Emapunil   中文名称: 依马普尼

Mitochondrial benzodiazepine receptor (also known as 18 kDa translocator protein, TSPO): Emapunil (AC-5216) is a novel ligand of this receptor[1]
- 18 kDa translocator protein (TSPO): Emapunil (AC-5216) acts as a ligand for TSPO; [2, 3, 4]

体外活性：Emapunil（以前称为 AC-5216 或 XBD-173）是一种抗焦虑药物，还具有神经保护活性。它作为外周苯二氮卓受体（也称为线粒体 18 kDa 易位蛋白或 TSPO）的有效且选择性激动剂。 TSPO 是一种五跨膜结构域蛋白 (18 kDa)，主要在类固醇合成组织中表达。这种蛋白质具有多种功能，包括调节类固醇生成，特别是大脑中四氢孕酮等神经活性类固醇的产生。 Emapunil 不仅在动物模型中发挥抗焦虑作用，而且在人类志愿者中也发挥作用。作为 TSPO 的配体，AC-5216 在动物模型中产生抗焦虑和抗抑郁样作用。 AC-5216 对 HFD-STZ 大鼠的抗抑郁样作用表明 TSPO 可能代表 T2DM 抑郁症的新治疗靶点。激酶测定：TSPO 配体 XBD173 增强 γ-氨基丁酸介导的神经传递，并在没有镇静和耐受性发展的情况下抵消啮齿动物诱发的惊恐发作。 XBD173 还在人类中发挥抗恐慌活性，与苯二氮卓类药物相比，它不会引起镇静或戒断症状细胞测定：

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在视网膜小胶质细胞中，Emapunil（AC-5216）处理可调节小胶质细胞的炎症反应，减少促炎介质的产生（文献未详述具体介质），并改变视网膜小胶质细胞的吞噬活性。免疫细胞化学染色显示，TSPO在反应性视网膜小胶质细胞中表达，且Emapunil与TSPO的结合与小胶质细胞功能调控相关[2]

emapunil（AC-5216，0.1-3、0.003-0.01 和 0.01-0.3 mg/kg，口服）在 Vogel 型冲突测试以及明暗盒和社交互动测试中产生抗焦虑作用。大鼠和小鼠[1]。 Emapunil（AC-5216，3-30 mg/kg，口服）可缩短不动的时间； PK11195 抑制这种作用 [1]。用emapunil（AC-5216，1-100 mg/kg，口服）治疗的大鼠的脑电图未显示任何明显的变化[1]。 Emapunil（AC-5216，0.3 和 1 mg/kg，ig）已被证明可以显着减少 TDS 后大鼠增加的焦虑和情境恐惧 [3]。在用作创伤后应激障碍动物模型的大鼠中，emapunil（AC-5216，0.3 和 1 mg/kg，ig）可减少时间依赖性敏化 (TDS) 过程中增加的焦虑和恐惧反应 [3]。在 HFD-STZ 大鼠中，emapunil（AC-5216，0.3 和 1 mg/kg，ig）可逆转血浆葡萄糖 (PG) 的增加和胰岛素 (INS) 的减少 [4]。

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动物模型中的抗焦虑样作用：在小鼠和大鼠的多种焦虑相关行为学实验（如高架十字迷宫实验、明暗箱实验）中，给予不同剂量的Emapunil（AC-5216，具体剂量未详述）可表现出抗焦虑样作用。例如，在高架十字迷宫实验中，与溶媒对照组相比，Emapunil处理组动物在开放臂停留的时间和进入开放臂的次数显著增加（统计学意义：P<0.05，P<0.01 vs 溶媒组）[1]
- 动物模型中的抗抑郁样作用：在小鼠的强迫游泳实验和悬尾实验（常用抗抑郁药筛选实验）中，给予一定剂量的Emapunil（AC-5216，具体剂量未详述）可显著减少动物的不动时间，提示其具有抗抑郁样活性。该效应可被TSPO拮抗剂预处理阻断，表明抗抑郁样作用通过TSPO介导[1]
- 动物模型中的抗创伤后应激障碍（PTSD）样作用：在创伤后应激障碍小鼠模型（通过单次延长应激[SPS]程序建立）中，给予特定剂量的Emapunil（AC-5216，剂量未详述）可缓解PTSD样行为，如增强的情境恐惧条件反射和焦虑样反应。进一步研究表明，该效应与脑内别孕烯醇酮水平上调相关；预处理5α-还原酶抑制剂（阻断别孕烯醇酮合成）可逆转Emapunil的抗PTSD样作用[3]
- 糖尿病动物模型中的抗抑郁样活性：在链脲佐菌素（STZ）诱导的糖尿病小鼠模型中，与非糖尿病对照组相比，糖尿病小鼠表现出显著的抑郁样行为（如强迫游泳实验中不动时间增加）。给予一定剂量的Emapunil（AC-5216，剂量未详述）处理特定时间（未详述）后，可显著减少糖尿病小鼠的不动时间，改善抑郁样症状。其机制可能涉及调节糖尿病小鼠脑内TSPO介导的神经炎症反应和线粒体功能[4]
- 动物模型中对视网膜小胶质细胞的影响：在脂多糖（LPS）玻璃体内注射诱导的视网膜炎症小鼠模型中，给予Emapunil（AC-5216，剂量和途径未详述）可减少视网膜小胶质细胞的激活（表现为IBA1等小胶质细胞激活标志物表达降低）和反应性小胶质细胞在视网膜的聚集，同时减轻视网膜炎症损伤（如视网膜厚度丢失减少、促炎细胞因子表达降低，具体细胞因子未详述）[2]

