GlyT1 (IC50=18 ~ 30 nM)

In human SK-N-MC and rat C6 cells, SSR504734 (15 nM-86 μM; 10 min) decreases glycine uptake[1].

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Blockade by SSR504734 of In Vitro [14C]glycine Uptake [1]
Southern blot analysis of RT-PCR products revealed that human SK-N-MC cells only expressed the GlyT1 mRNA isoform a. The other GlyT1 mRNA isoforms b and c were not detected in this cell line (data not shown). [14C]glycine uptake in SK-N-MC cells was saturable, sodium- and chloride-dependent (data not shown), and exhibited a Km=21±4 μM and a Vmax=667±186 pmol/min/106 cells (n=4 independent experiments). Specific uptake was typically 90% of total radioactivity. This is consistent with characteristics described previously for these transporters (Mallorga et al, 2003).
SSR504734 inhibited [14C]glycine uptake in human SK-N-MC and rat C6 cells, with IC50 values of 18±6 and 15±2 nM, respectively (Table 1). The (R,R) enantiomer SSR506204 was approximately 10-fold less potent in blocking the uptake of [14C]glycine, both in human and rat GlyT1.
SSR504734 was inactive (inhibition lower than 50% at 1 μM) against ca 120 targets, including glycine, glutamate, DA, 5-HT, adrenaline, noradrenaline, histamine and muscarinic receptors, enzymes such as MAO, and uptake systems such as DA, 5-HT and noradrenaline transporters (assays performed by Cerep, Celle l'Evescault, France, data available upon request). SSR504734 also had no effect (IC50>1 μM) on human GlyT2 and D-serine transporters, and on murine proline, glutamate, and GABA transporters (data available upon request).

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Antagonism by SSR504734 of the Decrease in Firing Rate of Prefrontal Cortex Neurons Induced by the CB1 Receptor Agonist WIN55212-2 [1]
Baseline firing of medial PFC cells occurred mainly in bursts of 2–4 spikes of approximately 1.5 ms duration and 0.2 mV amplitude at a spontaneous (control) frequency ranging from 0.3 to 6.0 Hz (mean±SEM: 2.5±0.3 Hz, n=20; Figure 7a, foremost left bars of the left panel). The i.v. administration of WIN55212-2 (0.1 mg/kg) elicited inhibition of firing rate in four out of the five PFC cells recorded. Inhibition was maximal (52.5±6.6%; Figure 7b, second bar from left) 5 min after administration and remained statistically significant (F(8,24)=9.69, P<0.01) for the following 20 min, before returning to baseline.

Oral bioavailability of SSR504734 (ip and po; 1-100 mg/kg; once) is good[1]. Specific glycine uptake is rapidly and significantly reduced by SSR504734 (ip; 30 mg/kg; once)[1]. (ip; 10 mg/kg; once) raises the extracellular Glycine levels in the rats' freely moving prefrontal cortex (PFC)[1].

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Noncompetitive N-methyl-D-aspartate (NMDA) blockers induce schizophrenic-like symptoms in humans, presumably by impairing glutamatergic transmission. Therefore, a compound potentiating this neurotransmission, by increasing extracellular levels of glycine (a requisite co-agonist of glutamate), could possess antipsychotic activity. Blocking the glycine transporter-1 (GlyT1) should, by increasing extracellular glycine levels, potentiate glutamatergic neurotransmission. SSR504734, a selective and reversible inhibitor of human, rat, and mouse GlyT1 (IC50=18, 15, and 38 nM, respectively), blocked reversibly the ex vivo uptake of glycine (mouse cortical homogenates: ID50: 5 mg/kg i.p.), rapidly and for a long duration. In vivo, it increased (minimal efficacious dose (MED): 3 mg/kg i.p.) extracellular levels of glycine in the rat prefrontal cortex (PFC). This resulted in an enhanced glutamatergic neurotransmission, as SSR504734 potentiated NMDA-mediated excitatory postsynaptic currents (EPSCs) in rat hippocampal slices (minimal efficacious concentration (MEC): 0.5 microM) and intrastriatal glycine-induced rotations in mice (MED: 1 mg/kg i.p.). It normalized activity in rat models of hippocampal and PFC hypofunctioning (through activation of presynaptic CB1 receptors): it reversed the decrease in electrically evoked [3H]acetylcholine release in hippocampal slices (MEC: 10 nM) and the reduction of PFC neurons firing (MED: 0.3 mg/kg i.v.). SSR504734 prevented ketamine-induced metabolic activation in mice limbic areas and reversed MK-801-induced hyperactivity and increase in EEG spectral energy in mice and rats, respectively (MED: 10-30 mg/kg i.p.). In schizophrenia models, it normalized a spontaneous prepulse inhibition deficit in DBA/2 mice (MED: 15 mg/kg i.p.), and reversed hypersensitivity to locomotor effects of d-amphetamine and selective attention deficits (MED: 1-3 mg/kg i.p.) in adult rats treated neonatally with phencyclidine. Finally, it increased extracellular dopamine in rat PFC (MED: 10 mg/kg i.p.). The compound showed additional activity in depression/anxiety models, such as the chronic mild stress in mice (10 mg/kg i.p.), ultrasonic distress calls in rat pups separated from their mother (MED: 1 mg/kg s.c.), and the increased latency of paradoxical sleep in rats (MED: 30 mg/kg i.p.). In conclusion, SSR504734 is a potent and selective GlyT1 inhibitor, exhibiting activity in schizophrenia, anxiety and depression models. By targeting one of the primary causes of schizophrenia (hypoglutamatergy), it is expected to be efficacious not only against positive but also negative symptoms, cognitive deficits, and comorbid depression/anxiety states [1].

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Blockade by SSR504734 of Ex Vivo [14C]glycine Uptake in Mouse Cortical Homogenate.
Increase by SSR504734 of Extracellular Levels of Glycine in the Prefrontal Cortex of Freely Moving Rats.
Potentiation by SSR504734 of Evoked NMDA-Mediated Excitatory Postsynaptic Currents in Rat Hippocampal Slices.
Augmentation by SSR504734 of the Number of Contralateral Rotations Induced by Intrastriatal Microinjection of Glycine in Mice.
Antagonism by SSR504734 of the Decrease of [3H]acetylcholine Release Induced by the CB1 Receptor Agonist WIN55212-2 in Rat Hippocampal Slices.
Antagonism by SSR504734 of the Decrease in Firing Rate of Prefrontal Cortex Neurons Induced by the CB1 Receptor Agonist WIN55212-2.
Increase by SSR504734 of Extracellular Levels of Dopamine in the Prefrontal Cortex of Freely Moving Rats.
Blockade by SSR504734 of Ketamine-Induced Increase of Brain Metabolic Activity in Mice.
Antagonism by SSR504734 of Locomotor Hyperactivity Induced by MK-801 in Mice.
Reversal by SSR504734 of MK-801-Induced Increase of the Absolute Power of the Alpha1 Band in Rat Cortex.
Antagonism by SSR504734 of the Impairment of Novelty Discrimination and the Hypersensitivity to an Acute Challenge with d-Amphetamine, in Adult Rats Treated with Phencyclidine at the Neonatal Stage.
Reversion by SSR504734 of a Spontaneous Deficit of Prepulse Inhibition of the Startle Reflex in DBA/2 Mice.
Reduction by SSR504734 of Ultrasonic Distress Vocalizations in Rat Pups.
Improvement by SSR504734 of Physical State Degradation in Mice Subjected to a Chronic Mild Stress.
Reduction by SSR504734 of Paradoxical Sleep in Rats [1].

