivo

2.2.1. Maximal electroshock (MES), tractionreflex and rotarod tests

2.2.1.1. Subjects. MES, traction reflex and rotarod testswere performed in NMR female mice (18–28 g, Janvier,Le Genest-Isle, France) housed 5 per cage. All animals

were kept with water and food ad libitum under a12-h light-dark cycle (light on: 06:00) and at a con-trolled temperature (20G 0.5 �C). Experiments wereperformed according to the animal rights commissionallowance #F 77-51 (Hessen, Germany).

The MES test was performed together with tests formyorelaxant action (traction reflex) and motor co-ordination (rotarod, Ugo Basile, Italy). For the tractionreflex test, mice were placed with their forepaws ona horizontal rod and were required to place all 4 pawson the wire within 10 s. To test ataxia (motor co-ordination) mice were placed on rotarod (5 r.p.m.) andwere required to remain on the rod for 1 min. Only micenot achieving the criteria in all three repetitions(performed with 1 min intervals) of each test wereconsidered to exhibit myorelaxation or ataxia, respec-tively. In case of reaching the criterium on the 1st or 2ndtrial, no further trials were performed. These tests werefollowed 1–2 min later by MES (100 Hz, 0.5 s shockduration, 50 mA shock intensity, 0.9 ms impulse dura-tion, Ugo Basile) applied through corneal electrodes.The presence of tonic convulsions was scored (tonicextension of hind paws with minimum angle to the bodyof 90 �). The aim was to obtain ED50s for all parametersscored (anticonvulsive activity and motor side effects)with use of the Litchfield Wilcoxon test for quantal doseresponses. Tested agents were dissolved in distilled waterand injected 15–30 min before MES.

An additional experiment was designed to exclude thepossibility that after chronic treatment, MRZ 2/596accumulates in the brain sufficiently to block centralNMDA receptors. Mice were treated for 7 days twicedaily with saline or MRZ 2/596 (10 mg/kg), 20 min afterthe last injection mice were subjected to MES and theoccurrence of tonic seizures was scored. Additionally,MRZ 2/576 was injected at a dose of 20 mg/kg asa positive control since this agent penetrates sufficientlyto the CNS.

2.2.2. Morphine tolerance

2.2.2.1. Subjects. Male Swiss mice (25–30 g, Institute ofPharmacology breeding facility, Krakow, Poland) weregroup-housed in standard laboratory cages and kept ina temperature-controlled colony room (21G 2 �C) witha 12-h light/dark cycle (light on: 07:00). Similarlyhoused and maintained male C57/Bl mice (25–30 g)were obtained from the Institute of Immunology andExperimental Therapy, Wroclaw, Poland. Commercialfood and tap water were available ad libitum. Eachexperimental group consisted of at least 7 mice pertreatment in morphine tolerance experiments and atleast 5 mice per treatment in acute morphine antinoci-ception experiment. All mice were used only once.

Morphine tolerance studies were carried out accord-ing to the National Institutes of Health Guide for Care


and Use of Laboratory Animals (revised 1996) and wereapproved by the Institute of Pharmacology PANAnimal Care and Use Bioethics Commission.

The tail-flick tests were performed as describedpreviously (Popik et al., 2000). The assessments ofantinociceptive ED50 of morphine (test #1 and test #2)were carried out without pretreatment with glutamateand opioid antagonists.

2.2.2.2. Experimental design. The first experiment wascarried out on Swiss mice to determine whether thedevelopment of tolerance to the antinociceptive effectsof morphine could be inhibited by NMDA receptor/glycineB site antagonists not penetrating (MRZ 2/596)or weakly penetrating (MRZ 2/576) the BBB.

On day 1 the first measurement of morphineantinociceptive potency was performed (test #1), fol-lowed by 6 days of b.i.d. morphine injections (10 mg/kg,s.c., 9:00 and 17:30) (Elliott et al., 1994; Popik et al.,2000b). MRZ 2/596 (0.05, 0.1, 0.3, and 1 mg/kg) andMRZ 2/576 (0.01, 0.03, 0.1, 0.3, 1, 3 and 10 mg/kg) weregiven s.c. 30 min prior to each morphine dose on days2–7. On day 8 the second measurement of theantinociceptive potency of morphine was performed(test #2). The degree of morphine tolerance was assessedby comparing the antinociceptive potency of morphine(ED50) observed in tests #1 and #2.

