TRAM-34   中文名称: 1-(2-氯苯基-二苯甲基)-1H-吡唑

Calcium-activated K+ channel (IKCa1) (Kd=20 nM)

TRAM-34 选择性阻断 IKCa1 电流 (Kd=25 nM)，TRAM-34 还以相同的功效阻断人 T84 结肠上皮细胞中的 IKCa1 电流 (Kd= 22 nM)。 TRAM-34 可阻断人类 T 细胞中的克隆和天然 IKCa1 通道，Kd 为 20-25 nM，选择性比其他离子通道高 200 至 1500 倍。剂量响应曲线表明，移液器中 1 μM 钙的 Kd 为 20±3 nM，希尔系数为 1.2 [1]。 TRAM-34 是 KCa 3.1 通道的选择性抑制剂，可根据浓度促进或降低细胞增殖。在中等浓度 (3-10 μM) 下，TRAM-34 可增强细胞增殖，但在较高浓度 (20-100 μM) 下，TRAM-34 会降低细胞增殖。雌激素受体拮抗剂 ICI182,780 和他莫昔芬可阻止 TRAM-34 产生的细胞增殖增强。 TRAM-34 还增强孕激素受体 mRNA 表达，降低雌激素受体-α mRNA 表达，并减少放射性标记雌激素与 MCF-7 雌激素受体的结合，在每种情况下都模仿 17β-雌激素受体乙二醇的参与 [2]。

连续 7 天单次静脉注射 TRAM-34（0.5 mg/kg；29 μM）的小鼠 (n = 5) 没有表现出临床表现异常。 TRAM-34治疗组的体重数据（第1天：17.8克；第7天：27.0克）与注射媒介物的对照小鼠的体重数据（第1天：17.4克；第7天：23.4克）相匹配。总体而言，根据这些小毒性实验的结果，TRAM-34 的剂量是阻断通道所需量的 500-1,000 倍时，并不会造成严重危险 [1]。 TRAM-34 治疗后，病变区域的苏木精和伊红 (H&E) 测量值显着降低；平均梗塞面积从对照组（n=8）的22.6±3.6%下降到10 mg/kg TRAM-34。在用 40 mg/kg TRAM-34 治疗的大鼠中，该百分比从大鼠的 11.3±2.8% 降至 8.1±1.9% (n=8；P=0.004) (n=6，平均值±sem，P=0.039)。此外，这种治疗有助于减少大脑萎缩。但只有40 mg/kg TRAM-34组的结果有统计学意义（P=0.013），而10 mg/kg组的结果则没有统计学意义（P=0.11）[3]。

ER竞争性结合试验[2]
竞争性结合试验如下进行。MCF-7细胞蛋白（250µg）在室温下在TEDG缓冲液中在0.1 nM[2,4,6,7,16,17-3H（N）]-雌二醇（[3H]-E2）（110 Ci·mmol−1）（总终体积为500µL）的存在下孵育2小时。在非放射性E2过量100倍的情况下评估非特异性结合TRAM-34和E2标准品在添加到细胞质蛋白之前，在不含酚红的5%DCC-FBS MEM补充剂中稀释。载体对照由5%DCC-FBS MEM组成，含有0.7%DMSO的补充剂。为了将ER结合的[3H]-E2与未结合的[3H]-E2分离，加入250µL羟基磷灰石（HAP，60%在TEDG缓冲液中），在15分钟内每5分钟涡旋一次混合物，并在1000×g下离心10分钟。用TEDG缓冲溶液洗涤HAP-[3H]-E2-ER复合物，离心并重复洗涤步骤。为了从HAP-[3H]-E2-ER复合物中洗脱[3H]-E2，加入500µL 100%乙醇，然后将混合物孵育15分钟，并在1034×g下离心10分钟。分离出的[3H]-E_2被移除并加入2 mL闪烁液中。使用Beckman LS 5000TA闪烁计数器对放射性进行定量。在四次独立的蛋白质提取中，对[3H]-E2与TRAM-34的竞争进行了四次测定。Scatchard分析确定表观解离常数为0.135±0.034 nM（n=3），最大结合容量为48.3±5.4 fmol·mg−1（=3）。

