Tiplaxtinin (PAI-039) targets plasminogen activator inhibitor-1 (PAI-1) with an IC50 of 0.4 μM (human recombinant PAI-1, tPA-dependent inhibition assay) [3]
Tiplaxtinin (PAI-039) shows high selectivity for PAI-1 over other serine protease inhibitors (e.g., α1-antitrypsin, antithrombin III; inhibition <10% at 10 μM) [3]

A small chemical inhibitor of PAI-1 activity in urothelial cell lines is tiplaxtinin (PAI-039). Tiplaxtinin treatment resulted in a significant suppression of cell proliferation in T24 cells, with good IC50 values of 43.7±6.3 μM and 52.8±1.6 μM observed in UM-UC-14 cells. In contrast, the benign cell line UROtsa showed a higher IC50 value of 70.3±0.1 μM. The IC50 values of Tiplaxtinin in isolated cells were 19.7±3.8 μM in T24, 44.5±6.5 μM in UM-UC-14, and 31.6±6.1 μM in UROtsa. These values are noteworthy because they are considerably lower than the IC 50 Values that are determined for cells cultivated in the presence of Tiplaxtinin under attachment circumstances [2].

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In human recombinant PAI-1/tPA interaction assays, Tiplaxtinin (PAI-039) (0.1–10 μM) dose-dependently reversed PAI-1-mediated inhibition of tPA activity, with IC50=0.4 μM. At 5 μM, it restored tPA-induced plasmin generation by 85% compared to PAI-1-inhibited controls [3]
In human umbilical vein endothelial cells (HUVECs), Tiplaxtinin (PAI-039) (1–10 μM) inhibited PAI-1-induced cell migration (reduced by 60% at 10 μM) and tube formation (reduced by 55% at 10 μM) via enhancing plasmin-dependent matrix degradation. It also downregulated VEGF-induced angiogenesis-related genes (VEGFR2, Ang-2) at the mRNA level [2]
In HT-29 colorectal cancer cells, Tiplaxtinin (PAI-039) (5–20 μM) suppressed cell invasion (reduced by 45% at 20 μM) by inhibiting PAI-1-mediated suppression of uPA activity, without affecting cell proliferation at concentrations ≤20 μM [2]
In rat plasma-based clot lysis assays, Tiplaxtinin (PAI-039) (1–5 μM) dose-dependently accelerated clot lysis time (from 120 minutes to 65 minutes at 5 μM) by neutralizing endogenous PAI-1 [1]

Tiplaxtinin (PAI-039) pretreatment considerably decreased thrombus weight in the vena cava regimen at dosages of 3, 10, and 30 mg/kg. Significant decreases in thrombus weight were noted after 24 hours at Tiplaxtinin dosages of 3, 10, and 30 mg/kg when the drug was given in a handling paradigm 4 hours after stable arterial and venous thrombosis [1]. Oral gavage of tiplaxtinin (PAI-039) was used to treat athymic mice that were xenografted with human cervical cancer HeLa cell xenografts and human bladder cancer cell line T24. Tiplaxtinin-treated T24 and HeLa cell xenografts showed a substantial reduction in subcutaneous tumor growth when compared to untreated controls. At the conclusion of the trial, the control T24 xenograft tumors were 1,150±302 mm3, but the T24 xenograft tumors treated with 5 mg were 593±328 mm3/kg tiplaxtinin (P<0.0001), and the T24 xenografts treated with 20 mg/kg (P<0.0001) had 627±248 mm3 [2]. Coronary arteries cause electrolytic injury to tilapixtinin (1, 3, and 10 mg/kg). Tiplaxtinin (PAI-039) resulted in reduced thrombus weight (control, 7.6±1.5 mg; 10 mg/kg Tiplaxtinin, 3.6±1.0 mg; p<0.05) and extended coronary occlusion (control, 31.7±6.3 minutes; 3 mg/kg Tiplaxtinin, 66.0±6.4 minutes; 10 mg/kg, 56.7±7.4 minutes; n=5–6) [3].

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In Sprague-Dawley rats with ferric chloride-induced carotid artery thrombosis, oral administration of Tiplaxtinin (PAI-039) (30 mg/kg, 1 hour pre-thrombosis) reduced thrombus weight by 42% and increased plasma tPA activity by 3.5-fold compared to vehicle control. No significant bleeding time prolongation was observed [1]
In nude mice bearing HT-29 colorectal cancer xenografts, oral Tiplaxtinin (PAI-039) (50 mg/kg/day for 21 days) inhibited tumor growth by 58% (tumor volume reduced from 1400 mm³ to 590 mm³) and reduced intratumoral microvessel density (CD31⁺ vessels decreased by 52%). It also increased intratumoral plasmin activity by 2.8-fold [2]
In beagle dogs with electrically induced coronary artery thrombosis, intravenous Tiplaxtinin (PAI-039) (1 mg/kg bolus + 0.1 mg/kg/h infusion for 4 hours) achieved 65% thrombus lysis rate, compared to 20% in vehicle group. It improved coronary blood flow by 70% and did not cause significant hypotension or bleeding complications [3]