The subsequent experiments were carried out severalmonths later and due to technical reasons C57/Bl micewere used. In order to confirm results obtained in Swissmice, the effect of MRZ 2/596 (0.3 mg/kg), MRZ 2/576(1 mg/kg) on the development of morphine tolerancewas tested in C57/Bl mice. To further confirm theusefulness of C57/Bl mice in the present experimentalsettings, we also used the NMDA channel blockermemantine (2.5 and 7.5 mg/kg) that has been shownpreviously to inhibit morphine tolerance in Swiss mice(Popik et al., 2000a). Since the effects obtained withSwiss mice were also observed in C57/Bl mice, furtherexperiments were conducted using of C57/Bl mice. Toinvestigate the possibility that the effects of MRZ 2/596on the development of morphine tolerance were due toan inhibition of morphine’s antinociceptive action(naloxone-like effect), MRZ 2/596 was administered ina single injection of 1 mg/kg s.c. 30 min before singlemorphine 3 mg/kg s.c. administration. The antinocicep-tive effects of morphine were investigated 30, 60 and120 min later. In another control experiment, weinvestigated if the inhibitory effects of MRZ 2/596 onthe development of morphine tolerance could beattributed to an accumulation of this compound. Tothis end, groups of mice were treated for 7 days twicedaily with saline or MRZ 2/596 (1 mg/kg). Twelve hoursafter the last injection, mice were injected with 3 mg/kgs.c. of morphine and tested in the tail-flick apparatus 30,60 and 120 min.

The next experiment was carried out to find doses ofopioid antagonists to affect acute morphine antinoci-ception. Thus, naloxone hydrochloride (1, 5, 10 and50 mg/kg) and naloxone methiodide (5, 10 and 50 mg/kg) were administered s.c. 15 min before 10 mg/kg of s.c.morphine (the dose used in chronic experiments) and thetail-flick test was conducted 30 min after morphineinjection.

Further, it was determined if (a) another NMDAreceptor/glycineB site antagonist, MDL 105,519, struc-turally dissimilar from both MRZ 2/576 and MRZ2/596 and also weakly penetrating the CNS and (b)opioid receptor antagonists, naloxone hydrochlorideand its quaternary derivative, naloxone methiodideaffect the development of morphine antinociceptivetolerance. MDL 105,519 (0.1 and 1 mg/kg) was givens.c. 30 min prior to each morphine dose. Naloxonehydrochloride (1, 5 and 50 mg/kg) and naloxonemethiodide (1, 5 and 50 mg/kg) were given s.c. 15 minprior to each morphine dose during its chronicadministration. Doses of morphine and memantinewere used based on previous observations (Popik andSkolnick, 1996; Popik et al., 2000b).

2.2.2.3. Data presentation and statistics. Latencies (in s)of the tail-flick responses were converted to %MaximumPossible Effect values [%MPE (Paronis and Holtzman,1991)], according to the formula: 100[(post-injectionlatency� baseline latency)/(cut-off latency� baselinelatency)]. %MPE values were used to construct mor-phine cumulative dose–response curves by non-linearregression; these curves were used to calculate anti-nociceptive ED50 values using GraphPad Prism ver. 3.00(GraphPad Software, CA, USA) software. The ED50

values obtained for tests #1 and #2 were comparedamong groups, as were the fold shifts (determined bydividing individual test #2 ED50 values by the test #1ED50 values) with one-way ANOVAs and post hocNewman-Keul’s and LSD tests. Data are presented asmeanG S.E.M.

2.2.3. DrugsMRZ 2/501 (8-chloro-1,4-dioxo-1,2,3,4-tetrahydro-

pyridazino (4,5-b) quinoline), MRZ 2/596 (8-chloro-1,4-dioxo-1,2,3,4-tetrahydropyridazino (4,5-b) quinolinecholine salt), MRZ 2/502 (8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazino (4,5-b) quinoline-5-oxide), andMRZ 2/576 (8-chloro-4-hydroxy-1-oxo-1,2-dihydropyr-idazino (4,5-b) quinoline-5-oxide choline salt) andmemantine (1-amino-3,5-dimethyladamantane) werefrom Merz Pharmaceuticals GmbH, Frankfurt/M,Germany. Morphine sulphate was obtained from Sigma,Taufkirchen, Germany.

[3H]-morphine was purchased from NEN-ResearchProducts, Koln, Germany, [3H]-MDL 105,519 ((E )-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1H-indole-2-


carboxylic acid) and [14C]-sucrose purchased fromAmersham Life Science, Freiburg, Germany.

