Functional in vitro characterization of CR 3394: A novel voltage dependent N-methyl-d -aspartate (NMDA) receptor antagonist

Neuropharmacology 50 (2006) 277e285 www.elsevier.com/locate/neuropharm

Functional in vitro characterization of CR 3394: A novel voltage dependent N-methyl-D-aspartate (NMDA) receptor antagonist Gabriele Losi a, Marco Lanza b, Francesco Makovec b,c, Roberto Artusi c, Gianfranco Caselli b, Giulia Puia a,* a

Department of Pharmaceutical Sciences, University of Modena and Reggio Emilia, via Campi 183, 41100 Modena, Italy b Department of Preclinical Pharmacology, Rottapharm S.p.A., Monza (MI), Italy c Department of Chemistry, Rottapharm S.p.A., Monza (MI), Italy Received 12 May 2005; received in revised form 31 August 2005; accepted 2 September 2005

Abstract Using the patch-clamp technique, we studied the effect of two novel adamantane derivatives, N-[2-(3,5-dimethyl-1-adamantyl)ethyl] guanidine (CR 3391) and N-[2-(3,5-dimethyl-1-adamantyl) ethyl]acetamidine (CR 3394), on NMDA receptors expressed in cortical neuron cultures. Our data show that CR 3391 and CR 3394 reduce NMDA-evoked currents (IC50 Z 1.7 G 0.6 mM and 6.7 G 1.5 mM, respectively). This antagonism is non-competitive and is completely reversible. The effect of CR 3394, like that of memantine, was strongly voltage dependent. HEK293 cells expressing NR1a/NR2B recombinant NMDA receptors and immature neurons (DIV 8e9) were more sensitive to CR 3394 antagonism than NR1a/NR2A expressing cells and DIV 15 neurons. CR 3394 also reduced the duration and amplitude of miniature excitatory post-synaptic currents mediated exclusively by NMDA receptors (NMDA-mEPSCs). Both memantine and CR 3394 inhibited NMDA-evoked [3H]norepinephrine release from rat hippocampal slices in a concentration-dependent manner with similar potency. CR 3394, but not memantine, increased cathecholamine resting release at low micromolar concentrations. Moreover, in an in vitro model of neurotoxicity, CR 3394 strongly reduced glutamate- and NMDA-induced neuronal death. Taken together, our data highlight pharmacological features of CR 3394 in vitro that prompt us to further evaluate it as a candidate for the treatment of neurodegenerative disorders. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: NMDA receptors; NMDA antagonists; Patch-clamp; Neurodegenerative disorders; Electrophysiology

1. Introduction Glutamate is the most abundant excitatory amino acid in the brain and acts on both metabotropic and ionotropic receptors. N-methyl- D-aspartate (NMDA) receptors belong to the ionotropic glutamate receptor family and are involved in many physiological processes such as neuronal development, learning and memory, motor activity and nociception. Functional NMDA receptors are heteromeric complexes containing NR1 and NR2 subunits (2A-B-C-D subtypes), and possibly the NR3A subunit (McBain and Mayer, 1994; Dingledine et al., * Corresponding author. Tel.: C059 2055138; fax: C059 2055131. E-mail address: [email protected] (G. Puia). 0028-3908/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2005.09.002

1999 for reviews). The pharmacology of the channel is mainly determined by the type of NR2 subunits (Cull-Candy et al., 2001) and by alternative splicing of NR1 subunits (Durand et al., 1993; Traynelis et al., 1995). Most NMDA receptors show high calcium permeability and strong voltage dependence. NMDA receptors play an important role in neuronal plasticity (Bliss and Collingridge, 1993), acting as the ‘‘coincidence detector’’ postulated by Donald Hebb’s theory (Hebb, 1949). However in pathological conditions, excessive NMDA activation may lead to neuronal cell death due to high intracellular CaCC (Choi, 1992). For this reason, many NMDA receptor antagonists have been developed for the treatment of pathologies such as Alzheimer’s disease (AD),

