Clozapine N-oxide (CNO)
(Synonyms: 氯氮平N-氧化物) 目录号 : GC10822
clozapine和受体DREADD激动剂的代谢产物
Cas No.:34233-69-7
Sample solution is provided at 25 µL, 10mM.
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- Purity: >98.00%
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| Cell experiment [1]: | |
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Cell lines |
Primary rat cortical cells |
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Preparation Method |
Receptor parameters (density affinity and mRNA levels) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels in rat primary cortical cells after 24 h of Clozapine N-oxide(CNO) (200/400 ng/ml) treatment under in vitro conditions |
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Reaction Conditions |
Clozapine N-oxide(CNO) (200/400 ng/ml)for 24h |
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Applications |
A significant decrease was found in primary cortical cells for 5-HT(2) receptor density (Clozapine N-oxide(CNO) 200/ 400/ 200 and Cloz-N-oxide 200 vs. control) and 5-HT(2A) receptor mRNA levels (Clozapine N-oxide(CNO) 200 vs. control). |
| Animal experiment [2]: | |
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Animal models |
β-RQ and β-RS Tg mice |
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Preparation Method |
To determine the in vivo effects of acute activation of β-cell G q /11 or G S signaling on glucose homeostasis, mice were injected with increased doses of Clozapine N-oxide(CNO) (0.0001-10 mg/kg) and blood glucose levels were monitored for 2 h. Before the experiment began, all mice were free to eat and were given. |
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Dosage form |
0.0001-10 mg/kg Clozapine N-oxide(CNO) for 2 h |
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Applications |
In both β-R-q and β-R-s Tg mice, Clozapine N-oxide(CNO) treatment resulted in a dose-dependent decrease in blood glucose levels and increased plasma insulin concentrations, demonstrating that the degree of β cell G protein signaling could be titrated according to the Clozapine N-oxide(CNO) dose administered. |
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References: [1]: Heiser P, Schulte E, et,al. Effects of clozapine and its metabolites on the 5-HT2 receptor system in cortical and hippocampal cells in vitro. Prog Neuropsychopharmacol Biol Psychiatry. 2004 Mar;28(2):297-302. doi: 10.1016/j.pnpbp.2003.10.008. PMID: 14751426. |
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Clozapine N-oxide(CNO) is a potent dopamine antagonist and also a potent and selective muscarinic M4 receptor (EC50=11 nM) agonist[5，6].Clozapine N-oxide also is a major metabolite of Clozapine and a human muscarinic designer receptors (DREADDs) agonist[1,2]. Clozapine N-oxide activates the DREADD receptor hM3Dq and hM4Di. Clozapine N-oxide(CNO) is a pharmacologically inert molecule that lacks a significant (<1 μm) affinity for the receptor[3]. Clozapine N-oxide(CNO) is highly bioavailable in rodents and humans[4]
Clozapine N-oxide(CNO) significantly reduces the density of the 5-HT2 receptor in rat cortical cells, A significant decrease was found in primary cortical cells for 5-HT(2) receptor density and 5-HT(2A) receptor mRNA levels[7].
In both β-R-q and β-R-s Tg mice, Clozapine N-oxide(CNO) treatment resulted in a dose-dependent decrease in blood glucose levels and increased plasma insulin concentrations, demonstrating that the degree of β cell G protein signaling could be titrated according to the Clozapine N-oxide(CNO) dose administered[8].Oral Clozapine N-oxide(CNO) administration via drinking water to mice expressing rM3Dq in pancreatic β-cells led to robust metabolic phenotypes, indicating that significant amounts of Clozapine N-oxide(CNO) are absorbed from the gastrointestinal tract[9].It created transgenic mice expressing an evolved G protein-coupled receptor (hM3Dq) selectively activated by the pharmacologically inert, orally bioavailable drug Clozapine N-oxide (CNO).Local field potential and single-neuron recordings revealed that peripheral administration of Clozapine N-oxide(CNO) activated hippocampal neurons selectively in hM3Dq-expressing mice. Behavioral correlates of neuronal activation included increased locomotion, stereotypy, and limbic seizures[10].