Morphine HCl (morphine tolerance studies) wasobtained from Polfa, Krakow, Poland and naloxonehydrochloride, naloxone methiodide, and MDL 105,519((E )-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1H-in-dole-2-carboxylic acid) from Sigma–Aldrich, USA. Allother compounds were obtained from Tocris, U.K. Formorphine tolerance experiments morphine, MDL105,519, memantine, naloxone hydrochloride and nal-oxone methiodide were dissolved in physiological saline(placebo). MRZ 2/596 and MRZ 2/576 were dissolved insterile water. All injections were administered ina volume of 10 ml/kg. All NMDA receptor/glycineBsite antagonist solutions were prepared fresh the daybefore the experiment started, aliqoted and stored at4 �C but administered at room temperature. The dose ofmorphine is expressed as the base, the doses of all othercompounds as their respective salts.

3. Results

3.1. In vitro

3.1.1. Receptor bindingIn Scatchard analysis [3H]MDL 105,519 had a Kd of

6.03G 0.14 nM and a Bmax of 4.03 pmol/mg protein.MRZ 2/576 and MRZ 2/596 displaced [3H]MDL-105,519 binding to rat cortical membranes with Kis of126 and 160 nM, respectively (Table 1, IC50 correctedaccording to the Cheng-Prussoff relationship for 2 nM[3H]MDL 105,519). MDL-105,519 displaced its ownbinding with a Ki of 11.6 nM. Non-specific bindingdetermined with glycine 100 mM was only 15–20% and

Table 1

Effects of tested agents on glycineB site as evidenced by binding and

patch clamp experiments

Substance [3H]MDL

105,519

binding Ki (n)

Patch clamp

peak IC50 (nM)

Patch clamp

plateau IC50

(nM)

MRZ 2/576 126G 24 1008G 60 540G 30

MRZ 2/596 160G 12 2280G 250 700G 35

MDL 105,519 11.6G 5.3 136G 22 106G 13

In case of binding, membranes were incubated with [3H]MDL 105,519

(2 nM) in 50 mM Tris–HCl, pH 8.0 for 45 min at 4 �C. Non-specific

binding (13.2G 0.2%) was defined by the addition of unlabeled

glycine (100 mM). The effects of each concentration of displacer were

assessed in duplicate in 3–6 individual experiments. MeansG S.E.M.

values were plotted as % specific binding against concentration.

Estimation of IC50s and curve fitting were made according to the 4

parameter logistic equation (GraFit, Erithacus Software). For

electrophysiological experiments, peak and plateau (steady state)

NMDA current responses of cultured hippocampal neurons were

normalised to control levels and shown as means (GS.E.M.).

Estimation of IC50s and curve fitting were made according to the 4

parameter logistic equation.

standard compounds displaced binding to non-specificlevels with potencies similar to the literature and Hillcoefficients close to unity.

3.1.2. Patch clampSteady-state inward current responses of cultured

hippocampal neurons to NMDA (200 mM with glycine1 mM) were antagonized by MDL-105-519 with IC50

of 106G 13 nM (Table 1) whereas MRZ 2/576 andMRZ 2/596 with IC50s of 540G 30 and 700G 35 nM,respectively, were 5–7-fold less potent (Table 1).When corrected for the affinity of glycine underthese conditions (399 nM) all three antagonists hadvery similar Kb values to their Kis assessed inthe [3H]MDL-105,519 binding experiments – Kbs:MRZ 2/576Z 154 nM, MRZ 2/596Z 199 nM, MDL105,519Z 30.3 nM.

3.1.3. BBB permeability studiesAs expected, the impermeant marker sucrose showed

a low permeability (pe) coefficient of 2.27! 10�5 cm/s(Fig. 1) in the established BBB model. The lipophylicNMDA channel blocker memantine had goodpenetration through the in vitro BBB (pe-coefficient50.32! 10�5 cm/s) (Fig. 1). In contrast, the hydrophylicNMDA receptor/glycineB site antagonist MDL 105,519diffused slowly through the endothelial monolayer (pe-coefficient 2.00! 10�5 cm/s) (Fig. 1).

MRZ 2/576 and its free base MRZ 2/502 showed pe-coefficients of 24.33! 10�5 and 25.07! 10�5 cm/s,respectively. The quinoline, MRZ 2/596 and its free baseMRZ 2/501 demonstrated pe-coefficients of 3.60! 10�5

and 3.33! 10�5 cm/s, respectively (Fig. 1). In the case ofMRZ 2/596, the accuracy of the calculated pe-coefficientwas limited by the sensitivity of the HPLC-method used.The concentrations of most analytical samples taken

Sucros

e

MDL 105

,519

MRZ 2/50

1

MRZ 2/59

6

MRZ 2/50

2

MRZ 2/57

6

Meman

tine

Perm

eabi

lity

coef

ficie

nt [*

10-5

cm

/sec

]

0

10

20

30

40

50

60

Fig. 1. In vitro BBB penetration of NMDA receptor/glycineB site

antagonists in comparison to standards – sucrose poor penetration,

memantine good penetration.


from the BBCEC monolayers were under the detectionlimit (100 nM). Therefore the concentration of thedetection limit had to be used for pe-coefficientcalculation which leads to a slightly higher pe-value asthe actual one.