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stroke, neuronal trauma, Parkinson’s disease (PD), chronic pain, depression, anxiety and epilepsy (for review see Stone and Addae, 2002; Kemp and McKernan, 2002). Despite encouraging preliminary data, most of these compounds failed clinical trials because of serious side effects. Complete block of NMDA receptors impairs normal synaptic transmission, and this likely causes the side effect profile. In contrast, low levels of antagonism are less likely to produce unwanted effects. This is the case of memantine and amantadine, two NMDA receptor antagonists with good therapeutic profiles (Bormann, 1989; Danysz et al., 1997; Parsons et al., 1993, 1999a,b; Rogawski and Wenk, 2003). Memantine is currently used to treat dementia in chronic neurodegenerative disorders like AD (Danysz and Parsons, 2003) while amantadine is employed for PD (Danysz et al., 1997). A recent paper demonstrated that the mechanism of amantadine is atypical for a channel blocker. Amantadine stabilizes the channel in the closed state (Blanpied et al., 2005), in contrast to most channel blockers which act on the open channel. By using patch-clamp techniques, we investigated the effects of CR 3391 and CR 3394, two novel compounds that include the adamantane nucleus, on native NMDA receptors expressed in cortical neurons in culture. To probe the selectivity of CR 3394 we also measured its effect on recombinant NR1a/NR2A and NR1a/NR2B receptors expressed in HEK293 cells. We then performed experiments using perfused cerebral slices to evaluate the activity of CR 3394 as a modulator of NMDA-evoked norepinephrine release and of the cathecolamines basal release pathway. Finally, the potential neuroprotective effect of CR 3394 against glutamate- and NMDA-induced toxicity was assessed in cultured cerebellar and cortical neurons. Preliminary reports relating to some data in this manuscript have been published in abstract form (Lanza et al., 2004; Losi et al., 2004).

2.2. HEK293 cells culture and transient transfection HEK293 cells were grown at 37  C and 5% CO2 in MEM medium (with Earle’s salt supplemented with 10% foetal bovine serum and glutamine) and transfected with NMDAR subunit and GFP cDNAs using calcium precipitation method (Chen and Okayama, 1987). Briefly, a dish with three to four cover slips with HEK293 cells was incubated with DNA/Ca2C mixture containing 3 mg cDNAs in 1.5 ml transfection medium (pH 7.85 pre-incubated at 37  C and 5% CO2) for 8e12 h at 37  C in 5% CO2. After double wash with PBS, fresh medium (MEM w/glutamine and Earle’s salts; 10% FBS) was added to the dish and incubated overnight (37  C in 5% CO2). All plasmids were kindly donated by Dr. Stefano Vicini (Washington DC, USA).

2.3. Electrophysiological recordings Recordings were performed on single neuron in culture at room temperature, under voltage-clamp in the whole-cell configuration of the patch-clamp technique (Hamil et al., 1981). Electrodes were pulled from borosilicate glass (Hidelberg, FRG) on a vertical puller (PB-7, Narishige) and had a resistance of 5e7 U when filled with KCl internal solution. Currents were amplified with an Axopatch 1D amplifier (Axon Instruments, Foster City, CA), filtered at 5 kHz, and digitised at 10 kHz. 2.3.1. Solutions and drugs The recording chamber was continuously perfused at 5 ml/min with an extracellular medium composed of (mM): 145 NaCl, 5 KCl, 1 CaCl2, 5 Hepes, 5 glucose, 20 sucrose, pH 7.4 with NaOH. Intracellular solution contains (mM): 140 KCl, 3 MgCl2, 5 EGTA, 5 Hepes, 2 ATP-Na, pH 7.3 with KOH. NMDA and glycine were purchased from Sigma Aldrich (Milan, Italy). CR 3391 and CR 3394 were synthesized by Rottapharm S.p.A (Monza, Italy). 2.3.1.1. Drug application. CR 3391, CR 3394, NMDA and glycine were dissolved from 10 mM frozen stocks. All drugs were applied directly by gravity through a Y-tube perfusion system (Murase et al., 1989) every 60 s. Drug application had a fast onset and achieved a complete local perfusion of the recorded cell. NMDA-mEPSCs were recorded in the presence of 0.1 mM tetrodotoxin citrate (TTX, Sigma) and pharmacologically isolated with bicuculline methiodide (BMI, 20 mM, Sigma) and 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX, 5 mM, Tocris).

2.4. Release experiments from cerebral slices

2. Methods 2.1. Primary cultures of cerebellar granule cells Primary cultures of cortical neurons were prepared from newborn (postnatal day 1) SpragueeDawley rats. Briefly cells from cortex were dispersed with trypsin (0.24 mg/ml; Sigma Aldrich, Milan, Italy) and plated at a density of 0.8 ! 106 cells/ml on 35 mm Falcon dishes coated with poly-L-lysine (10 mg/ml, Sigma Aldrich). Cells were plated in basal Eagle’s Medium (Celbio, Milan, Italy), supplemented with 10% foetal bovine serum (Celbio), 2 mM glutamine, and 100 mg/ml gentamycin (Sigma Aldrich) and maintained at 37  C in 5% CO2. After 24 h in vitro, the medium was replaced with 1:1 mixture of BME and Neurobasal medium (Celbio, Milan) containing 2% B27 supplement, 1% antibiotic, and 0.25% glutamine (Invitrogen). At 5 days in vitro (DIV5), cytosine arabinofuranoside (Ara-C) was added at a final concentration of 10 mM. Thereafter, half of the medium was replaced twice a week with Neurobasal medium containing 2% B27 supplement, 1% antibiotic, and 0.25% glutamine. Primary cultures of cerebellar granule cells were prepared from postnatal day 7 SpragueeDawley rats following the same procedure as for cortical neurons. On day 2 the medium was replaced with BME containing 25 mM KC and 10 mM Ara-C. Recordings were made from DIV 8e16 neurons in culture.

