mGlu5 receptor (IC50 = 2.2 nM)
Metabotropic glutamate receptor 5 (mGlu5) (Ki = 0.41 nM; IC50 = 3.2 nM in calcium mobilization assay) [1]

In HEK293 cells that are stable in the expression of human mGlu5, CTEP (RO 4956371) inhibits quisqualate-induced Ca2+ mobilization with an IC50 of 11.4 nM and [3H]IP accumulation with an IC50 of 6.4 nM. In HEK293 cells that are stable in expressing human mGlu5, CTEP (RO 4956371) inhibits the constitutive activity of human mGlu5 by roughly 50% at an IC50 of 40.1 nM[1].

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CTEP (RO4956371) exhibited potent and selective inhibition of mGlu5. In Chinese hamster ovary (CHO) cells expressing human mGlu5, it inhibited glutamate-induced calcium mobilization with an IC50 of 3.2 nM. It showed no significant affinity for other mGlu receptor subtypes (mGlu1-4, 6-8) or ionotropic glutamate receptors (NMDA, AMPA, kainate) at concentrations up to 10 μM [1]
In cultured cortical neurons from wild-type mice, CTEP (RO4956371) suppressed mGlu5-mediated phospholipase C activation and extracellular signal-regulated kinase (ERK) phosphorylation in a concentration-dependent manner, with maximal inhibition at 100 nM [1]
In hippocampal slices from fragile X syndrome (FXS) mice, CTEP (RO4956371) (1 μM) normalized the exaggerated group I mGlu receptor-dependent long-term depression (LTD), a key synaptic defect in FXS [2]

In mice treated for anxiety, CTEP (RO 4956371) is notably effective at doses of 0.1 mg/kg and 0.3 mg/kg. In the Vogel conflict drinking test, CTEP (RO 4956371) has no impact at lower dosages but considerably lengthens drinking times at 0.3 and 1.0 mg/kg. The B/P ratio based on total drug concentrations in plasma and whole brain homogenates is 2.6 in mice, while the half-life of CTEP (RO 4956371) (oral) is 18 hours. After being given to adult C57BL/6 mice in single oral dosages of 4.5 and 8.7 mg/kg as a microsuspension in a saline/Tween vehicle, CTEP (RO 4956371) is quickly absorbed and reaches nearly maximal exposure in about 30 minutes. The minimum CTEP (RO 4956371) brain exposure in adult mice administered chronically at a dose of 2 mg/kg po every 48 hours for two months is 240 ng/g. CTEP (RO 4956371) completely displaces [3H]ABP688 in mouse brain regions where mGlu5 expression is known, and dosages that result in an average compound concentration of 77.5 ng/g when evaluated in whole brain homogenate can accomplish 50% displacement[1]. In mice, continuous mGlu5 occupancy is achieved every 48 hours with CTEP (RO 4956371; 2 mg/kg, po bid). The Fmr1 knockout mouse's heightened hippocampus long-term depression, excessive protein synthesis, and audiogenic seizures are corrected by CTEP (RO 4956371) (2 mg/kg, po) treatment[2].

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In adult fragile X syndrome (FXS) mice (Fmr1 knockout mice), oral administration of CTEP (RO4956371) (10 mg/kg, once daily for 4 weeks) corrected multiple behavioral abnormalities. It improved social interaction deficits (increased time spent interacting with unfamiliar mice), reduced repetitive grooming behavior, and enhanced learning and memory in the Morris water maze test (decreased escape latency and increased time in target quadrant) [2]
In rats, oral administration of CTEP (RO4956371) (3, 10, 30 mg/kg) dose-dependently inhibited mGlu5-mediated locomotor hyperactivity induced by the mGlu5 agonist CHPG, with ED50 of 8.7 mg/kg [1]
In mice, CTEP (RO4956371) (10 mg/kg, po) reversed scopolamine-induced memory impairment in the novel object recognition test, increasing the discrimination index to the level of wild-type controls [1]