Clozapine N-oxide（CNO）是一种强效的多巴胺拮抗剂，也是一种选择性肌动蛋白M4受体（EC50=11 nM）激动剂。Clozapine N-oxide还是氯氮平的主要代谢产物和人类肌动蛋白设计受体（DREADDs）激动剂。Clozapine N-oxide可以激活DREADD受体hM3Dq和hM4Di。Clozapine N-oxide（CNO）是一种药理学上惰性分子，缺乏对受体的显着亲和力(
Clozapine N-oxide（CNO）显著降低了大鼠皮层细胞中5-HT2受体的密度。在初级皮层细胞中发现5-HT(2)受体密度和5-HT(2A)受体mRNA水平明显下降[7]。
在β-R-q和β-R-s Tg小鼠中，克氏培南氧化物（CNO）治疗导致血糖水平降低和血浆胰岛素浓度增加，表明β细胞G蛋白信号的程度可以根据给予的CNO剂量进行调节。通过口服含有Clozapine N-oxide(CNO)的饮用水给表达rM3Dq的胰岛β细胞小鼠进行处理，产生了强大的代谢表型，这表明从肠道吸收了大量Clozapine N-oxide(CNO)。创建了转基因小鼠，它们表达一种进化后的G蛋白偶联受体（hM3Dq），只能被药理学上惰性、口服可利用药物Clozapine N-oxide (CNO)激活。局部场电位和单个神经元记录显示，在hM3Dq表达小鼠中外周给予Clozapine N-oxide(CNO)会选择性地激活海马神经元。神经元激活行为相关包括运动增加、刻板行为和边缘癫痫发作。
References:
[1]: Wess J, Nakajima K, et,al. Novel designer receptors to probe GPCR signaling and physiology. Trends Pharmacol Sci. 2013 Jul;34(7):385-92. doi: 10.1016/j.tips.2013.04.006. Epub 2013 Jun 13. PMID: 23769625; PMCID: PMC3758874.
[2]: Manvich DF, Webster KA, et,al. The DREADD agonist clozapine N-oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice. Sci Rep. 2018 Mar 1;8(1):3840. doi: 10.1038/s41598-018-22116-z. PMID: 29497149; PMCID: PMC5832819.
[3]: Weiner DM, Meltzer HY, et,al. The role of M1 muscarinic receptor agonism of N-desmethylclozapine in the unique clinical effects of clozapine. Psychopharmacology (Berl). 2004 Dec;177(1-2):207-16. doi: 10.1007/s00213-004-1940-5. Epub 2004 Jul 16. PMID: 15258717.
[4]: Bender D, Holschbach M, et,al. Synthesis of n.c.a. carbon-11 labelled clozapine and its major metabolite clozapine-N-oxide and comparison of their biodistribution in mice. Nucl Med Biol. 1994 Oct;21(7):921-5. doi: 10.1016/0969-8051(94)90080-9. PMID: 9234345.
[5]: Silva RR, Parreiras-E-Silva LT, et,al. Evaluation of Functional Selectivity of Haloperidol, Clozapine, and LASSBio-579, an Experimental Compound With Antipsychotic-Like Actions in Rodents, at G Protein and Arrestin Signaling Downstream of the Dopamine D2 Receptor. Front Pharmacol. 2019 Jun 4;10:628. doi: 10.3389/fphar.2019.00628. PMID: 31214037; PMCID: PMC6558205.
[6]: Zorn SH, Jones SB, et,al. Clozapine is a potent and selective muscarinic M4 receptor agonist. Eur J Pharmacol. 1994 Nov 15;269(3):R1-2. doi: 10.1016/0922-4106(94)90047-7. PMID: 7895765.
[7]: Heiser P, Schulte E, et,al. Effects of clozapine and its metabolites on the 5-HT2 receptor system in cortical and hippocampal cells in vitro. Prog Neuropsychopharmacol Biol Psychiatry. 2004 Mar;28(2):297-302. doi: 10.1016/j.pnpbp.2003.10.008. PMID: 14751426.
[8]: Guettier JM, Gautam D, et,al. A chemical-genetic approach to study G protein regulation of beta cell function in vivo. Proc Natl Acad Sci U S A. 2009 Nov 10;106(45):19197-202. doi: 10.1073/pnas.0906593106. Epub 2009 Oct 26. PMID: 19858481; PMCID: PMC2767362.
[9]: Jain S, Ruiz de Azua I, et,al. Chronic activation of a designer G(q)-coupled receptor improves β cell function. J Clin Invest. 2013 Apr;123(4):1750-62. doi: 10.1172/JCI66432. Epub 2013 Mar 8. PMID: 23478411; PMCID: PMC3613926.
