Rap1A ( pIC50 = 3 μM ); Histamine H1 receptor ( Ha-Ras > 20 μM )

RhoA parent (GGTI298) 和 ROCK parent (H1152) 均显着降低 cAMP 激动剂刺激的 IK(ap)，而夜间还降低 T84WT 细胞中 KCNN4c 与顶膜标记麦芽凝集素的共定位[1]。的敲低会消除 NF-κB 的激活，从而导致 GGTI298 和 TRAIL 联合感应的 DR5 促使细胞激活敏感。 GGTI298/TRAIL 激活 NF-κB 并抑制 Akt。敲除 DR5，可阻止 GGTI298/TRAIL 感应的 IκBα 和 p-Akt 减少，表明 DR5 介导 GGTI298/TRAIL 感应的 IκBα 和 p-Akt 减少。相比之下，DR4 敲低进一步促进 GGTI298/TRAIL 感应的 p-Akt 减少[2]。

体内模型回肠环实验表明，当TRAM-34、GGTI298或H1152与霍乱毒素一起注射到环中时，液体体积集中以剂量依赖性方式减少[1]。

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研究药物对骨巨细胞瘤（GCT）影响的主要障碍是缺乏动物模型。在这项研究中，我们创建了一种动物模型，其中GCT基质细胞存活并作为增殖肿瘤细胞发挥作用。使用GCT基质细胞的增殖细胞系来创建稳定的萤光素酶转导细胞系Luc-G33。对细胞系进行了表征，发现与野生型GCT基质细胞相比，细胞增殖率和单核细胞募集没有显著差异。我们将Luc-G33细胞皮下注射到裸鼠的背部或胫骨。使用IVIS 200生物发光成像（BLI）系统的实时活体成像评估活Luc-G33细胞的存在。肿瘤细胞最初在皮下肿瘤模型中增殖并在现场存活7周。我们还测试了唑来膦酸盐（ZOL）和香叶基转移酶-I抑制剂（GGTI-298）单独或其组合在Luc-G33移植裸鼠体内的抗肿瘤作用。400µg/kg的单独ZOL以及400µg/kg ZOL和1.16mg/kg GGTI-298的联合治疗降低了模型中的肿瘤细胞存活率。此外，ZOL、GGTI-298的抗肿瘤作用以及皮下肿瘤模型中的联合治疗也通过免疫组织化学（IHC）染色得到证实。总之，我们建立了一个GCT基质细胞的裸鼠模型，该模型可以无创、实时评估肿瘤的发展，并测试不同佐剂治疗GCT的体内效果。

将指定的细胞在报告裂解缓冲液中裂解，然后使用荧光素酶测定系统在光度计中进行荧光素酶活性测定。相对荧光素酶活性根据蛋白质浓度进行校准。

将细胞接种到 96 孔细胞培养板中，并在第二天用指定的试剂对其进行处理。磺胺罗丹明 B 测定用于计算活细胞的数量。

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细胞凋亡检测[2]
根据制造商的说明，使用市售的Annexin V-PE凋亡检测试剂盒通过Annexin V染色评估细胞凋亡。通过蛋白质印迹法（如下所述）也检测到半胱天冬酶激活，作为凋亡的另一个指标。

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蛋白质印迹分析[2]
如前所述，制备全细胞蛋白裂解物并通过蛋白质印迹进行分析。

Mice aged 6 to 8 weeks are used in the ileal loop experiment, which uses a modified rabbit ileal loop assay. The animals are gut sterilized, fed only water ad libitum, and fasted for twenty-four hours before surgery. A combination of xylazine (5 mg/kg body weight) and ketamine (35 mg/kg body weight) is used to induce anesthesia. A laparotomy is done, and non-absorbable silk is used to tie the 5-cm-long experimental loops at the terminal ileum. Each loop is filled with the following fluids using a tuberculin syringe equipped with a disposable needle through the ligated end of the loop as the ligature is tightened: pure CT (1 μg; positive control), saline (negative control), CT (1 μg) + TRAM-34 (varying concentrations in μM as shown in Fig. 7), CT (1 μg) + H1152 (1 μM), and CT (1 μg) + GGTI298 (variable concentrations in μM), a specific inhibitor of Rap1A. The mice are sutured back into their cages and the intestine is reattached to the peritoneum. After six hours, the loops are removed and the animals are sacrificed by cervical dislocation. The fluid from every loop is collected, and the ratio of the fluid volume inside the loop to its length (fluid accumulation ratio in g/cm) is computed to show how effective different inhibitors are.