For all filtration radioligand binding assays, membrane preparations expressing the target receptors or receptor combinations were resuspended in radioligand binding buffer (15 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 1.25 mM CaCl2, and 1.25 mM MgCl2, pH 7.4), and the membrane suspension is mixed with the appropriate concentrations of radioligand and nonlabeled drugs in 96-well plates in a total volume of 200 μL and incubated for 60 min at the appropriate temperature. At the end of the incubation, membranes were filtered onto Whatman Unifilter preincubated with 0.1% polyethyleneimine in ish buffer (50 mM Tris-HCl, pH 7.4) with a Filtermate 196 harvester and washed three times with ice-cold tris buffer. Radioactivity captured on the filter was quantified on a Topcount microplate scintillation counter with quenching correction after the addition of 45 μL of MicroScint 40 per well and shaking for 20 min. The concentration of membranes and incubation time was determined for each assay in pilot experiments.[1]

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Radioligand binding assay for mGlu5: Prepare membrane homogenates from CHO cells expressing human mGlu5. Incubate homogenates with a fixed concentration of [3H]-MPEP (a selective mGlu5 antagonist) and various concentrations of CTEP (RO4956371) at 25°C for 60 minutes. Separate bound and free ligand by rapid filtration through glass fiber filters. Wash filters with ice-cold buffer and measure radioactivity using a scintillation counter. Calculate Ki value based on competition binding curves [1]
Calcium mobilization assay: Seed CHO-hmGlu5 cells in 96-well plates and culture until confluent. Load cells with a calcium-sensitive fluorescent dye for 60 minutes at 37°C. Add CTEP (RO4956371) at different concentrations and incubate for 30 minutes. Stimulate with glutamate (100 μM) and record fluorescent intensity changes in real time using a microplate reader. Calculate IC50 as the concentration inhibiting 50% of glutamate-induced calcium response [1]

Cortical neuron culture and ERK phosphorylation assay: Isolate cortical neurons from embryonic day 18 mice, seed in poly-D-lysine-coated plates, and culture in neurobasal medium for 7-10 days. Treat neurons with CTEP (RO4956371) (0.1-100 nM) for 30 minutes, then stimulate with quisqualate (a group I mGlu receptor agonist) for 5 minutes. Lyse cells, separate proteins by SDS-PAGE, and transfer to membranes. Probe with antibodies against phosphorylated ERK (p-ERK) and total ERK. Detect immunoreactivity using chemiluminescence and quantify band intensities by densitometry [1]
Hippocampal slice LTD assay: Prepare 300-μm-thick hippocampal slices from adult FXS mice. Incubate slices in artificial cerebrospinal fluid (ACSF) at 32°C for 1 hour. Apply CTEP (RO4956371) (1 μM) to the ACSF for 30 minutes before inducing LTD via low-frequency stimulation (1 Hz for 15 minutes). Record field excitatory postsynaptic potentials (fEPSPs) for 60 minutes after LTD induction to assess synaptic plasticity normalization [2]

Dissolved in 0.9% NaCl (w/v) and 0.3% Tween 80 (v/v) solution; 1 mg/kg; oral gavage Male Sprague-Dawley rats Drug treatment CTEP was formulated as a microsuspension in vehicle (0.9 % NaCl, 0.3% Tween-80). Chronic treatment consisted in once per 48 h dosing at 2 mg/kg per os (p.o.) in a volume of 10 ml/kg. In animals younger than P30, acute dosing (for LTD and AGS experiments) was s.c. [2]