Effects of SSR504734 and Its (R,R) Enantiomer SSR506204 on In Vitro [14C]glycine Uptake [1]
Human neuroblastoma (SK-N-MC) and rat astrocytoma (C6) cell lines were maintained at 37°C, in humidified air with 5% CO2, in monolayer culture in growth medium (MEM for SK-N-MC cells and HAM-F12 for C6 cells) containing 10% fetal calf serum. Cells, collected by trypsination, were subcultured twice a week. The presence of GlyT1 in C6 cells has been reported by Gomeza et al (1995). In SK-N-MC cells, the presence of GlyT1 was assessed as follows: total RNA of cells was isolated using Trizol reagent, and then reverse-transcribed and submitted to PCR amplification (initial denaturing step at 93°C for 1 min, and 35 cycles consisting of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min) in the presence of two primers specific for one of the three known GlyT1 isoforms (Kim et al, 1994). Sense primers were 5′-TGC CAA AGG GAT GCT GAA TG-3′ for isoform a and 5′-GCG GCT CAT GGA CCT GTG-3′ for both isoforms b and c. The antisense primer sequence, common to the three isoforms, was 5′-CGC AGA TGA GCA TGA TG-3′. For confirmation, RT-PCR products were analyzed by Southern blot using a 32P-labeled internal oligonucleotide probe specific for each human GlyT1 isoform. Blots were scanned with a Storm Phosphoimager.

At 48 h before [14C]glycine uptake experiments, cells were plated at a density of 20 000–30 000 per well in 96-well culture dishes previously coated with fibronectin for SK-N-MC cells or poly-D-lysine for C6 cells. Assays were performed at 37°C in 200 μl of HEPES buffer (HB) containing (in mM) NaCl (147), KCl (5), MgCl2 (2), CaCl2 (2), HEPES (10), D-glucose (10), and L-alanine (5) with pH 7.4. Growth medium was removed and after washing with HB, cells were incubated for 10 min with test compounds. Uptake was started by adding 10 μM [14C]glycine (112.4 mCi/mmol; NEN Life Science Products, Paris, France). Nonspecific uptake was determined with 10 mM unlabeled glycine. After 10 min, cells were washed twice with HB, and scintillation fluid was added to the wells. Radioactivity was measured by liquid scintillation in a Wallac MicroBeta counter. Results are expressed as the drug concentration required to inhibit 50% (IC50) of specific [14C]glycine uptake, and were obtained by nonlinear regression analysis.

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Receptor Selectivity of SSR504734 [1]
Interaction of SSR504734 with about 120 different receptors, ion channels, enzymes, or transporters was evaluated by use of standard protocols or by internal studies.

Cell Viability Assay[1]
Cell Types: Human neuroblastoma (SK-N-MC) and rat astrocytoma (C6) cells
Tested Concentrations: 15 nM-86 μM
Incubation Duration: 10 min
Experimental Results: demonstrated IC50 values of 18 and 15 nM for human SK-N-MC and rat C6 cells, respectively.

Animal/Disease Models: Male SD (Sprague-Dawley) rats[1]
Doses: 1-100 mg/kg
Route of Administration: intraperitoneal (ip)injection and po (oral gavage).; 1-100 mg/kg; once
Experimental Results: demonstrated ID50 values of 5.0 and 4.6 mg/kg for ip and po treatments, respectively.

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Animal/Disease Models: Male SD (Sprague-Dawley) rats[1]
Doses: 30 mg/kg
Route of Administration: intraperitoneal (ip)injection; 30 mg/kg; once
Experimental Results: Maintained at about 80% inhibition from 1 to 7 h after administration.

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Animal/Disease Models: Male SD (Sprague-Dawley) rats[1]
Doses: 10 mg/kg
Route of Administration: intraperitoneal (ip)injection; 10 mg/kg; once
Experimental Results: Produced a rapid and sustained increase in PFC extracellular levels of glycine.\n

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\n\nEffects of SSR504734 and SSR506204 on Ex Vivo [14C]glycine Uptake [1]
\nMale OF1 mice (20–25 g) were killed 30 or 60 min after i.p. or p.o. administration, respectively. Cortical tissues were rapidly dissected and homogenized on ice using a polytron apparatus (1 g tissue for 10 volumes of cold HB). The assay performed on fresh homogenates was started by adding 10 μM [14C]glycine to 20 μl of tissue in HB (final protein concentration: 0.8–0.9 mg/ml). Nonspecific uptake was determined with 10 mM unlabeled glycine. The uptake was performed for 10 min at 25°C, and stopped by aspiration onto MultiScreen glass fiber filter plate using a MultiScreen vacuum manifold. The filter was washed twice with ice-cold HB, dried, and soaked with scintillation fluid. Radioactivity was measured by liquid scintillation in a Wallac MicroBeta counter.\n
\nA time-course study was performed at 5 min, 15 min, 1 h, 4 h, 7 h, 16 h, and 24 h after oral treatment with 30 mg/kg of SSR504734, using the protocol described above.\n
\nResults are expressed as the percentage of [14C]glycine uptake vs the control (vehicle-treated) group and as the dose of the compound that inhibits 50% of uptake (ID50) calculated by nonlinear regression analysis. For the time-course experiment, data were analyzed with a one-way ANOVA, followed by post hoc Dunnett's tests.\n

