mAChR3/4; impurity/metabolite of clozapine

由设计药物独家激活的设计受体（DREADD）是一种对远程集体自由移动动物的神经活动进行化学关联的方法。 DREADD 是一类明显改变的 G 偶联受体 (GPCR)，其本身对内源性神经递质无反应，但能接受其他“非”外源化学物质 [2]。

当给予去氯氮平（0.3 mg/kg；手术注射）时，恒河猴（5至6岁，体重5.5-7.9 kg）表现出工作记忆能力受损[3]。在体内，去氯氮平（0.1 mg/kg；肌肉注射）可逆地感知猴子的行为影响并有效激活 DREADD 受体 [3]。

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最常见的化学遗传神经调节系统，即仅由设计药物激活的设计受体（DREADDs），使用非内源性执行器配体激活对乙酰胆碱不敏感的修饰毒蕈碱乙酰胆碱受体。在使用这些系统的研究中，在任何DREADD转导之前测试DREADD致动器的潜在作用至关重要，这样DREADD的作用就可以归因于化学遗传系统而不是致动器药物，特别是在使用非人灵长类动物的实验中。我们研究了在任何DREADD转导发生之前，在空间延迟反应任务中测试的四只雄性恒河猴中注射三种DREADD致动器（氯氮平、奥氮平和去氯氯氮平）后的工作记忆性能。在四名受试者中，0.1 mg/kg氯氮平和0.1 mg/kgDeschloroclozapine/去氯氯氮平的表现与赋形剂没有差异。0.2mg/kg氯氮平损害了四只猴子中的三只的工作记忆功能。两只猴子在服用0.1mg/kg奥氮平后受损，两只猴子在服用0.3mg/kgDeschloroclozapine/去氯氯氮平后受损。我们推测，前额叶皮层功能的独特神经药理学使灵长类前额叶皮层特别容易受到DREADD致动器药物的脱靶效应的影响，这些药物对内源性单胺类受体系统具有亲和力。这些发现强调了DREADD促动剂药物在特定研究任务中的受试者内对照的重要性，以确认DREADD受体转导后的影响不是由促动剂本身引起的。他们还表明，DREADD促动器的脱靶效应可能会限制化学遗传神经调控的翻译应用。

放射性配体结合分析[2]
人胚胎肾（HEK-293，ATCC）细胞在添加了2 mM L-谷氨酰胺、抗生素/抗真菌剂（均来自Gibco）和10%热灭活胎牛血清的Dulbecco改良Eagle培养基中生长，并保存在37°C和5%CO2的培养箱中。对细胞进行常规检测，以检测其是否受到霉菌污染。转染前24小时，细胞以4×106个细胞/皿的速度接种在60 cm2的培养皿上。用5μg/皿编码hM3Dq、hM4Di的AAV包装质粒或对照载体转染细胞，并在转染后48小时收获。将细胞悬浮在补充有蛋白酶抑制剂混合物的Tris-HCl 50 mM pH 7.4中。HEK-293细胞用Polytron均质器破碎。匀浆在48000 g（50分钟，4°C）下离心，并在相同条件下洗涤两次，以分离膜部分。蛋白质用双辛可宁酸法定量。对于竞争实验，在室温下2小时内，将膜悬浮液（50μg蛋白质/mL）在含有10 mM MgCl2、2.5 nM[3H]氯氮平（3070 GBq/mmol（83 Ci/mmol））和竞争药物浓度增加的50 mM Tris-HCl（pH 7.4）中孵育。在10μM氯氮平存在的情况下测定非特异性结合。在所有情况下，通过在96孔板收集器中快速过滤500μL等分试样来分离游离和膜结合的放射性配体，并用2mL冰冷的Tris-HCl缓冲液洗涤。将Microscint-20闪烁液（65μL/孔）添加到滤板中。将平板在室温下孵育过夜，在MicroBeta2平板计数器中测定放射性计数，效率为41%。使用Prism 7拟合了一个地点的竞争曲线。Ki值使用Cheng-Prusoff方程计算。

