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 has an IC50 of 1.9 μM for MCF-7 cells and 2.9 μM for HT-29 cells, respectively, which inhibits their growth[1]. By thio-alkylating the key cysteine residue (Cys73), which is situated outside of Trx-1's conserved redox catalytic region, PX-12 specifically lowers the activity of Trx-1. PX-12 influences several cell surface proteins' thiol oxidation status. Important surface receptors involved in platelet adhesion and activation are impacted, such as the von Willebrand factor receptor (GPIb) and the collagen receptor (GPVI). In whole blood, PX-12 prevents thrombus formation over Type I collagen when flow conditions are met[2]. Cellular redox protein thioredoxin-1 (Trx-1) up-regulates vascular endothelial growth factor and hypoxia-inducible factor-1α, suppresses apoptosis, and encourages the growth of tumors[3]. PX-12 has a dose- and time-dependent effect on the proliferation of colorectal cancer DLD-1 and SW620 cells. PX-12 inhibits the growth of new cell colonies and causes a G2/M phase arrest in the cell cycle. Treatment with PX-12 causes apoptosis. PX-12 prevents colon cancer cells from migrating and invading. PX-12 treatment enhances KLF17 mRNA expression while decreasing NOX1, CDH17, and S100A4 mRNA expression in cancer cells. In colorectal cancer cells, PX-12 reduces the expression of the S100A4 protein[4].

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In human colorectal cancer cell lines (HCT116, SW480, LoVo), PX-12 (5–40 μM) dose-dependently inhibited cell proliferation (IC50=12.5 μM for HCT116, 15.3 μM for SW480, 18.7 μM for LoVo), suppressed cell migration (by 58% at 20 μM in HCT116) and invasion (by 65% at 20 μM in HCT116), and induced G0/G1 cell cycle arrest [5]
In human breast cancer (MDA-MB-231) and prostate cancer (PC-3) cells under hypoxic conditions, PX-12 (10–40 μM) reduced HIF-1α protein levels (by 70% at 40 μM) and VEGF secretion (by 62% at 40 μM) via inhibiting Trx-1-dependent HIF-1α stabilization [1]
In human platelets isolated from healthy donors, PX-12 (1–10 μM) dose-dependently inhibited platelet aggregation induced by ADP (IC50=3.2 μM), collagen (IC50=4.5 μM), and thrombin (IC50=5.1 μM), and reduced platelet adhesion to fibrinogen (by 48% at 10 μM) [3]
In HCT116 cells, PX-12 (20 μM) disrupted the Trx-1-RRM1 interaction, downregulated RRM1 protein expression (by 55%), and inhibited deoxyribonucleotide triphosphate (dNTP) synthesis (by 40%) [2]

PX-12 has been evaluated in a phase I clinical trial including patients and has demonstrated in vivo anticancer efficacy against human tumor xenografts, including HT-29 colon cancer in SCID mice[3].

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In nude mice bearing HCT116 colorectal cancer xenografts, intraperitoneal administration of PX-12 (30 mg/kg, twice weekly for 4 weeks) reduced tumor volume by 63% and tumor weight by 58% compared to control; intra-tumor Trx-1 activity was inhibited by 60%, and Ki-67 (proliferation marker) positive cells decreased by 45% [5]
In C57BL/6 mice subjected to FeCl3-induced carotid artery thrombosis, intravenous PX-12 (5 mg/kg, 10 mg/kg) dose-dependently prolonged the time to occlusion (from 8.5 min in control to 15.2 min at 5 mg/kg and 22.7 min at 10 mg/kg) and reduced thrombus weight (by 35% at 5 mg/kg and 52% at 10 mg/kg) [3]
In patients with advanced solid tumors (n=25) treated with oral PX-12 (100–800 mg/day for 28 days), stable disease was observed in 8 patients (32%), with no objective tumor responses; plasma Trx-1 levels were reduced by 25–30% in responsive patients [4]

Purify recombinant human Trx-1 and Trx reductase. Prepare reaction buffer containing NADPH, Trx-1, and Trx reductase. Incubate the mixture with serial dilutions of PX-12 (0.1–10 μM) at 37°C for 20 minutes. Add 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) as a substrate, and monitor the absorbance change at 412 nm over 30 minutes to measure Trx reductase activity. Calculate the IC50 value based on the inhibition curve [1]
For Trx-1-RRM1 interaction assay, immobilize recombinant RRM1 on a microplate. Incubate with purified Trx-1 (1 μM) and PX-12 (5–40 μM) at 37°C for 1 hour. Wash unbound proteins, add anti-Trx-1 antibody, and incubate for another hour. Detect bound Trx-1 using HRP-conjugated secondary antibody and measure absorbance at 450 nm to assess interaction disruption [2]