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\n\nEffect of SSR504734 on Extracellular Levels of Glycine Measured in the Prefrontal Cortex of Freely Moving Rats [1]
\nMale Sprague–Dawley rats (280–330 g) were housed two per cage. At 2 days before the dialysis measurements, they were anesthetized with chloral hydrate (400 mg/kg i.p., 10 ml/kg of body weight) and placed in a stereotaxic apparatus. Anesthesia was maintained throughout surgery as necessary with supplementary doses of chloral hydrate. Body temperature was monitored by a rectal probe and adjusted (37±1°C) by a homeothermic blanket. The skull and the dura were opened to allow the implantation of a guide cannula in the medial prefrontal cortex (PFC). The coordinates were 2.5 mm anterior to bregma, 0.6 mm lateral to the midline, and 1.3 mm below the dural surface. A dental cement cap held the cannula in place, and three screws anchored the cap to the skull. The rats were individually housed postsurgery and allowed 2 days of recovery before the start of the experiment. On the day of the experiment, animals were placed in a microdialysis bowl, the obturator of the cannula was removed, and a 3 mm microdialysis probe was inserted into the guide cannula. The probe was perfused at a constant flow rate of 1 μl/min using a microinjection pump with a gassed Ringer's solution containing (in mM) NaCl (145), KCl (2.7), CaCl2 (1.2), MgCl2 (1), Na2HPO4 (2.3), and NaH2PO4 (0.45); pH 7.4. Microdialysis sampling started 120 min after probe placement into the PFC. The outlet of the probe was connected to an online derivatization system as described previously (Bert et al, 1996), allowing direct analysis of dialysate samples collected every 15 min.\n
\n\nGlycine levels were measured in 15 μl dialysate samples using capillary electrophoresis (CE) with laser-induced fluorescence detection. Before analysis, the samples were derivatized using naphtalene-2,3-dicarboxaldehyde and sodium cyanide, as described previously (Bert et al, 1996). CE experiments were performed on a P/ACE MDQ capillary electrophoresis system coupled to an external Zetalif fluorescence detector. The excitation was performed by an Omnichrome helium–cadmium laser at a wavelength of 442 nm with a 30 mW excitation power. The emission intensity was measured at a wavelength of 490 nm. Separations were carried out with a fused-silica capillary of 50 μm i.d. and 375 μm o.d. having a total length of 55 cm and an effective length of 38.9 cm with an applied voltage of 25 kV (ie 65 μA current). Borate buffer (75 mM) containing β-cyclodextrin (1 mM), pH 10.5, was used for CE running.\n
\nAt the end of the experiments, an injection of sky blue solution was performed through the probe and animals were killed with an overdose of pentobarbital. The brain was removed, frozen, and 50-μm-thick sections were cut with a cryostat to verify correct placement of the microdialysis probe.\n
\nGlycine levels in fractional samples were converted to a percentage of the mean value of the 90 min baseline measurements before treatment. Time-course effects of SSR504734 and SSR506204 on glycine levels were analyzed by two-way ANOVAs, with treatment as a between-subjects factor and time of sampling as a within-subjects factor, followed by Dunnett's post hoc tests. Dose effects of SSR504734 were evaluated by comparing the area under the curve during the first 180 min after i.p. injection of the drug or vehicle. Statistical analysis was carried out by a one-way ANOVA followed by Dunnett's post hoc tests.\n

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\n\nEffect of SSR504734 on Evoked NMDA-Mediated Excitatory Postsynaptic Currents in Rat Hippocampal Slices [1]
\nSprague–Dawley rats (17–22 days old) were killed and their brains removed and sectioned (coronal 0.3-mm-thick slices) with a Campden 752M microslicer in an artificial cerebrospinal fluid (aCSF) solution containing (in mM) NaCl (126), KCl (3), MgCl2 (1), KH2PO4 (1), CaCl2 (1), NaHCO3 (25), and glucose (11), pH 7.35, at approximately 0°C and aerated with 95% O2 and 5% CO2. After at least 1 h in aCSF at room temperature, slices were transferred to the recording chamber and superfused continuously with aCSF at 30–32°C. Patch-clamp whole-cell recordings of layer CA1 pyramidal cells were obtained with borosilicate glass electrodes (resistance: 4–7 MΩ), filled with a 290 mosmol solution containing (in mM) KMeSo4 (130), EGTA (10), ATP (2), GTP (0.5), and QX314 (5); pH 7.2.\n \n\nEffects of SSR504734 and SSR506204 on Contralateral Rotations Induced by Intrastriatal Microinjection of Glycine in Mice [1]
\nAt 1 week before experiments, female CD1 mice (25–27 g) were housed in groups of 10 per cage. Details of the microinjection procedure have been published elsewhere (Poncelet et al, 1993).\n\nSSR504734, SSR506204, or vehicle was injected i.p. 30 min before intrastriatal microinjection of glycine (0.001 ng, a subliminal dose). In another experiment, MK-801 (0.03 mg/kg i.p.) or vehicle was administered 15 min before SSR504734 (1 mg/kg i.p.) or vehicle, followed 30 min later by a microinjection of 0.001 ng glycine.\n

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\n\n\nEffects of SSR504734 and SSR506204 on the Decrease of [3H]acetylcholine Release Induced by WIN55212-2 in Rat Hippocampal Slices [1]
\nMale OFA rats (250–350 g) were decapitated, their brains quickly removed, and the hippocampus dissected on an ice-cold aluminum block. Following dissection, 350 μm tissue slices were cut with a vibratome, and the slices transferred to 20 ml of Krebs buffer (pH 7.4) saturated with 95% O2 and 5% CO2 and containing 20 μM [3H]choline (86 Ci/mmol). Following incubation with the radiolabel for 30 min at 37°C, slices were transferred to 18 superfusion chambers (three slices per chamber). Slices were superfused at 37°C, at a rate of 0.5 ml/min, with oxygenated Krebs buffer containing 10 μM hemicholinium to prevent the reuptake of [3H]choline. Sample collection was started after a 45 min wash period. Two 3 min fractions were subsequently collected in order to measure basal release. The cannabinoid-1 (CB1) receptor agonist WIN55212-2 (10−6 M) was introduced during the fifth collection period and was present until the end of the experiment. SSR504734 was added 9 min prior to WIN55212-2. Release of [3H]acetylcholine ([3H]ACh) was electrically evoked (rectangular pulses, 2 Hz, 2 ms, 1 mA) for 3 min, after which the original Krebs buffer was introduced to re-establish basal release. Results are expressed as percent of inhibition of controls. Data were analyzed by means of one-way ANOVAs followed by Dunnett's post hoc tests. An IC50 for the effects of SSR504734 was determined with a four-parameter logistic model using a weighed linear curve fitting program.\n