Male Wistar rats weighing 250e300 g (Harlan, Milan, Italy) were used for all release experiments. Animals were housed in plastic cages, with free access to standard laboratory food and water, under controlled conditions of lighting, humidity and temperature. All experiments were carried out in accordance with the principles and guidelines of the Local Government and the regulations of the European Communities Council Directive of 24 November 1986 (86/ 609/EEC). Authorization for experimental procedures was granted by Italian Ministry of Health. Rats were killed by decapitation and brains were quickly removed and immediately transferred in ice-cold artificial cerebrospinal fluid (aCSF) having the following composition (mM): 125 NaCl, 3 KCl, 1.2 CaCl2, 1.2 MgSO4, 1 NaH2PO4, 22 NaHCO3 and 10 glucose (aerated with 95% O2 and 5% CO2 at 37  C), pH 7.2e7.4. 2.4.1. NMDA-evoked [3H]noradrenaline release Hippocampi were removed on ice and coronal slices chopped (0.40 mm thick). Slices were then labelled with 0.01 mM [3H]noradrenaline (20 min at 37  C). The incubation medium contained 0.1 mM of the serotonin uptake inhibitor 6-nitroquipazine to prevent possible false labelling of serotonergic terminals (Pittaluga et al., 1995). After washing with tracer-free medium, slices were transferred to parallel superfusion chambers e one slice/chamber e and perfused at 0.5 ml/min (37  C) with the same medium from which MgCC ions were omitted. After 48 min of perfusion to equilibrate the system, a series of seven fractions of 5 min each were collected. Eighteen minutes before

G. Losi et al. / Neuropharmacology 50 (2006) 277e285 starting collection, compounds under study were added and perfused till the end of the experiment. The first two fractions represented the basal release of [3H]noradrenaline in the absence or presence of antagonists. NMDA was added only during the 5 min fraction starting 15 min after the end of the equilibration period of perfusion. At the end of the experiment samples and perfused slices (dissolved with soluene) were counted for radioactivity in a liquid scintillation counter. The fractional release of tritium was calculated as the amount of radioactivity in each sample divided by the total amount in the slice at that particular time point. 2.4.2. Resting release studies from rat brain slices Slices labelled with [3H]noradrenaline or [3H]dopamine were both incubated in the presence of 0.1 mM of serotonin uptake inhibitor, 6-nitroquipazine, to prevent possible false labelling of serotonergic terminals (Pittaluga et al., 1995). Striatal slices labelled with [3H]dopamine were also incubated in the presence of 0.1 mM of the noradrenaline uptake inhibitor, nisoxetine to avoid false labelling of noradrenergic terminals. After washing with tracer-free medium, slices were transferred to parallel superfusion chambers and perfused with saline at 0.5 ml/min (37  C). After 45 min of superfusion to equilibrate the system, nine 5 min fractions were collected. Memantine and CR 3394 were added starting 10 min after the end of the equilibration period of perfusion.

2.5. Toxicity experiments For neurotoxicity experiments cerebellar granule cells and cortical neurons were plated in 24 wells multiplates. At the day of experiment wells were washed once with phosphate buffered saline (PBS), then incubated for 1 h in sterile Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 3.6 mM NaHCO3, 5.5 mM D-glucose, 5 mM Hepes, pH 7.4) containing 100 mM glutamate or 500 mM NMDA and 10 mM glycine with or without CR 3394. Control group was treated with buffer solution only. Twenty-four hours later a solution containing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide (MTT; 3 mg/ml) was added. MTT is converted to an insoluble purple formazan by mitochondrial dehydrogenases of living cells. Formazan was solubilized with isopropanol plus HCl (333 ml of HCl 37% in 50 ml of isopropanol). Absorbance was measured with Labsystem Multiskan MCC/30 with dual wavelength lecture (570 and 630 nM). Data values are mean G SEM.