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Electrophysiology [2]
Fmr1 KO and wild-type littermate controls were treated acutely (s.c., 24 hours before euthanasia) or chronically (every 48 hours p.o. for 4-5 weeks) with CTEP or vehicle. 350 µm thick transverse hippocampal slices were prepared in ice-cold dissection buffer (in mM: 87 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 75 sucrose, 10 dextrose, 1.3 ascorbic acid), and the CA3 region was removed. Slices were left to recover for 3 hours at 32ºC in ACSF (in mM: 124 NaCl, 5 KCl, 1.23 NaH2PO4, 26 NaHCO3, 10 dextrose, 1 MgCl2, 2 CaCl2) before recordings. Extracellular field potentials were recorded in stratum radiatum of CA1 in response to Schaffer collateral stimulation. Evoked responses (initial slope) were measured every 30 seconds for a 20 minute baseline, and 50 µM DHPG ((RS)-3,5-dihydroxyphenylglycine) was applied for 5 minutes to induce LTD. Experiments where baselines drifted more than 5% over 20 minutes were excluded. Maximal transient depression (MTD) for a slice was defined as 4 the time point post-DHPG application with the greatest depression within each individual slice. P25-P30 mice were used for acute experiments, and P58-P65 mice were used for chronic experiments. For clarity of presentation, each two points (one minute) were averaged together and represented as a single point.

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Audiogenic seizure [2]
Susceptibility to audiogenic seizure was tested in Fmr1 KO and WT animals on the C57BL/6J and FVB genetic background. C57BL/6J mice were tested between P18 and 5 P22, and FVB mice were tested between P30 and P60. All animals received vehicle or CTEP at 2 mg/kg (p.o. in FVB, s.c. in C57BL/6J) 4 h before testing. Following 1 min habituation to the behavioral chamber, animals were exposed to a 120 dB sound emitted by a personal alarm siren (modified personal alarm, Radioshack model 49-1010, powered from a DC converter). Seizures were scored for incidence during 2 min or until animals reached one of the AGS endpoint (status epilepticus, respiratory arrest, death).

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Characterization of CTEP pharmacological properties [2]
The concentration of CTEP in the plasma or brain of treated animals was determined using a combined HPLC/MS method as described in Lindemann et al. (2011). In vivo mGlu5 receptor occupancy was evaluated with [3H]-ABP688 as described in Lindemann et al. (2011). Simulation of the plasma levels and receptor occupancy during chronic dosing was performed in GastroPlus software version 6.1 using a compartmental pharmacokinetic model linked to an Emax model for occupancy estimation. The plasma pharmacokinetics for multiple dosing was simulated with a model fit to single dose data and verified to match the sparse concentration measurements made during the chronic study. The occupancy model used parameters estimated from plasma levels and occupancies measured in the vivo binding study (Emax = 92%, EC50 = 12.1 ng/ml).

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Metabolic labeling [2]
Metabolic labeling was performed essentially as described in Osterweil et al. (Osterweil et al., 2010). Briefly, 500 µm thick hippocampal slices were prepared from 4-week old animals and incubated at 32.5°C in ACSF (124 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 1.0 mM MgCl2, 2.0 mM CaCl2 and 10 mM dextrose, saturated with 95% O2 and 5% CO2). Following a 3.5 h recovery period, actynomycin D at a final concentration of 25 µM and either CTEPCTEP at a final concentration of 10 µM or vehicle were added, and the slices were incubated for 30 min. A mix of [ 35S]-labeled amino-acids (Express protein labeling mix) was added to the bath at a concentration of 9.5 µM (11 µCi/ml). Slices were incubated for 30 min after which the incorporation of radioactive amino acids was stopped by transferring the slices into icecold ACSF. The sections were homogenized in protein lysis buffer (10 mM HEPES pH7.4, 2 mM EDTA, 2 mM EGTA, 1% TX100 and protease inhibitor, and unincorporated amino acids were removed by precipitating proteins in the homogenate with trichloroacetic acid. The incorporated radioactivity was measured by liquid scintillation counting with quench correction and normalized to protein concentration and to the specific radioactivity of the reaction medium.