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\n\nEffects of SSR504734 and SSR506204 on the Decrease of Firing Rate of Prefrontal Cortex Neurons Induced by WIN55212-2 [1]
\nMale Sprague–Dawley rats (280–400 g) were anesthetized with chloral hydrate (400 mg/kg i.p.) and placed in a stereotaxic apparatus. Adequate level of anesthesia was maintained throughout the experiment with supplementary doses of choral hydrate. The animal's temperature was maintained at 37±0.1°C by an electronic heating pad device. After appropriate surgery, small burr holes were drilled into the skull bone above the areas to be explored. Extracellular recordings were made with 1-μm-tipped glass micropipettes filled either with 1 M NaCl, or 0.5 M sodium acetate containing 2% Pontamine Sky Blue (impedance 4–7 MΩ at 100 Hz) aimed at medial PFC cells: 2.9–3.2 and 0.6–0.8 mm anterior and lateral to bregma, respectively, and 2.2–3.6 mm below the cortical surface (Paxinos and Watson, 1998). A hydraulic microdrive allowed fine 3-D electrode movement. Action potentials (spikes) were amplified, filtered (400 Hz–5 kHz), and monitored on a digital oscilloscope and an audio amplifier. The potentials were fed into a window discriminator and analyzed online using a ‘μ1401 Intelligent Laboratory Interface’ connected to a PC running the CED ‘Spike2’ software.\n
\nFiring rate histograms were run in a continuous mode (10 s bin size). The firing rate was allowed to stabilize for 10–15 min before the first i.v. injection of SSR504734, SSR506204, or vehicle (50 μl of 1 N HCl+distilled water for 1 ml/kg v/w). WIN55212-2 was administered (0.1 mg/kg i.v.) 10 min after the test drug (or vehicle). The firing rate was then monitored for at least an additional 30 min. A single cell was recorded per rat. All i.v. injections were performed over a duration of 1 min.\n

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\n\n\nEffect of SSR504734 on Extracellular Levels of Dopamine in the Prefrontal Cortex of Freely Moving Rats [1]
\nNote: The capacity of some atypical antipsychotics to augment dopaminergic neurotransmission in the PFC has been suggested by some authors (Kapur and Remington, 1996) to form the basis for their beneficial impact on negative symptoms and cognitive deficits in schizophrenic patients. For this reason, we also measured the ability of SSR504734 to augment extracellular DA tone in this structure.\n\n

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\n\nEffect of SSR504734 on Ketamine-Induced Increase of Brain Metabolic Activity in Mice [1]
\nMale OF1 mice (28–37 g) were housed 10 per cage, 1 week before the experiment. On the day of the experiment, they were housed in individual cages at least 1 h before treatment. Vehicle or SSR504734 was injected i.p. 30 min before administration of ketamine (30 mg/kg i.p.) or saline (ketamine was chosen instead of PCP or MK-801 since it was the compound used in the princeps study by Duncan et al, 1998a). The solution of 2-deoxy-D-[1-14C]glucose (2-DG) was slowly injected (13 μCi in 0.3 ml of saline, over 20 s) into the tail vein 2 min after injection of ketamine or saline. Mice were killed by decapitation 5 min after the i.v. injection of 2-DG. Brains were removed and frozen by immersion in cooled isopentane (−45°C), and then stored at about −80°C until sectioned.\n

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\n\nEffects of SSR504734 and SSR506204 on Locomotor Hyperactivity Induced by MK-801 in Mice [1]
\nMale Swiss mice (18–21 g) were individually isolated in boxes and pretreated i.p. with SSR504734, SSR506204, or vehicle, immediately followed by vehicle or MK-801 (0.2 mg/kg i.p.). At 30 min after the second injection, they were placed in actimeters (20 cm diameter, 9.5 cm height; Apelex, France) equipped with two perpendicular light beams 1.5 cm above the floor. Locomotor activity (number of interrupted light beams) was recorded for a period of 30 min after placing the mouse into the actimeter. Data were analyzed with one-way ANOVAs, followed by Dunnett's post hoc tests.\n

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\n\nEffect of SSR504734 on the Increase of the Alpha1 Band Spectral Energy Induced by MK-801 in Rats [1]
\nMale Sprague–Dawley rats (220–240 g) were anesthetized with sodium pentobarbital (50 mg/kg i.p.) and mounted in a stereotaxic apparatus. Cortical electrodes (small stainless-steel screw 0.9 mm in diameter) were screwed into the bone at the level of the sensorimotor cortex (1.5 mm lateral to the median suture and 1.5 mm behind the fronto-parietal suture), the visual cortex (1.5 mm lateral to the median suture and 1.5 mm in front of the parieto-occipital suture), and the cerebellum (reference electrode). Cortical electrodes were soldered to a miniature connector fixed with dental cement to the cranium.\n
\nAfter 3 weeks of postoperative recovery, animals were placed in Plexiglas cylinders (60 cm diameter) with free access to food and water. For studying the effect of SSR504734 on the increase of the alpha1 spectral energy induced by MK-801, after 15 min of EEG recording (first period: control), vehicle or SSR504734 was administered i.p., followed 15 min later by MK-801 (0.2 mg/kg i.p.). EEG signals were amplified and filtered (1–100 Hz). Sequential spectral analysis was performed automatically by means of a computerized system (‘Coherence 32’ software, Deltamed, Paris, France). Signals were sampled (128 Hz) for the Fourier transformation, which allowed calculation of the power variable (μV2). Absolute power spectra of EEG signals were computed every 30 s from 0.5 to 32 Hz in steps of 0.5 Hz and were calculated for each of the following frequency bands: delta band (0.5–3.5 Hz), theta band (theta1: 4–6 Hz; theta2: 6.5–8 Hz), alpha band (alpha1: 8.5–11 Hz; alpha2: 11.5–13.5 Hz), and beta band (14–32 Hz). MK-801 was tested at 0.2 mg/kg i.p., because it induced a large increase of the absolute power within the alpha1 band at this dose.\n
\nThe EEG spectral power of the alpha1 band was averaged for 15 min before administration of MK-801, and every 10 min for 1 h after injection of MK-801. Drug-induced changes were evaluated by calculating the ratio of the absolute power after i.p. injection of SSR504734 or vehicle on the absolute power during the control period (taken as 100%) and expressed as percent of the power during the control period.\n

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\n\n\nEffect of SSR504734 on the Impairment of Novelty Discrimination in Adult Rats Treated with Phencyclidine at the Neonatal Stage [1]
\nFemale Wistar Han rats were obtained with 10 male pups on postnatal day 3 (PN 3). Pups were treated on PN 7, 9, and 11 with 10 mg/kg of PCP (s.c. administration, 1 ml/100 g body weight) or vehicle. Pups from the same litter received an identical treatment. The mother and pups were housed together until weaning at PN 21, at which stage pups where housed five per cage until 2 weeks before the beginning of behavioral experiments, when they were housed individually. Behavioral experiments were performed once they reached the adult stage (between PN 60 and PN 108).\n
\n \n\nExperiments were performed during the dark phase, under infrared illumination (15 lux). Juvenile rats were isolated 30 min before being placed into the home cage of an adult rat. The cage was placed underneath a video camera and the mesh top was removed and replaced by a Plexiglas cover. A first (familiar) juvenile was placed inside the home cage containing one adult rat for a period of 30 min. A second (novel) juvenile was introduced at the end of this period. Durations of investigation behavior (nosing, sniffing, grooming, close chase of the juvenile rat) between the adult rat and each of the two juveniles were recorded manually for a period of 5 min following the introduction of the novel juvenile, by an observer located in an adjacent room fitted with a video monitor. SSR504734 or vehicle was administered i.p. to the adult rat 30 min before exposure to the first juvenile. Each adult rat was subjected to four treatments: one vehicle and three doses of SSR504734. Treatments were administered with 1 or 2 days between each treatment.\n