279

3. Results 3.1. Both CR 3391 and CR 3394 inhibit NMDA-evoked currents Using the patch-clamp technique in the whole-cell configuration we recorded NMDA-evoked currents (100 mM NMDA and 10 mM glycine) in cultured cortical neurons. Increasing concentrations of CR 3391 and CR 3394 reduced NMDA steady state currents in a dose-dependent fashion (Fig. 1). The IC50 values were 1.7 G 0.6 mM and 6.7 G 1.5 mM for CR 3391 and CR 3394, respectively (Fig. 1B). CR 3391 and CR 3394 block was complete at concentrations higher than 100 mM and was easily reversible with washout. After application of agonist with either CR 3394 or memantine, drug was washed off for 60 s, and then agonist response was evoked again. The recovery from antagonist was calculated as the percent of response after washout compared to the control response. After application and washout of CR 3394 (10 mM), CR 3391 (10 mM) and memantine (10 mM) the recovery was 87 G 4%, 77 G 4% and 83 G 4%, respectively. In the same conditions the recovery from MK801 (10 mM) was 20 G 2%. 3.2. CR 3394 is a non-competitive antagonist with strong voltage dependence To investigate the nature of CR 3391 and CR 3394 antagonism we tested their effects on currents evoked by increasing concentrations of NMDA. CR 3391 and CR 3394 reduction of NMDA current was not prevented by high agonist concentrations, instead the effect was significantly stronger (Fig. 2B). Subsequently we studied NMDA inhibition by CR 3391 and

2.6. Data analysis

A

+CR 3394 (µM) 5

NMDA

10

100

NMDA

100 pA 5s

B

control NMDA- current

Data were expressed as the mean G standard error of mean (SEM) of n experiments. For the electrophysiological experiments after data normalisation, a fitting of the doseeresponse relationship was performed using the logistic equation %Imax Z 100/Imax (1 C (IC50/[ANTAGO]nh)) where Imax is the maximal current elicited by the agonist, IC50 is the antagonist concentration eliciting the half-maximal response, nh is the Hill coefficient. The ratio of the current amplitude in the absence and presence of CR 3394 10 mM (L) measured at different holding potentials gives an estimate of the voltage dependence of the block (d Z fraction of the total electric field experienced by the antagonist at its binding site). The value of d was determined using the following equation: zd Z (RT/F )Ve where z is the charge of the molecule; R, T and F have their usual thermodynamic meanings and Ve is the voltage required for an e-fold change in the membrane potential and it was calculated as in Kloda and Adams (2005). For release experiments in slices concentrationeresponse curves were analyzed by means of a computerized curve fitting program using the following four-parameter logistic equation: y Z a C [(b ÿ a)/(1 C (10c/10x)d)] where a was the minimum and b the maximum value of the data; c the EC50 or the IC50 and d the slope of the curve. The statistical significance was determined by Student’s t-tests or one-way analysis of variance followed by the Dunnett’s t-test. The accepted level of significance was p ! 0.05. Off-line data analysis, curve fitting, and figure preparation were performed with Clampfit 8 (Axon Instruments), Origin 6.1 (Microcal, Northampton, MA), SigmaPlot 3.0 (Jandel software), SigmaStat 2.0 (Jandel software), Minianalysis (Synaptosoft, Decatur, GA) and Microsoft Office (Microsoft).

CR 3394 IC50 = 6.7 ± 1.5 µM nh = 1

100 80 60 40 20

CR 3391 IC50 = 1.7 ± 0.6 µM nh = 0.7

0 0.001

0.01

0.1

1

10

100

[drug](µM) Fig. 1. CR 3391 and CR 3394 inhibit NMDA-evoked currents. (A) Whole-cell currents evoked by NMDA 100 mM C glycine 10 mM alone or together with CR 3394 from a cortical neuron held at ÿ60 mV (DIV 8). (B) Sigmoidal fitting of the doseeresponse curves of the modulation of CR 3391 (empty squares) and CR 3394 (filled squares) on NMDA steady state currents.

G. Losi et al. / Neuropharmacology 50 (2006) 277e285

[NMDA](µM)

B

A NMDA 600

10

current inhibition

100 pA 2s

C

100

30

600

20 40 60 80

+ 60 mV

+ CR 3394

current inhibition

5 4 3 2 1 20 40 60

Vh

CR 3394

CR 3391

2s

MEM

D

CR 3394

50 pA

-60 -40 -20

*

*

+ MEM

- 60 mV

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CR 3394

**

**

CR 3391 100 + CR 3391

1000

CR 3391

NMDA 30

MEM

280

20 **

40

**

60 * 80

+ 60 mV

100 - 60 mV

Fig. 2. CR 3394 is a non-competitive antagonist with strong voltage dependence. (A) Currents evoked by NMDA 30 mM and 600 mM (Cglycine 10 mM) alone and with CR 3394 10 mM (traces marked with diamond) from a cortical neuron (Vh Z ÿ60 mV) at DIV 8. (B) Histogram summarizing the effect (% of inhibition) induced by CR 3391 (5 mM) and CR 3394 (10 mM) on currents evoked by increasing concentrations of NMDA. Data are expressed as mean G SEM (n R 6; *p ! 0.05; **p ! 0.01, t-test vs NMDA 10 mM). (C) Effect on NMDA-evoked currents by CR 3391, CR 3394 and memantine (all 10 mM; traces marked with diamond) at Vh Z ÿ60 and C60 mV. (D) Histogram summarizing the effect induced by the compounds at two different holding potentials. Data are expressed as mean G SEM (n R 6; **p ! 0.01, t-test vs effect at ÿ60 mV). (E) Ratio of current amplitudes obtained in the absence and presence of CR 3394 10 mM (L) plotted against the holding potential (Vh) fitted by linear regression (r Z ÿ0.989; p ! 0.01; slope ÿ0.014 G 0.002).