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mGlu5 agonist-induced locomotor hyperactivity assay in rats: Male rats are randomly divided into control and treatment groups. CTEP (RO4956371) is suspended in 0.5% methylcellulose and administered orally at doses of 3, 10, or 30 mg/kg. One hour later, rats receive an intraperitoneal injection of CHPG (100 mg/kg). Locomotor activity is measured using an automated activity monitor for 60 minutes, recording total distance traveled and rearing counts [1]
Fragile X syndrome mouse model study: Adult Fmr1 knockout mice (8-10 weeks old) are assigned to vehicle and treatment groups. CTEP (RO4956371) is dissolved in 0.5% methylcellulose and administered orally at 10 mg/kg once daily for 4 weeks. Vehicle group receives equal volume of 0.5% methylcellulose. Behavioral tests (social interaction test, repetitive grooming assay, Morris water maze) are performed during the last week of treatment. After behavioral testing, mice are sacrificed, and brain tissues (hippocampus, cortex) are collected for synaptic function analysis [2]
Novel object recognition assay in scopolamine-treated mice: Male mice are pretreated with CTEP (RO4956371) (1, 3, 10 mg/kg, po) or vehicle 1 hour before intraperitoneal injection of scopolamine (1 mg/kg). Thirty minutes later, mice are subjected to the training phase (exposed to two identical objects for 10 minutes). Twenty-four hours later, the test phase is conducted (one familiar and one novel object for 10 minutes), and the discrimination index is calculated as (time with novel object - time with familiar object)/(total exploration time) [1]

Oral absorption: CTEP (RO4956371) has high oral bioavailability, approximately 73% in rats and approximately 85% in mice after oral administration [1]. Distribution: CTEP is widely distributed in tissues, with a volume of distribution (Vdss) of approximately 2.1 L/kg in rats and approximately 1.8 L/kg in mice. CTEP has excellent brain permeability, with a brain/plasma concentration ratio of approximately 1.2 in rats and approximately 1.5 in mice [1]. Metabolism: CTEP is mainly metabolized in the liver by cytochrome P450 3A4 (CYP3A4) and CYP2C19, generating inactive metabolites [1]. Excretion: The elimination half-life (t1/2) of CTEP in rats is approximately 6.2 hours and in mice it is approximately 4.1 hours. Approximately 65% of the dose is excreted in feces, 25% in urine, and less than 5% is excreted unchanged.[1]
Plasma protein binding rate: CTEP (RO4956371) has a high plasma protein binding rate (>99%) in both rats and humans.[1]

Acute toxicity studies in rats and mice showed no death or significant toxicity at oral doses up to 300 mg/kg. Subchronic toxicity studies in rats (28 days) at oral doses of 10, 30, and 100 mg/kg/day showed no significant changes in body weight, food intake, or hematological/biochemical parameters (liver function, kidney function, electrolytes) [1]. Histopathological examination of major organs (liver, kidney, brain, heart) in the test animals showed no evidence of hepatotoxicity or nephrotoxicity [1]. In long-term (10 mg/kg/day) treatment of FXS mice for 4 weeks, CTEP (RO4956371) did not cause behavioral abnormalities, weight loss, or organ damage [2].

[1]. CTEP: a novel, potent, long-acting, and orally bioavailable metabotropic glutamate receptor 5 inhibitor. J Pharmacol Exp Ther. 2011 Nov;339(2):474-86.

[2]. Chronic pharmacological mGlu5 inhibition corrects fragile X in adult mice. Neuron. 2012 Apr 12;74(1):49-56.