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\n\nEffect of SSR504734 on the Hypersensitivity to an Acute Challenge with d-Amphetamine in Adult Rats Treated with Phencyclidine at the Neonatal Stage [1]
\nAdult rats used in this experiment were those used in the novelty discrimination experiment. Animals were first placed into the experiment room 1 h before the experiment was started. They were then placed in activity chambers (Plexiglas boxes 40 × 40 × 30 cm), fitted with infrared beams (2.5 cm apart, 2.5 cm above the floor). Their basal locomotor activity was first evaluated during 30 min (habituation period). At the end of this period, they were injected i.p. with SSR504734 or vehicle, and replaced into the actimeters. After 30 min, they were injected i.p. with d-amphetamine (2 mg/kg) or vehicle, and their locomotor activity was measured during 90 min. Each rat was subjected to five treatments: two vehicle (alone and with d-amphetamine) and three doses of SSR504734 in combination with d-amphetamine. Treatments were administered with a minimum of 3 days between each treatment.\n\nData are expressed as the mean number of infrared beam interruptions, and were analyzed with a two-way ANOVA for repeated measures, with treatment at the neonatal stage as the between-subjects factor and acute treatment at the adult stage as the within-subjects factor, followed by appropriate post hoc tests.\n

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\n\nEffect of SSR504734 on a Spontaneous Deficit of Prepulse Inhibition of the Startle Reflex in DBA/2 Mice [1]
\nMale DBA/2J mice (22–26 g) were housed in groups of four per cage. They were subjected to a selection session (see below) 13 days after their arrival. Mice were tested in six startle boxes. The startle reflex was detected and transduced by a piezoelectric accelerometer positioned underneath the startle platform, and digitized and stored by a microcomputer running the SR-LAB software (that also controlled all other events). Mice were first placed inside a Plexiglas restraint cylinder (3.7 × 12.8 cm long) fixed on top of the startle platform. The selection session was then started: after 5 min of habituation, 20 stimuli (separated by a variable interval: 5–25 s, by steps of 5 s) were delivered—17 startling pulses stimuli (P; 40 ms duration, 120 dB intensity) intertwined with three prepulses at 30 dB followed by a pulse stimulus (p30/P). For these last three stimuli, prepulses were of 20 ms duration and 88 dB (that is 30 dB above background noise of 58 dB), and pulses were as above, with 100 ms between the end of the prepulse and the beginning of the pulse. Audio stimuli were calibrated (±2 dB, A scale) with a sound level meter (model CDA 830, Chauvin Arnoux, France). Startle platforms were calibrated (SR-LAB calibrator standardization unit), with less than 3% variation between platforms. The startle reflex was recorded during the 100 ms following onset of the startling stimulus (sampling interval: 1 ms). Startle amplitude was defined as the peak amplitude of the downward force exerted by the startled mouse on the platform. Throughout the experiment, mice were in the dark. This selection session was used to distribute mice across groups with similar levels of basal startle for subsequent testing.\n
\nOn the second day (test session), mice were injected i.p. with vehicle or SSR504734, 30 min before being placed into the restraint cylinders. The test session started with a 5 min habituation period, followed by delivery of four pulses alone (P). These were followed by two identical blocks of stimuli: each block comprised a sequence of 26 stimuli, divided into five conditions: 10 startle pulses without prepulses (P), four startle pulses preceded by 6 dB prepulses (p6/P), four startle pulses preceded by 18 dB prepulses (p18/P), four startle pulses preceded by 30 dB prepulses (p30/P), and four stimuli with no prepulses and no pulses (nop/noP). The five conditions were delivered in a pseudo-random order. Otherwise, all other parameters were similar to those in effect for the selection session described above.\n\n

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\n\nEffects of SSR504734 and SSR506204 on Ultrasonic Distress Vocalizations in Rat Pups [1]
\nFemale Sprague–Dawley rats were obtained with 10 male pups on PN 3–4. The procedure, adapted from the one described by Gardner (1985), was as follows: each pup (PN 7) was first separated from its mother and littermates, injected s.c. (0.1 ml) with SSR504734 or vehicle, and returned to its mother. After 30 min, the pup was placed in a soundproof cage. The Ultravox system was used to record ultrasonic vocalizations (UV, in the 40 kHz range). First, a modified ultrasound detector (Mini-3 bat model) connected to an electret microphone (positioned next to the pup) was used to transform ultrasonic sound into audible sound. The signal was then filtered (user-defined frequency range and amplitude threshold) and sent to a PC, where the UltraVox software recorded each bout of ultrasonic vocalizations during the 3 min test session.\n

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\n\n\nEffect of SSR504734 in a Chronic Mild Stress Procedure in Mice [1]
\nThe chronic mild stress (CMS) protocol, originally described by Willner et al (1992) for rats, was adapted from the one described by Kopp et al (1999) for mice. It consisted in the sequential application of a variety of mild stressors, including restraint, forced swimming, water and/or food deprivation, pairing with another stressed animal, each applied for a period ranging from 2 to 24 h, in a schedule lasting 3 weeks, which was repeated as necessary until the end of the experiment.\n
\nAdministration of SSR504734 (10 mg/kg i.p.) was started 2 weeks after the beginning of the CMS. The dose chosen was one that had been shown to have efficacy in mice in neurochemical and behavioral models. Animals (BALB/c male mice, 21–28 g at the start of the experiment) were injected i.p. once a day until all experiments were completed (33 days).\n

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\n\nEffects of SSR504734 and SSR506204 on the Sleep/Wakefulness Cycle in Freely Moving Rats [1]
\nDetails of the surgical procedure were as described in ‘Effect of SSR504734 on the Increase of the Alpha1 Band Spectral Energy Induced by MK-801 in Rats’.\n
\nRecording sessions took place from 1100 to 1700 during 3 consecutive days: precontrol (Day 1), drug challenge (Day 2), and postcontrol (Day 3). EEG activity in sensorimotor and visual cortices was recorded with the cerebellar electrode as the reference. Three stages were differentiated: wakefulness (W; characterized by low-voltage EEG activity), slow-wave sleep (SWS; characterized by an increase in EEG activity, that is, high-amplitude slow waves with some bursts of sleep spindles), and paradoxical sleep (PS; characterized by hypersynchronization of the theta rhythm in the visual cortex). Analysis of the EEG signal was performed automatically by means of a computerized system discriminating between the various sleep phases using spectral frequency analysis (‘Coherence 32’ software, Deltamed, Paris, France). Vehicle (Days 1 and 3), SSR504734, or SSR506204 (Day 2) was administered i.p. 15 min before recording. The effects of compounds on the time spent in W, in SWS, in PS, the number of episodes of PS, and the latency time to enter PS were analyzed over a 6 h period and are expressed as the percentage of the control values obtained on Day 1. For each treatment, statistical analysis was carried out using one-way ANOVAs for repeated measures on raw values, followed by Dunnett's post hoc tests (Day 1 as control).

