CTEP (RO4956371)

mGlu5 receptor (IC50 = 2.2 nM)

在稳定表达人 mGlu5 的 HEK293 细胞中，CTEP (RO 4956371) 抑制君子氨酸诱导的 Ca2+ 动员，IC50 为 11.4 nM，并抑制 [3H]IP 积聚，IC50 为 6.4 nM。在稳定表达人 mGlu5 的 HEK293 细胞中，CTEP (RO 4956371) 可抑制人 mGlu5 的组成活性大约 50%，IC50 为 40.1 nM[1]。

在接受焦虑治疗的小鼠中，CTEP (RO 4956371) 在 0.1 mg/kg 和 0.3 mg/kg 剂量下显着有效。在 Vogel 冲突饮酒测试中，CTEP (RO 4956371) 在较低剂量下没有影响，但在 0.3 和 1.0 mg/kg 时显着延长饮酒时间。小鼠中基于血浆和全脑匀浆中总药物浓度的 B/P 比为 2.6，而 CTEP (RO 4956371)（口服）的半衰期为 18 小时。将 CTEP (RO 4956371) 以生理盐水/吐温载体中的微悬浮液形式单次口服剂量 4.5 和 8.7 mg/kg 给予成年 C57BL/6 小鼠后，CTEP (RO 4956371) 被迅速吸收，并在约 30 分钟内达到接近最大暴露量。成年小鼠以每 48 小时口服 2 mg/kg 的剂量长期服用两个月，其脑部暴露的最低 CTEP (RO 4956371) 为 240 ng/g。 CTEP (RO 4956371) 在 mGlu5 表达已知的小鼠大脑区域中完全取代 [3H]ABP688，在全脑匀浆中评估时，导致平均化合物浓度为 77.5 ng/g 的剂量可以实现 50% 的取代[1]。在小鼠中，使用 CTEP（RO 4956371；2 mg/kg，po bid）每 48 小时可实现连续 mGlu5 占据。 Fmr1 敲除小鼠的海马长期抑郁加剧、蛋白质合成过多和听源性癫痫发作可通过 CTEP (RO 4956371)（2 mg/kg，口服）治疗得到纠正[2]。

对于所有滤过性放射性配体结合试验，表达目标受体或受体组合的膜制剂在放射性配体结合缓冲液（15 mM Tris-HCl, 120 mM NaCl, 5 mM KCl， 1.25 mM CaCl2和1.25 mM MgCl2, pH 7.4）中重悬，膜悬液与适当浓度的放射性配体和未标记药物混合在96孔板中，总量为200 μL，在适当温度下孵育60分钟。在孵育结束时，将膜过滤到Whatman unfilter上，用0.1%聚乙烯亚胺在ish缓冲液（50 mM tris - hcl, pH 7.4）中预孵育，用Filtermate 196收集机洗涤，并用冰冷的tris缓冲液洗涤三次。每孔加入45 μL MicroScint 40，振荡20 min后，在Topcount微孔板闪烁计数器上定量过滤上捕获的放射性。在中试实验中确定每个检测的膜浓度和孵育时间。[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

39.94

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

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮和光照。

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

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.38 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.5 mg/mL (6.38 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.38 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 30% propylene glycol, 5% Tween 80, 65% D5W: 6 mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。