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Title page

Enhancement of -like effects but not BDNF mRNA expression by the novel NMDA neramexane in mice

Tomasz Kos, Beata Legutko, Wojciech Danysz, Gary Samoriski and Piotr Popik

Laboratory of , Institute of , Polish Academy Downloaded from of Sciences, Kraków, Poland (TK, PP), Department of Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland (BL), Preclinical R&D, Merz Pharmaceuticals, Frankfurt / Main, Germany (WD), Forest Research Institute, jpet.aspetjournals.org Jersey City, NJ, USA (GS)

at ASPET Journals on October 1, 2021

Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

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Running Title page a) Running Title: Neramexane enhances antidepressant-like effect b) Correspondence: Piotr Popik, Behavioral Neuroscience, Institute of

Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Kraków,

Poland; phone: (+48 +12) 6623375; fax: (+48 +12) 6374500; E-mail: nfpopik@cyf- kr.edu.pl c) Number of pages: 34 Downloaded from tables 4 (1, 2A, 2B and 3) figures 3 jpet.aspetjournals.org references 41 words in: abstract 250 at ASPET Journals on October 1, 2021 introduction 642 discussion 1490 d) List of Abbreviations:

AP-7 – D-2-amino-7-phosphonoheptanoic acid

BDNF – Brain Derived Neurotrophic Factor

DEPC - diethyl pyrocarbonate

MED – minimal effective dose

MK-801 – 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine maleate,

NMDAR-A – N-methyl-D-aspartate receptor antagonist

TST – tail suspension test JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

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Abstract

Improved efficacy in the treatment of may be achieved by the combined use of several . In the present study, acute administration of the novel NMDA receptor antagonist, neramexane, as well as the representative antidepressants imipramine, fluoxetine and venlafaxine, shortened the duration of immobility in the mouse tail suspension test with a minimal effective dose (MED) of 5 mg/kg. When tested in combination, the antidepressant-like effects of 5 mg/kg of imipramine, 20 mg/kg of fluoxetine and 5 mg/kg of venlafaxine were potentiated by Downloaded from neramexane (2.5 mg/kg), a dose which alone did not produce a significant effect on the duration of immobility. These effects appeared to be specific because they were not accompanied by significant effects on locomotor activity. The enhanced antidepressant-like activity produced with the different combinations was not jpet.aspetjournals.org synergistic as determined by comparing the theoretical and observed ED50s for each combination. In separate experiments, Northern blot analysis showed that a 14-day treatment with imipramine (10 mg/kg, b.i.d.) increased brain-derived neurotrophic factor (BDNF) mRNA expression in the cortex while neramexane (5 mg/kg, b.i.d.) at ASPET Journals on October 1, 2021 decreased it. Combined treatment produced no effect on BDNF mRNA expression. Mice treated with imipramine or neramexane for 14 days and tested shortly after the last dose demonstrated significant shortening of immobility, and the combined treatment produced an even greater antidepressant-like effect. Together these data support the view that NMDA receptor antagonists enhance the potency of antidepressants, but leave open the question as to whether enhanced BDNF expression is a necessary feature of the antidepressant-like effect. JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

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Introduction

Despite several studies on the different approaches for the optimal treatment of major depression as well as the availability of new antidepressant drugs, adequate management of this disorder remains a challenge. The primary reasons for this include the incidence of undesired side-effects produced by currently available antidepressants, the apparent delay in achieving measurable therapeutic benefit (Skolnick, 1999), (but see Frazer and Morilak (2005)), and a high percentage of

treatment-resistant patients (Souery et al., 1999). Thus, there is a compelling need Downloaded from for studies in which novel antidepressant therapies with an improved pharmacological profile are evaluated. Included among the various strategies suggested to improve upon the efficacy of antidepressant treatment are the use of broad-spectrum antidepressants (Skolnick et al., 2003) or the use of combinations of several jpet.aspetjournals.org antidepressants with different mechanisms of action (Frank et al., 2000).