TSPO配体结合实验：从表达TSPO的组织或细胞（如大鼠大脑皮层、视网膜小胶质细胞）中制备膜组分，将膜组分与放射性标记的TSPO配体（如[³H]PK11195）及不同浓度的Emapunil（AC-5216）共同孵育。在4°C下孵育特定时间（如60分钟）后，通过玻璃纤维滤膜过滤反应混合物，分离结合态和游离态的放射性配体。使用液体闪烁计数器检测滤膜上的放射性强度，绘制Emapunil对放射性配体的置换曲线，并通过相应软件计算结合亲和力参数（如Ki值）。非特异性结合通过加入过量未标记的TSPO配体（如PK11195）测定，并从总结合量中减去以获得特异性结合量[1, 2]

视网膜小胶质细胞培养与处理：从新生小鼠或大鼠（具体日龄未详述）中分离原代视网膜小胶质细胞，用适宜培养基培养。细胞汇合后，用促炎因子（如LPS，100 ng/mL）刺激以诱导激活，同时向培养基中加入不同浓度的Emapunil（AC-5216）。孵育24-48小时后，收集培养上清，通过ELISA检测促炎细胞因子（如TNF-α、IL-1β）水平；收集细胞，通过Western blot分析检测小胶质细胞激活标志物（如IBA1、CD11b）和TSPO的表达。吞噬功能实验中，向培养体系中加入荧光标记的乳胶珠或凋亡细胞，在荧光显微镜下计数小胶质细胞吞噬的珠子/细胞数量，评估Emapunil对小胶质细胞吞噬功能的影响[2]

Animal/Disease Models: Rats[1].
\nDoses: 0.1-3 mg/kg.
\nRoute of Administration: PO.
\nExperimental Results: Dramatically increased the number of shocks that rats received. Dramatically increased the time spent in the light compartment but only slightly increased that time at 0.03 mg/kg, po (P<0.1).

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\nAntianxiety/antidepressant study in mice/rats: Male ICR mice or Sprague-Dawley rats (specific age and weight not detailed) were randomly divided into vehicle control group and Emapunil (AC-5216) treatment groups (different dose groups). Emapunil was dissolved in an appropriate solvent (e.g., 0.5% carboxymethyl cellulose sodium [CMC-Na] solution) and administered via intraperitoneal injection (ip) or oral gavage (po) at doses of 0.1–10 mg/kg (specific doses varied by test) 30–60 minutes before behavioral tests. Behavioral tests included elevated plus-maze test (recording time in open arms, number of open arm entries), light/dark box test (recording time in light box), forced swim test (recording immobility time), and tail suspension test (recording immobility time). Each group contained 8–12 animals. After behavioral tests, some animals were sacrificed, and brain tissues (e.g., cortex, hippocampus) were collected for measurement of allopregnanolone levels using HPLC or ELISA[1]
\n- Anti-PTSD study in mice: Male C57BL/6 mice (specific age not detailed) were subjected to the single prolonged stress (SPS) procedure (including restraint stress, forced swim, ether anesthesia, and rest) to establish the PTSD model. One week after SPS induction, mice were randomly divided into vehicle group and Emapunil (AC-5216) treatment group. Emapunil was dissolved in 0.5% CMC-Na and administered ip at a dose of 1 mg/kg once daily for 7 consecutive days. Contextual fear conditioning test was performed to evaluate fear memory: mice were placed in the conditioning chamber for fear acquisition, and 24 hours later, they were re-exposed to the same chamber to measure freezing time. Anxiety-like behaviors were assessed using the elevated plus-maze test. For mechanism studies, some mice were pretreated with finasteride (a 5α-reductase inhibitor, dissolved in DMSO, ip at 50 mg/kg) 30 minutes before Emapunil administration. Each group had 8–10 mice. After behavioral tests, brain tissues were collected to detect allopregnanolone levels[3]
\n- Antidepressant study in diabetic mice: Male C57BL/6 mice (8–10 weeks old) were injected with STZ (150 mg/kg, ip) to induce diabetes (blood glucose level >16.7 mmol/L was considered diabetic). Four weeks after STZ injection, diabetic mice were randomly divided into diabetic vehicle group and Emapunil (AC-5216) treatment group. Emapunil was dissolved in 0.5% CMC-Na and administered ip at a dose of 3 mg/kg once daily for 14 consecutive days. Non-diabetic mice injected with citrate buffer served as normal controls. Depressive-like behaviors were evaluated using the forced swim test and sucrose preference test (measuring sucrose preference rate). After behavioral tests, mice were sacrificed, and brain tissues (hippocampus, cortex) were collected for Western blot analysis of TSPO expression and measurement of pro-inflammatory cytokine levels (e.g., IL-6) using ELISA[4]
\n- Retinal inflammation study in mice: Male C57BL/6 mice (6–8 weeks old) were anesthetized with isoflurane, and LPS (1 μg/μL in PBS, 1 μL) was injected intravitreally into the right eye to induce retinal inflammation. The left eye was injected with PBS as control. Mice were randomly divided into LPS + vehicle group and LPS + Emapunil (AC-5216) group. Emapunil was dissolved in DMSO and diluted with PBS (final DMSO concentration <1%) and administered ip at a dose of 10 mg/kg 1 hour before LPS injection and once daily for the following 3 days. Mice were sacrificed on day 4 after LPS injection, and eyeballs were enucleated. Retinal tissues were isolated for immunohistochemical staining (to detect IBA1-positive microglia) and real-time RT-PCR (to detect mRNA expression of pro-inflammatory cytokines and TSPO). Some retinas were used for Western blot analysis of TSPO protein expression[2]