[3H]胸苷掺入试验。[1]
将静息或2天活化（10 nM PMA或5 ng/ml抗CD3 Ab）的PBMC以每孔2×105个细胞的速度接种在平底96孔板（最终体积200μl）的培养基中。用药物预孵育的细胞（60分钟）用丝裂原（10 nM PMA+175 nM离子霉素或5 ng/ml抗CD3抗体）刺激48小时。在最后6小时加入三联体胸苷（[3H]TdR）（每孔1μCi）。将细胞收集到玻璃纤维过滤器上，并在闪烁计数器中测量放射性。

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细胞活力的流式细胞术测量。[1]
细胞以5×105个细胞/ml（Jurkat E6-1，MEL细胞，人T淋巴细胞）或105个细胞/ml（C2F3成肌细胞，CHO，COS-7，L929，NGP和NLF神经母细胞瘤，RBL-2H3）接种在12孔板中。药物（5μM）以0.1%的DMSO终浓度加入，发现不会影响细胞存活率。48小时后，通过抽吸（悬浮细胞）或胰蛋白酶消化（贴壁细胞系）收集细胞，离心，重新悬浮在含有1μg/ml碘化丙啶（PI）的0.5ml PBS中，并在FACScan流式细胞仪上测量红色荧光。死细胞的百分比是通过其PI摄取来确定的，每个样本中有104个细胞被分析。

Acute in Vivo Toxicity Determinations.[1]
Five CF-1BR mice (17–19 g) were injected intravenously with a single 1.0-ml dose of 0.5 mg/kg TRAM-34 (in mammalian Ringer solution with 1% ethanol and 2.5% BSA). Five control mice were injected with an equal volume of the vehicle. Mice were observed for adverse effects immediately after dosing, at 4 h after injection and daily for 7 days.

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The filament was kept in place for 90 minutes and then withdrawn and removed from the blood vessel to restore blood supply. Rats received TRAM-34 at 10 mg/kg, 40 mg/kg or vehicle (Miglyol 812 neutral oil at 1 μL/g) twice daily intraperitoneally for 7 days starting 12 hours after reperfusion.[3]