Drugs were prepared fresh daily, at concentrations so that monkeys received 0.1 ml/kg for injection (e.g., for a 0.2 mg/kg dose of drug, drug solution was prepared at a concentration of 2.0 mg/ml). Solutions were filtered through a 0.22 µm syringe filter and pH was determined before injection. Acetic acid, sodium acetate, and sodium hydroxide (NaOH) were all obtained from Fisher Scientific. Concentrations used for glacial acetic acid, sodium acetate, and NaOH were 99.7% (v/v), 1 m, and 0.2 m, respectively. Clozapine was stored at room temperature. Clozapine was given at 0.1 or 0.2 mg/kg, intramuscularly. For 0.1 mg/kg, clozapine powder was first dissolved in acetic acid and sodium acetate then diluted with NaOH to a final concentration in 0.25/50/49.75 acetic acid/sodium acetate/NaOH (v/v/v). For 0.2 mg/kg, the same reagents were used but the final concentration was 0.5/50/49.5 acetic acid/sodium acetate/NaOH (v/v/v). Olanzapine was stored at room temperature and given at 0.05 or 0.1 mg/kg, intramuscularly. Olanzapine solutions were made using the same method as above for the 0.1 mg/kg clozapine dose. Deschloroclozapine was stored at 4 °C and was given at 0.1 mg/kg and 0.3 mg/kg, intramuscularly. Low dose of Deschloroclozapine was made using the same method as low-dose clozapine and high-dose Deschloroclozapine was made using the same method as the higher dose of clozapine. Vehicle injections consisted of 0.25/50/49.75 acetic acid, sodium acetate, and NaOH and were given at 0.1 ml/kg. Actuator injections were never administered more than twice in 1 week and never on adjacent test days to allow for a washout period, accounting for the half-life of clozapine (14.2 h on average) and olanzapine (33 h on average). There were vehicle or no-injection test days on other days of the test week. Actuators were not counterbalanced; we tested clozapine, olanzapine, and Deschloroclozapine in that order in each monkey. Within drug conditions, however, doses were shuffled in order across drug test days. We also interpolated additional clozapine test days during testing with the other two actuators. Throughout the study, we did not observe any order effects or obvious effects on behavior on the days following actuator injections. Clozapine and olanzapine were given in the home cage 10 min before the start of testing and Deschloroclozapine was given in the home cage 30 min before the start of testing. Clozapine and olanzapine both get into the brain fairly quickly and previous DREADD studies using these actuators have started behavior 10 min post injection. Deschloroclozapine shows a slightly slower timescale with higher plasma concentration ~15–30 min after intramuscular injection and CSF concentration continues to rise between 30 min to 90 min post injection. Accordingly, beginning the delayed response task 30 min post injection for Deschloroclozapine would allow sufficient time for the actuator to get into the brain.[3]

[1]. The metabolic formation of reactive intermediates from clozapine, a drug associated with agranulocytosis in man. J Pharmacol Exp Ther. 1995;275(3):1463-1475.

[2]. 18F-labeled radiotracers for in vivo imaging of DREADD with positron emission tomography. Eur J Med Chem. 2021;213:113047.

[3]. Effect of chemogenetic actuator drugs on prefrontal cortex-dependent working memory in nonhuman primates. Neuropsychopharmacology. 2020;45(11):1793-1798.

Clozapine, a dibenzodiazepine antipsychotic, is associated with a 0.8% incidence of agranulocytosis. This clinically restrictive toxicity has been attributed to its chemically reactive metabolites. The generation of such metabolites--assessed via covalent binding and formation of thioether adducts--was investigated using human, rat and mouse liver microsomes and human neutrophils and bone marrow cells. In every instance, one major glutathione adduct of clozapine--C-6 glutathionyl clozapine--was formed in the presence of added glutathione. Adduct formation by the neutrophils and myeloid cells was dependent on cell activation by phorbol myristate acetate. Small fractions of drug underwent covalent binding to microsomes (1-6.8%) and to protein coincubated with neutrophils (0.47%) and myeloid cells (0.21%). Clozapine did not deplete intracellular glutathione in activated neutrophils. Clozapine was also metabolized in vivo to glutathione conjugates in rats and mice, the conjugates eliminated in bile over a 3-hr period representing 38% and 33% of the dose, respectively. In addition to the principal clozapine adduct found in vitro, the C-8 glutathionyl derivative of Deschloroclozapine was excreted by both species. It is concluded that clozapine undergoes bioactivation in several tissues and considerable bioactivation in vivo. The reactive metabolites generated by neutrophils and myeloid cells may play an important role in the metabolic causation of clozapine-induced agranuiocytosis. [1]

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Designer Receptors Exclusively Activated by Designer Drugs (DREADD) are a preclinical chemogenetic approach with clinical potential for various disorders. In vivo visualization of DREADDs has been achieved with positron emission tomography (PET) using 11C radiotracers. The objective of this study was to develop DREADD radiotracers labeled with 18F for a longer isotope half-life. A series of non-radioactive fluorinated analogs of clozapine with a wide range of in vitro binding affinities for the hM3Dq and hM4Di DREADD receptors has been synthesized for PET. Compound [18F]7b was radiolabeled via a modified 18F-deoxyfluorination protocol with a commercial ruthenium reagent. [18F]7b demonstrated encouraging PET imaging properties in a DREADD hM3Dq transgenic mouse model, whereas the radiotracer uptake in the wild type mouse brain was low. [18F]7b is a promising long-lived alternative to the DREADD radiotracers [11C]clozapine ([11C]CLZ) and [11C]Deschloroclozapine ([11C]DCZ).

C18H22CL2N4

365.30

364.1221521

Deschloroclozapine;1977-07-7

166610795

Light yellow to yellow solid powder

30.9 Ų

3

3

1

24

413

0

CN1CCN(CC1)C2=NC3=CC=CC=C3NC4=CC=CC=C42.Cl.Cl

ZMDCCOPUWCVMFM-UHFFFAOYSA-N

InChI=1S/C18H20N4.2ClH/c1-21-10-12-22(13-11-21)18-14-6-2-3-7-15(14)19-16-8-4-5-9-17(16)20-18;;/h2-9,19H,10-13H2,1H3;2*1H

6-(4-methylpiperazin-1-yl)-11H-benzo[b][1,4]benzodiazepine;dihydrochloride

Deschloroclozapine (dihydrochloride); DCZ; 1977-07-7 (free base);

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。