CR 3394 at different holding potentials and compared it to that of memantine, the well known voltage dependent NMDA antagonist (Fig. 2C). Similar to memantine antagonism, CR 3394 (10 mM) antagonism was greatly reduced at positive holding potentials (Fig. 2D), being ÿ66 G 3% at ÿ60 mV and ÿ22 G 5% at C60 mV, ÿ60 G 7% at ÿ40 mV and ÿ34 G 4% at C40 mV. CR 3391 effect at 10 mM was also affected by voltage but to a lesser extent, being ÿ84 G 7% at ÿ60 mV and ÿ62 G 6% at C60 mV. In Fig. 2E we plotted L (ratio of the amplitude of steady state current obtained in the absence and presence of CR 3394) versus the holding potential and calculated (see Section 2) the voltage dependence of CR 3394 effect (zd Z 0.81). We considered strong voltage dependence as an important requirement for the effectiveness of NMDA antagonism, and therefore we focused our further study only on CR 3394. 3.3. CR 3394 reduces NMDA miniature excitatory post-synaptic currents (mEPSCs) We recorded NMDA mediated miniature EPSCs (NMDAmEPSCs) in cortical neurons at 15e16 days in vitro in control conditions and after bath perfusion of CR 3394. At 10 mM the compound significantly reduced peak amplitude (ÿ35 G 8%, n Z 10) and deactivation kinetics (ÿ31 G 5%, n Z 8) of

NMDA-mEPSCs as shown in Fig. 3. Although not statistically significant, we also observed a reduction in NMDA-mEPSCs frequency. The background noise measured as standard deviation of the baseline current was decreased by CR 3394 of 36 G 5% (n Z 7). 3.4. CR 3394 is more potent on NR1a/NR2B than NR1a/NR2A NMDA recombinant receptors To determine a possible subunit selectivity of CR 3394 we tested its effect on HEK293 cells transiently transfected with NR1a/NR2B or NR1a/NR2A subunits and compared CR 3394 to ifenprodil (Fig. 4A). CR 3394 was significantly more active on NR1a/NR2B than NR1a/NR2A receptors (IC50 NR2B Z 3 G 1 mM and IC50 NR2A Z 11 G 1 mM) (Fig. 4B). Given the subunit selectivity of CR 3394 for NR2B-containing receptors, we wondered if a change in the expression of this subunit in culture could correlate with changes in CR 3394 activity. We compared the effect of CR 3394 (5 mM and 10 mM) on cortical neurons at two different developmental stages, because the NR2B subunit is more highly expressed early in development. As shown in Fig. 4C, CR 3394 blocks a significantly larger percent of NMDA current in immature (DIV 8e9) than in older neurons (DIV 15).

G. Losi et al. / Neuropharmacology 50 (2006) 277e285

281

NMDA-mEPSCs

A

50 pA 2s

+CR3394 10 µM

B τw 94 ms

τw 120 ms

15 pA 200 ms

Peak Amplitude (pA)

τw (ms)

140

**

100 60 20

0.14

50 40

Frequency (Hz)

C

**

30 20 10 CTRL

CTRL +CR 3394

0.10 0.06 0.02

+CR 3394

CTRL

+CR 3394

Fig. 3. CR 3394 reduces NMDA miniature excitatory post-synaptic currents (mEPSCs). (A). Whole-cell recordings of NMDA-mEPSCs from a cortical neuron (DIV 15; Vh Z ÿ60 mV) in control condition (TTX 0.1 mM, BIC 20 mM and NBQX 5 mM) and with CR 3394 10 mM. (B) Averaged NMDA-mEPSCs from individual cell under control (n Z 21 events) or with CR 3394 (n Z 14 events) with superimposed decay fitting curves and indication of the weighted time constant of decay (tw). (C) Summary of the effect of CR 3394 10 mM on NMDA-sEPSCs tw, peak amplitude and frequency of occurrence. Each data point is the mean G SEM of 10 cells (**p ! 0.01, t-test vs control).

modulating NMDA receptors. NMDA (10e3000 mM, in the absence of MgCC) evoked a concentration-dependent increase in [3H]noradrenaline (EC50 Z 98.3 G 4.8 mM; n Z 6) (Fig. 5A). Both memantine and CR 3394 antagonized [3H]noradrenaline release induced by 100 mM NMDA with IC50s in the low micromolar range: 0.71 G 0.07 mM (range: 0.03e30 mM; n Z 4) and 1.64 mM G 0.11 (range: 0.3e30 mM; n Z 6), respectively. This

