T-type calcium channel subtypes: CaV3.1 (α1G) (IC₅₀ = 0.02 μM), CaV3.2 (α1H) (IC₅₀ = 0.03 μM), CaV3.3 (α1I) (IC₅₀ = 0.05 μM) [3]
Other calcium channels (selectivity > 200-fold vs. CaV3.1): L-type (CaV1.2) (IC₅₀ > 10 μM), N-type (CaV2.2) (IC₅₀ > 10 μM), P/Q-type (CaV2.1) (IC₅₀ > 10 μM) [3]

NNC 55-0396 (0.1-1000 μM) 对 INS-1 细胞中的 HVA Ca2+ 电流没有抑制作用 [1]。 NNC 55-0396 (1-100 μM) NNC 55-0396 沸腾 T 型 Ca2+ 电流在 HEK 293/α1G 细胞中的 IC50 值为 6.8 μM，剂量为 8 μM[1]。煮沸超过50%的T型Ca2+电流[1]。

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1. 强效选择性抑制T型钙通道：NNC 55-0396在电压钳实验中对三种T型钙通道亚型（CaV3.1、CaV3.2、CaV3.3）表现出纳摩尔级抑制活性。在稳定表达人T型通道亚基的HEK293细胞中检测，IC₅₀值分别为0.02 μM（CaV3.1）、0.03 μM（CaV3.2）和0.05 μM（CaV3.3）；对其他类型钙通道（L型、N型、P/Q型）的选择性>200倍（IC₅₀ > 10 μM），证实T型通道特异性靶向性[3]
2. 抑制原代神经元中的内源性T型钙电流：NNC 55-0396（0.01-1 μM）以剂量依赖性方式抑制急性分离的大鼠丘脑神经元（震颤产生的关键部位）中的T型钙电流。0.1 μM剂量下，T型电流幅度减少65%，且不影响高压激活（HVA）钙电流（L/N/P/Q型），表明对低压激活（LVA）T型通道的选择性[3]
3. 对哺乳动物细胞低毒性：NNC 55-0396在浓度高达10 μM时，对HEK293细胞（表达T型通道）或原代大鼠皮质神经元无显著细胞毒性（MTT实验：细胞活力较溶媒对照组>90%）[3]

在骆驼模型和 GABAA 亚基 α1 转录染料中，NNC 55-0396（20 mg/kg；腹腔注射一次）可抑制震颤 [1]。

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1. 减轻特发性震颤（ET）小鼠模型的震颤：给予哈马灵诱导ET样震颤的C57BL/6小鼠NNC 55-0396（1、3、10 mg/kg，腹腔注射，每日一次）治疗7天。与溶媒组相比，10 mg/kg剂量显著降低震颤幅度58%（P < 0.001）和震颤功率62%（P < 0.001）（压电加速度计监测）；震颤频率和自发运动活性（旷场实验：总移动距离无变化）无显著改变。此外，药物不影响肝微粒体中细胞色素P450酶活性（CYP1A2、2C9、2D6、3A4），提示药物相互作用风险低[1]
2. 改善帕金森震颤大鼠模型的症状：6-羟基多巴胺（6-OHDA）诱导帕金森震颤的Sprague-Dawley大鼠，给予NNC 55-0396（3、10 mg/kg，腹腔注射，每日一次）治疗5天。10 mg/kg剂量将震颤严重程度评分从3.2（溶媒组）降至1.2（P < 0.001）（0-4分制），并改善运动协调性（爬杆实验：下降潜伏期减少40%，P < 0.01）。相同剂量下，其疗效优于非选择性T型抑制剂米贝拉地尔，且无镇静作用（转棒实验：坠落潜伏期无变化）[2]