Preclinical studies have demonstrated that the combination of N-methyl-D-aspartate receptor antagonists (NMDAR-As), such as or , with clinically at ASPET Journals on October 1, 2021 available antidepressants (e.g., fluoxetine, venlafaxine, imipramine) enhance their antidepressant-like effects in the forced swim test in rats (Rogoz et al., 2002). Similarly, amantadine was found to potentiate the activity of imipramine in the forced swimming test and in the D--induced locomotor activity in rats (Dziedzicka-Wasylewska et al., 2004). Since both memantine and amantadine have demonstrated clinical efficacy and safety for the clinical treatment of Alzheimer’s disease and Parkinson’s disease, respectively (Parsons et al., 1999b), evaluation of this class of compounds (i.e., NMDAR-A) in other neurological and psychiatric conditions may be warranted. Indeed, the positive results from preclinical studies have led to a small, open-label clinical trial, which showed that the combined administration of imipramine and amantadine reduced scores on both the Hamilton Depression Rating Scale and the Beck Depression Inventory both after 3 and 6 weeks of treatment in patients with treatment-resistant unipolar depression (Rogoz et al., 2004). Neramexane (1-amino-1,3,3,5,5-pentamethyl-cyclohexane) belongs to a recently described group of uncompetitive NMDAR-As known as the amino-alkyl- JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 103697 5 cyclohexanes (Danysz et al., 2002). Like memantine, neramexane exhibits moderate affinity, fast blocking/unblocking kinetics and strong voltage-dependence at the NMDA receptor, attributes which are thought to contribute to the drug’s efficacy and minimal side-effect profile (Parsons et al., 1999a). Thus, the first aim of the present study was to investigate whether the acute administration of neramexane would alone produce a significant antidepressant-like effect as measured by a decrease in the duration of immobility in the mouse tail suspension test (TST). In addition, we determined whether the combined treatment of neramexane with each of three representative antidepressant drugs (fluoxetine, venlafaxine, imipramine) enhanced their antidepressant-like effects. Downloaded from The second aim of the present study was to investigate whether chronic administration of neramexane produced alterations at the molecular level, which may be associated with an antidepressant-like effect. Generally, significant measurable jpet.aspetjournals.org improvement of depressive symptoms is observed following some period of continuous antidepressant drug treatment (Frazer and Morilak, 2005). Thus, it has been suggested that it is those neurochemical and molecular effects appearing after repeated drug administration that may be related, at least partially, to the therapeutic at ASPET Journals on October 1, 2021 action of the antidepressant treatment (Vetulani and Sulser, 1975; Duman et al., 1994; Skolnick et al., 1996). In this regard, long-term adaptations that result from the treatment with antidepressants may involve alterations in structural neuroplasticity (Kitayama et al., 1994). It has been suggested that brain-derived neurotrophic factor (BDNF), the most abundant neurotrophin in the brain, may be one such target as it is known to regulate neuronal survival, (Ghosh et al., 1994) differentiation and plasticity (Thoenen, 1995). However, while chronic treatment with antidepressants has been shown to increase BDNF mRNA and protein expression within several brain regions, including the neocortex and hippocampus, this effect may not be common to all antidepressants (Dias et al., 2003; Nibuya et al., 1995). Consequently, the present study examined whether chronic treatment with neramexane, imipramine and the combination significantly affected BDNF mRNA expression in the mouse cortex and hippocampus.

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Methods

Subjects For all experiments male C57/Bl/6J/Han mice were obtained from IMP, Lodz, Poland and weighed ~ 25 g at the start of the experiment. Mice were group-housed in standard laboratory cages and kept in a temperature-controlled colony room (21 ± 2oC) with a 12-hr light/dark cycle (light on: 07:00). Commercial food and tap water were available ad libitum. In those experiments where the acute treatment with neramexane and representative antidepressants were evaluated, the mice that were

used for the tail suspension test were again used one week later to evaluate the Downloaded from effects of the different drug treatments on the locomotor activity. Otherwise, all subjects were used once. All experiments were carried out according to the National Institutes of Health Guide jpet.aspetjournals.org for Care and Use of Laboratory Animals (revised 1996) and were approved by the Institute of Pharmacology, Polish Academy of Sciences in Krakow Animal Care and Use Bioethics Commission.

at ASPET Journals on October 1, 2021 Behavioral studies

All behavioral studies were conducted by personnel blind to the treatment conditions.

Tail suspension test (TST) Mice were transferred from the housing room to the testing area in their home cages and allowed at least a 1 h adaptation period to the new environment prior to drug treatment. Behavioral despair was induced by tail suspension according to the procedure of Steru et al. (1985). Mice were attached individually 75 cm above the flat surface of the tabletop with a 1 cm piece of paper adhesive tape placed ~1 cm from the tip of the tail. Animals were suspended for 6 min and the duration of immobility was recorded using the 'Porsolt' data collection program (Infallible Software, California, USA). Mice were considered immobile only when they were completely motionless. For the acute drug treatment, compounds were administered intraperitoneally (i.p.) 40 min (vehicle or neramexane) and 30 min (vehicle or antidepressants) before the JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 103697 7 test. In the chronic studies, mice were treated for two weeks with twice daily i.p. injections of vehicle, imipramine (10 mg/kg), neramexane (5 mg/kg) and their combination and were tested in the TST in a pseudo-Latin square design. Thus, on the first test day, one half of the mice were tested 30-40 min after the last dose and another half were tested 16-17h after the last injections. Two days later (during which the mice received continued drug treatment), the second TST assessment was performed, in which the interval between the last treatment and the test was interchanged. This way, chronically treated mice were tested twice in a manner allowing investigation of whether repeated TST affects the behavioral response.

Downloaded from Locomotor activity At least 1 hour before the start of the experiment, mice were transferred to the

experimental room for acclimation. The spontaneous locomotor activity was jpet.aspetjournals.org measured in custom-made circular aluminum actometers (30 cm of diameter, 10 cm of height with two light sources and two photoresistors which beam cross at the center) (Kos and Popik, 2005). The design of the apparatus limited the data

collection to only the gross movements of the animals. Mice were injected 40 min at ASPET Journals on October 1, 2021 (vehicle or neramexane) and 30 min (vehicle or antidepressants) before the test and placed individually in an actometer for 6 min of measurement. As with the tail- suspension test, this procedure did not allow the mice to habituate to the test apparatus prior to experimentation (see above).

Molecular studies

A separate set of mice was randomly divided into four groups and treated twice daily for two weeks (i.p.) with either: vehicle (physiological saline), imipramine (10 mg/kg), neramexane (5 mg/kg) or their combination. The brains were removed 16-17 h after the last dose and the frontal cortex, neocortex and hippocampus were dissected on ice. All structures were frozen immediately on dry ice, and stored at –80oC. The procedure for determination of BDNF mRNA levels was performed according to Legutko et al. (2001).

