mGlu5 receptor (IC50 = 2.2 nM)

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].

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].

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]

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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Dissolved in 0.9% NaCl (w/v) and 0.3% Tween 80 (v/v) solution; 1 mg/kg; oral gavage

Male Sprague-Dawley rats

[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.

The metabotropic glutamate receptor 5 (mGlu5) is a glutamate-activated class C G protein-coupled receptor widely expressed in the central nervous system and clinically investigated as a drug target for a range of indications, including depression, Parkinson's disease, and fragile X syndrome. Here, we present the novel potent, selective, and orally bioavailable mGlu5 negative allosteric modulator with inverse agonist properties 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP). CTEP binds mGlu5 with low nanomolar affinity and shows >1000-fold selectivity when tested against 103 targets, including all known mGlu receptors. CTEP penetrates the brain with a brain/plasma ratio of 2.6 and displaces the tracer [(3)H]3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-methyl-oxime (ABP688) in vivo in mice from brain regions expressing mGlu5 with an average ED(50) equivalent to a drug concentration of 77.5 ng/g in brain tissue. This novel mGlu5 inhibitor is active in the stress-induced hyperthermia procedure in mice and the Vogel conflict drinking test in rats with minimal effective doses of 0.1 and 0.3 mg/kg, respectively, reflecting a 30- to 100-fold higher in vivo potency compared with 2-methyl-6-(phenylethynyl)pyridine (MPEP) and fenobam. CTEP is the first reported mGlu5 inhibitor with both long half-life of approximately 18 h and high oral bioavailability allowing chronic treatment with continuous receptor blockade with one dose every 48 h in adult and newborn animals. By enabling long-term treatment through a wide age range, CTEP allows the exploration of the full therapeutic potential of mGlu5 inhibitors for indications requiring chronic receptor inhibition.[1]

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Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. Previous studies have implicated mGlu5 in the pathogenesis of the disease, but a crucial unanswered question is whether pharmacological mGlu5 inhibition is able to reverse an already established FXS phenotype in mammals. Here we have used the novel, potent, and selective mGlu5 inhibitor CTEP to address this issue in the Fmr1 knockout mouse. Acute CTEP treatment corrects elevated hippocampal long-term depression, protein synthesis, and audiogenic seizures. Chronic treatment that inhibits mGlu5 within a receptor occupancy range of 81% ± 4% rescues cognitive deficits, auditory hypersensitivity, aberrant dendritic spine density, overactive ERK and mTOR signaling, and partially corrects macroorchidism. This study shows that a comprehensive phenotype correction in FXS is possible with pharmacological intervention starting in young adulthood, after development of the phenotype. It is of great interest how these findings may translate into ongoing clinical research testing mGlu5 inhibitors in FXS patients.[2]

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.)