DREADD; Dopamine receptor; muscarinic M4 receptor

氯氮平 N-氧化物 (CNO) 可以在 DREADD 激活所需的浓度下与非 DREADD 吸收结合，并经过逆转解读母体化合物氯平，氯氮平是一种非典型抗精神病药物，可作用于多种药理靶点并产生多种生理和行为影响[2]。

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在确认本文合成的氯氮平-N-氧化物/CNO和提取的氯氮平的生物活性的研究中，我们使用ERK1/2磷酸化测定作为与该受体相关的信号终点，测量了它们在细胞中激活M1 DREADD的能力。CNO和氯氮平产生了强效和有效的反应（pEC50 8.31 ± 0.12 10.32 ± 0.18;电子邮件74.8 ± 2.8 77.0 ± 3.7,分别见图3）。观察到的效力与该细胞系报告的商业CNO和氯氮平（Sigma-Aldrich）的效力相当（pEC50 8.50 ± 0.13 9.68 ± 0.28,分别）[4]。

在向小鼠单次腹膜内 (ip) 注射Clozapine-N-oxide (1 mg/kg) 后，Clozapine-N-oxide (CNO) 神经细胞水平在 15 分钟内达到高峰，2 小时后非常低。 CNO在小鼠体内的胚胎半期衰退，但对表达DREADD的实验动物进行迅速处理后所描述的生物学效应通常要长分裂（6-10小时）[1]。

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长期以来，Clozapine-N-oxide/氯氮平-N-氧化物（CNO）一直是选择性激活仅由设计药物激活的设计受体（DREADDs）的首选配体。然而，最近的研究对长期以来认为CNO在药理学上是惰性的说法提出了质疑。本研究旨在1）确定氯氮平-N-氧化物/CNO是否在小鼠体内逆代谢为其母体化合物氯氮平（如最近在大鼠中报道的那样），2）确定CNO是否在大鼠和/或小鼠体内产生氯氮平样的感受内刺激作用。在给予10.0 mg/kg的CNO后，药代动力学分析重复了最近关于大鼠反向转化为氯氮平的报告，并表明这种现象也发生在小鼠身上。在训练区分1.25 mg/kg氯氮平和赋形剂的大鼠和小鼠中，CNO（1.0-20.0 mg/kg）平均对氯氮平刺激产生部分替代，在经常用于激活DREADDs的剂量下，在两种物种的一些个体动物中都检测到完全替代。目前的证明表明，CNO转化为氯氮平，并在小鼠和大鼠中产生类似氯氮平的行为效应，这进一步强调了在使用DREADD的研究中需要适当的对照组，并强调了药物鉴别程序作为筛查新型DREADD激动剂脱靶效应的工具的实用性。[1]

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体内活性[4]
在证实体内合成的氯氮平-N-氧化物/CNO的生物活性的研究中，我们研究了NI在大鼠体内的化学活化作用。使用具有强鸡β-肌动蛋白（CAG）启动子-AVA1/2-sCAG-hM3Dq-mCherry的腺相关病毒载体表达系统，在NI神经元中表达与mCherry荧光蛋白融合的修饰Gq偶联的人毒蕈碱受体hM3Dq。使用AAV1/2-sCAG-mCherry载体作为对照。将这种病毒载体微量注射到NI中导致NI胞体和近端突起中hM3Dq表达（以下简称NI-hM3Dq），如荧光mCherry puncta的存在所示（图4A，B）。相比之下，对照AAV1/2-sCAG-mCherry载体（以下称为NI mCherry）在NI细胞中产生大量红色细胞质荧光（数据未显示），如所述。我们已经证明，NI-hM3Dq大鼠的化学遗传NI激活会导致皮质觉醒、睡眠减少、运动活动增加和风险评估行为增加，所有这些都与一般觉醒的增加是一致的。此外，这些研究还表明，与对照NI mCherry大鼠相比，氯氮平-N-氧化物/Clozapine-N-oxide/CNO在体外激活hM3Dq导致NI神经元具有动作电位的长期去极化，并且外周注射CNO（3 mg/kg）显著增加了NI-hM3Dq大鼠神经元激活的即时早期基因标志物c-Fos的免疫染色。这些数据证实了通过CNO激活hM3Dq在体内对NI的功能性激活，以及CNO在对照NI-mCherry大鼠中缺乏作用。