3.1.4. BBB integrity studyBBCEC treated with morphine on the luminal side

(3 mg/ml for 1 h, 3 mg/ml for 6 h, 6 mg/ml for 6 h and10 mg/ml for 24 h) showed no significant increase in[14C]-sucrose permeability in comparison to untreatedendothelial cells (data not shown).

3.2. In vivo

3.2.1. Maximal electroshock (MES), tractionreflex and rotarod tests

Memantine and MRZ 2/576 inhibited MES withED50s of 5.93 and 20.8 mg/kg, respectively, while bothMRZ 2/596 andMDL 105,519 were ineffective (Table 2).

Memantine disturbed the traction reflex with anED50Z 12.5 mg/kg and ataxia was observed with anED50Z 14.2 mg/kg. MRZ 2/576 produced myorelax-ation with an ED50Z 21.1 mg/kg and ataxia with anED50Z 22.3 mg/kg. MRZ 2/596 and MDL 105, 519administered even at very high doses did not show anyactivity in these tests (Table 2).

All mice treated semi-chronically with MRZ 2/596(10 mg/kg) for 7 days showed tonic convulsions in theabsence of drug as in control animals (8/8 mice). Incontrast, in mice treated acutely with MRZ 2/576(20 mg/kg) occurrence of tonic seizures was much lower(1/8 mice).

3.2.2. Morphine toleranceThe initial experiment carried out on Swiss mice

demonstrated no differences among groups in antinoci-ceptive morphine ED50 values in test #1. The followingED50 values (mg/kg) were observed in test #1 forplaceboC placebo: 5.40G 0.68; for placeboCmor-phine: 5.17G 0.62; for MRZ 2/596 (at 0.05, 0.1, 0.3,1 mg/kgCmorphine): 5.83G 1.97, 4.76G 0.70, 5.03G1.36, 4.87G 0.95, respectively; and for MRZ 2/576

Table 2

Effect of selected NMDA receptor antagonists on convulsions induced

by maximal electroshock (MES), traction reflex and ataxia (rotarod

test) in mice

Agent MES ED50

(mg/kg)

Traction ED50

(mg/kg)

Ataxia ED50

(mg/kg)

Memantine 5.93 (3.7–9.5) 12.5 (8.0–19.4) 14.2 (10.2–19.9)

MRZ 2/576 20.8 (15.2–28.4) 21.1 (17.2–25.9) 22.3 (19.9–25.6)

MRZ 2/596 O50 O50 O50

MDL 105,519 O50 O50 O50

Values are ED50s in mg/kg (95% confidence limits are shown in

parentheses).

(at 0.01, 0.03, 0.1, 0.3, 1, 3, 10 mg/kg)Cmorphine:3.43G 0.62, 5.10G 0.75, 3.95G 0.49, 4.99G 0.67,2.88G 0.65, 4.53G 0.61, 2.99G 0.50, respectively(ANOVA F(12, 158)Z 0.92). Six-day b.i.d. treatmentwith 10 mg/kg of morphine produced a 3.47-foldincrease in the morphine antinociceptive ED50 valuesas determined in test #2. MRZ 2/596 and MRZ 2/576inhibited morphine antinociceptive tolerance at dosesstarting at 0.1 and 0.03 mg/kg, respectively. This wasrevealed by smaller differences between tests #1 and #2(ED50 test #2/ED50 test #1 fold changes, presented inFig. 2) in respective groups as compared to controlgroup that received placeboCmorphine. The raw dataof the representative groups are also shown in Fig. 3.

For reasons explained in the methods, all thesubsequent experiments were carried out in C57/Blmice. In the acute morphine antinociception experiment,naloxone hydrochloride at doses of 5, 10 and 50 mg/kgand naloxone methiodide at a dose of 50 mg/kgprevented acute antinociception induced by morphine(10 mg/kg) (Fig. 4).

Six-day administration of morphine induced a2.67G 0.42 fold change in morphine potency in test

Fold

cha

nge

in E

D50

Fold

cha

nge

in E

D50

0

1

2

3

4

5

0

1

2

3

4

5

MRZ 2/596 00

100

100.05

100.1

100.3

101

mg/kg

mg/kgMorphineMRZ 2/576

00

100

100.03

100.1

100.3

101

100.01

103

1010

**

*

**

***

**

**

*

A

B

**

**

Morphine

Fig. 2. Effects of NMDA receptor/glycineB site antagonists on the

development of morphine antinociceptive tolerance in Swiss mice.