3.5. CR 3394 inhibits NMDA-evoked release from rat hippocampal slices As we characterized CR 3394 as a low-affinity NMDA receptor antagonist, we tested its effect on the release of [3H]noradrenaline evoked by NMDA from rat hippocampal slices, a classical paradigm used to evaluate in vitro affinity of drugs

B

CR 3394 IFENP 5 µM 10 µM 10 µM

MAX EFFECT

current inhibition

A

20 40 60 80

**

100 NR2A

**

100

**

80 60 40

NR1a/NR2A IC50 = 11±1 µM nh = 1.8 ±0.3

20

0.0

NR2B

NR1a/NR2B IC50 = 3 ±1 µM nh =1.4 ±0.3

0.1

1

10

100

[CR 3394](µM)

current inhibition

C

+CR 3394 5 µM

+CR 3394 10 µM

20 40

** *

60 DIV 8-9

DIV 15

Fig. 4. CR 3394 is more potent on HEK293 cells expressing NR1a/NR2B than NR1a/NR2A NMDA recombinant receptors. (A) Comparison between the effects of CR 3394 (5 and 10 mM) and ifenprodil (10 mM; IFENP) on recombinant NR1a/NR2A and NR1a/NR2B receptors transiently expressed in HEK293 cells. (n R 6; **p ! 0.01; t-test vs NR2A). (B) Sigmoidal fitting of the doseeresponse curves of CR 3394 effect on NR1a/NR2A (empty squares) and NR1a/NR2B (filled squares) normalized to the maximal effect. (C) Histogram showing the percentage of reduction induced by CR 3394 on cortical neurons at 8e9 DIV and 15 DIV. Data are expressed as mean G SEM (n R 6; *p ! 0.05; **p ! 0.01; t-test vs effect at 8e9 DIV).

G. Losi et al. / Neuropharmacology 50 (2006) 277e285

282

100 80 60 40 EC50 = 98 ±1 µM

20

of 100 µM NMDA increase)

B 100 80 60

Memantine IC50 = 0.7 ± 0.1 µM

40 20

CR 3394 IC50 = 1.6 ± 0.1 µM DNQX

0

0

(

NMDA-evoked [³H]NE release ( of maximal response)

A

1

100

1000

[NMDA] (µM)

0.01

0.1

1

10

[Drug concentration] (µM)

Fig. 5. CR 3394 inhibits NMDA-evoked release from rat hippocampal slices. (A) Doseeresponse effect of NMDA () in a MgCC free medium on [3H]noradrenaline release. (B) NMDA (100 mM) was used to stimulate [3H]noradrenaline release (462.8 G 18.5%; n Z 15) in the presence of increasing concentrations of memantine (,), CR 3394 (:) and DNQX (>). Each data point is the mean G SEM of four to six experiments performed in duplicate.

inhibition of release was not mediated by non-NMDA receptor activation because antagonism of AMPA receptors by DNQX had no effect (Fig. 5B).

3394. At 10 mM the reduction was 29 G 6% and at 30 mM 48 G 8%. In the same conditions, memantine (10 mM) decreased glutamate (70 G 5%) and NMDA (75 G 9%) toxicity.

3.6. CR 3394 enhances noradrenaline and dopamine basal release from rat hippocampal and striatal slices

4. Discussion

Since adamantane derivatives were first characterized as catecholamino-mimetic compounds, we investigated whether memantine and CR 3394 could affect the catecholamine resting release in perfused rat cerebral slices. In this assay system, we found marked differences between the two compounds. When memantine was perfused at concentrations that inhibit the NMDA receptor current, it caused no change in outflow of hippocampal [3H]noradrenaline and striatal [3H]dopamine. On the other hand, CR 3394 perfusion increased both hippocampal [3H]noradrenaline and striatal [3H]dopamine outflow in the low micromolar range (Table 1) at concentrations consistent with those that inhibit the NMDA receptor current. 3.7. CR 3394 reduces glutamate- and NMDA-induced toxicity Finally we tested the neuroprotective activity of CR 3394 against glutamate- and NMDA-induced toxicity in cultured neurons (Fig. 6). CR 3394 at concentrations of 5, 10 and 30 mM significantly reduced glutamate-induced damage (ÿ36 G 9% of cell viability) in cerebellar granule cells (DIV 8e9) in four different experiments. The average reduction in toxicity was 35 G 6% at 5 mM, 87 G 10% at 10 mM and 85 G 8% at 30 mM. Concentrations of CR 3394 higher than 30 mM were not used since they produced a weak toxic effect per se (ÿ19 G 9% at 60 mM). Treatment with CR 3394 before or after the induction of toxicity did not result in any added protection. Subsequently, given the low sensitivity of cerebellar granule cells to NMDA toxicity (reduction of cell viability: 10 G 8%; n Z 5 data not shown), we used cortical neurons at 10e11 DIV (Fig. 6B). NMDA-induced toxicity (reduction of cell viability: 43 G 5%, n Z 4) was decreased by CR