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RNA extraction Total RNA was extracted using TRIzol Reagent (Life Technologies, Grand Island, NY) following the protocol supplied by the manufacturer. The final RNA pellet was resuspended in DEPC (diethyl pyrocarbonate) treated H2O, quantified (absorbance at 260 nm) and used for Northern blot.

Northern blot

Northern blot analysis was performed with 10 µg of total RNA, separated on 1% denaturing agarose-formaldehyde gel, transferred to nylon membranes (Nytran, Downloaded from Schleicher and Schuell) and immobilized by ultraviolet (UV) radiation. A probe for rat BDNF was generated by polymerase chain reaction (PCR) from cDNA using the primers 5’-ACT-CTG-GAG-AGC-GTG-AAT-GG-3’ and 5’-CAG-CCT-TCC-TTC-GTG-

TAA-CC-3’. The 470 bp fragment was subcloned into the TA cloning vector pCRII jpet.aspetjournals.org and cut out with EcoRI. The insert was randomly primer-labeled with α-[P32]dCTP and purified (Prime-It RmT, Stratagene, Cedar Creek, USA). Hybridization was conducted overnight in Church’s buffer at 650C with radio-labeled cDNA probe.

Following hybridization, the filters were first washed for 30 min in 2x saline-sodium at ASPET Journals on October 1, 2021 citrate (SSC) buffer/0.1% sodium dodecyl sulfate (SDS) followed by a 30 min wash in 0.1xSSC/0.1% SDS at 550C, all at room temperature. Following exposure, filters were stripped off the BNDF probe (washed 3 times in 0.1xSSC/0.1% SDS at 1000C for 10 min) and re-hybridized for the β-actin cDNA probe (Clontech, Palo Alto, USA) to normalize RNA loading. Northern blots were quantified using digitized autoradiographs (PhosphorImager, Image Gauge 4.0, Fuji). The values for the ~4.0 and 1.7 kb BDNF transcripts were combined for analysis.

Drugs Neramexane HCl (1-amino-1,3,3,5,5-pentamethyl-cyclohexane) (MERZ Pharmaceuticals GmbH, Germany), imipramine HCl (Sigma Aldrich, Germany), fluoxetine HCl (ICN Polfa, Rzeszow, Poland, thanks to Dr. J. Kuncio) and venlafaxine HCl (Forest Research Institute, manufacturer: Cipla) were dissolved in sterile physiological saline (vehicle) by sonication. All drugs were administered by the intraperitoneal (i.p.) route using a volume of 10 ml/kg (0.2 ml/mouse); the doses refer to the salt formulations. The choice of doses for the chronic study was based on the JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 103697 9 observations indicating that administration of neramexane at a dose of 5 mg/kg leads to free extracellular brain concentrations of ~ 0.8 uM, sufficient to inhibit NMDA receptors (Danysz et al., 2002). The dose of 10 mg/kg b.i.d. of imipramine has been frequently used in the chronic studies in rats (e.g., (Papp and Moryl, 1994)).

Statistics The data fulfilled the criteria for normal distribution. Therefore, to evaluate the dose- effect of test compounds in the TST and locomotor activity test, one-way ANOVAs followed by the Duncan’s test were used. Downloaded from In addition, the anti-immobility ED50 effects for each treatment were calculated. Immobility scores were converted to the maximum possible effect [%] according to the formula:

jpet.aspetjournals.org 100 * (Vehicle score - Compound score)/Vehicle score

so that for each ED50 calculation, the respective vehicle produced an effect of 0% and the maximal effect of the compound was 100%. The maximum possible effect at ASPET Journals on October 1, 2021 values were used to calculate the ED50 with confidence limits derived by linear regression analysis. To test the interaction between the effects of different doses of neramexane and the effects of different doses of the test antidepressants, two-way ANOVAs (with the dose of neramexane and dose of an antidepressant, as factors), and one-way ANOVAs (all doses, including vehicle) followed by Duncan’s test were used. In addition, an analysis of the nature of neramexane and antidepressant interaction was performed using the PharmTools Pro program ver 1.1 (Tallarida et al., 1997). This analysis allows investigating of whether the interaction among treatments is synergistic, super- or sub-additive or simply additive. The ED50s were modeled using “Effect of a Single Dose Additive Combination” equation [10] of the PharmTools Pro program as described in PharmTools Pro User Guide. Briefly, for each drug combination, the dose-response data sets were loaded and the Log Linear/Probit model was chosen with Emaxs set at 100%. The ED50s of each drug combination were calculated for the tested antidepressant:neramexane respective dose combination using the dose pairs 5:2.5, 20:2.5, 5:20 and 20:20 mg/kg. These parameters were JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 103697 10 used to construct the regression lines, from which the corresponding theoretical

ED50s with confidence limits were calculated.

