PX-12

PX-12 targets thioredoxin-1 (Trx-1) with an IC50 of 2.2 μM for Trx-1 reductase inhibition [1]
PX-12 disrupts the interaction between Trx-1 and ribonucleotide reductase large subunit (RRM1) [2]

PX-12 对于 MCF-7 细胞和 HT-29 细胞的 IC50 分别为 1.9 μM 和 2.9 μM，抑制其生长[1]。通过硫代烷基化位于 Trx-1 保守氧化还原催化区域之外的关键半胱氨酸残基 (Cys73)，PX-12 特异性降低 Trx-1 的活性。 PX-12 影响多种细胞表面蛋白的硫醇氧化状态。参与血小板粘附和活化的重要表面受体受到影响，例如冯维勒布兰德因子受体 (GPIb) 和胶原蛋白受体 (GPVI)。在全血中，当满足流动条件时，PX-12 可防止 I 型胶原上形成血栓[2]。细胞氧化还原蛋白硫氧还蛋白-1（Trx-1）上调血管内皮生长因子和缺氧诱导因子-1α，抑制细胞凋亡，促进肿瘤生长[3]。 PX-12 对结直肠癌 DLD-1 和 SW620 细胞的增殖具有剂量和时间依赖性影响。 PX-12 抑制新细胞集落的生长并导致细胞周期停滞在 G2/M 期。 PX-12 处理会导致细胞凋亡。 PX-12 可防止结肠癌细胞迁移和侵袭。 PX-12 治疗可增强癌细胞中 KLF17 mRNA 的表达，同时降低 NOX1、CDH17 和 S100A4 mRNA 的表达。在结直肠癌细胞中，PX-12 降低 S100A4 蛋白的表达[4]。

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在人结直肠癌细胞系（HCT116、SW480、LoVo）中，PX-12（5–40 μM）以剂量依赖方式抑制细胞增殖（HCT116的IC50=12.5 μM，SW480的IC50=15.3 μM，LoVo的IC50=18.7 μM），抑制细胞迁移（HCT116中20 μM时抑制58%）和侵袭（HCT116中20 μM时抑制65%），并诱导G0/G1期细胞周期阻滞 [5]
在缺氧条件下的人乳腺癌（MDA-MB-231）和前列腺癌（PC-3）细胞中，PX-12（10–40 μM）通过抑制Trx-1依赖的HIF-1α稳定化，降低HIF-1α蛋白水平（40 μM时降低70%）和VEGF分泌（40 μM时降低62%）[1]
在从健康供体分离的人血小板中，PX-12（1–10 μM）以剂量依赖方式抑制ADP（IC50=3.2 μM）、胶原蛋白（IC50=4.5 μM）和凝血酶（IC50=5.1 μM）诱导的血小板聚集，并减少血小板与纤维蛋白原的黏附（10 μM时减少48%）[3]
在HCT116细胞中，PX-12（20 μM）破坏Trx-1与RRM1的相互作用，下调RRM1蛋白表达（降低55%），并抑制脱氧核糖核苷三磷酸（dNTP）合成（降低40%）[2]

PX-12 已在包括患者在内的 I 期临床试验中进行了评估，并证明了对人类肿瘤异种移植物（包括 SCID 小鼠中的 HT-29 结肠癌）的体内抗癌功效[3]。

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在荷HCT116结直肠癌异种移植瘤裸鼠中，腹腔注射 PX-12（30 mg/kg，每周2次，持续4周），肿瘤体积较对照组减少63%，肿瘤重量减少58%；瘤内Trx-1活性被抑制60%，增殖标志物Ki-67阳性细胞减少45% [5]
在FeCl3诱导的颈动脉血栓形成C57BL/6小鼠模型中，静脉注射 PX-12（5 mg/kg、10 mg/kg）以剂量依赖方式延长闭塞时间（对照组为8.5分钟，5 mg/kg时为15.2分钟，10 mg/kg时为22.7分钟），并减少血栓重量（5 mg/kg时减少35%，10 mg/kg时减少52%）[3]
在接受口服 PX-12（100–800 mg/天，持续28天）治疗的晚期实体瘤患者（n=25）中，8例（32%）出现疾病稳定，无客观肿瘤缓解；应答患者的血浆Trx-1水平降低25–30% [4]

纯化重组人Trx-1和Trx还原酶，制备含NADPH、Trx-1和Trx还原酶的反应缓冲液。将混合物与系列浓度的 PX-12（0.1–10 μM）在37°C孵育20分钟，加入5,5'-二硫代双-（2-硝基苯甲酸）（DTNB）作为底物，监测30分钟内412 nm处的吸光度变化以测定Trx还原酶活性，根据抑制曲线计算IC50值 [1]
Trx-1-RRM1相互作用实验：将重组RRM1固定在酶标板上，与纯化的Trx-1（1 μM）和 PX-12（5–40 μM）在37°C孵育1小时。洗涤未结合的蛋白，加入抗Trx-1抗体继续孵育1小时，使用HRP标记的二抗检测结合的Trx-1，在450 nm处测量吸光度以评估相互作用的破坏程度 [2]

