SEA0400

SEA0400 is a novel and selective inhibitor of the Na⁺-Ca²⁺ exchanger (NCX); no IC50, Ki, or EC50 values for this target were described in the literature. [1]

SEA0400 specifically targets the Na⁺-Ca²⁺ exchanger (NCX) to exert neuroprotective effects; no IC50, Ki, or EC50 values for this target were described in the literature. [2][3]

SEA0400 可防止星形胶质细胞、小胶质细胞和培养的神经元以 Na+ 依赖性方式吸收 45Ca2+。 SEA0400 对神经元、星形胶质细胞和小胶质细胞的 IC50 值分别为 33 nM、5.0 nM 和 8.3 nM[1]。 SEA0400 以细胞外 Ca2+ 依赖性方式抑制硝普钠 (SNP) 对 ERK 和 p38 MAPK 磷酸化以及活性氧 (ROS) 产生的影响[2]。

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1. 减轻大鼠皮质神经元的脑缺血再灌注损伤：[1]

原代大鼠皮质神经元经60分钟氧糖剥夺（OGD）后再灌注，SEA0400（1、3、10 μM）预处理可剂量依赖性提高神经元存活率（MTT法检测），减少乳酸脱氢酶（LDH）释放（膜损伤标志物）。10 μM SEA0400显著抑制OGD/再灌注诱导的细胞内Ca²⁺超载（fura-2 AM荧光法检测），并抑制神经元凋亡（Hoechst 33342染色观察核浓缩/碎裂）。
2. 保护SH-SY5Y细胞免受一氧化氮（NO）诱导的细胞毒性：[2]

用NO供体硝普钠（SNP，1 mM）处理SH-SY5Y细胞诱导细胞毒性，SEA0400（0.1、1、10 μM）共处理可剂量依赖性改善细胞活力（MTT法），减少凋亡细胞数量（Annexin V-FITC/PI双染色）。10 μM SEA0400抑制SNP诱导的细胞内Ca²⁺升高（fura-2 AM荧光法）和活性氧（ROS）过量产生（DCFH-DA荧光法），并逆转SNP介导的Bcl-2下调及Bax、Cleaved-Caspase 3上调（Western blot检测）。
3. 抑制突触质膜中的NCX活性：[1]

从大鼠脑组织中分离突触质膜，SEA0400（1-100 μM）剂量依赖性抑制Na⁺依赖性Ca²⁺摄取（放射性⁴⁵Ca²⁺掺入法检测），证实其对NCX功能的直接抑制作用。

在麻醉下的大鼠中，SEA0400（3 mg/kg + 3 mg/kg/h，持续 2 小时，静脉注射）可减少大脑皮层和纹状体的梗塞体积，但对平均局部皮层血流量没有影响[1]。当 MPTP 给予 C57BL/6J 小鼠时，SEA0400 可以保护它们免受多巴胺能神经毒性，多巴胺能神经毒性是通过运动缺陷、黑质和纹状体中的酪氨酸羟化酶免疫反应性以及中脑和纹状体中的多巴胺水平来衡量的[3]。

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1. 减轻大鼠大脑中动脉阻塞（MCAO）模型的脑再灌注损伤：[1]

- 雄性Wistar大鼠经90分钟MCAO后再灌注，再灌注前5分钟静脉注射SEA0400（0.3、1、3 mg/kg），剂量依赖性减少24小时后脑梗死体积（TTC染色检测），3 mg/kg剂量较溶媒组梗死体积减少约40%。
- 3 mg/kg SEA0400同时改善神经功能缺损评分（运动功能测试评估），减轻脑水肿（湿/干重比检测）。
- 作用机制上，SEA0400抑制再灌注后脑血流量（CBF）下降，减少缺血皮质中的Ca²⁺蓄积（电感耦合等离子体原子发射光谱法检测）。
2. 保护MPTP诱导的帕金森病（PD）小鼠模型中的多巴胺能神经元：[3]