Presented data show fold shifts in morphine antinociceptive ED50

values (meanG S.E.M.) between test #1 and test #2, before and after

6-day morphine administration (ANOVA F(12, 158)Z 4.27,

p! 0.001). Asterisks indicate a statistically significant difference

toward ‘‘PlaceboCMorphine’’ group that received saline and

morphine during the development of morphine tolerance (*p! 0.05,

**p! 0.01).


Fig. 3. The figures present morphine dose–response curves obtained before (test #1) and after (test #2) 6-day twice daily morphine administration

with MRZ 2/596 (the lowest and the highest dose among administered). The titles describe the chronic treatment between tests #1 and #2. Chronic

morphine administration produced right shift of morphine dose–response curve in test #2 (B) as compared to placebo treated group (A). MRZ 2/596

at the dose of 1 mg/kg given prior to each morphine injection prevented the right shift of morphine dose–response curve (D). MRZ 2/596 at 0.05

mg/kg was ineffective (C).

0

25

50

75

100

10 101010101010

10

1010

55

50501

******

**

******

mg/kg000 0 0 0 0 0

0 000

MPE

[%]

MorphineNaloxone HCl

Naloxone Methiodide

Fig. 4. The effects of naloxone hydrochloride and naloxone methiodide

on acute morphine (10 mg/kg)-induced antinociception in the tail-flick

test in C57/Bl mice. The opioid receptor antagonists were adminis-

tered 15 min before morphine and the morphine effect was tested

30 min after its injection (ANOVA F(8, 48)Z 23.00, p! 0.001).

Asterisks indicate a statistically significant difference toward

‘‘PlaceboC Morphine’’ group that received saline and morphine

(**p! 0.01, ***p! 0.001).

#2 (as compared to test #1) that was significantly higherthan the placebo treated group (fold change1.04G 0.13). MRZ 2/576 at 0.3 mg/kg and MRZ 2/596at 1 mg/kg given prior to each morphine dose inhibitedthe development of morphine antinociceptive tolerance(fold changes 1.52G 0.16 and 1.44G 0.17, respectively)(ANOVA F(3, 54)Z 6.45, p! 0.001). This confirmsthat the phenomenon previously observed in albinoSwiss mice can also be demonstrated in C57/Bl mice.Thus, further experiments were continued on C57/Blmice using the same morphine tolerance paradigm.

In the next set of experiments, there were no differ-ences among C57/Bl mice in the antinociceptive mor-phine ED50 values in test #1. Test #1 ED50 values (mg/kg) were obtained: for placeboC placebo, 1.92G 0.16;placeboCmorphine 1.51G 0.13; memantine (2.5 and7.5 mg/kg)Cmorphine 1.40G 0.22, 1.59G 0.29, re-spectively; MDL 105,519 (0.1, 1 mg/kg)Cmorphine1.53G 0.22, 1.86G 0.31, respectively; naloxone hydro-chloride (1, 5, 50 mg/kg)Cmorphine 1.66G 0.23,


1.66G 0.30, 1.51G 0.16, respectively; naloxone methio-dide (1, 5, 50 mg/kg)Cmorphine 1.63G 0.24, 1.56G0.16, 1.60G 0.22, respectively (ANOVA F(11, 158)Z0.67).

Treatment with 10 mg/kg b.i.d. of morphine pro-duced a 4.55-fold increase in the morphine antinocicep-tive ED50 values as determined in test #2. Morphinetolerance was attenuated by memantine at a dose of 7.5but not at 2.5 mg/kg, and MDL 105,519 at the dose of 1but not at 0.1 mg/kg (Fig. 5A).

Naloxone hydrochloride prevented the developmentof morphine antinociceptive tolerance at doses of 5 and50 but not 1 mg/kg. Naloxone methiodide preventedmorphine tolerance at doses of 50 but not 5 and 1 mg/kg(Fig. 5B).

Additionally, the possible acute effect of MRZ 2/596on morphine-induced antinociceptive activity was in-vestigated. Area Under the Curve calculated on %MPEvalues for 1 mg/kg MRZ 2/596C saline, salineC 3 mg/kg morphine and 1 mg/kg MRZ 2/596C 3 mg/kgmorphine were 470G 302, 3260G 743 and 3990G 691,respectively (NZ 8–10 mice per group). Thus, there wasno significant effect of MRZ 2/596 on morphine action.