Peripheral administration of SSR504734 dose-dependently inhibited the uptake of isolated [14C]glycine by the mouse cerebral cortex. The ID50 values for intraperitoneal and oral administration were 5.0 and 4.6 mg/kg, respectively, and the oral/intraperitoneal ratio was close to 1, indicating good oral bioavailability (Figure 2a). [1]

Safety profile of SSR504734 [1]
SSR504734 did not cause rigidity in mice at intraperitoneal doses up to 30 mg/kg (data not shown), indicating that it does not cause extrapyramidal symptoms in patients; in this respect, it is consistent with commonly used atypical antipsychotics such as clozapine, amisulpride, olanzapine and quetiapine. In addition, it did not increase prolactin levels in rats (intraperitoneal doses up to 30 mg/kg; data not shown), while increased prolactin is a common side effect of most antipsychotics.

[1]. Neurochemical, electrophysiological and pharmacological profiles of the selective inhibitor of the glycine transporter-1 SSR504734, a potential new type of antipsychotic. Neuropsychopharmacology. 2005 Nov;30(11):1963-85.

SSR504734 is a potent, selective, and orally effective GlyT1 inhibitor that has shown activity in both schizophrenia and animal models of anxiety/depression. Extensive in vitro, ex vivo, and in vivo studies have demonstrated enantioselectivity of its activity, as its (R,R) enantiomer, SSR506204, exhibits low or no activity. Its mechanism of action targets glutamatergic hypoactivity, considered a major cause of schizophrenia. Therefore, it is expected to be effective not only for positive symptoms but also for negative symptoms, cognitive deficits, and comorbid depression and anxiety. A recent double-blind, placebo-controlled study further reinforces the potential use of GlyT1 inhibitors as a novel treatment for schizophrenia. This study demonstrated that the combination of the weak GlyT1 inhibitor sarcosine and risperidone significantly reduced positive, negative, and cognitive symptoms of schizophrenia, with efficacy comparable to that previously observed with the complete glycine site agonist D-serine (Tsai et al., 2004). [1] This is the first report to elaborate in detail the neurochemical, electrophysiological, and pharmacological properties of the novel selective reversible GlyT1 inhibitor SSR504734. Other GlyT1 blockers have been reported in the literature, but their low affinity (sarcosine, dodecylglycamide) or irreversible blocking effect (ALX5407) makes them unattractive to pharmacological studies of GlyT1.

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SSR504734 is an in vitro and ex vivo selective GlyT1 blocker [1]
SSR504734 effectively blocks the uptake of [14C]glycine in natural human SK-N-MC cell lines expressing the GlyT1a subtype and in rat C6 cell lines containing GlyT1. This effect of SSR504734 is stereoselective because its (R,R) enantiomer SSR506204 is much less potent than the former. SSR504734 inhibits glycine transport on human and murine GlyT1 transporters (IC50 approximately 20 nM), with efficacy between ALX5407 (3 nM, Atkinson et al., 2001; 220 nM, Herdon et al., 2001, both targeting hGlyT1c; 26 nM, targeting hGlyT1b, Smith et al., 2004; 10 nM, targeting rGlyT1a, Kinney et al., 2003) and ORG 24598 (120 nM, targeting hGlyT1b, Brown et al., 2001), significantly higher than sarcosine or dodecylglycylamide, two earlier GlyT1 inhibitors (IC50 greater than 10 μM; results from this study; Javitt and Frusciante, 1997). SSR504734 also exhibited similar potency in mice (IC50: 38±5 nM, in cortical homogenate, not reported in the “Materials and Methods” and “Results” sections), which we validated using mice in multiple experiments. SSR504734 was inactive against GlyT2, glutamate, GABA, DA, and serotonin transporters in vitro. Unlike current antipsychotics, it has no affinity for DA receptors and other receptors associated with various side effects, such as weight gain (5-HT2c, histamine H1 receptors), hypotension (α-adrenergic receptors), dry mouth/constipation/cognitive impairment (muscarinic receptors), and sedation (histaminergic receptors), etc.
In vitro experiments showed that SSR504734 blocked glycine uptake in mouse cortical homogenate after intraperitoneal injection and oral administration. Analysis of the time-course progression of glycine uptake inhibition in vitro showed that, unlike ALX5407 (unpublished data), the inhibition by SSR504734 was reversible. The reversibility of SSR504734 was also observed in in vitro experiments (enhanced EPSC in hippocampal slices), while ALX5407 did not exhibit this reversibility. The reversibility of SSR504734's GlyT1 blockade is crucial, as irreversible blockade could constitute a potential drawback for the clinical application of this compound.

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The blocking effect of SSR504734 on GlyT1 enhances extracellular levels of glycine and dopamine in the prefrontal cortex[1]
In microdialysis experiments, the potency and efficacy of SSR504734 (10 mg/kg, intraperitoneal injection) in increasing basal glycine levels in the prefrontal cortex (PFC) were at least comparable to ALX5407 (10 mg/kg, intraperitoneal injection or oral administration; unpublished data; Atkinson et al., 2001). The ability of SSR504734 to increase extracellular levels of glycine and dopamine in the rat prefrontal cortex (PFC) is expected to have a variety of consequences. The prefrontal cortex (PFC) occupies a special place among brain structures closely associated with the manifestations of schizophrenia. Increasing evidence suggests that impaired PFC function is closely associated with the development of negative symptoms and cognitive deficits (Goldman-Rakic and Selemon, 1997), which are increasingly considered to be at the heart of the pathology of schizophrenia. At least two neurotransmitter systems are considered deficiencies to contribute to PFC dysfunction: the NMDA/glutamatergic system and the dopaminergic system (Goldman-Rakic and Selemon, 1997). SSR504734, which enhances prefrontal glycinergic tone, is expected to enhance NMDA/glutamatergic transmission (see below) and further balance the deficiency in this neurotransmitter transmission. Furthermore, the ability of existing antipsychotic drugs to enhance prefrontal dopamine (DA) tone is considered a neurochemical marker for alleviating negative and cognitive symptoms in patients (Kapur and Remington, 1996). In summary, these data suggest that SSR504734, with its dual-mode neurochemical mechanism, may be effective in combating negative/cognitive deficits in patients with schizophrenia. Finally, insufficient activity of the prefrontal cortex (PFC) dopamine system is thought to lead to hyperactivity of the subcortical dopaminergic system, thereby triggering positive symptoms in schizophrenia (Grace, 1991). Since SSR504734 can enhance dopamine (DA) transmission in the prefrontal cortex (PFC) (thereby reducing subcortical DA tension), it is expected to have a direct benefit for positive symptoms, a hypothesis that has been further confirmed by behavioral data (see below).