Overactivation of NMDA receptors occurs in several pathological situations and can lead to cell death through an excessive calcium influx (Arundine and Tymianski, 2003). Neurons with reduced metabolism appear to be highly sensitive to this excitotoxicity (Wieloch, 1985; Monyer et al., 1989). Thus, a rationale approach to block excitotoxicity would be the administration of NMDA antagonists. Unfortunately, the great efforts spent in the last two decades to develop NMDA antagonists for the treatment of acute or chronic disorders have so far produced very few molecules with good therapeutic index. Several potent and selective NMDA antagonists failed in clinical trials because of their side effects (Abi-Saab et al., 1998; Ikonomidou et al., 1999). However two aminoadamantane derivatives, memantine and amantadine, have features that make them almost devoid of side effects (Parsons et al., 1999a,b). The improved therapeutic safety of these NMDA antagonists has been ascribed to their moderate potency, strong voltage

Table 1 Basal outflow of [3H]cathecolamines

Saline CR 3394 1 mM CR 3394 3 mM CR 3394 10 mM Memantine 30 mM

Hippocampal rat slices [3H]NE release (%)

Striatal rat slices [3H]DA release (%)

100.0 G 1.1 120.7 G 7.7 148.6 G 8.5* 207.9 G 9.0** 104.4 G 3.3

100.0 G 1.8 146.9 G 6.8* 223.6 G 15.7** 285.0 G 10.8** 116.3 G 7.0

Basal outflow of [3H]noradrenaline from hippocampal rat slices or [3H]dopamine from striatal rat slices calculated after 30 min of perfusion in the presence of indicated drug concentration. Results are expressed as percentage of mean G SEM from three to five independent experiments performed in duplicate. Basal 5 min fractional release was 1.42 G 0.06% and 2.23 G 0.11% of total tritium for [3H]noradrenaline and [3H]dopamine, respectively. (* Z p ! 0.05; ** Z p ! 0.01; t-test).

G. Losi et al. / Neuropharmacology 50 (2006) 277e285

120

CTRL

**

100 80 60

**

* GLUT

40 20 5

10

30

GLUT + [CR 3394](µM)

of cell viability

B

A of cell viability

283

120 CTRL 100 80

*

NMDA

**

60 40 20 5

10

30

NMDA + [CR 3394](µM)

Fig. 6. CR 3394 reduces glutamate- and NMDA-induced toxicity. Representative experiments showing the neuroprotective effect of CR 3394 against (A) glutamate (100 mM C glycine 10 mM) induced toxicity on cerebellar granule cells at DIV 8 and (B) NMDA (500 mM C glycine 10 mM) induced toxicity on cortical neurons at DIV 11. The percentage of cell viability is measured with the MTT assay (see Section 2). Each data point is the mean G SEM of eight wells (*p ! 0.05; **p ! 0.01, t-test vs glutamate or NMDA group).

dependence and fast blocking/unblocking kinetics. These features allow them to reduce low tonic glutamate excitation and toxicity, but not to block normal synaptic transmission. The fact that normal glutamatergic transmission is retained in the presence of these drugs increases the signal to noise ratio of treated neurons and is likely why these antagonists show improved clinical profiles. Thus the pharmacological profile of memantine, the most studied aminoadamantane, can provide selection criteria for the preclinical development of new NMDA antagonists. Both CR 3394 and CR 3391 belong to a new series of adamantyl derivatives that are chemical modifications of memantine having amidine and guanidine moieties, respectively. These modifications were aimed to combine the NMDA receptor antagonism of the parent compound with selective antagonism of the inducible enzyme nitric oxide synthase (iNOS), in order to obtain new chemical entities exhibiting efficacy for the treatment of both neuropathic pain and neurological disorders. In this paper, we focus only on the modulation of NMDA receptor. Our data show that CR 3391 and CR 3394 reduce NMDAactivated currents in primary culture of cortical neurons. The antagonistic effects elicited by these compounds are dose-dependent and easily reversed with IC50s in the micromolar range. Both compounds are non-competitive antagonists of NMDA receptor complex and their effect is more pronounced at high agonist concentrations suggesting an openchannel blocker activity. However, we have to keep in mind that at low agonist concentrations, given the low open probability of channel opening, the drug effect could be underestimated. CR 3394 antagonism shows strong voltage dependence while that of CR 3391 is weakly affected by voltage. Evaluation of the voltage dependence of CR 3394 effect (Fig. 2E) revealed a zd value similar to that reported for memantine (zd Z 0.80 G 0.2; Parsons et al., 1999a,b). Fast offset kinetics and strong voltage dependence are two important characteristics of aminoadamantanes like memantine. Parsons et al. (1993, 1995) suggest that the combination of these features allows the compounds to rapidly leave the NMDA receptor during transient physiological activation by high glutamate concentrations released in the synaptic cleft. These same