0

1

2

3

4

5

6

0

1

2

3

4

5

6

000

1000

102.50

107.50

100

0.1

1001

mg/kg

mg/kg000

1000

1050

10500

1001

1005

1010

10050

* * *

*

*

*

A

B

**

* *

Fold

cha

nge

in E

D50

Fold

cha

nge

in E

D50

MorphineMemantine

MDL 105,519

MorphineNaloxone HCl

NaloxoneMethiodide

Fig. 5. Effects of memantine, MDL 105,519, naloxone hydrochloride

and naloxone methiodide on the development of morphine antinoci-

ceptive tolerance in C57/Bl mice. Presented data show fold shifts in the

morphine antinociceptive ED50 values (meanG S.E.M.) between test

#1 and test #2, before and after 6-day morphine administration

(ANOVA F(11, 158)Z 2.87, p! 0.01). Asterisks indicate a statistically

significant difference toward ‘‘Placebo C Morphine’’ group that

received saline and morphine during the development of morphine

tolerance (*p! 0.05, **p! 0.01).

It is unlikely that repeated administration of MRZ2/596 for 7 days has an affect on the pharmacokineticsof morphine as there was no change in the AUC formorphine antinociception (values for saline and 1 mg/kgof MRZ 2/596 were, respectively, 4100G 401 and2930G 440, t(18)Z 1.97, NS).

4. Discussion

NMDA receptor binding and patch clamp studiesconfirmed that MRZ 2/576 and MRZ 2/596 bind withmoderate (nanomolar) affinity to NMDA receptor/glycineB site whereas MDL 105,519 was some 5–10-foldmore potent. Because of the use of glycine (1 mM) in thepatch clamp studies, IC50 values obtained in theseexperiments were 4–9-fold higher than the Ki valuesobtained in binding experiments. However, correctionof these IC50 values to Kbs taking into account a Kd forglycine in these patch clamp experiments of 399 nMrevealed very similar potency of all three antagonists inboth assays. The effective concentrations in vivo willclearly depend on the endogenous levels of glycine andcould differ in the CNS and the periphery. Certainly,considerable evidence suggests that the glycineB site isnot saturated in the CNS in vivo (Danysz and Parsons,1998).

The antagonism observed with MRZ 2/576 and MRZ2/596 was typical for moderate affinity NMDA re-ceptor/glycineB antagonists, i.e. they induced glycine-sensitive desensitization whereas MDL-105,519 wasalmost equally effective against peak and plateau – aneffect probably related to its higher affinity – see Parsonset al. (1997).

Neurochemical studies using radiolabelled MDL105,519 demonstrate that its brain penetration (plasmavs. brain levels) is low, and the uptake of radioactivityinto the brain is in a range of 0.01–0.08% of the injecteddose (Opackajuffry et al., 1998). The MES experiment inthe present study is in line with this data and alsoindicates that MRZ 2/596 has negligible CNS effects.Thus, both MRZ 2/596 and MDL 105,519 seem to bedevoid of CNS activity, at least at the doses used. Thisassumption could also be confirmed by the present BBBpermeability study where both compounds pass the BBBvery slowly as compared with memantine. The effect ofmorphine on BBB integrity was also investigated asOishi et al. (1989) reported that morphine treatmentaffects BBB permeability. The present results indicatethat morphine treatment did not affect BBB integrity invitro since there was no difference in sucrose perme-ability after incubation with morphine.

In principle, the present data indicate that structur-ally similar substances with different penetration to theCNS can be discriminated in the BBB in vitro model.Of the glycineB antagonists tested, only those with a


NO-substitution could pass the blood–brain barrier(MRZ 2/502 and MRZ 2/576) in contrast to thequinolines (MRZ 2/501 and MRZ 2/596) and MDL105,519 confirming results of functional studies such asMES.

However, it should be stressed that this model canonly be regarded as a ‘‘first line screen’’ for CNSpenetration since it does not reflect all aspects ofgoverning CNS bioavailability in vivo such as e.g.probenecid sensitive transport out of the brain in thechoroid plexus.

The present study revealed that NMDA receptorantagonists acting at the NMDA receptor/glycineB sitewith modest (MRZ 2/576) or likely no CNS activitysuch as MRZ 2/596 and MDL 105,519 inhibitedtolerance to the antinociceptive effects of morphine.The effects of the structurally similar moderate affinityagents MRZ 2/576 and MRZ 2/596 were compared tothe structurally different high affinity NMDA receptor/glycineB site antagonist MDL 105,519. All three NMDAreceptor/glycineB site antagonists inhibited morphinetolerance suggesting that the inhibition of morphinetolerance by both MRZ 2/576 and MRZ 2/596 is relatedto their common pharmacodynamic action, i.e. block-ade of NMDA receptors at the glycineB site, and not viaactions at an unrelated site due to chemical similarity.