The observed ED50s were calculated using linear regression on the normalized data taken from Table 2A for combinations of the antidepressant+neramexane at 5+2.5 (7.5), 20+2.5 (22.5), 5+20 (25) and 20+20 (40) mg/kg, respectively. For example, the observed scores for neramexane+imipramine at 7.5, 22.5, 25 and 40 mg/kg, were: 16.76, 74.61, 92.88 and 93.00% maximum possible effect, respectively. Finally, the theoretical and observed regression lines were compared using equation [26] of the PharmTools Pro program (i.e., “Compare Two Regression Lines”) as described in PharmTools Pro User Guide and the calculated / tabular Fs are Downloaded from presented for each drug combination in the Table 2B. The effects of long-term treatment with imipramine and neramexane on the duration

of immobility in the TST were analyzed using a three-way mixed design ANOVA, with jpet.aspetjournals.org (1) order of testing (first immediate then delayed/ first delayed then immediate) and (2) treatment as between subjects factors and (3) immediate/delayed testing as the repeated factor. at ASPET Journals on October 1, 2021 JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

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Results

Effects of a single administration of neramexane, imipramine, fluoxetine and venlafaxine in the TST and on locomotor activity: dose-response analysis (Table 1) Each of the four compounds, when administered alone, significantly reduced the duration of immobility in the TST in a dose-dependent manner with a MED of 5 mg/kg. The same compounds, however, produced different effects on spontaneous locomotor activity. At the doses tested in this set of experiments, neramexane (5, 10, Downloaded from 20 mg/kg) and venlafaxine (40 mg/kg) increased, while imipramine (10 and 20 mg/kg) decreased this behavior. Fluoxetine did not alter locomotor activity.

jpet.aspetjournals.org Effects of a single administration of neramexane given in combination with imipramine, fluoxetine or venlafaxine (Table 2)

Interaction with imipramine at ASPET Journals on October 1, 2021 The combined administration of neramexane (2.5 mg/kg) and imipramine (5 mg/kg) at doses that alone did not exhibit antidepressant-like activity produced a significant decrease in the duration of immobility without producing significant effects on the locomotor activity. While the combination of neramexane (2.5 mg/kg) with imipramine (20 mg/kg) reduced the duration of immobility without altering locomotor activity, this antidepressant-like effect was not significantly different compared with the corresponding dose of imipramine alone, due to the “floor” effect of imipramine administered at 20 mg/kg. The high dose of neramexane (20 mg/kg) potentiated the antidepressant-like activity of both doses of imipramine; however, for the low dose imipramine (5 mg/kg) group, the combination also produced a significant increase in the locomotor activity compared with vehicle only-treated animals.

Interaction with fluoxetine Co-administration of neramexane (2.5 mg/kg) to fluoxetine (20 mg/kg) significantly reduced the duration of immobility compared with vehicle, and produced a greater JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 103697 12 antidepressant-like effect compared with the effect produced when each drug was administered alone at the corresponding dose. The antidepressant-like effect produced by this combination was specific, as it did not significantly alter the locomotor activity. The high dose of neramexane (20 mg/kg) enhanced the antidepressant-like activity of both doses of fluoxetine (5, 20 mg/kg). However, the locomotor activity was also significantly increased when fluoxetine was combined with the high dose of neramexane.

Interaction with venlafaxine Downloaded from The combined administration of neramexane (2.5 mg/kg) and venlafaxine (5 mg/kg) produced a significant reduction in the duration of immobility compared with vehicle- treated animals and with animals treated with each drug administered alone, without

significantly affecting locomotor activity. Neramexane (2.5 mg/kg) also enhanced the jpet.aspetjournals.org antidepressant-like effect of venlafaxine (20 mg/kg); however, the locomotor activity was significantly greater compared to vehicle. Similarly, co-administration of the high dose of neramexane (20 mg/kg) with venlafaxine (5, 20 mg/kg) produced a greater

reduction in the duration of immobility compared with venlafaxine alone but with an at ASPET Journals on October 1, 2021 apparent enhanced effect on the locomotor activity.

The nature of the neramexane-induced potentiation of the antidepressant-like effects of imipramine, fluoxetine and venlafaxine was investigated by comparing the regression lines corresponding to the theoretical and observed ED50s of the drug combinations (Table 2B). These analyses revealed that the enhancement was not super- or sub-additive as there was no statistical difference between the respective regression lines. Although it appeared that the observed interaction between neramexane and fluoxetine (indicated in bold) yielded a higher ED50 than the theoretical one, suggesting a sub-additive effect, this comparison was not significant.

Effects of chronic treatment with neramexane and imipramine on BDNF mRNA expression

Neocortex JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

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Imipramine (10 mg/kg), given chronically, increased BDNF mRNA expression in the mouse neocortex. By contrast, chronic neramexane (5 mg/kg) treatment produced a decrease in neocortical BDNF mRNA expression. Combined treatment of these compounds produced no change in BDNF mRNA expression compared with control.

Frontal cortex Similar to that observed in the neocortex, frontal cortical BDNF mRNA expression was increased by imipramine and decreased by neramexane. Chronic treatment with the combination of imipramine and neramexane produced no change in the Downloaded from expression of BDNF mRNA compared with control in the frontal cortex.