features also cause aminoadamantanes to block the prolonged activation by low glutamate concentrations that is distinctive of chronic excitotoxic injuries. This is in contrast to antagonists that bind persistently to the channel, like MK801, which exert pronounced psychotomimetic side effects. Even though the kinetic of CR 3394 modulation was not analyzed in detail, our data on the recovery of the response to agonist suggest that this compound, similar to memantine and unlike MK801, is not persistently trapped in the channel since its effect was easily washed. The study of CR 3394 on miniature synaptic currents revealed that the compound reduced both duration and peak amplitude of events (the latter to the same extent of evoked whole-cell currents). The decrease in frequency, even though not significant, could be due to a reduced detection of small NMDA events caused by the decrease in peak amplitude. Our data on recombinant NMDA receptors expressed in HEK293 cells showed that CR 3394 is more potent (almost four-folds) on NR2B containing NMDA receptors. Furthermore in neurons from 8 to 9 DIV cultures when NMDA receptors are mainly comprised of NR2B subunits (Monyer et al., 1994; Akazawa et al., 1994), the stronger antagonism of CR 3394 confirms the preference of the compound for this receptor composition. Increasing evidence suggests that in preclinical trials NR2B-selective antagonists have lower incidence of side effects (Kemp and McKernan, 2002 for review). NR2B receptors in mature cells are predominatly extrasynaptic (Tovar and Westbrook, 1999; Rumbaugh and Vicini, 1999) and are believed to play a pro-apoptotic role when activated by excessive glutamate release (Hardingham et al., 2002). NR2A subunits, whose expression increases during the second postnatal week, instead localize preferentially at synaptic sites. We speculate that in mature neurons CR 3394 plays an important neuroprotective role by blocking mainly NR2B -containing extrasynaptic receptors while less effectively inhibiting NR2A-containing synaptic receptors. We saw that CR 3394 blocks NMDA-evoked noradrenaline release in slices of rat hippocampus at the same concentrations that reduce NMDA-evoked currents in primary cultures. However, CR 3394 had greater potency in blocking noradrenaline release compared to its effect in electrophysiological experiments. This could be due to an interaction with a different

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type of receptor or with an NMDA receptor of a different subunit composition (such as NR2D). As opposed to memantine, CR 3394 at low micromolar concentrations surprisingly was able to increase the basal release of noradrenaline and dopamine from hippocampal and striatal slices, respectively. The molecular mechanism underlying this effect is still unknown even though some preliminary experiments suggest a Ca2C-independent mechanism (unpublished observation). Since it was observed that memantine can affect nicotinic as well as 5HT3 receptor responses at low micromolar concentrations (Buisson and Bertrand, 1998; Aracava et al., 2005; Rammes et al., 2001) we cannot rule out the possibility that CR 3394 interacts with other membrane channels structurally similar to NMDA receptors. Finally we observed that CR 3394 protects from glutamateand NMDA-induced damage at concentrations that blocked NMDA-evoked currents in electrophysiological experiments. CR 3394 (10 mM) reduces glutamate-induced neurotoxicity more efficaciously than NMDA-evoked cell death (about ÿ87% vs ÿ30%, respectively). Glutamate activates also AMPA, KA and metabotropic receptors whose activity could be affected by CR 3394. We observed indeed a reduction of AMPA/KA mediated currents at high concentrations of CR 3394 (data not shown). Memantine was more effective than CR 3394 in protecting against NMDA-induced damage in cortical cells. The different magnitude of protection could be ascribed to a different potency of the two drugs or to a diverse affinity for other receptor types. Taken together our data show that CR 3394 is an NMDA antagonist with a good pharmacological profile in vitro. Like memantine, CR 3394 has a moderate affinity and strong voltage dependence. In addition CR 3394 shows subtype selective activity and increases basal catecholamine release. All these features render CR 3394 a novel and interesting candidate for the study and treatment of neurodegenerative disorders.

Acknowledgements We thank Prof. Giorgio Racagni for critically reading the manuscript and giving us the opportunity to put the basis of a fruitful collaboration with Rottapharm. We also thank Dr Kate Prybylowski and Dr Fabrizia Manicardi for proofreading the manuscript. G.L. was supported by Consorzio SPINNER (Bologna, Italy).

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