The inhibition of morphine tolerance by glycineBantagonists has been well documented. The full glycineBantagonist, ACEA-1328 (20 mg/kg) completely blockstolerance to morphine-induced antinociception in thetail-flick test in CD-1 mice (Lutfy et al., 1999). Similarly,another antagonists GV 196771A prevented develop-ment of tolerance to morphine antinociception in theformalin test in mice (Quartaroli et al., 2001). Even thelow intrinsic activity partial agonist, (R,C)HA966, wasable to inhibit morphine tolerance in neuropathic rats(Christensen et al., 2000).

It is certain that the doses of MRZ 2/576 thateffectively inhibited morphine tolerance (starting at0.03 mg/kg) do not reach sufficient concentrations inthe brain to block CNS NMDA receptors. Brainmicrodialysis experiments revealed that only 2% ofsystemically applied MRZ 2/576 is found in theextracellular fluid in the CNS (Hesselink et al., 1999b).Despite good BBB permeability properties, as describedalso in our study in the in vitro BBB model, MRZ 2/576is quickly removed from the brain by probenecid-sensitive transporters (Hesselink et al., 1999b).Moreover, a number of behavioral studies showed thatCNS-related effects of MRZ 2/576 occur at doses muchhigher than the minimal effective dose in the presentreport. MRZ 2/576 shows neuroprotective properties at5 mg/kg (Wenk et al., 1998), anticataleptic effects at10 mg/kg (Karcz-Kubicha et al., 1999) and generaliza-tion to ethanol cue in drug discrimination test at 5 mg/kg (Bienkowski et al., 1998). In addition, MRZ 2/576

inhibited firing produced by iontophoretic applicationof NMDA to the dorsal horn spinal neurons with anED50Z 2.8 mg/kg after intravenous administration(Parsons et al., 1997). In the present study, anticonvul-sive, ataxia-producing and traction reflex disturbingED50s were w20 mg/kg, which is over 200 times higherthan the dose inhibiting morphine tolerance. In contrast,doses of memantine inhibiting MES convulsions andmorphine tolerance were very similar and there was only50% difference in the case of ataxia and traction reflexdisturbance. Thus, it seems very unlikely that MRZ 2/576 given s.c. at the dose of 0.03 mg/kg is present in thebrain at a concentration sufficient to block NMDAreceptors to the degree producing inhibition of mor-phine tolerance. With regard to MRZ 2/596 and MDL105,519, these compounds show no CNS-related anti-convulsive activity against maximal electroshock at50 mg/kg in adult mice (present study). This apparentdissimilarity of the potencies in morphine tolerance andother behavioral measures (ataxia, suppression ofconvulsions) as well as the lack of parallelism inpotencies in various tests (patch clamp and bindingstudies: MDL 105,519OMRZ 2/576ZMRZ 2/596;morphine tolerance: MRZ 2/576OMRZ 2/596OMDL 105,519) may suggest that different neuronalsystems are involved in mediating these effects. At facevalue, the effectiveness of glycine site NMDA receptorantagonists not penetrating to the brain in inhibitingmorphine tolerance appears counterintuitive. However,these findings do not contradict a bulk of earlier findingsdemonstrating that centrally active NMDA receptorantagonists produce the same effects. This is becausedifferent, redundant neuronal targets may mediateinteracting effects of various glutamate antagonists.

The importance of peripheral mechanisms for toler-ance to the analgesic action of systematically adminis-tered morphine has been suggested in the past.Raghavendra and Kulkarni (1999) reported that mela-tonin reverses the development of tolerance and de-pendence to morphine and suggested that peripheralbenzodiazepine receptors are involved. Possible mecha-nisms have been suggested by a recent study by Patiernoet al. (submitted for publication). Opioid mu receptorendocytosis in enteric neurons induced by abdominallaparotomy was significantly inhibited by NMDAreceptor antagonists including MRZ 2/576 and MRZ2/596 (with similar potency), but not by the AMPAreceptor antagonist CNQX. Also mu receptor endocy-tosis in neurons from tissue exposed to NMDA and fromelectrically stimulated preparations was pronounced andprevented by NMDA receptor antagonists. It has beensuggested that such receptor internalization is evoked bylocal release of endogenous opioids and that this ismodulated by NMDA receptors. Such a mechanismcould play a role in the peripheral component oftolerance to the analgesic effects of morphine.


It was also previously reported that systemic admin-istration of morphine for 4 days (10 mg/kg) producesdownregulation of mu receptor mRNA in dorsal rootganglia accompanied by tolerance to the analgesiceffects in the hot plate, and the authors suggested thatthe peripheral nervous system may be important site ofopioid tolerance development (Meuser et al., 2003). Thisform of tolerance is also not surprising when consideringthat peripheral mu receptors seem to contribute toanalgesia produced by systemically administered mor-phine (Lewanowitsch and Irvine, 2002).