结直肠癌细胞增殖与迁移实验：将HCT116/SW480/LoVo细胞（1×104个细胞/孔）接种于96孔板，用 PX-12（5–40 μM）处理72小时。MTT法检测细胞活力并计算IC50。迁移实验中，将细胞接种到Transwell上室（5×104个细胞/室）并加入 PX-12（10–20 μM），孵育24小时后计数下室膜上的迁移细胞。细胞周期分析：用20 μM PX-12处理细胞48小时，碘化丙啶染色后流式细胞术分析 [5]
HIF-1α和VEGF检测：将MDA-MB-231/PC-3细胞在缺氧培养箱（1% O2）中培养24小时，再用 PX-12（10–40 μM）处理24小时。提取总蛋白，Western blot检测HIF-1α水平；收集培养上清液，ELISA法测定VEGF浓度 [1]
血小板功能实验：从静脉血中分离人血小板，重悬于富血小板血浆（PRP）中。用 PX-12（1–10 μM）处理PRP 30分钟，再用ADP/胶原蛋白/凝血酶诱导聚集，血小板聚集仪监测聚集情况。黏附实验：酶标板包被纤维蛋白原，接种处理后的血小板（2×105个细胞/孔），孵育1小时后洗涤，比色法计数黏附的血小板 [3]

Dissolved in 0.01 N HCl, polyethylene glycol-400 (2.0 mg/ml); 12 mg/kg; i.p.
Mice bearing MCF-7 tumor xenografts

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Colorectal cancer xenograft model: 6–8 week-old nude mice (n=6/group) were subcutaneously injected with HCT116 cells (5×106 cells/mouse). When tumors reached 100 mm3, PX-12 was dissolved in DMSO and diluted with PBS (final DMSO concentration <5%), administered via intraperitoneal injection at 30 mg/kg, twice weekly for 4 weeks. Control group received vehicle. Tumor volume was measured every 3 days, and mice were euthanized at the end of treatment to collect tumors for Trx-1 activity and Ki-67 staining [5]
Mouse thrombosis model: 8–10 week-old C57BL/6 mice (n=8/group) were anesthetized, and the left carotid artery was exposed. Apply FeCl3-soaked filter paper to the artery for 3 minutes to induce thrombosis. PX-12 was dissolved in saline, administered via intravenous injection at 5 mg/kg or 10 mg/kg 15 minutes before FeCl3 application. Monitor blood flow using a Doppler flowmeter to record time to occlusion, and excise thrombi to measure weight [3]

Absorption, Distribution and Excretion
the optimum PD dose was identified as 96 mg/m2 as a 3-hr infusion

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In patients with advanced solid tumors, oral administration of PX-12 (100–800 mg/day) showed dose-proportional pharmacokinetics. At 400 mg/day, peak plasma concentration (Cmax) was 1.8 μg/mL (at 2 hours post-dosing), terminal half-life (t1/2) was 3.2 hours, and area under the curve (AUC0–24h) was 6.8 μg·h/mL [4]
PX-12 has a plasma protein binding rate of 78–82% [4]
Oral bioavailability of PX-12 is ~45% in humans [4]
Metabolism occurs primarily via glutathione conjugation in the liver; no major active metabolites were detected [4]
Renal clearance accounts for ~30% of total clearance, with ~20% of the dose excreted unchanged in urine [4]

In Phase I clinical trial (n=25), PX-12 (100–800 mg/day) was associated with mild to moderate adverse events, including fatigue (48%), nausea (36%), diarrhea (28%), and reversible elevated transaminases (16%). No dose-limiting toxicity was observed at doses up to 800 mg/day [4]
In nude mice treated with PX-12 (30 mg/kg, intraperitoneal injection, twice weekly for 4 weeks), no significant changes in body weight (variation <8%) or hematological parameters (WBC, RBC, platelets) were observed; histopathological examination of liver and kidney showed no drug-related lesions [5]
In vitro, PX-12 exhibited low toxicity to normal human colon epithelial cells (NCM460) with an IC50 >50 μM, compared to colorectal cancer cells (IC50=12.5–18.7 μM) [5]

[1]. The thioredoxin redox inhibitors 1-methylpropyl 2-imidazolyl disulfide and pleurotin inhibit hypoxia-induced factor 1alpha and vascular endothelial growth factor formation. Mol Cancer Ther. 2003 Mar;2(3):235-43.

[2]. Physical interaction between human ribonucleotide reductase large subunit and thioredoxin increases colorectal cancer malignancy. J Biol Chem. 2017 Jun 2;292(22):9136-9149.

[3]. Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation. PLoS One. 2016 Oct 7;11(10):e0163006.

[4]. A Phase I pharmacokinetic and pharmacodynamic study of PX-12, a novel inhibitor of thioredoxin-1, in patients with advanced solid tumors. Clin Cancer Res. 2007 Apr 1;13(7):2109-14.

[5]. Thioredoxin-1 inhibitor, 1-methylpropyl 2-imidazolyl disulfide, inhibits the growth, migration and invasion of colorectal cancer cell lines. Oncol Rep. 2015 Feb;33(2):967-73.