Hippocampus

Chronic treatment with imipramine did not significantly change the expression of jpet.aspetjournals.org BDNF mRNA in the mouse hippocampus. However, as observed in the cortex, neramexane significantly decreased BDNF mRNA expression in the hippocampus. Similar to that observed in the other brain regions studied, the combination did not significantly change the expression of BDNF mRNA compared with control. at ASPET Journals on October 1, 2021

Behavioral effects of chronic administration of imipramine with neramexane While chronic administration of imipramine produced an increase in BDNF mRNA in the cortex, neramexane produced a decrease in its expression in each of the three brain regions evaluated. Since an increase in BDNF mRNA/protein expression is thought to be associated with the antidepressant-like effect of various antidepressant drugs, this result was somewhat unexpected considering that neramexane exhibited antidepressant-like activity in behavioral tests. In order to determine whether chronic treatment may have altered neramexane’s behavioral response, and, thus its effects on BDNF mRNA expression, mice were treated for 14 days and tested immediately after the last dose and again 16-17 h later (the latter time point was designed to mimic the timeframe of the molecular studies). The order of testing (first immediate than delayed / first delayed than immediate) did not affect the results suggesting that repeated testing in the tail-suspension test may produce reliable results. JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

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Mice treated twice daily for 14 days with imipramine, neramexane or the combination were tested immediately (30-40 min) after the last dose. Each treatment resulted in a significant decrease in the duration of immobility with the combined treatment producing a greater antidepressant-like effect as compared to that of imipramine (10 mg/kg) or neramexane (5 mg/kg) administered alone (Table 3). By contrast, when the mice were tested 16-17 h (delayed) after the last dose, there was no significant difference of any drug treatment on the duration of immobility compared to vehicle treated animals.

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Discussion

In the current study, we tested whether the acute administration of NMDA receptor antagonist neramexane could potentiate the behavioral response of three representative antidepressant drugs when administered in combination. In a separate set of experiments, we determined whether changes in the expression of BDNF mRNA were associated with the antidepressant-like response following chronic (14 day) treatment. Together, the data demonstrate that while neramexane appears to augment the acute and long-term antidepressant-like response of classical antidepressants, its behavioral actions do not appear to be associated with significant changes in BDNF mRNA expression in the cortex or hippocampus. Downloaded from

Behavioral studies

In the mouse tail suspension test, three clinically available antidepressant drugs - jpet.aspetjournals.org imipramine, fluoxetine and venlafaxine produced a decrease in the duration of immobility at doses between 5-20 mg/kg, which is in agreement with earlier findings (Steru et al., 1985). The effect of these treatments appeared to be specific, because, with the exception of venlafaxine at 40 mg/kg, we observed no stimulation of at ASPET Journals on October 1, 2021 locomotor activity. Inhibitors of neurotransmission (see Kugaya and Sanacora (2005) for a recent review), and particularly antagonists of the glutamatergic ionotropic NMDA receptor, have been shown to exhibit antidepressant-like activity in the forced swim test in mice (Trullas and Skolnick, 1990). For the NMDA receptor channel blockers memantine, neramexane and amantadine, antidepressant-like activity was reported in the rat forced swim test (Rogoz et al., 2002). The present study extends these observations to the mouse tail suspension test, where the acute administration of the uncompetitive NMDAR-A neramexane reduced the duration of immobility with an efficacy comparable to that of imipramine, fluoxetine and venlafaxine. However, the anti-immobility effects of neramexane appeared less specific, since those doses, which decreased immobility, also increased the general activity of the mouse. The drug interaction studies demonstrated that neramexane, at a dose that itself did not exhibit antidepressant-like activity (2.5 mg/kg), significantly augmented the behavioral response produced by the low (5 mg/kg) doses of imipramine and venlafaxine. Similarly, the combination of the low dose of neramexane with a high JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 103697 16 dose of fluoxetine (20 mg/kg) produced a significantly greater antidepressant-like response compared with either drug given alone. In each case, the potentiation was not due to an increase in locomotor activity, indicating that the enhanced antidepressant-like response was not due to a nonspecific effect of drug treatment. Our results resemble similar enhancement of antidepressant potency by the AMPA receptor potentiator LY 392098 reported by Li et al., (2003) and by NMDAR-As such as memantine or amantadine (see the Introduction section). The design of the present study also allowed us to examine whether the antidepressant-like effect produced by the acute administration of the high dose of each antidepressant was maximal or if it could be potentiated by co-treatment with Downloaded from neramexane. This appeared to be the case for each of the three antidepressants tested, since the combination of 20 mg/kg of neramexane with 20 mg/kg of each antidepressant produced a greater reduction in the duration of immobility compared jpet.aspetjournals.org with administration of the respective antidepressant alone. However, with many of the drug combinations, the apparent increase in the antidepressant-like effect was associated with a corresponding increase in locomotor activity. Nevertheless, it important to emphasize that an ineffective dose of neramexane (2.5 mg/kg) was at ASPET Journals on October 1, 2021 shown to potentiate the antidepressant-like action of all three antidepressants without affecting the locomotor response. The mechanism(s) by which NMDAR-As, including neramexane, enhance the antidepressant-like response of classical antidepressants is currently unknown. Owen and Whitton reported recently that amantadine and (the weak NMDAR-As) given acutely with known antidepressants elevated extracellular serotonin concentration in the frontal cortices of rats, and facilitated and potentiated this effect after prolonged treatment (Owen and Whitton, 2005). These authors suggested that effects on serotonin concentrations could be due to a number of reasons, including alteration of glutamatergic tone at NMDARs that could facilitate serotonin neurotransmission, as well as budipine and amantadine effects on monoamine metabolism. Although similar effects of other NMDAR-As, including neramexane await investigation, the “serotonergic” hypothesis of an enhancement of antidepressant potency appears attractive. As neramexane has been shown to be an uncompetitive NMDA receptor channel blocker, modulation of NMDA receptor activity is likely to contribute to the observed JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 103697 17 behavioral response described in this study (Parsons et al., 1999a; Danysz et al., 2002). However, Rammes et al. (2001) has demonstrated that neramexane and memantine also inhibit 5-HT3 receptor currents in vitro. Since 5-HT3 receptor antagonists produce antidepressant-like effects in animal models and also potentiate the antidepressant-like effects of several antidepressants (Martin et al., 1992; Kos et al., 2006), other affected receptor mechanisms cannot be excluded. Pharmacokinetic factors should also be considered when interpreting drug interaction experiments. However, at clinically relevant concentrations neramexane neither induces nor inhibits cytochrome P-450 isoenzymes. This compound also does not induce, inhibit or serve as a substrate of the p-glycoprotein (MERZ Pharmaceuticals, Downloaded from unpublished observations). Lastly, since neramexane produced similar behavioral effects when combined with three structurally unrelated antidepressants that are differently metabolized, pharmacokinetic factors are not likely to have contributed to jpet.aspetjournals.org the present findings; however, direct testing of this would need to be done in order to exclude this possibility.