In contrast to studies demonstrating behavioralactivity of MRZ 2/576 at doses R 5 mg/kg, Olivarand Laird (1999) reported that this compound inhibitedthe effects of visceral noxious stimuli (as measured by anincrease in blood pressure) with an ED50 of 0.2 mg/kg.These findings support the notion that MRZ 2/576 hasperipheral actions, at least in the case of processing ofnociceptive signaling.

Another surprising aspect of the present findings isthat despite the short half life (of about 15 min) and alsothe short duration of anticonvulsive activity (Parsonset al., 1997; Hesselink et al., 1999b), MRZ 2/576 wasactive in the present study when given 30 min beforemorphine. It is possible that administration of MRZ2/576 leaves ‘‘finger prints’’, an effect that outlasts thepresence of the compound in the body. The ‘‘fingerprint’’ concept is supported by the data of Belozertsevaet al. (2000a), demonstrating that MRZ 2/576 at a doseof 1 mg/kg inhibited spontaneous morphine withdrawalin mice 45–60 min after injection when less than 25% ofthe injected dose is still present in the body (Parsonset al., 1997; Hesselink et al., 1999b). The ‘‘finger print’’effect of MRZ 2/576 was also described by Chizh andcolleagues in rats in the chronic constriction nerve injurymodel. In this study MRZ 2/576 (1–10 mg/kg, i.p.)produced an antiallodynic effect more than 24 h afteradministration of the drug despite the fact that theinhibition of responses to iontophoretically appliedNMDA in the spinal cord only lasted about 10–15 min(Chizh et al., 2001). All these and the present findingssuggest that even a short lasting blockade of the NMDAreceptor/glycineB site may lead to long lasting con-sequences.

In the present study, both naloxone hydrochlorideand methiodide prevented the development of morphinetolerance with minimal effective doses of 5 and 50 mg/kg,respectively. These doses also inhibited acute morphineantinociception although the former agent was moreeffective in this regard (Fig. 4). The inhibitory effect ofnaloxone methiodide on morphine antinociceptive tol-erance may additionally suggest that this phenomenoninvolves peripheral sites considering that its purity was atleast 99% (Sigma–Aldrich communication). The differ-ence in potencies between naloxone hydrochloride andnaloxone methiodide can be attributed to the difference

in the affinity at mu-opioid receptors, for which naloxonemethiodide has much lower affinity than naloxonehydrochloride ca. 50-fold in mu-opioid receptor bindingassay and ca. 26 in functional assay (Valentino et al.,1983).

One could also consider the possibility that theNMDA receptor/glycineB site antagonists used in thepresent study mediate their effects of morphinetolerance in a similar manner to opioid antagonists,but they should then have an inhibitory effect onmorphine-induced antinociception per se (‘‘naloxone-like’’ effect). However, in previous reports, NMDAreceptor/glycineB site antagonists, either did not affect,or even potentiated morphine antinociceptive effects inthe mice tail-flick test (Lutfy et al., 1999; Belozertsevaet al., 2000b), for review see Kozela and Popik (2002),thus making this possibility unlikely. Nevertheless, weinvestigated this possibility for MRZ 2/596 and thiscompound did not change morphine’s antinociceptiveactivity.

Moreover, one cannot exclude the possibility thatglycine site NMDA receptor antagonists not penetratingto the brain after acute administration, could showtissue accumulation after chronic administration, lead-ing to concentrations that have central activity. This wasinvestigated in two types of experiments. Treatment withMRZ 2/596 for 7 days did not affect morphineantinociception in the absence of acute MRZ 2/596and similarly such treatment even at a much higher dosedid not influence MES convulsions suggesting insuffi-cient NMDA receptor blockade in the CNS. Thus, thepossibility of brain accumulation leading to brain levelssufficient to block NMDA receptors can be ratherexcluded.

In conclusion, it appears that both the NMDAreceptor/glycineB site and opioid receptors in the PNSplay a role in the development of antinociceptivetolerance to morphine applied systemically. This furthersupports previous findings about peripheral (topical)components of morphine tolerance by Kolesnikov andPasternak group (Kolesnikov et al., 1996; Kolesnikovand Pasternak, 1999a,b).

It can be further hypothesized that the use of NMDAreceptor antagonists to inhibit morphine antinociceptivetolerance could be restricted to centrally inactiveNMDA receptor antagonists e.g. acting at the NMDAreceptor/glycineB sites. Such an approach would allowthe avoidance of centrally-mediated side effects knownfor many NMDA receptor antagonists which penetrateto the brain well.

Acknowledgements

The study was supported in part by Institute ofPharmacology Statutory Activity.


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