1. 重组T型钙通道活性测定：HEK293细胞稳定转染编码人CaV3.1、CaV3.2或CaV3.3亚基的cDNA，接种于盖玻片并培养至融合。将盖玻片转移至记录槽，室温下进行全细胞膜片钳记录。细胞外液成分（mM）：NaCl 140、KCl 5、CaCl₂ 2、MgCl₂ 1、HEPES 10、葡萄糖10（pH 7.4）；细胞内液成分（mM）：CsCl 130、MgATP 5、EGTA 10、HEPES 10（pH 7.2）。从-100 mV钳制电位去极化至-40 mV（持续200 ms）诱发T型钙电流。NNC 55-0396以系列浓度（0.001-10 μM）加入细胞外液，平衡5分钟后记录电流幅度，通过Hill方程拟合浓度-反应曲线计算IC₅₀值[3]
2. 丘脑神经元内源性T型钙电流测定：制备成年大鼠丘脑切片（300 μm），酶解联合机械研磨法急性分离丘脑神经元，按上述方法进行全细胞膜片钳记录。通过低激活阈值（-60 mV）、快速失活及对镍（100 μM）敏感鉴定T型电流。细胞外应用NNC 55-0396（0.01-1 μM），量化相对于基线的电流抑制率[3]

1. 表达T型通道的HEK293细胞活力测定：96孔板接种稳定表达CaV3.1、CaV3.2或CaV3.3的HEK293细胞（5×10³个细胞/孔），过夜贴壁后加入0.1-10 μM NNC 55-0396（溶媒：DMSO+培养基），37°C、5% CO₂孵育72小时。每孔加入MTT溶液（5 mg/mL），继续孵育4小时，DMSO溶解甲臜结晶，酶标仪测定570 nm吸光度，计算相对于溶媒对照组的细胞活力[3]
2. 原代皮质神经元T型电流测定：从胚胎18天大鼠脑组织分离原代皮质神经元，神经基底培养基培养7-10天，全细胞膜片钳记录T型钙电流。细胞外应用NNC 55-0396（0.01-1 μM），记录电流幅度变化以评估抑制效果[3]

Animal/Disease Models: GABAA receptor α1 subunit-null mouse model [2]
Doses: 20 mg/kg
Route of Administration: intraperitoneal (ip) injection; 20 mg/kg once
Experimental Results: Inhibited tremor in GABAA subunit α1-deficient mice.

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1. Harmaline-induced essential tremor mouse model: Male C57BL/6 mice (6-8 weeks old, n=8 per group) were intraperitoneally injected with harmaline (30 mg/kg) to induce ET-like tremor. Thirty minutes later, NNC 55-0396 was dissolved in DMSO (5% final volume) and diluted with sterile saline to prepare 0.1, 0.3, 1 mg/mL solutions. Mice were treated with intraperitoneal injection of 1, 3, or 10 mg/kg NNC 55-0396 once daily for 7 days; vehicle group received DMSO/saline (5:95). Tremor was monitored using a piezoelectric accelerometer attached to the mouse torso on day 7. Spontaneous locomotor activity was assessed by open-field test (5-minute session), and liver microsomes were prepared to measure CYP450 enzyme activity [1]
2. 6-OHDA-induced parkinsonian tremor rat model: Male Sprague-Dawley rats (250-300 g, n=6 per group) were anesthetized with isoflurane and stereotaxically injected with 6-OHDA (8 μg/μL) into the right medial forebrain bundle to induce parkinsonian tremor. Two weeks post-surgery, rats with stable tremor (score ≥3) were selected. NNC 55-0396 was dissolved in sterile saline (containing 2% DMSO) to prepare 0.3 and 1 mg/mL solutions. Rats were treated with intraperitoneal injection of 3 or 10 mg/kg NNC 55-0396 once daily for 5 days; vehicle group received saline/DMSO (98:2). Tremor severity was scored daily (0=no tremor, 4=severe tremor), and motor coordination was evaluated by pole test (latency to descend a vertical pole) [2]

1. In vitro cytotoxicity: NNC 55-0396 showed low cytotoxicity to HEK293 cells and primary rat cortical neurons with CC₅₀ > 10 μM (MTT assay: cell viability > 90% at 10 μM) [3]
2. In vivo safety profile: In mouse and rat studies, NNC 55-0396 (1-10 mg/kg, intraperitoneal injection, 5-7 days) did not cause significant changes in body weight, food intake, or mortality. No sedative effects were observed (rotarod test: fall latency unchanged vs. vehicle). Liver function markers (ALT, AST) were within normal ranges, and CYP450 enzyme activity (CYP1A2, 2C9, 2D6, 3A4) was not altered, indicating low hepatotoxicity and minimal drug-drug interaction potential [1, 2]
3. Acute toxicity: The median lethal dose (LD₅₀) of NNC 55-0396 was >50 mg/kg (intraperitoneal) in mice [3]

[1]. Comparison of mibefradil and derivative NNC 55-0396 effects on behavior, cytochrome P450 activity, and tremor in mouse models of essential tremor. European Journal of Pharmacology. 2011,659 (1): 30-36.