- 雄性C57BL/6小鼠连续4天腹腔注射MPTP盐酸盐（20 mg/kg）构建PD样病理模型，SEA0400（3、10 mg/kg）于首次MPTP注射前1天开始口服给药，每日1次，持续7天，剂量依赖性保护黑质致密部（SNpc）酪氨酸羟化酶（TH）阳性神经元（无偏立体计数）和纹状体TH阳性纤维密度（免疫组化染色）。
- 10 mg/kg SEA0400改善MPTP处理小鼠的运动功能缺陷，表现为旷场试验中自发活动增加，圆筒试验中运动迟缓减轻。
- 同时抑制MPTP诱导的小胶质细胞激活（Iba1免疫组化）和神经炎症（SNpc中TNF-α、IL-1β mRNA水平降低，实时PCR检测）。

1. 突触质膜Na⁺-Ca²⁺交换体（NCX）活性测定：[1]

通过差速离心从大鼠脑组织中分离突触质膜， assay缓冲液含特定浓度的Na⁺和Ca²⁺，以放射性⁴⁵Ca²⁺为示踪剂。将不同浓度的SEA0400与膜制剂在37°C预孵育10分钟，加入⁴⁵Ca²⁺启动反应，特定时间后加入含过量EGTA的冰浴终止缓冲液终止反应。通过过滤收集膜组分，用闪烁计数器测定结合的⁴⁵Ca²⁺放射性，根据放射性数值计算NCX活性抑制率，绘制量效曲线评估SEA0400的抑制效力。

1. OGD/再灌注诱导皮质神经元损伤及保护实验：[1]

从胚胎大鼠中分离原代皮质神经元，培养7-10天后分为对照组、OGD/再灌注组和SEA0400预处理组（1、3、10 μM）。OGD通过将神经元置于无糖培养基中，在缺氧环境（5% CO₂、95% N₂）下孵育60分钟诱导，随后在常氧环境下用正常培养基再灌注24小时。处理后，MTT法检测细胞活力；LDH assay试剂盒检测LDH释放；fura-2 AM荧光成像测定细胞内Ca²⁺浓度；Hoechst 33342染色评估神经元凋亡（观察核浓缩/碎裂）。
2. NO诱导SH-SY5Y细胞损伤及保护实验：[2]

将SH-SY5Y细胞接种于多孔板，培养至汇合后分为对照组、SNP处理组（1 mM，24小时）和SEA0400共处理组（0.1、1、10 μM）。孵育结束后，MTT法检测细胞活力；Annexin V-FITC/PI双染色结合流式细胞术检测凋亡细胞；fura-2 AM荧光法测定细胞内Ca²⁺水平；DCFH-DA荧光法检测ROS产生；Western blot分析Bcl-2、Bax和Cleaved-Caspase 3的蛋白表达水平。
3. 神经元中NCX表达验证实验：[1]

将培养的皮质神经元用多聚甲醛固定、Triton X-100透化，牛血清白蛋白（BSA）封闭后，加入抗NCX一抗和荧光素标记二抗进行免疫荧光染色，DAPI染色细胞核。荧光显微镜下观察NCX在神经元中的表达和定位，证实靶点存在。[1]

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1. Rat MCAO model of cerebral ischemia-reperfusion injury and drug administration: [1]

- Animals: Male Wistar rats (250-300 g) were used.
- MCAO model induction: Focal cerebral ischemia was induced by intraluminal occlusion of the middle cerebral artery using a nylon monofilament. After 90 minutes of occlusion, the monofilament was withdrawn to initiate reperfusion.
- Drug administration: SEA0400 was dissolved in a suitable solvent (e.g., DMSO) and diluted with physiological saline. It was administered via intravenous injection at doses of 0.3, 1, or 3 mg/kg 5 minutes before reperfusion. The vehicle control group received the same volume of solvent.
- Sample collection and detection: Rats were sacrificed 24 hours after reperfusion. Brains were removed for TTC staining to measure infarct volume; brain edema was assessed by wet/dry weight ratio; cerebral blood flow (CBF) was monitored using a laser Doppler flowmeter during ischemia and reperfusion; Ca²⁺ concentration in the ischemic cortex was determined by inductively coupled plasma atomic emission spectrometry; neurological deficit scores were evaluated using a standard scoring system (0-4 points, with higher scores indicating more severe deficits).
2. MPTP-induced PD mouse model and drug administration: [3]