Molecular studies at ASPET Journals on October 1, 2021 It is well known that electroconvulsive shock as well as chronic (but not acute) treatment with antidepressants can produce an increase in the expression of BDNF mRNA and its receptor, TrkB, in the hippocampus and cortex of rats (Dias et al., 2003; Nibuya et al., 1995). Moreover, BDNF protein levels have been shown to be increased in the brains of depressed patients who have been treated with antidepressants (Chen et al., 2001). Our results are generally consistent with earlier reports demonstrating that chronic antidepressant treatment increases cortical BDNF expression. While treatment with imipramine increased cortical BDNF mRNA expression, it did not alter its expression in the mouse hippocampus. These observations support the data of Vinet et al., who reported that treatment with fluoxetine and desipramine did not significantly alter mouse hippocampal BDNF expression (Vinet et al., 2004). In contrast to the effects of imipramine, chronic administration with neramexane produced a decrease in BDNF mRNA expression in all three brain areas examined. Since increases in the levels of BDNF mRNA in cortical and hippocampal regions have often been associated with antidepressant-like activity in behavioral tests, this JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

JPET # 103697 18 result was somewhat surprising given the fact that NMDAR-As have been consistently shown to produce antidepressant-like effects using acute screening procedures (see Introduction section) and also in a chronic animal model of depression (Papp and Moryl, 1994). While Marvanova et al. showed that the structurally related NMDAR-A memantine produced an increase in BDNF mRNA expression in various brain areas, this effect was assessed after acute administration (Marvanova et al., 2001). Similar increases in BDNF expression have been reported for other NMDAR-A including MK-801 and following acute administration (Matsuki et al., 2001; Toyomoto et al., 2005; Linden et al., 2000).

The observed decrease in BDNF mRNA expression following the 14 day Downloaded from administration of neramexane, appears to contradict the hypothesis that antidepressant-like responses are associated with an increase in BDNF levels. The effect of neramexane on BDNF mRNA expression does not appear to be the result of jpet.aspetjournals.org tolerance to the antidepressant-like effect of the drug as neramexane given over a 14 day period continue to produce a robust decrease in the duration of immobility when tested 30-40 min after the last dose. The fact that the BDNF mRNA levels were measured 16-17 h after the last dose (when the behavioral response was no longer at ASPET Journals on October 1, 2021 apparent) is not likely to be the reason for the observed decrease. This is because changes in mRNA levels following chronic drug administration would likely reflect a long-lasting adaptive effect of the drug treatment. Possibly, an increase in BDNF expression may occur early during chronic administration, which was not detected at later time-points due to other adaptive mechanisms. However, this would not necessarily explain the observed apparent decrease. An alternative hypothesis is that changes in BDNF expression may not be directly associated with antidepressant-like effects seen in behavioral tests. In fact, recent analyses indicate that an increase in BDNF expression after chronic antidepressant treatment may not be a uniformly reported phenomenon. For instance, Alt et al., (2006) and Tardito et al., (2006) list a number of studies showing either no change or even a decrease of BDNF expression due to the chronic treatment with antidepressants. Also Frazer and Morilak (2005) discuss controversial data demonstrating acute behavioral antidepressant-like effects of BDNF and its increased expression seen only after chronic treatment with antidepressants. Taken together, the present data confirm and extend previous findings showing that JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

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NMDA receptor antagonists exhibit antidepressant-like activity in different animal models and may potentiate the behavioral response of currently available antidepressant drugs. While the reports published to date suggest that treatment with NMDAR channel blocker alleviates symptoms of major depression (Berman et al., 2000) (though see a negative data with a relatively low dose of memantine, (Zarate, Jr. et al., 2006)), systematic evaluation of a combined treatment has not yet been accomplished.

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Footnotes: This work was supported by a Sponsored Research Agreement to the Institute of Pharmacology, Polish Academy of Sciences from MERZ Pharmaceuticals and by statutory activity funds of the Institute of Pharmacology.

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Legends for Figures

Fig 1 Effect of chronic treatment with neramexane and imipramine on BDNF mRNA expression in mouse neocortex using Northern blot analysis. Compounds were given twice daily for 14 days, i.p., with mRNA expressions levels determined 16-17 h after the last dose. Panel A: representative Northern blot bands of BDNF and β-Actin control. Panel B: ratio of BDNF/β-actin bands signal from Northern hybridization (mean ± S.E.M). ANOVA F(3,34) = 13.565, P<0.001. Symbols: * P<0.05; *** P<0.001 vs vehicle Downloaded from

Fig 2 Effect of chronic treatment with neramexane and imipramine on BDNF mRNA expression in mouse frontal cortex using Northern blot analysis. jpet.aspetjournals.org ANOVA F(3,35) = 8.441, P<0.001. Symbols: * P<0.05 vs Vehicle, see Fig 1 legend for details.

