5-HT7 receptors (Ki = 2.5nM); 5-HT1A (Ki = 700nM)

对于所使用的5-HT（7）受体激动剂，还评估了结合谱和刺激表达人5-HT（6）受体的HEK-293F细胞中cAMP形成的内在功效。AS-19和E-55888 HCL对5-HT（7）受体具有选择性。E-55888 HCL是一种完全激动剂，而AS-19和MSD-5a表现为部分激动剂，最大作用分别对应于完全激动剂5-HT诱发的cAMP反应的77%和61%[1]。

体内结果显示，全身给药5-HT（7）受体激动剂具有明显的剂量依赖性镇痛作用，5-HT（8）受体拮抗剂可以阻止这种作用，但5-HT（1A）受体拮抗药不能阻止这种作用。疗效顺序（E-55888 HCL>AS-19>MSD-5a）与它们作为5-HT（7）受体激动剂的体外疗效相匹配。与激动剂相反，在施用5-HT（7）受体拮抗剂后，观察到机械超敏反应的剂量依赖性促进，证实了5-HT（8）受体参与了辣椒素诱导的机械超敏的控制。这些发现表明，血清素通过激活5-HT（7）受体在控制伤害感受中发挥抑制作用，并指出5-HT（8）受体激动剂在镇痛领域的新的潜在治疗用途[1]。

E-55888 HCL和AS-19的结合亲和力由MDS Pharma和/或CEREP根据其标准测定方案通过商业放射性配体结合测定确定，如所示。进行了两次测定，并对两次浓度进行了测试。（有关特定受体和实验条件的详细信息，请参阅http://discovery.mdsps.com/Catalog/OnlineCatalog/Profiling/Assays/AssayList.aspx?id=5和http://www.cerep.fr/Cerep/Users/pages/catalog/binding/catalog.asp).
为了确定本研究中使用的5-HT7受体激动剂的功效/效力，使用基于均匀时间分辨荧光（HTRF）的系统进行cAMP测量，该系统应用于稳定表达人5-HT7（a）受体的人胚胎肾（HEK）-293F细胞，如前所述。该测定基于细胞产生的cAMP与cAMP-XL665结合物之间的竞争，以结合单克隆抗cAMP隐窝结合物。HTRF cAMP试剂盒按照制造商的说明使用。简而言之，在无血清培养基中孵育过夜后，将细胞加入含有1mM 3-异丁基-1-甲基黄嘌呤（IBMX）和20μM帕吉林的Ham's F12孵育缓冲液（40μl/孔）中的96孔板（20000个细胞/孔）中。然后，加入10μl不同浓度的受试化合物，在37°C下孵育30分钟。使用制造商提供的裂解缓冲液中制备的25μl穴状物和25μl XL-665的混合物停止反应。然后将板在室温下再孵育一小时，使用RubyStar板读数器根据665nm/620nm的比率计算cAMP含量[1]。

E-55888 HCL是一种新型的高选择性、高效的5-HT7受体激动剂。它对5-HT7受体（Ki=2.5nM）具有高亲和力，对5-HT1A （Ki=700nM）具有显著亲和力，而对其他5-HT受体亚型和170个额外靶点（包括受体、转运体和离子通道）没有显著亲和力（Ki>1μ m或在10 - 6M时抑制率低于50%）（CEREP和MDS Pharma Services的体外结合筛选包）（表1）。E-55888 HCL浓度依赖性地增加HEK-293F/h5-HT7细胞cAMP的形成，并表现为完全激动剂，其功效和效价（Emax=99±1%,EC50=16±1nM）与5-HT （Emax=100, EC50=11nM）相似。[1]

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该化合物/ as -19也在功能试验中进行了测试（cAMP刺激HEK-293F/h5-HT7细胞），发现其表现为一种有效的（EC50=9±1nM）但部分的5-HT7受体激动剂，其最大效果达到5-HT的77% [1]