细胞外信号调节激酶1/2磷酸化（pERK1/2）测定[4]
按照制造商的说明，使用基于Alpha Screen的Sure Fire试剂盒进行了测量M1 DREADD介导的ERK1/2磷酸化刺激的检测。简而言之，将稳定表达M1 DREADD的FlpIn-CHO细胞以每孔40000个细胞的速度接种到96孔培养板中，并使其粘附。然后用磷酸盐缓冲盐水冲洗细胞，并在37℃下在无血清培养基中保持过夜 °C，5%二氧化碳。第二天，用激动剂刺激细胞。进行了初步的pERK1/2时间过程实验，以确定Clozapine-N-oxide/氯氮平-N-氧化物/CNO和氯氮平的ERK1/2最大磷酸化时间（发现为5 两种配体的最小值）。然后使用峰值激动剂反应的时间来建立浓度反应曲线。在所有实验中，10%（v/v）胎牛血清（FBS）用作pERK1/2的阳性对照。通过移除培养基和加入裂解缓冲液终止反应。样品按照试剂盒说明进行处理。使用POLARstar Omega板读数器测量荧光信号。将数据归一化为5%时10%（v/v）FBS引起的最大反应 min.

standard laboratory rats and mice
1.0, 3.2, 10.0 mg/kg for rats; 1.25, 2.5, 5.0, 10.0, 20.0 mg/kg for mice
i.p.

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Substitution tests occurred only when animals had satisfied strict performance criteria (see Supplementary Methods and Materials). To confirm the selectivity of the clozapine stimulus and its control over behavioral responding, rats were tested with vehicle or multiple doses of clozapine (0.0395, 0.125, 0.395, 1.25 mg/kg), the mixed dopamine/serotonin/norepinephrine antagonists olanzapine (1.0 mg/kg) and risperidone (0.56 mg/kg), the α1-adrenergic receptor antagonist prazosin (0.56 mg/kg), and the β-adrenergic receptor antagonist propranolol (10.0 mg/kg). Similarly, mice were tested with vehicle and multiple doses of clozapine (0.156, 0.3125, 0.625, 0.88, 1.25 mg/kg), olanzapine (0.5 mg/kg), the nonselective serotonin 5-HT2 receptor antagonist ritanserin (16.0 mg/kg), prazosin (10.0 mg/kg), and the nonselective dopamine D2-like receptor antagonist haloperidol (0.1 mg/kg). The doses of olanzapine, ritanserin, prazosin, and haloperidol were used because we have observed that higher doses produce nonspecific rate-suppressant effects. To test for Clozapine-N-oxide/CNO-induced clozapine-like effects, animals were administered Clozapine-N-oxide/CNO (rats −1.0, 3.2, 10.0 mg/kg; mice −1.25, 2.5, 5.0, 10.0, 20.0 mg/kg) prior to a test session. All drugs and doses were administered in a randomized order to each subject. [1]

Blood Sample Collection and Analysis [1]
Rats were surgically prepared with chronic indwelling intrajugular catheters as described previously24 to allow for rapid and repeated blood sampling. Blood collections began at least two weeks following surgery. On a test day, rats were administered either clozapine (1.25 mg/kg) or Clozapine-N-oxide/CNO (1.0, 10.0 mg/kg) and returned to the home cage. Blood samples (0.4–0.5 ml per sample, ~0.1 ml withdrawn per 5 s) were collected via aspiration from the intravenous catheter 30 and 60 min following drug administration. Catheters were flushed with bacteriostatic saline and locked with 0.1 ml of heparinized saline (300 heparin IU/ml) when not in use to maintain catheter patency between collections and on days between tests. Tests were separated by a minimum of 2 weeks and were performed in the following order for all subjects: (1) 10.0 mg/kg CNO, (2) 1.25 mg/kg clozapine, (3) 1.0 mg/kg CNO.