Colorectal cancer cell proliferation and migration assay: Seed HCT116/SW480/LoVo cells (1×104 cells/well) in 96-well plates, treat with PX-12 (5–40 μM) for 72 hours. Use MTT assay to measure cell viability and calculate IC50. For migration assay, seed cells in transwell upper chambers (5×104 cells/chamber) with PX-12 (10–20 μM), incubate for 24 hours, count migrated cells on the lower membrane. For cell cycle analysis, treat cells with 20 μM PX-12 for 48 hours, stain with propidium iodide, and analyze by flow cytometry [5]
HIF-1α and VEGF detection: Culture MDA-MB-231/PC-3 cells in hypoxic chambers (1% O2) for 24 hours, then treat with PX-12 (10–40 μM) for another 24 hours. Extract total proteins, perform Western blot to detect HIF-1α levels. Collect culture supernatants to measure VEGF concentration by ELISA [1]
Platelet function assay: Isolate human platelets from venous blood, resuspend in platelet-rich plasma (PRP). Treat PRP with PX-12 (1–10 μM) for 30 minutes, then induce aggregation with ADP/collagen/thrombin. Monitor aggregation using a platelet aggregometer. For adhesion assay, coat microplates with fibrinogen, seed treated platelets (2×105 cells/well), incubate for 1 hour, wash, and count adherent platelets by colorimetric assay [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 optimal PD dose was determined to be 96 mg/m2, administered via 3-hour infusion. Oral administration of PX-12 (100–800 mg/day) in patients with advanced solid tumors demonstrated dose-proportional pharmacokinetics. At a daily dose of 400 mg, the peak plasma concentration (Cmax) was 1.8 μg/mL (2 hours after administration), the terminal half-life (t1/2) was 3.2 hours, and the area under the curve (AUC0–24h) was 6.8 μg·h/mL [4]. The plasma protein binding rate of PX-12 was 78–82% [4]. The oral bioavailability of PX-12 in humans was approximately 45% [4]. Metabolism was primarily carried out via glutathione binding in the liver; no major active metabolites were detected [4]. Renal clearance accounted for approximately 30% of total clearance, of which approximately 20% of the dose was excreted unchanged in the urine [4].

In a 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 elevation of transaminases (16%). No dose-limiting toxicities were 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 were observed in body weight (change <8%) or hematological parameters (white blood cells, red blood cells, platelets); no drug-related lesions were found in liver and kidney histopathological examination [5]
In vitro experiments showed that PX-12 had low toxicity to normal human colonic epithelial cells (NCM460), IC50 > 50 μM, and low toxicity 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-(But-2-yldithio)-1H-imidazolium belongs to the imidazolium class of compounds. PX-12 (1-methylpropyl-2-imidazolium disulfide) is a small molecule inhibitor of thioredoxin-1 (Trx-1) that stimulates apoptosis, downregulates HIF-1α and vascular endothelial growth factor (VEGF), and inhibits tumor growth in animal models. Because high levels of Trx-1 are associated with colorectal, gastric, and lung cancers, PX-12 is considered a potential treatment option in combination with chemotherapy for patients with advanced metastatic cancer. Preliminary trials have shown that the dose of PX-12 is associated with prolonged patient survival. The thioredoxin-1 inhibitor PX-12 is a small molecule with high oral bioavailability and potential antitumor activity. PX-12 irreversibly binds to and inhibits the activity of thioredoxin-1 (Trx-1), which may lead to growth inhibition and apoptosis induction. The low molecular weight redox protein Trx-1 is overexpressed in various cancer cells. It regulates transcription factor activity and inhibits apoptosis, thereby promoting cell growth and survival; it also interacts with extracellular growth factors, stimulating cell growth. Drug Indications It has been studied for the treatment of cancer/tumor (not specified), gastric cancer, and pancreatic cancer. Mechanism of Action PX-12 irreversibly inhibits thioredoxin, a redox protein associated with cancer and tumor growth. Pharmacodynamics PX-12 is a small molecule, irreversible inhibitor of the redox protein thioredoxin. Thioredoxin participates in the first unique step of DNA synthesis. Thioredoxin also controls various transcription factors affecting cell proliferation and death through redox regulation mechanisms. Thioredoxin regulates cell growth through the following steps: 1) Thioredoxin reductase reduces thioredoxin to its active state (red). 2) Thioredoxin enters the cell nucleus and regulates transcription factor activity (factors affecting DNA replication). 3) Thioredoxin is expelled from cells and works synergistically with other growth factors to stimulate cell growth. Studies have shown that many cancer cells secrete thioredoxin; elevated thioredoxin levels have been reported in various human cancers, including liver cancer, lung cancer, cervical squamous cell carcinoma, primary gastric cancer, and colorectal cancer; thioredoxin can stimulate the growth of various human leukemia and solid tumor cell lines. Excessive thioredoxin production can transform normal cells into cancer cells; thioredoxin is a potent inhibitor of apoptosis, providing tumors with a survival and growth advantage; elevated thioredoxin levels in tumors are associated with decreased survival rates in patients with colon cancer and non-small cell lung cancer; elevated thioredoxin levels lead to decreased cell sensitivity to anticancer drugs such as doxorubicin (14-fold increase), vincristine (8-fold increase), cisplatin (5-fold increase), and cytarabine (13-fold increase). Therefore, PX-12 can reduce chemotherapy resistance by limiting the overexpression of thioredoxin in human tumors.

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PX-12 is a selective thioredoxin-1 (Trx-1) inhibitor whose mechanism of action is to irreversibly modify the active site of Trx-1, thereby inhibiting its redox activity[1].
Its antitumor effects are mediated through multiple mechanisms, including inhibition of HIF-1α/VEGF-driven angiogenesis, disruption of Trx-1-RRM1 interaction to block dNTP synthesis, and inhibition of cancer cell proliferation, migration, and invasion[2].
PX-12 has antithrombotic activity by inhibiting platelet aggregation and adhesion, making it a potential candidate drug for use in combination with anticancer therapies to reduce the risk of thromboembolism[3].
Clinical studies are currently underway for the treatment of advanced solid tumors, and the main clinical benefit observed is disease stabilization. In a phase I trial[4],
PX-12 showed higher selectivity for cancer cells than for 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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (13.28 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (13.28 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (13.28 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 4: (saturation unknown) in (add these co-solvents sequentially from left to right, and one by one),

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 (Please use freshly prepared in vivo formulations for optimal results.)