AM-6545

Cannabinoid receptor/CB receptor

AM6545以1.7 nM的K（i）结合CB（1）受体，以523 nM的K（i）与CB（2）受体结合。AM6545是一种中性拮抗剂，对转染细胞中的cAMP水平没有影响，并且其中心渗透性不如AM4113，AM4113是一种类似的CB（1）受体拮抗剂[1]。

AM6545在结肠运动测定中逆转了WIN55212-2的作用。与AM251相比，AM6545在大鼠中没有产生条件性张口或条件性味觉回避。在大鼠和小鼠中，AM6545剂量依赖性地减少了食物摄入，并诱导体重持续下降。在完全膈下迷走神经切断术的大鼠中，对食物摄入的影响得以维持。AM6545抑制CB（1）受体基因缺陷小鼠的食物摄入，但不抑制CB（1/CB（2）受体双敲除小鼠的食物摄取[1]。

大麻素受体结合试验[1]
使用由表达小鼠CB2（mCB2）或人CB2（hCB2）和[3H]CP-55940的大鼠脑（CB1）或HEK293细胞制备的膜制剂测试AM6545对大麻素受体的亲和力，如前所述（Morse等人，1995；Lan等人，1999；McLaughlin等人，2006）[1]。

cAMP测定
反向激动剂与大麻素受体的结合提高了细胞内环AMP（cAMP）的水平，而中性拮抗剂对cAMP水平没有影响（Janero和Makriyannis，2009）。我们观察了AM6545对毛喉素诱导的cAMP水平的影响，以确定它是否是一种中性拮抗剂。作为阳性对照，还研究了反向激动剂AM251的作用。如前所述（McLaughlin等人，2006），使用表达hCB1或hCB2的完整HEK293细胞通过竞争性结合试验测量细胞内cAMP水平。裂解细胞并离心后，使用cAMP测定试剂盒（Sigma-Aldrich，St.Louis，MO，USA）测定所得上清液中释放的cAMP。[1]

Brain penetration assay[1]
Rats (280–310 g) were killed (sodium pentobarbital, 80–100 mg·kg−1, i.p.) 1, 3 and 5 h after receiving an i.p. injection of vehicle (4% DMSO, 1% Tween 80 in physiological saline; n = 2), AM6545 (10 mg·kg−1; n = 4) or AM4113 (10 mg·kg−1; n = 4). In further experiments, female C57BL/6 mice (18.5–21.0 g) were administered an i.p. injection of vehicle (n = 2) or AM6545 (5 mg·kg−1; n = 4) and were killed 1, 3 and 17 h post-injection. From both rats and mice, blood was collected and centrifuged for plasma, and brains were removed. All samples were flash-frozen in liquid nitrogen and stored at −80°C. Tissues (plasma or brain) were extracted following published procedures (Folch et al., 1957) and analysed in SRM mode after APCI+ ionization using a Thermo-Finnigan Quantum Ultra triple quadrupole mass spectrometer with an Agilent 1100 HPLC front-end. Compounds were eluted from the Phenomenex Gemini C18 column (2 × 50 mm, 5 µ) with a C18 guard column using a gradient elution consisting of 0.1% formic acid in both methanol (A) and water (B). The detection limits in this assay for AM6545 were: rat plasma, 9 ng·mL−1; mouse plasma, 2.6 ng·mL−1 and for AM4113, in rat plasma, 1.5 ng·mL−1. For brain samples, the corresponding lower limits were AM6545 (rat) 7.5 ng·g−1 (mouse) 5.4 ng·g−1 and AM4113 (rat) 5.3 ng·g−1.