Male CD1 mice aged from 6 to 8 weeks old were used. Animals were housed in groups of five, provided with food and water ad libitum and kept under controlled laboratory conditions (temperature: 21±1°C; 12h light/dark cycles with light on at 07:00h). Experiments were carried out in a soundproof and air-regulated room. The number of mice ranged from 9 to 16 in each experimental group as 3–4 independent experiments using 3–4 mice per group were always performed. All experimental procedures and animal husbandry were conducted according to ethical principles for the evaluation of pain in conscious animals and to ethical guidelines of the European Community on the Care and Use of Laboratory Animals (European Communities Council Directive of 24 November 1986, 86/609/ECC), and received approval by the Local Ethical Committee.[1]
When fixed at 1g, the pressure exerted by the filament is insufficient to induce timely pain responses in normal control mice (in the absence of capsaicin), and thus prompt withdrawal responses found 15min after sensitization with 1μg capsaicin reflect mechanical hypersensitivity (see Section 3 and Fig. 2). This approach enabled us to evaluate the possible antinociceptive effect of 5-HT7 and 5-HT1A receptor ligands.

[1]. 5-HT7 receptor activation inhibits mechanical hypersensitivity secondary to capsaicin sensitization in mice. Pain. 2009 Feb;141(3):239-47. https://pubmed.ncbi.nlm.nih.gov/19118950/

This work aimed to evaluate the potential role of the 5-HT(7) receptor in nociception secondary to a sensitizing stimulus in mice. For this purpose, the effects of relevant ligands (5-HT(7) receptor agonists: AS-19, MSD-5a, E-55888 HCL; 5-HT(7) receptor antagonists: SB-258719, SB-269970; 5-HT(1A) receptor agonist: F-13640; 5-HT(1A) receptor antagonist: WAY-100635) were assessed on capsaicin-induced mechanical hypersensitivity, a pain behavior involving hypersensitivity of dorsal horn neurons (central sensitization). For the 5-HT(7) receptor agonists used, binding profile and intrinsic efficacy to stimulate cAMP formation in HEK-293F cells expressing the human 5-HT(7) receptor were also evaluated. AS-19 and E-55888 HCL were selective for 5-HT(7) receptors. E-55888 HCL was a full agonist whereas AS-19 and MSD-5a behaved as partial agonists, with maximal effects corresponding to 77% and 61%, respectively, of the cAMP response evoked by the full agonist 5-HT. Our in vivo results revealed that systemic administration of 5-HT(7) receptor agonists exerted a clear-cut dose-dependent antinociceptive effect that was prevented by 5-HT(7) receptor antagonists, but not by the 5-HT(1A) receptor antagonist. The order of efficacy (E-55888 HCL>AS-19>MSD-5a) matched their in vitro efficacy as 5-HT(7) receptor agonists. Contrary to agonists, a dose-dependent promotion of mechanical hypersensitivity was observed after administration of 5-HT(7) receptor antagonists, substantiating the involvement of the 5-HT(7) receptor in the control of capsaicin-induced mechanical hypersensitivity. These findings suggest that serotonin exerts an inhibitory role in the control of nociception through activation of 5-HT(7) receptors, and point to a new potential therapeutic use of 5-HT(7) receptor agonists in the field of analgesia.[1]

C16H23N3.HCL

293.83

1034142-33-0

E55888

Solid powder if stored under nitrogen but this product is extremely hydroscopic and can become oily (ointment) when exposed to air

MFUWRMRKXKCSPL-UHFFFAOYSA-N

InChI=1S/C16H23N3/c1-12-16(13(2)19(5)17-12)15-8-6-7-14(11-15)9-10-18(3)4/h6-8,11H,9-10H2,1-5H3

N,N-dimethyl-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)phenethylamine

E-55888 HCL; E 55888 HCL; 1034142-33-0; E55888 HCL; E-55888 HCL; CHEMBL4205349; N,N-dimethyl-2-[3-(1,3,5-trimethylpyrazol-4-yl)phenyl]ethanamine; N,N-dimethyl-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)phenethylamine; Benzeneethanamine, N,N-dimethyl-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)-; dimethyl-{2-[3-(1,3,5-trimethyl-1H-pyrazol-4-yl)-phenyl]-ethyl}-amine; E55888 HCL

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保护的环境中，避免暴露在潮湿环境中，因为本产品吸湿性极强，暴露在空气中会变成油状（软膏）。

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。