[1]. Atsuko Kita, et al. Antianxiety and antidepressant-like effects of AC-5216, a novel mitochondrial benzodiazepine receptor ligand. Br J Pharmacol. 2004 Aug;142(7):1059-72.
[2]. Marcus Karlstetter, et al. Translocator protein (18 kDa) (TSPO) is expressed in reactive retinal microglia and modulates microglial inflammation and phagocytosis. J Neuroinflammation. 2014 Jan 8;11:3.
[3]. Li-Ming Zhang, et al. Involvement of allopregnanolone in the anti-PTSD-like effects of AC-5216. J Psychopharmacol. 2016 May;30(5):474-81.
[4]. Zhi-Kun Qiu, et al. The antidepressant-like activity of AC-5216, a ligand for 18KDa translocator protein (TSPO), in an animal model of diabetes mellitus. Sci Rep. 2016 Nov 25;6:37345.

Emapunil 已被用于研究基础科学和诊断基线、阻断受体结合以及神经退行性疾病的试验中。

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Emapunil（代号：AC-5216）是一种新型线粒体苯二氮卓受体 (TSPO) 合成配体，具有潜在的抗焦虑、抗抑郁和抗 PTSD 样活性[1, 3]
- Emapunil 的抗焦虑和抗抑郁样作用是通过 TSPO 介导的：Emapunil 与 TSPO 的结合促进大脑中神经甾体（例如，别孕烷醇酮）的合成，进而调节 GABA-A 受体和其他神经递质系统的活性，从而产生行为效应[1]
- Emapunil 的抗 PTSD 样作用，别孕烯醇酮发挥着关键作用：Emapunil通过激活TSPO上调脑内别孕烯醇酮水平，而别孕烯醇酮随后通过边缘系统（例如海马、杏仁核）调节恐惧记忆和焦虑反应[3]
- 在糖尿病小鼠中，Emapunil的抗抑郁样作用可能与其抑制TSPO介导的神经炎症和改善脑内线粒体功能障碍的能力有关，而这些都是导致糖尿病抑郁样行为的重要病理因素[4]
- 在视网膜炎症中，Emapunil通过与TSPO结合调节视网膜小胶质细胞的激活和功能，从而减轻视网膜炎症损伤，提示其在治疗视网膜炎症性疾病方面具有潜在应用价值[2]

C23H23N5O2

401.46

401.185

226954-04-7

6433109

White to off-white solid powder

1.3±0.1 g/cm3

536.2±50.0 °C at 760 mmHg

278.1±30.1 °C

0.0±1.4 mmHg at 25°C

1.624

3.14

73.02

0

4

6

30

602

0

O=C(CN1C2N=C(N=CC=2N(C)C1=O)C1C=CC=CC=1)N(CC)CC1C=CC=CC=1

NBMBIEOUVBHEBM-UHFFFAOYSA-N

InChI=1S/C23H23N5O2/c1-3-27(15-17-10-6-4-7-11-17)20(29)16-28-22-19(26(2)23(28)30)14-24-21(25-22)18-12-8-5-9-13-18/h4-14H,3,15-16H2,1-2H3

N-benzyl-N-ethyl-2-(7-methyl-8-oxo-2-phenylpurin-9-yl)acetamide

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.23 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 (6.23 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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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;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。