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Mouse Ileal Loop Experiment[1]
The ileal loop experiment was performed in 6–8-week-old mice by a modified rabbit ileal loop assay originally described by De and Chatterje. Following gut sterilization, the animals were kept fasted for 24 h prior to surgery and fed only water ad libitum. Anesthesia was induced by a mixture of ketamine (35 mg/kg of body weight) and xylazine (5 mg/kg of body weight). A laparotomy was performed, and the experimental loops of 5-cm length were constricted at the terminal ileum by tying with non-absorbable silk. The following fluids were instilled in each loop by means of a tuberculin syringe fitted with a disposable needle through the ligated end of the loop as the ligatured was tightened: pure CT (1 μg; positive control), saline (negative control), CT (1 μg) + TRAM-34 (different concentrations in μm as indicated in Fig. 7), CT (1 μg) + H1152 (1 μm), and CT (1 μg) + GGTI298 (different concentrations in μm), a specific inhibitor of Rap1A. The intestine was returned to the peritoneum, and the mice were sutured and returned to their cages. After 6 h, these animals were sacrificed by cervical dislocation, and the loops were excised. The fluid from each loop was collected, and the ratio of the amount of fluid contained in the loop with respect to the length of the loop (fluid accumulation ratio in g/cm) was calculated as a reflection of the efficacy of various inhibitors.

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dissolved in DMSO, dilute in saline; 1.16 mg/kg; s.c. injection

Nude mice

[1]. The Epac1 signaling pathway regulates Cl- secretion via modulation of apical KCNN4c channels in diarrhea. J Biol Chem. 2013 Jul 12;288(28):20404-15.

[2]. Dissecting the roles of DR4, DR5 and c-FLIP in the regulation of geranylgeranyltransferase I inhibition-mediated augmentation of TRAIL-induced apoptosis. Mol Cancer. 2010 Jan 29;9:23.

[3]. Platelet-derived growth factor receptor tyrosine phosphorylation requires protein geranylgeranylation but not farnesylation. J Biol Chem. 1996 Nov 1;271(44):27402-7.

[4]. A mouse model of luciferase-transfected stromal cells of giant cell tumor of bone. Connect Tissue Res. 2015 Nov;56(6):493-503.

(2S)-2-[[[4-[[(2R)-2-amino-3-mercaptopropyl]amino]-2-(1-naphthalenyl)phenyl]-oxomethyl]amino]-4-methylpentanoic acid methyl ester is a leucine derivative.

C27H33N3O3S

479.634

479.22

C, 67.61; H, 6.93; N, 8.76; O, 10.01; S, 6.69

180977-44-0

GGTI298 Trifluoroacetate; 1217457-86-7; 180977-44-0; 205590-41-6 (HCl)

9811606

White solid powder

6.474

173.04

4

6

11

34

661

2

S([H])C([H])([H])[C@@]([H])(C([H])([H])N([H])C1C([H])=C([H])C(C(N([H])[C@]([H])(C(=O)OC([H])([H])[H])C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])=O)=C(C=1[H])C1=C([H])C([H])=C([H])C2=C([H])C([H])=C([H])C([H])=C12)N([H])[H].FC(C(=O)O[H])(F)F

XVWPFYDMUFBHBF-CLOONOSVSA-N

InChI=1S/C27H33N3O3S/c1-17(2)13-25(27(32)33-3)30-26(31)23-12-11-20(29-15-19(28)16-34)14-24(23)22-10-6-8-18-7-4-5-9-21(18)22/h4-12,14,17,19,25,29,34H,13,15-16,28H2,1-3H3,(H,30,31)/t19-,25+/m1/s1

methyl (2S)-2-[[4-[[(2R)-2-amino-3-sulfanylpropyl]amino]-2-naphthalen-1-ylbenzoyl]amino]-4-methylpentanoate

GGTI 298; GGTI298; GGTI-298; GGTI298; 180977-44-0; Ggti 298; (S)-methyl 2-(4-(((R)-2-amino-3-mercaptopropyl)amino)-2-(naphthalen-1-yl)benzamido)-4-methylpentanoate; ELA97V8Q7P; CHEMBL282748; L-Leucine, N-[4-[[(2R)-2-amino-3-mercaptopropyl]amino]-2-(1-naphthalenyl)benzoyl]-, methyl ester; GGTI-298

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)

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。