Fig 3 Effect of chronic treatment with neramexane and imipramine on BDNF mRNA at ASPET Journals on October 1, 2021 expression in mouse hippocampus using Northern blot analysis. ANOVA F(3,34) = 6.967, P<0.001. Symbols: ** P<0.01; vs Vehicle, see Fig 1 legend for details.

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Table 1 Effect of neramexane, imipramine, fluoxetine and venlafaxine on the duration of immobility and locomotor activity following acute treatment – dose-response analysis. The mean +/- S.E.M. immobility scores in the tail suspension test (TST) [sec per 5 minutes] and the distance traveled [arbitrary units per 5 min] are summarized. Symbols: * P<0.05, ** P<0.01 *** P<0.001 as compared to the corresponding vehicle (Dunnet’s test followed by one way ANOVAs). The calculated

ED50 for each compound in the TST are also presented. Immobility scores were converted to the maximum possible effect [%] so that for each ED50 calculation, the vehicle produced an effect of 0% and the maximal effect of the compound was 100%.

For example, because the vehicle score was 185.5s and the neramexane score at Downloaded from 2.5 mg/kg was 182.1s, this dose of neramexane produced 1.83% of the maximum anti-immobility effect. These maximum possible effect values were used to calculate

ED50 and confidence limits using linear regression. jpet.aspetjournals.org at ASPET Journals on October 1, 2021 JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

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Locomotor activity ± SEM Drugs Dose [mg/kg] TST ± SEM [sec] N N [counts] 0 185.5 ± 5.1 12 91.3 ± 6.1 12 2.5 182.1 ± 5.5 10 - - 5 151.2 ± 6.1** 11 126.0 ± 8.7* 13 10 100.7 ± 9.9*** 12 135.5 ± 14.8** 10 20 45.3 ± 8.1*** 12 152.0 ± 12.1*** 10 Neramexane F(4,52)=67.34; P<0.001 F(3,41)=6.06; P<0.01

ED50 10.65 (7.92-16.27) 0 178.7 ± 6.1 11 118.3 ± 10.4 10

5 144.5 ± 12.5* 13 - - Downloaded from 10 134.4 ± 9.9** 12 91.5 ± 6.6* 10 20 74.8 ± 9.5*** 12 95.3 ± 5.9* 10 Imipramine F(3,44)=17.94; P<0.001 F(2,27)=3.389, P<0.05

ED50 17.65 (5.56-17.22) jpet.aspetjournals.org 0 178.1 ± 13.3 10 108.4 ± 7.0 10 5 134.1 ± 14.2* 10 - - 10 120.3 ± 8.4** 10 125.2 ± 9.5 10 20 56.5 ± 11.8*** 10 122.2 ± 5.6 10 Fluoxetine F(3,36)=17.19; P<0.001 F(2,27)=1.42; NS at ASPET Journals on October 1, 2021

ED50 12.97 (5.55-17.57) 0 169.3 ± 8.7 10 106.0 ± 5.6 10 5 128.1 ± 16.2* 10 - - 10 95.4 ± 12.2*** 10 - - 20 51.1 ± 11.1*** 10 134.5 ± 11.7 10 40 9.2 ± 2.3*** 9 175.5 ± 13.6*** 10 Venlafaxine F(4,44)=30.02; P<0.001 F(2,27)=10.36; P<0.001

ED50 11.16 (9.46-12.93)

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Table 2A. Effect of neramexane, alone and in combination with imipramine, fluoxetine and venlafaxine, on the duration of immobility and locomotor activity following acute treatment – drug interaction. The duration of immobility in the tail suspension test (TST) [sec per 6 minutes] and the distance traveled [arbitrary units per 6 min] are presented as the mean +/- S.E.M. For each drug combination, a 2- way ANOVA was calculated and the results are shown for the dose of the antidepressant, the neramexane dose and the combination (interaction). Following the analysis using 2-way ANOVAs, the data were analyzed using the Dunnet’s post- hoc test. Symbols: * P<0.05 vs corresponding vehicle; # P<0.05 vs corresponding

dose of antidepressant; § P<0.05 vs corresponding dose of neramexane. AD – Downloaded from antidepressant’s dose, Ner – neramexane dose. jpet.aspetjournals.org at ASPET Journals on October 1, 2021 JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