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Colonic propulsion assay[1]
Cannabinoid agonists slow GI transit and thus the actions of AM6545 on WIN55212-2-induced slowing of colonic propulsion was used to confirm the functional blockade of peripheral CB1 receptors by AM6545 (Pinto et al., 2002). Male C57BL/6 mice (19–26 g) were lightly anesthetized with isoflurane (4% in air) before a 2.5 mm spherical glass bead was inserted 2 cm intrarectally. The time to the expulsion of the bead was recorded. AM6545 (10 mg·kg−1, n = 5–10 or 20 mg·kg−1, n = 6–7) or vehicle (4% DMSO, 1% Tween 80 in physiological saline, n = 9–18) was injected i.p. 60 min prior to the administration of WIN55212-2 (1 mg·kg−1, n = 7–16), loperamide (1 mg·kg−1, n = 5–9) or vehicle (n = 7–18). Twenty minutes later, colonic propulsion was measured. Doses of WIN55212-2 (Pinto et al., 2002) and loperamide (Yamada and Onoda, 1993) were based on previous work.

[1]. A novel peripherally restricted cannabinoid receptor antagonist, AM6545, reduces food intake and body weight, but does not cause malaise, in rodents. Br J Pharmacol. 2010 Oct;161(3):629-42.

Background and purpose: Cannabinoid CB(1) receptor antagonists reduce food intake and body weight, but clinical use in humans is limited by effects on the CNS. We have evaluated a novel cannabinoid antagonist (AM6545) designed to have limited CNS penetration, to see if it would inhibit food intake in rodents, without aversive effects. Experimental approach: Cannabinoid receptor binding studies, cAMP assays, brain penetration studies and gastrointestinal motility studies were carried out to assess the activity profile of AM6545. The potential for AM6545 to induce malaise in rats and the actions of AM6545 on food intake and body weight were also investigated. Key results: AM6545 binds to CB(1) receptors with a K(i) of 1.7 nM and CB(2) receptors with a K(i) of 523 nM. AM6545 is a neutral antagonist, having no effect on cAMP levels in transfected cells and was less centrally penetrant than AM4113, a comparable CB(1) receptor antagonist. AM6545 reversed the effects of WIN55212-2 in an assay of colonic motility. In contrast to AM251, AM6545 did not produce conditioned gaping or conditioned taste avoidance in rats. In rats and mice, AM6545 dose-dependently reduced food intake and induced a sustained reduction in body weight. The effect on food intake was maintained in rats with a complete subdiaphragmatic vagotomy. AM6545 inhibited food intake in CB(1) receptor gene-deficient mice, but not in CB(1)/CB(2) receptor double knockout mice. Conclusions and implications: Peripherally active, cannabinoid receptor antagonists with limited brain penetration may be useful agents for the treatment of obesity and its complications. [1]

C26H23CL2N5O3S

556.463522195816

555.09

1245626-05-4

46912919

White to off-white solid powder

5.778

119.96

1

6

6

37

1020

0

CC1=C(N(N=C1C(NN2CCS(=O)(CC2)=O)=O)C3=C(Cl)C=C(Cl)C=C3)C4=CC=C(C=C4)C#CCCC#N

XBHQLFVDGLPBCK-UHFFFAOYSA-N

InChI=1S/C26H23Cl2N5O3S/c1-18-24(26(34)31-32-13-15-37(35,36)16-14-32)30-33(23-11-10-21(27)17-22(23)28)25(18)20-8-6-19(7-9-20)5-3-2-4-12-29/h6-11,17H,2,4,13-16H2,1H3,(H,31,34)

5-[4-(4-cyanobut-1-ynyl)phenyl]-1-(2,4-dichlorophenyl)-N-(1,1-dioxo-1,4-thiazinan-4-yl)-4-methylpyrazole-3-carboxamide

AM6545; AM 6545; AM-6545; CHEMBL3341898; 5-(4-[4-cyanobut-1-ynyl]phenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(1,1-dioxo-thiomorpholino)-1H-pyrazole-3-carboxamide; 5-[4-(4-Cyanobut-1-yn-1-yl)phenyl]-1-(2,4-dichlorophenyl)-N-(1,1-dioxo-1lambda~6~,4-thiazinan-4-yl)-4-methyl-1H-pyrazole-3-carboxamide; 5-[4-(4-cyanobut-1-ynyl)phenyl]-1-(2,4-dichlorophenyl)-N-(1,1-dioxo-1,4-thiazinan-4-yl)-4-methylpyrazole-3-carboxamide;

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。