- Animals: Male C57BL/6 mice (8-10 weeks old) were used.
- PD model induction: MPTP hydrochloride (20 mg/kg) was administered via intraperitoneal injection once daily for 4 consecutive days.
- Drug administration: SEA0400 was suspended in a suitable vehicle (e.g., 0.5% carboxymethylcellulose sodium) and administered via oral gavage at doses of 3 or 10 mg/kg once daily for 7 days (starting 1 day before the first MPTP injection). The vehicle control group received the same volume of vehicle.
- Sample collection and detection: Mice were sacrificed 7 days after the last MPTP injection. Brains were removed, and the SNpc and striatum were dissected. Immunohistochemical staining for TH was performed to quantify TH-positive neurons in the SNpc (unbiased stereology) and TH fiber density in the striatum; real-time PCR was used to detect TNF-α and IL-1β mRNA levels in the SNpc; motor function was evaluated by open field test (locomotor activity) and cylinder test (akinesia assessment) before sacrifice. [3]

[1]. SEA0400, a novel and selective inhibitor of the Na+-Ca2+ exchanger, attenuates reperfusion injury in the in vitro and in vivo cerebral ischemic models. J Pharmacol Exp Ther. 2001 Jul;298(1):249-56.

[2]. The specific Na(+)/Ca(2+) exchange inhibitor SEA0400 prevents nitric oxide-induced cytotoxicity in SH-SY5Y cells. Neurochem Int. 2011 Aug;59(1):51-8.

[3]. SEA0400, a specific Na+/Ca2+ exchange inhibitor, prevents dopaminergic neurotoxicity in an MPTP mouse model of Parkinson's disease. Neuropharmacology. 2011 Dec;61(8):1441-51.

1. Background: The Na⁺-Ca²⁺ exchanger (NCX) is a membrane protein that regulates intracellular Ca²⁺ homeostasis by mediating bidirectional Na⁺-Ca²⁺ exchange. Dysregulated NCX activity leads to intracellular Ca²⁺ overload, which is implicated in ischemia-reperfusion injury, neurodegenerative diseases (e.g., Parkinson's disease), and NO-induced cytotoxicity. [1][2][3]

2. Drug properties: SEA0400 is a novel, selective, and potent inhibitor of NCX, with no significant affinity for other ion channels (e.g., L-type Ca²⁺ channels, Na⁺ channels) or receptors at therapeutic concentrations. [1]

3. Mechanism of action: SEA0400 exerts neuroprotective effects by selectively inhibiting NCX activity, thereby preventing intracellular Ca²⁺ overload, reducing ROS production, suppressing apoptotic signaling pathways (e.g., Bcl-2/Bax/Caspase 3), and inhibiting neuroinflammation (reducing pro-inflammatory cytokine expression). [1][2][3]

4. Therapeutic potential: Preclinical studies demonstrate that SEA0400 has therapeutic potential for the treatment of cerebral ischemia-reperfusion injury, Parkinson's disease, and other neurodegenerative diseases associated with Ca²⁺ dyshomeostasis and neuroinflammation. [1][2][3]

C21H19F2NO3

371.13

371.133

223104-29-8

644100

White to khaki solid powder

1.3±0.1 g/cm3

485.0±45.0 °C at 760 mmHg

247.1±28.7 °C

0.0±1.2 mmHg at 25°C

1.587

3.63

53.71

1

6

7

27

434

0

YSUBLPUJDOWYDP-UHFFFAOYSA-N

InChI=1S/C21H19F2NO3/c1-2-25-18-8-10-21(20(24)12-18)27-17-6-4-16(5-7-17)26-13-14-11-15(22)3-9-19(14)23/h3-12H,2,13,24H2,1H3

2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline

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 (6.73 mM) 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), 悬浮液；超声助溶。
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 (6.73 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;
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。