[10]:Alexander GM, Rogan SC, et,al. Remote control of neuronal activity in transgenic mice expressing evolved G protein-coupled receptors. Neuron. 2009 Jul 16;63(1):27-39. doi: 10.1016/j.neuron.2009.06.014. PMID: 19607790; PMCID: PMC2751885.
| Cas No. | 34233-69-7 | SDF | |
| 别名 | 氯氮平N-氧化物 | ||
| 化学名 | 3-chloro-6-(4-methyl-4-oxidopiperazin-4-ium-1-yl)-5H-benzo[b][1,4]benzodiazepine | ||
| Canonical SMILES | C[N+]1(CCN(CC1)C2=C3C=CC=CC3=NC4=C(N2)C=C(C=C4)Cl)[O-] | ||
| 分子式 | C18H19ClN4O | 分子量 | 342.82 |
| 溶解度 | ≥ 17.15 mg/mL in DMSO, ≥ 49 mg/mL in Water with gentle warming | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.917 mL | 14.5849 mL | 29.1698 mL |
| 5 mM | 0.5834 mL | 2.917 mL | 5.834 mL |
| 10 mM | 0.2917 mL | 1.4585 mL | 2.917 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Chemogenetics revealed: DREADD occupancy and activation via converted clozapine
Science2017 Aug 4;357(6350):503-507.PMID: 28774929DOI: 10.1126/science.aan2475
The chemogenetic technology DREADD (designer receptors exclusively activated by designer drugs) is widely used for remote manipulation of neuronal activity in freely moving animals. DREADD technology posits the use of "designer receptors," which are exclusively activated by the "designer drug" clozapine N-oxide (CNO). Nevertheless, the in vivo mechanism of action of CNO at DREADDs has never been confirmed. CNO does not enter the brain after systemic drug injections and shows low affinity for DREADDs. Clozapine, to which CNO rapidly converts in vivo, shows high DREADD affinity and potency. Upon systemic CNO injections, converted clozapine readily enters the brain and occupies central nervous system-expressed DREADDs, whereas systemic subthreshold clozapine injections induce preferential DREADD-mediated behaviors.
The DREADD agonist clozapine N-oxide (CNO) is reverse-metabolized to clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice
Sci Rep2018 Mar 1;8(1):3840.PMID: 29497149DOI: 10.1038/s41598-018-22116-z
Clozapine-N-oxide (CNO) has long been the ligand of choice for selectively activating Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). However, recent studies have challenged the long-held assertion that CNO is otherwise pharmacologically inert. The present study aimed to 1) determine whether CNO is reverse-metabolized to its parent compound clozapine in mice (as has recently been reported in rats), and 2) determine whether CNO exerts clozapine-like interoceptive stimulus effects in rats and/or mice. Following administration of 10.0 mg/kg CNO, pharmacokinetic analyses replicated recent reports of back-conversion to clozapine in rats and revealed that this phenomenon also occurs in mice. In rats and mice trained to discriminate 1.25 mg/kg clozapine from vehicle, CNO (1.0-20.0 mg/kg) produced partial substitution for the clozapine stimulus on average, with full substitution being detected in some individual animals of both species at doses frequently used to activate DREADDs. The present demonstration that CNO is converted to clozapine and exerts clozapine-like behavioral effects in both mice and rats further emphasizes the need for appropriate control groups in studies employing DREADDs, and highlights the utility of the drug discrimination procedure as a tool with which to screen the off-target effects of novel DREADD agonists.
Gram scale preparation of clozapine N-oxide (CNO), a synthetic small molecule actuator for muscarinic acetylcholine DREADDs
MethodsX2018 Mar 23;5:257-267.PMID: 30038895DOI: 10.1016/j.mex.2018.03.003
Chemogenetics uses engineered proteins that are controlled by small molecule actuators, allowing in vivo functional studies of proteins with temporal and dose control, and include Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). One major class of DREADDs are mutated muscarinic receptors that are unresponsive to acetylcholine, and are activated by administration of clozapine N-oxide (CNO). However, CNO is available in only small amounts and large scale studies involving animals and multiple cohorts are prohibitively expensive for many investigators. The precursor, clozapine, is also expensive when purchased from specialist suppliers. Here we report: ?A simple extraction method of clozapine from commercial tablets;?A simple preparation of CNO from clozapine, and for the first time its single-crystal X-ray structure; and?That the CNO prepared by this method specifically activates the DREADD receptor hM3Dq in vivo. This method provides large quantities of CNO suitable for large-scale DREADD applications that is identical to commercial material.