Mice were administered either clozapine (1.25 mg/kg) or Clozapine-N-oxide/CNO (10.0 mg/kg) and returned to their home cage. 2–3 min prior to the desired time point of blood sampling, each mouse was placed in a Plexiglas anesthesia induction chamber and exposed to 4–5% isoflurane until loss of movement and then transferred to a nosecone that continued to supply isoflurane (1–2%). Once deep anesthesia was verified, the heart was exposed and a 23 g needle attached to a 1 ml syringe was inserted into the left ventricle. 0.4–0.5 ml of blood was withdrawn and handled identical to the description above for rat blood sample collections.

All blood samples were deposited into a 1.7 ml tube containing 10 μl of heparinized saline (500 heparin IU/ml) and stored on ice until centrifugation at room temperature at 800 g for 10 min. The plasma was then removed, placed into a separate sterile 1.7 ml tube, and stored at −80 °C until subsequent analysis via UPLC-LC/MS/MS (see Supplementary Materials and Methods).

Clozapine, olanzapine, risperidone, haloperidol, prazosin, propranolol, and ritanserin were each dissolved in distilled water with 2–3 drops of lactic acid and pH-adjusted to 6.0–7.0 with NaOH. For mouse drug discrimination studies, CNO was also dissolved in this vehicle. For rat drug discrimination studies and for mouse and rat pharmacokinetic analyses, Clozapine-N-oxideCNO was dissolved in bacteriostatic saline containing v/v 2.5–5.0% dimethyl sulfoxide (Sigma-Aldrich) and 10% Cremophor EL.

For mouse drug discrimination studies, all drugs were administered s.c. at a volume of 10 ml/kg, 30 min prior to session onset. For rat drug discrimination studies, all drugs were administered i.p. at a volume of 1 ml/kg. Clozapine was administered 60 min prior to session onset, while olanzapine, risperidone, prazosin, and propranolol were administered 30 min prior to session onset. Clozapine-N-oxide/CNO was tested at both 30 and 60 min pretreatment times. All drug doses are expressed as the salt weight.

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In vivo test of food and water intake in rats [4]
On the day of use, CNO/Clozapine-N-oxide was dissolved in sterile saline at 1 mg/ml and administered by intraperitoneal (i.p.) injection in rats at 1 or 3 mg/kg, or rats were injected with an equivalent volume of sterile saline. For behavioral testing, rats received an i.p. injection of sterile saline on Day 1 and placed back into their home cage with access to pre-weighed food and water, and consumption was recorded 4 h post-injection. Testing was repeated on Day 2, where rats received an i.p. injection of CNO at 1 or 3 mg/kg. Food and water intake following CNO/Clozapine-N-oxide injection were normalized to intake following saline injection, where a value of 1 represents equivalent intake. Unpaired t-tests were used to calculate statistical differences between groups.

Metabolism / Metabolites
Clozapine-N-oxide is a known human metabolite of clozapine.

mouse LD50 oral 245 mg/kg BEHAVIORAL: ATAXIA Collection of Czechoslovak Chemical Communications., 43(309), 1978

[1]. Sci Rep. 2018 Mar 1;8(1):3840.

[2]. Front Pharmacol. 2019 Jun 4:10:628.

[3]. Eur J Pharmacol. 1994 Nov 15;269(3):R1-2.

[4]. MethodsX. 2018 Mar 23:5:257-267.

Clozapine N-oxide is a dibenzodiazepine.

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LASSBio-579, an N-phenylpiperazine antipsychotic lead compound, has been previously reported as a D2 receptor (D2R) ligand with antipsychotic-like activities in rodent models of schizophrenia. In order to better understand the molecular mechanism of action of LASSBio-579 and of its main metabolite, LQFM 037, we decided to address the hypothesis of functional selectivity at the D2R. HEK-293T cells transiently coexpressing the human long isoform of D2 receptor (D2LR) and bioluminescence resonance energy transfer (BRET)-based biosensors were used. The antagonist activity was evaluated using different concentrations of the compounds in the presence of a submaximal concentration of dopamine (DA), after 5 and 20 min. For both signaling pathways, haloperidol, clozapine, and our compounds act as DA antagonists in a concentration-dependent manner, with haloperidol being by far the most potent, consistent with its nanomolar D2R affinity measured in binding assays. In our experimental conditions, only haloperidol presented a robust functional selectivity, being four- to fivefold more efficient for inhibiting translocation of β-arrestin-2 (β-arr2) than for antagonizing Gi activation. Present data are the first report on the effects of LASSBio-579 and LQFM 037 on the β-arr2 signaling pathway and further illustrate that the functional activity could vary depending on the assay conditions and approaches used. [2]