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SSR504734 enhances central glycinergic tension and has a functional effect on central glutamatergic neurotransmission[1]
The increase in central glycine levels induced by SSR504734 has a functional effect on central glutamatergic neurotransmission, both in vitro and in vivo. In vitro, SSR504734 enhanced NMDA-mediated excitatory postsynaptic currents (eEPSC) in rat hippocampal slices. Consistent with these results, Wilcox and his collaborators (1996) showed that NMDA receptor-mediated eEPSC recorded from hippocampal neurons can be significantly enhanced by glycine. Some evidence suggests that the hippocampus of patients with schizophrenia is abnormal at both the cellular and neurochemical levels (Heckers and Konradi, 2002). The hippocampus is associated with various cognitive functions, including episodic memory, which is impaired in patients with schizophrenia (Rushe et al., 1999). Data from this study, combined with experimental results showing the long-term enhancing effects of SSR504734 (under writing) and two GlyT1 inhibitors, NFPS (Kinney et al., 2003) and CP 802,079 (Martina et al., 2004), on hippocampal enhancement in vivo, suggest that GlyT1 inhibitors may have a beneficial effect on hippocampal-dependent memory deficits in schizophrenia. Furthermore, in anesthetized rats, SSR504734 enhanced dopamine voltammetry signaling in the nucleus accumbens induced by amygdala electrical stimulation. Since this dopamine signaling has been shown to be dependent on NMDA receptors, this enhancement provides supplemental evidence for the enhanced NMDA neurotransmission following GlyT1 blockade by SSR504734 (Leonetti et al., under writing).
Systemic administration of SSR504734 dose-dependently increased the number of contralateral rotations induced by subthreshold doses of glycine injected into the striatum of mice, and it was also found that unilateral microinjection of SSR504734 into the striatum alone could also induce contralateral rotations. The effect of SSR504734 could be antagonized by MK-801, while MK-801 itself had no effect at this dose, further indicating that the enhanced behavioral output (increased number of rotations) is a result of enhanced glutamatergic/NMDA neurotransmission.
These in vitro and in vivo experimental results together reinforce the following view (Bergeron et al., 1998; Chen et al., 2003): the glycine concentration near the NMDA receptor is maintained at a subsaturated level by GlyT1, and increasing the glycine concentration by blocking this transporter leads to NMDA-mediated functional enhancement. SSR504734 can restore normal function of the NMDA/glutamatergic system in two brain structures suspected of being associated with schizophrenia[1]
Among the brain structures suspected of being associated with schizophrenia, the hippocampus and prefrontal cortex (PFC) play important roles in the etiology of schizophrenia. Studies have repeatedly shown subtle neuroanatomical abnormalities in the hippocampus in patients (Heckers and Konradi, 2002), while prefrontal cortex dysfunction is thought to play a role in the development of negative symptoms (Goff and Evins, 1998). Both are involved in the control of cognitive functions, particularly memory and attention processes, which are affected in the pathological process of schizophrenia. Presynaptic CB1 receptors negatively regulate excitatory synaptic transmission, which may be achieved by blocking the release of neurotransmitters, particularly glutamate (see Schlicker and Kathmann, 2001 for a review). CB1 receptor agonists, such as WIN55212-2, have been shown to inhibit the release of [3H]ACh from rat hippocampal slices (Gifford and Ashby, 1996). This study demonstrates that SSR504734 almost completely reversed the WIN55212-2-mediated reduction in [3H]ACh efflux in a similar in vitro hippocampal model. Similar reversal effects were obtained using 10⁻⁵ M NMDA or 10⁻⁴ M glycine (data not shown), strongly suggesting that the action of SSR504734 is related to NMDA receptor activation. Given the importance of the cholinergic system in memory processes (Gold, 2003), the ability of SSR504734 to restore normal levels of central cholinergic transmission suggests that this compound may have therapeutic activity for memory disorders, not only in schizophrenia but also potentially in other diseases such as Alzheimer's disease. In summary, these data complement the data obtained from eEPSC experiments (see above) and provide strong experimental evidence that SSR504734 can promote or normalize basal or reduced hippocampal neuronal activity, respectively. In anesthetized rats, the reduced spontaneous firing rate of WIN55212-2-induced prefrontal cortex (PFC) neurons could be completely reversed by administration of SSR504734. Normal PFC function is highly dependent on glutamatergic neurotransmission, primarily through the glutamate/NMDA system, and disruption of this system is associated with schizophrenia (see Konradi and Heckers, 2003 review). In vitro experiments have shown that CB1 receptor agonists can inhibit glutamatergic synaptic transmission in multiple brain regions, including the PFC (Auclair et al., 2000). The restoration of reduced PFC neuronal activity by SSR504734 further confirms that the functional decline of this structure (at least in terms of electrophysiological parameters) can be reversed by blocking GlyT1 to enhance glycinergic tone. Therefore, it can be expected that this compound can restore normal neuronal activity in brain structures (prefrontal cortex) suspected of functional decline in patients with schizophrenia. It should be emphasized that the beneficial effects of SSR504734 in these two experiments ([3H]ACh hippocampal efflux and PFC spontaneous firing rate) can be ruled out as being due to its direct antagonistic activity against CB1 receptors, as the compound did not show affinity for CB1 receptors (IC50>1 μM). SSR504734 showed activity in a variety of tests predicting antipsychotic activity[1] Evidence of potential antipsychotic activity of SSR504734 was obtained in a variety of neurochemical and behavioral tests that are considered to detect its efficacy against positive and/or negative symptoms of schizophrenia. Increased 2-DG uptake is thought to reflect an increase in metabolic rate, i.e., enhanced neuronal activity. SSR504734 restored the relative increase in 2-DG uptake in the prefrontal cortex (PFC) and other subcortical limbic regions induced by acute subanesthetic doses of ketamine in mice to normal. Literature reports a pattern of ketamine-induced 2-DG-like increased uptake (Duncan et al., 1998a, 1998b; Miyamoto et al., 2000). Acute administration of atypical antipsychotics (such as clozapine) completely blocks these increases, but typical antipsychotics (such as haloperidol) are ineffective (Duncan et al., 1998b). Clinical studies have shown a positive correlation between ketamine-induced psychotic symptoms in volunteers and increased metabolic activity in the frontal cortex (Breier et al., 1997; Vollenweider et al., 1997). SSR504734 reduces the increased cortical metabolic activity in mice (an increase caused by glutamatergic hypoactivity), suggesting that this compound may have activity against mottled symptoms. At the behavioral level, SSR504734 antagonizes MK-801-induced hyperactivity in mice and increases the α1 band spectral energy in the rat cortex. Harsing et al. (2003) reported similar effects of NFPS and/or ORG 24461 on PCP-induced hyperactivity in mice and EEG spectral power changes in rats. In our view, MK-801-induced hyperactivity and EEG pattern changes were sensitive to haloperidol and clozapine, which may indicate that activity in these tests only predicts efficacy for positive symptoms, as haloperidol is less effective for negative symptoms and cognitive deficits. As previously reported (Olivier et al., 2001; Kinney et al., 2003), DBA/2 mice exhibited low baseline PPI levels in the startle reflex. In other words, they displayed a spontaneous PPI deficiency. SSR504734 dose-dependently enhanced PPI levels, i.e., reduced this innate PPI deficiency. Similar reversal effects have been reported in DBA/2 mice after treatment with clozapine, haloperidol, and risperidone (McCaughran et al., 1997; Olivier et al., 2001), and more recently, the more active enantiomers (+) of NFPS have also shown similar reversal effects (Kinney et al., 2003). Furthermore, glycine and ORG 24598 (the active R(−) enantiomer of ORG 24461) have also been found to reverse PPI deficiency in adult rats with neonatal ventral hippocampal injury (Le Pen et al., 2003). All these data highlight the potential of glycinergic strategies in reversing PPI deficiencies of different natures. Prepulse inhibition (PPI) deficiency is known in patients with schizophrenia (Braff et al., 1978), which is considered an overt manifestation of underlying information processing abnormalities. The role of SSR504734 suggests that this compound may alleviate this abnormality, which is considered a key factor in this pathological manifestation.
The neonatal PCP model is based on the neurodevelopmental concept of schizophrenia (Weinberger, 1986; Lieberman et al., 1997). Studies by Wang et al. (2001, 2003) have shown that high-dose PCP injections in young rats lead to long-term behavioral changes associated with neuronal alterations in adolescence or adulthood. Studies have found that SSR504734 has beneficial effects on two pathology-related behaviors in neonatal PCP-treated adult rats: (1) hypersensitivity to acute stimulation of d-amphetamine (reported during acute psychotic episodes in patients with schizophrenia; Laruelle, 2000); (2) selective attention deficit (a cognitive process severely impaired in schizophrenia and considered a major feature of the disease; Brébion et al., 2000). The beneficial effects of SSR504734 on the former are consistent with recent findings that long-term use of NFPS or glycine can prevent the enhanced dopamine release in the striatum induced by long-term PCP administration (Javitt et al., 2004). If this hypersensitivity to dextroamphetamine reflects an abnormal sensitivity of the subcortical dopamine system, leading to obvious symptoms in schizophrenia patients (Laruelle, 2000), then the inhibitory effect of SSR504734 provides additional experimental evidence that this compound may have activity against positive symptoms. SSR504734 was able to restore selective attention deficit in rats and spontaneous PPI (a potential marker of information processing) deficit in DBA/2 mice to normal, suggesting that SSR504734 may have beneficial effects on impaired cognitive processes in schizophrenia patients. Furthermore, recent studies have shown that the direct glycine agonist D-serine can reverse the detrimental effects of PCP-like treatment in spatial memory tasks in newborn rats (Andersen and Pouzet, 2004), which further supports the idea that glycinergic strategies may have a positive effect on various cognitive deficits in schizophrenia. This aspect warrants further investigation.