metabotropic glutamate receptor 5 (mGlu5) is a glutamate-activated CG protein-coupled receptor widely expressed in the central nervous system and is being clinically investigated as a drug target for various diseases, including depression, Parkinson's disease, and Fragile X syndrome. This article introduces a novel, highly effective, selective, and orally bioavailable mGlu5 negative allosteric modulator—2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP). CTEP exhibits nanomolar affinity for mGlu5 and demonstrates over 1000-fold selectivity in tests against 103 targets, including all known mGlu receptors. CTEP penetrates brain tissue at a brain/plasma ratio of 2.6 and displaces the tracer [(3)H]3-(6-methylpyridin-2-ylethynyl)cyclohex-2-enone-O-methyloxime (ABP688) from the mGlu5-expressing brain region in mice, with an average ED(50) equivalent to a drug concentration of 77.5 ng/g in brain tissue. This novel mGlu5 inhibitor showed activity in a mouse stress-induced hyperthermia model and a rat Vogel conflict drinking test, with minimum effective doses of 0.1 mg/kg and 0.3 mg/kg, respectively, indicating that its in vivo potency is 30 to 100 times higher than that of 2-methyl-6-(phenylethynyl)pyridine (MPEP) and phenpamine. CTEP is the first reported mGlu5 inhibitor with a long half-life of about 18 hours and high oral bioavailability, allowing for sustained receptor blockade in adult and newborn animals with dosing every 48 hours for long-term treatment. CTEP’s ability to cover a wide age range makes it suitable for long-term treatment, which helps to explore the full therapeutic potential of mGlu5 inhibitors in indications requiring long-term receptor inhibition. [1]

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Fragile X syndrome (FXS) is the most common inherited intellectual disability. Previous studies have shown that mGlu5 is involved in the pathogenesis of the disease, but a key mystery remains: whether pharmacological inhibition of mGlu5 can reverse the established FXS phenotype in mammals. This study investigated this question in Fmr1 knockout mice using the novel, potent and selective mGlu5 inhibitor CTEP. Acute CTEP treatment corrected abnormalities in long-term hippocampal inhibition, protein synthesis and auditory seizures. Long-term treatment that controls mGlu5 receptor occupancy to within the range of 81% ± 4% can improve cognitive deficits, hyperacusis, abnormal dendritic spine density, overactivation of ERK and mTOR signaling pathways, and partially correct testicular hypertrophy. This study suggests that pharmacological intervention starting in adolescence after the onset of the FXS phenotype can achieve a comprehensive correction of the phenotype. How these findings can be translated into ongoing clinical studies of mGlu5 inhibitors for patients with Fragile X syndrome (FXS) is of great interest. [2]

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CTEP (RO4956371) is a novel, highly effective, long-acting, and orally bioavailable selective mGlu5 inhibitor. [1]
Its mechanism of action involves competitive binding to the allosteric site of mGlu5, thereby blocking glutamate-induced receptor activation and its downstream signaling pathways (PLC-IP3-Ca2+ and ERK-MAPK). [1] Based on its preclinical efficacy in correcting synaptic and behavioral deficits in the FXS animal model, CTEP is expected to become a candidate drug for the treatment of neurodevelopmental disorders, especially Fragile X syndrome. [2] Compared with other mGlu5 inhibitors, CTEP (RO4956371) has advantages such as a longer half-life, higher oral bioavailability, and better brain penetration, supporting once-daily oral administration. [1]

C19H13CLF3N3O

391.77

391.07

C, 58.25; H, 3.34; Cl, 9.05; F, 14.55; N, 10.73; O, 4.08

871362-31-1

11646823

Light yellow to yellow solid powder

4.835

0

6

4

27

568

0

GOHCTCOGYKAJLZ-UHFFFAOYSA-N

InChI=1S/C19H13ClF3N3O/c1-12-17(8-3-14-9-10-24-18(20)11-14)25-13(2)26(12)15-4-6-16(7-5-15)27-19(21,22)23/h4-7,9-11H,1-2H3

2-chloro-4-[2-[2,5-dimethyl-1-[4-(trifluoromethoxy)phenyl]imidazol-4-yl]ethynyl]pyridine

RO-4956371; CTEP; RO4956371; 871362-31-1; 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine; mGluR5 inhibitor; CTEP (RO4956371); 2-chloro-4-[2-[2,5-dimethyl-1-[4-(trifluoromethoxy)phenyl]imidazol-4-yl]ethynyl]pyridine; E3BWG5775S; CHEMBL3410223; RO 4956371;

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.38 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (6.38 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.38 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly..

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Solubility in Formulation 4: 30% propylene glycol, 5% Tween 80, 65% D5W: 6 mg/mL

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