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Pharmacokinetics, Brain Concentrations, and Plasma Protein Binding of TRAM-34[3]
TRAM-34 was synthesized in our laboratory as previously described (Wulff et al, 2000) and its chemical identity and purity checked by 1H NMR and high pressure liquid chromatography/mass spectrometry (HPLC/MS). For intravenous application, TRAM-34 was dissolved at 5 mg/mL in a mixture of 25% CremophorEL and 75% phosphate-buffered saline and then injected at 10 mg/kg into the tail vein of male Wistar rats. At various time points after the injection, ∼100 to 200 μL of blood was collected from a tail nick into EDTA blood sample collection tubes. For simultaneous determinations of plasma and brain concentrations, TRAM-34 was dissolved in Miglyol 812 neutral oil (caprylic/capric triglyceride) at 10 or 40 mg/mL and injected intraperitoneally at 10 or 40 mg/kg. Blood samples were taken by cardiac puncture under deep isoflurane anesthesia. The right atrium was then cut open and 20 mL of saline slowly injected into the left ventricle to flush the blood out of the circulation. The rats were then sacrificed and brains removed. Plasma was separated by centrifugation and samples stored at −80 °C for pending analysis. Plasma and homogenized brain samples were purified using C18 solid phase extraction cartridges. Elutioned fractions corresponding to TRAM-34 were dried under nitrogen and reconstituted in acetonitrile. LC/MS analysis was performed with a Hewlett-Packard 1100 series HPLC stack equipped with a Merck KGaA RT 250-4 LiChrosorb RP-18 column interfaced to a Finnigan LCQ Classic MS. The mobile phase consisted of acetonitrile and water, both containing 0.2% formic acid. With a flow rate of 1.0 mL/min, the gradient was ramped from 20/80 to 70/30 in 5 minutes, then to 80/20 over 11 minutes, to 5/95 till 16.5 minutes, and finally back to 80/20 till 38 minutes. With the column temperature maintained at 30 °C, TRAM-34 eluted at 14.4 minutes and was detected by a variable wavelength detector set to 190 nm and the MS in series. Using electrospray ionization/ion trap MS (capillary temperature 270 °C, capillary voltage 1 V, tube lens offset −15 V, positive ion mode), TRAM-34 was quantified by its base peak of 277 m/z (2-chlorotrityl fragment) and concentrations calculated with a 5-point calibration curve from 25 nmol/L to 2.5 μmol/L. Concentrations above 2.5 μmol/L were quantified by their UV absorption at 190 nm. The related compound TRAM-46 (base peak of 261 m/z, 2-fluorotrityl fragment) was used as an internal standard.
The percentage of plasma protein binding for TRAM-34 was determined by ultrafiltration. Rat plasma was spiked with 50 and 100 μmol/L TRAM-34 in 1% dimethylsulfoxide and the sample loaded onto a Microcon YM-100 Centrifugal Filter and centrifuged at 14,000 g for 15 minutes at room temperature. The centrifugate (=free TRAM-34) was directly analyzed for TRAM-34 by HPLC-MS. The retentate was collected by inverting the filter into an Eppendorf tube and spinning at 14,000 g for 15 minutes. The retentate then underwent sample preparation as per the above-described procedure for determining total TRAM-34 concentration in plasma. The plasma protein binding of TRAM-34 was found to be 98±0.5% (n=3) and the unbound (=free) fraction 2.0±0.4%.[3]

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Formulated in peanut oil; 120 mg/kg/day; s.c. injection

Sprague-Dawley rats subjected to BCI of the left CA by use of a 2F Fogarty embolectomy catheter

[1]. Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activated K+ channel, IKCa1: a potential immunosuppressant. Proc Natl Acad Sci U S A. 2000 Jul 5;97(14):8151-6.

[2]. The intermediate conductance Ca2+-activated K+ channel inhibitor TRAM-34 stimulates proliferation of breast cancer cells via activation of oestrogen receptors. Br J Pharmacol. 2010 Feb 1;159(3):650-8.

[3]. The KCa3.1 blocker TRAM-34 reduces infarction and neurological deficit in a rat model of ischemia/reperfusion stroke. J Cereb Blood Flow Metab. 2011 Dec;31(12):2363-74.

TRAM-34 is an organochlorine compound.

C22H17CLN2

344.84

344.108

C, 76.63; H, 4.97; Cl, 10.28; N, 8.12

289905-88-0

656734

White to off-white solid powder

1.1±0.1 g/cm3

510.2±50.0 °C at 760 mmHg

145-147ºC

262.4±30.1 °C

0.0±1.3 mmHg at 25°C

1.617

5.65

17.82

0

1

4

25

396

0

KBFUQFVFYYBHBT-UHFFFAOYSA-N

InChI=1S/C22H17ClN2/c23-21-15-8-7-14-20(21)22(25-17-9-16-24-25,18-10-3-1-4-11-18)19-12-5-2-6-13-19/h1-17H

1-[(2-chlorophenyl)-diphenylmethyl]pyrazole

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: ≥ 2.08 mg/mL (6.03 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 (6.03 mM) 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), 悬浊液; 超声助溶。
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 (6.03 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900 μL 玉米油中，混合均匀。

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配方 4 中的溶解度: 30% PEG400+0.5% Tween80+5% Propylene glycol : 30mg/mL

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配方 5 中的溶解度: 5 mg/mL (14.50 mM) in 20% SBE-β-CD in Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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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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。