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SSR504734 has additional antidepressant/anxiety-like activity[1]
SSR504734 showed activity in tests predicting antidepressant and/or anxiety activity. After acute administration, it reduced the ultrasonic distress signals of pups separated from their mothers, similar to the effects of the antidepressant fluoxetine and anxiolytics such as benzodiazepines and 5-HT1A receptor agonists (Gardner, 1985; Fish et al., 2000). SSR504734 also prolonged the latency to enter REM sleep, an effect commonly seen in antidepressants such as fluoxetine and imipramine (Slater et al., 1978; Kleinlogel, 1982). Furthermore, long-term administration of SSR504734 prevented physiological degeneration in mice subjected to chronic stress (CMS), an effect similar to that of other known or potential antidepressant/anxiety compounds such as fluoxetine or the corticotropin-releasing factor (1) receptor antagonist SSR125543 (Griebel et al., 2002). In summary, these pharmacological data are consistent with the results of a recent preliminary report indicating reduced anxiety levels in GlyT1 heterozygous knockout mice (Vanhoof et al., 2004). The antidepressant/anxiety potential of SSR504734 appears to contradict other preclinical data showing efficacy of antiglutaminase compounds, such as PCP and MK-801 (non-competitive antagonists), or CCP, CPPene, and CGS 19755 (competitive antagonists), in certain animal models of anxiety (see the most recent review by Bergink et al., 2004). However, the anxiety potential of these antiglutaminase compounds has not only not been confirmed at the clinical level, but there are even indications that these compounds may possess anxiolytic properties. An open-label trial of MK-801 in patients with anxiety disorders was discontinued due to worsening symptoms in most patients (Reimherr et al., 1986). Furthermore, CPPene has been reported to cause agitation, while CGS 19755 (selfotel) has been reported to cause dizziness, anxiety, irritability, and confusion (see Muir and Lees, 1995 review). Given these clinical considerations, it is not entirely unreasonable to suggest that compounds enhancing NMDA/glutamatergic transmission (e.g., SSR504734) may possess anti-anxiety/antidepressant activity. Further investigation is underway into the characterization of SSR504734 in other models predicting antidepressant and/or anti-anxiety activity. In any case, anxiety and depression are two common comorbidities in patients with schizophrenia and are considered key factors in the interictal period of the disease; therefore, actively treating these factors can delay and/or reduce the severity of subsequent episodes and contribute to better stabilization of the condition.

C20H20CLF3N2O

396.833814620972

396.121

C, 60.53; H, 5.08; Cl, 8.93; F, 14.36; N, 7.06; O, 4.03

742693-38-5

615571-23-8

9954540

White to off-white solid powder

4.7

2

5

4

27

496

2

ClC1C(C(F)(F)F)=CC=CC=1C(N[C@@H](C1C=CC=CC=1)[C@@H]1CCCCN1)=O

MEZRZVWPLXVLSO-WMZOPIPTSA-N

InChI=1S/C20H20ClF3N2O/c21-17-14(9-6-10-15(17)20(22,23)24)19(27)26-18(13-7-2-1-3-8-13)16-11-4-5-12-25-16/h1-3,6-10,16,18,25H,4-5,11-12H2,(H,26,27)/t16-,18-/m0/s1

2-chloro-N-[(S)-phenyl-[(2S)-piperidin-2-yl]methyl]-3-(trifluoromethyl)benzamide

615571-23-8; SSR504734; SSR 504734; SSR 504734 hydrochloride; SSR-504734; L297UZF32G; 2-chloro-3-(trifluoromethyl)-N-((S)-phenyl((S)-piperidin-2-yl)methyl)benzamide hydrochloride; 2-Chloro-N-[(S)-phenyl-[(2S)-piperidin-2-yl]methyl]-3-(trifluoromethyl)benzamide;hydrochloride;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)