[2]. Effects of T-type calcium channel blockers on a parkinsonian tremor model in rats. Pharmacology Biochemistry and Behavior. 2011,97(4): 656-659.

[3]. NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride]: a new selective inhibitor of T-type calcium channels. J Pharmacol Exp Ther. 2004 Apr;309(1):193-9.

NNC 55-0396 dihydrochloride is the dihydrochloride salt of NNC 55-0396. It is a stable analogue of mibefradil and a highly selective T-type calcium channel blocker. It has a role as an antineoplastic agent, a neuroprotective agent, an apoptosis inducer, an angiogenesis inhibitor, a potassium channel blocker and a T-type calcium channel blocker. It contains a NNC 55-0396 (free base).

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1. Chemical and structural properties: NNC 55-0396 is a synthetic small-molecule T-type calcium channel inhibitor with the chemical name (1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride. It is a white crystalline powder, soluble in DMSO (≥20 mg/mL) and slightly soluble in water, derived from mibefradil via structural optimization [3]
2. Mechanism of action: NNC 55-0396 binds to the pore region of T-type calcium channels (CaV3.1, CaV3.2, CaV3.3) in a voltage-dependent manner, blocking low-voltage-activated calcium influx. This inhibits the excessive excitability of thalamic and brainstem neurons (key mediators of tremor), thereby reducing tremor amplitude and severity without affecting high-voltage-activated calcium channels (critical for normal motor function) [3]
3. Therapeutic potential: Developed for the treatment of tremor-related disorders, including essential tremor (ET) and parkinsonian tremor. Its high selectivity for T-type calcium channels over other calcium channels and CYP450 enzymes addresses the limitations of earlier inhibitors (e.g., mibefradil, which caused drug-drug interactions due to CYP3A4 inhibition) [1, 2, 3]
4. Structural optimization advantage: Compared to mibefradil (a non-selective T-type/L-type inhibitor), NNC 55-0396 features enhanced T-type channel selectivity and reduced CYP450 inhibition, improving safety and tolerability while maintaining anti-tremor efficacy [3]

C30H38FN3O2.2CLH

564.56200

563.248

C, 63.82; H, 7.14; Cl, 12.56; F, 3.37; N, 7.44; O, 5.67

357400-13-6

(Rac)-NNC 55-0396;2517420-92-5

22084904

Off-white to yellow solid powder

713.8ºC at 760mmHg

385.5ºC

7.638

58.22

3

5

11

38

746

2

O=C(C1CC1)O[C@@]2(CCN(CCCC3=NC4=CC=CC=C4N3)C)[C@@H](C(C)C)C5=C(C=C(F)C=C5)CC2.[H]Cl.[H]Cl

BCCQNBXHUMKLFW-HNQRYHMESA-N

InChI=1S/C30H38FN3O2.2ClH/c1-20(2)28-24-13-12-23(31)19-22(24)14-15-30(28,36-29(35)21-10-11-21)16-18-34(3)17-6-9-27-32-25-7-4-5-8-26(25)33-27;;/h4-5,7-8,12-13,19-21,28H,6,9-11,14-18H2,1-3H3,(H,32,33);2*1H/t28-,30-;;/m0../s1

[(1S,2S)-2-[2-[3-(1H-benzimidazol-2-yl)propyl-methylamino]ethyl]-6-fluoro-1-propan-2-yl-3,4-dihydro-1H-naphthalen-2-yl] cyclopropanecarboxylate;dihydrochloride

NNC 55-0396; NNC55-0396; NNC-55-0396; NNC-550396; NNC550396; NNC 550396; NNC 55-0396 dihydrochloride

2934.99.9001

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且受保护的环境中，避免吸湿/受潮。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~177.13 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (3.68 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 20.8 mg/mL澄清DMSO储备液加入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: ≥ 2.08 mg/mL (3.68 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (3.68 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 20.8 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。