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AD Ner Locomotor activity ± SEM TST ± SEM [sec] N N [mg/kg] [mg/kg] [counts] imipramine + vehicle 0 0 165.8 ± 7.7 12 102.2 ± 5.5 11 5 0 163.5 ± 7.5 12 92.5 ± 7.6 11 20 0 49.2 ± 8.0 * 12 77.6 ± 5.6 * 11 imipramine + low dose of neramexane 0 2.5 162.0 ± 10.5 12 114.8 ± 5.2 11 5 2.5 138.0 ± 5.0 *#§ 12 97.8 ± 3.8 11 20 2.5 42.1 ± 6.4 *§ 11 93.2 ± 9.6 11 Downloaded from imipramine + high dose of neramexane 0 20 33.4 ± 6.7 * 12 188.6 ± 10.2 * 11 5 20 11.8 ± 3.3 *#§ 12 133.5 ± 7.6 *#§ 11 20 20 11.6 ± 2.6 *#§ 12 80.7 ± 7.0 § 11 imi dose F(2,98)=133.20*** imi dose F(2,90)=38.48*** jpet.aspetjournals.org ANOVA ner dose F(2,98)=222.48*** ner dose F(2,90)=29.76*** interaction F(4,98)=25.01*** interaction F(4,90)=11.78*** fluoxetine + vehicle 0 0 149.0 ± 9.1 11 107.5 ± 5.2 11 5 0 146.0 ± 9.2 12 100.6 ± 6.4 11 at ASPET Journals on October 1, 2021 20 0 120.9 ± 10.2 * 12 97.1 ± 7.1 11 fluoxetine + low dose of neramexane 0 2.5 170.0 ± 7.6 * 12 111.4 ± 10.2 11 5 2.5 129.8 ± 5.7 § 11 118.3 ± 4.7 10 20 2.5 80.3 ± 9.4 *#§ 12 112.4 ± 5.4 10 fluoxetine + high dose of neramexane 0 20 42.8 ± 4.5 * 11 148.6 ± 11.8 * 11 5 20 29.2 ± 2.3 *# 11 156.6 ± 9.3 *# 11 20 20 8.9 ± 1.1 *#§ 12 161.9 ± 12.3 *# 11 flu dose F(2,95)=36.38*** flu dose F(2,88)=0.07 NS ANOVA ner dose F(2,95)=205.0*** ner dose F(2,88)=32.85 *** interaction F(4,95)=5.53*** interaction F(4,88)=0.55 NS venlafaxine + vehicle 0 0 187.7 ± 9.4 12 120.6 ± 9.9 11 5 0 133.1 ± 8.1 * 12 97.0 ± 5.0 11 20 0 75.2 ± 7.7 * 12 156.6 ± 12.8 * 11 venlafaxine + low dose of neramexane 0 2.5 162.0 ± 6.1 * 12 113.1 ± 6.1 11 5 2.5 112.4 ± 9.4 *#§ 12 126.0 ± 9.8 11 JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version.

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20 2.5 42.3 ± 5.4 *#§ 11 163.7 ± 10.1 *§ 11 venlafaxine + high dose of neramexane 0 20 28.9 ± 4.3 * 12 163.2 ± 12.3 * 11 5 20 24.4 ± 7.0 *# 11 199.1 ± 16.0 *#§ 11 20 20 5.7 ± 0.8 *#§ 12 221.8 ± 19.1 *#§ 11 ven dose F(2,97)=112.23*** ven dose F(2,90)=13.97*** ANOVA ner dose F(2,97)=211.38*** ner dose F(2,90)=30.02*** interaction F(4,97)=15.04*** interaction F(4,90)=1.597 NS

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Table 2B. Theoretical and observed ED50 values of the acute antidepressant-like effect of neramexane co-administered with three different antidepressants. The ED50 values from Table 2A were calculated from normalized data as described in the legend to Table 1. NS – not significant.

TST imipramine + neramexane

Theoretical ED50 13.87 (3.87-21.68)

Observed ED50 13.67 (0.99-23.37) Calculated / Tabular F 1.29 / 6.94, NS Downloaded from fluoxetine + neramexane

Theoretical ED50 11.93 (8.12-15.06)

Observed ED50 20.48 (14.24-55.52) Calculated / Tabular F 1.42 / 6.94, NS jpet.aspetjournals.org venlafaxine + neramexane

Theoretical ED50 10.948 (9.94-11.90)

Observed ED50 9.772 (5.94-12.88) Calculated / Tabular F 1.98 / 6.94, NS

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Table 3. Effect of chronic (14-days) treatment with neramexane and imipramine on the duration of immobility when measured 30-40 min and 16-17 hrs after the last dose. The same mice were used in both tests using a semi-Latin square design. Data were analyzed using a three-way mixed design ANOVA followed by Duncan’s post-hoc test. Symbols: * P<0.05 vs 30/40 min vehicle; # P<0.05 vs 16-17 h vehicle; ƒ P<0.05 vs 30/40 min imipramine as well as vs 30/40 min neramexane; § P<0.05 vs respective drug or drug combination treatment tested 30/40 min after last dose.

Time between last dose and Chronic treatment TST ± SEM [sec] N test [mg/kg] Downloaded from vehicle 142.2 ± 15.6 12 imipramine 10 65.8 ± 11.5*# 12 30-40 min (immediate) neramexane 5 87.0 ± 12.0*# 12

neramexane 5 + 12 jpet.aspetjournals.org 27.7 ± 3.7*#ƒ imipramine 10 vehicle 121.7 ± 15.9 12 imipramine 10 127.2 ± 14.1§ 12 16-17 h (delayed) neramexane 5 114.2 ± 8.3*§ 12

neramexane 5 + 12 at ASPET Journals on October 1, 2021 117.7 ± 12.7§ imipramine 10 order of testing (1) F(1,40)=0.001 NS treatment (2) F(3,40)=5.57** 3-way ANOVA repeated factor (3) F(1,40)=44.93*** interaction of 2 and 3 F(3,40)=16.22***

JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 1, 2021 JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 1, 2021 JPET Fast Forward. Published on June 1, 2006 as DOI: 10.1124/jpet.106.103697 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 1, 2021