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Clozapine was studied in functional assays at human muscarinic M1-M5 receptors expressed in Chinese hamster ovary cells. Clozapine was a full agonist at the muscarinic M4 receptor (EC50 = 11 nM), producing inhibition of forskolin-stimulated cAMP accumulation. In contrast, clozapine potently antagonized agonist-induced responses at the other four muscarinic receptor subtypes. Selective stimulation of M4 receptors may, in part, explain the hypersalivation observed clinically with clozapine. Moreover, the unique overall muscarinic profile of clozapine may contribute to its atypical antipsychotic efficacy. [3]

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Increasing use of muscarinic acetylcholine receptor DREADDs has led to a growing demand for CNO, but the high cost from most commercial vendors has created difficulties for large-scale deployment of the technology. This work reports simple access to clozapine from commercially available (pharmacy) tablets, and its conversion to CNO by oxidation with Oxone. Based on raw material costs (clozapine is obtained at approx. $14/g from tablets; reagent costs are minimal), CNO can be produced in a 97% yield at a similar price, excluding labor costs. By comparison, commercial prices for CNO from representative suppliers range from $53,000/g , $24,000/g, $6700/g. The present route with some proprietary modifications allowing removal of methanol has been implemented by AMT Pty Ltd , and commercial material produced by this method is now available through AK Scientific for $420/g, representing a major reduction in the cost of this important reagent. It is hoped that this work will support the continuing development of DREADD technology and further insights into brain function and related behaviors.[4]

C18H19CLN4O

342.82266

342.124

C, 63.06; H, 5.59; Cl, 10.34; N, 16.34; O, 4.67

34233-69-7

Clozapine; 5786-21-0; Clozapine N-oxide dihydrochloride; 2250025-93-3; 54241-01-9 (HCl); 34233-69-7 (N-oxide); 5786-21-0

135445691

Light yellow to yellow solid powder

1.36g/cm3

517.4ºC at 760mmHg

190-248ºC

266.7ºC

1.685

0.76

57.06

1

3

1

24

491

0

[O-][N+]1(C)CCN(C2=NC3=CC(Cl)=CC=C3NC4=CC=CC=C42)CC1

OGUCZBIQSYYWEF-UHFFFAOYSA-N

InChI=1S/C18H19ClN4O/c1-23(24)10-8-22(9-11-23)18-14-4-2-3-5-15(14)20-16-7-6-13(19)12-17(16)21-18/h2-7,12,20H,8-11H2,1H3

3-chloro-6-(4-methyl-4-oxidopiperazin-4-ium-1-yl)-11H-benzo[b][1,4]benzodiazepine

Clozapine N-oxide; Clozapine N-oxide; 34233-69-7; N-Oxyclozapine; UNII-MZA8BK588J; MZA8BK588J; VUFB-12426; NSC-750266; 8-Chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo(b,e)(1,4)diazepine N-oxide;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: 68~100 mg/mL (198.4~291.7 mM)
Water: ~68 mg/mL
Ethanol: ~8 mg/mL

配方 1 中的溶解度: ≥ 2.5 mg/mL (7.29 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 (7.29 mM) (饱和度未知) 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), 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 (7.29 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: ≥ 0.5 mg/mL (1.46 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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 5 中的溶解度: ≥ 0.5 mg/mL (1.46 mM) (饱和度未知) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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 6 中的溶解度: 5%DMSO+ 40%PEG300+ 5%Tween 80+ 50%ddH2O: 3.4mg/ml (9.92mM)

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配方 7 中的溶解度: 2.44 mg/mL (7.12 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶 (<60°C).

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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；
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