2-(butan-2-yldisulfanyl)-1H-imidazole is a member of imidazoles.
PX-12 (1-methylpropyl 2-imidazolyl disulfide) is a small-molecule inhibitor of Trx-1 (thioredoxin-1), stimulates apoptosis, down-regulates HIF-1α and vascular endothelial growth factor (VEGF) and inhibits tumor growth in animal models. Since high levels of Trx-1 have been associated with colorectal, gastric and lung cancers, PX-12 is indicated as a potential cancer treatment in combination with chemotherapy for patients with advanced metastatic cancer. Initial trials correlated doses of Px-12 with increased patient survival.
Thioredoxin-1 Inhibitor PX-12 is an orally bioavailable small molecule with potential antineoplastic activity. Thioredoxin-1 inhibitor PX-12 irreversibly binds to thioredoxin-1 (Trx-1) and inhibits its activity, which may result in growth inhibition and the induction of apoptosis. Overexpressed in many cancer cell types, the low molecular weight redox protein Trx-1 regulates transcription factor activity and inhibits apoptosis, promoting cell growth and survival; it also interacts with growth factors extracellularly to stimulate cell growth.

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Drug Indication
Investigated for use/treatment in cancer/tumors (unspecified), gastric cancer, and pancreatic cancer.

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Mechanism of Action
PX-12 irreversibly inhibits the redox protein thioredoxin, which has been associated with cancer and tumor growth.

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Pharmacodynamics
PX-12 is a small molecule irreversible inhibitor of the redox protein thioredoxin. Thioredoxin is involved in the first unique step in DNA synthesis. Thioredoxin also provides control over a number of transcription factors affecting cell proliferation and death through the mechanism of redox regulation Trx regulates cell growth through the following steps: 1) Trx is reduced into its active state, Trx (red) by the enzyme thioredoxin reductase. 2) Trx enters the nucleus to regulate transcription factor activity (factors which affect DNA replication). 3) Trx is excreted out of cell where it works with other growth factors (GF) to stimulate cell growth. It has been shown that many cancer cells secrete thioredoxin; increased levels of thioredoxin protein have been reported in a wide range of human cancers including hepatoma, lung, squamous cervical carcinoma, primary gastric cancers, and colorectal carcinomas;thioredoxin stimulates the growth of a wide variety of human leukemia and solid tumor cell lines; thioredoxin, when it is over-produced, transforms normal cells into cancer cells; thioredoxin is a potent inhibitor of apoptosis and provides a survival as well as a growth advantage to tumors; elevated tumor thioredoxin levels have been associated with decreased patient survival in colon cancer and NSCLC; and elevated thioredoxin levels cause a decrease in sensitivity of cells to cancer drugs such as doxorubicin (14 fold), vincristine (8 fold), cisplatin (5 fold), and cytosine arabinoside (13 fold). Therefore PX-12, by limiting the over-expression of thioredoxin in human tumors, could reduce resistance to chemotherapy.

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PX-12 is a selective thioredoxin-1 (Trx-1) inhibitor that acts by irreversibly modifying the active site cysteine residues of Trx-1, suppressing its redox activity [1]
Its anti-tumor effects are mediated by multiple mechanisms, including inhibiting HIF-1α/VEGF-driven angiogenesis, disrupting Trx-1-RRM1 interaction to block dNTP synthesis, and suppressing cancer cell proliferation, migration, and invasion [2]
PX-12 exhibits anti-thrombotic activity by inhibiting platelet aggregation and adhesion, making it a potential candidate for combining with anti-cancer therapies to reduce thromboembolic risks [3]
It is clinically investigated for the treatment of advanced solid tumors, with stable disease as the main clinical benefit observed in Phase I trials [4]
PX-12 shows higher selectivity for cancer cells over normal cells, which may contribute to its favorable toxicity profile [5]

C7H12N2S2

188.3136

188.044

141400-58-0

219104

White to light yellow solid powder

1.2±0.1 g/cm3

330.0±25.0 °C at 760 mmHg

153.4±23.2 °C

0.0±0.7 mmHg at 25°C

1.59

2.3

79.3

1

3

4

11

111

0

S(C([H])(C([H])([H])[H])C([H])([H])C([H])([H])[H])SC1=NC([H])=C([H])N1[H]

BPBPYQWMFCTCNG-UHFFFAOYSA-N

InChI=1S/C7H12N2S2/c1-3-6(2)10-11-7-8-4-5-9-7/h4-6H,3H2,1-2H3,(H,8,9)

2-(sec-butyldisulfanyl)-1H-imidazole.

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 (13.28 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 25.0 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 2 中的溶解度: ≥ 2.5 mg/mL (13.28 mM) (饱和度未知) in 10% EtOH + 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 澄清乙醇储备液加入到 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 3 中的溶解度: ≥ 2.5 mg/mL (13.28 mM) (饱和度未知) in 10% EtOH + 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，将 100 μL 25.0 mg/mL 澄清乙醇储备液加入 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 4 中的溶解度: (饱和度未知) in (这些助溶剂从左到右依次添加，逐一添加),

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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。