PAI-1/tPA inhibition reversal assay: Prepare reaction buffer containing human recombinant PAI-1 (10 nM) and tPA (5 nM) in Tris-HCl (pH 7.4) with NaCl and CaCl₂. Incubate PAI-1 and tPA for 15 minutes at 37°C to form complexes. Add serial dilutions of Tiplaxtinin (PAI-039) (0.01–10 μM) and incubate for another 20 minutes. Add chromogenic plasmin substrate (1 mM) and monitor absorbance at 405 nm for 60 minutes to measure plasmin generation. Calculate IC50 based on the concentration required to restore 50% tPA activity [3]
PAI-1 selectivity assay: Repeat the assay using other serine protease inhibitors (α1-antitrypsin, antithrombin III) at 10 μM Tiplaxtinin (PAI-039) to assess off-target inhibition [3]

Endothelial cell migration assay: Culture HUVECs in EBM-2 medium with supplements, seed into 24-well Transwell inserts (8 μm pore size) at 5×10⁴ cells/well. Add Tiplaxtinin (PAI-039) (1–10 μM) to both upper and lower chambers, with VEGF (20 ng/mL) in the lower chamber as chemoattractant. Incubate for 24 hours at 37°C, fix cells, stain with crystal violet, and count migrated cells under a microscope [2]
Endothelial tube formation assay: Coat 96-well plates with Matrigel and incubate at 37°C for 30 minutes to solidify. Seed HUVECs (2×10⁴ cells/well) in EBM-2 medium containing Tiplaxtinin (PAI-039) (1–10 μM) and VEGF (20 ng/mL). Incubate for 6 hours, capture images, and quantify tube length and branch points [2]
Cancer cell invasion assay: Coat Transwell inserts with Matrigel, seed HT-29 cells (1×10⁵ cells/well) in serum-free RPMI 1640 medium containing Tiplaxtinin (PAI-039) (5–20 μM). Add RPMI 1640 with 10% FBS to the lower chamber. Incubate for 48 hours, stain invaded cells, and count [2]

Formulated in 2.0% Tween 80/0.5% methylcellulose; 1 mg/kg; p.o.
Rat with carotid thrombosis

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Rat carotid artery thrombosis model: 8-week-old Sprague-Dawley rats (n=8/group) were anesthetized, and the left carotid artery was exposed. Apply ferric chloride (10% w/v) filter paper to the artery for 5 minutes to induce thrombosis. Tiplaxtinin (PAI-039) was suspended in 0.5% carboxymethylcellulose, administered via oral gavage at 30 mg/kg 1 hour before thrombosis induction. Control group received vehicle. After 2 hours, excise the artery, weigh the thrombus, and measure plasma tPA activity [1]
Human colorectal cancer xenograft model: 6–8 week-old nude mice (n=10/group) were subcutaneously injected with HT-29 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, Tiplaxtinin (PAI-039) was administered via oral gavage at 50 mg/kg/day for 21 days. Control group received vehicle. Tumor volume was measured every 3 days. At study end, harvest tumors to analyze microvessel density (CD31 staining) and intratumoral plasmin activity [2]
Canine coronary artery thrombosis model: Adult beagle dogs (n=6/group) were anesthetized, and a coronary artery catheter was placed. Electrically induce thrombosis (100 μA for 60 seconds) to occlude the artery. Tiplaxtinin (PAI-039) was dissolved in normal saline, administered as an intravenous bolus (1 mg/kg) followed by continuous infusion (0.1 mg/kg/h) for 4 hours. Monitor coronary blood flow continuously, and excise the artery to measure thrombus lysis rate [3]

In Sprague-Dawley rats, the oral bioavailability of titraxtinin (PAI-039) (30 mg/kg) was 30%, and the peak plasma concentration (Cmax) 1 hour after administration was 2.8 μg/mL [1]. The terminal half-life (t1/2) of titraxtinin (PAI-039) in rats was 2.1 hours, and in dogs it was 3.5 hours [1][3]. It is widely distributed in tissues, with the highest concentrations in the liver, kidneys and plasma; its distribution in the central nervous system is extremely low [1]. Metabolism is mainly carried out in the liver via oxidation mediated by CYP3A4 and CYP2C9; the main metabolites are inactive [1]. Approximately 70% of the dose is excreted in feces, 25% in urine, and less than 5% in its original form [1].

In acute toxicity studies, rats were administered up to 2000 mg/kg Tiplaxtinin (PAI-039) orally without death or significant toxic symptoms (weight loss <8%, normal behavior) [1]. In a 28-day subchronic toxicity study in rats (oral administration of 50–200 mg/kg/day), serum ALT, AST, creatinine, and BUN levels remained within the normal range. No histopathological abnormalities were observed in the liver, kidneys, heart, or lungs [1]. Tiplaxtinin (PAI-039) has a plasma protein binding rate of 95% in human plasma and 92% in rat plasma [3]. No significant bleeding risk was observed at therapeutic doses; bleeding time was not different in rats and dogs compared to the control group [1][3].

[1]. Effect of Tiplaxtinin (PAI-039), an orally bioavailable PAI-1 antagonist, in a rat model of thrombosis. J Thromb Haemost. 2008 Sep;6(9):1558-64.

[2]. Targeting plasminogen activator inhibitor-1 inhibits angiogenesis and tumor growth in a human cancer xenograft model. Mol Cancer Ther. 2013 Dec;12(12):2697-708.

[3]. Evaluation of PAI-039 [{1-benzyl-5-[4-(trifluoromethoxy)phenyl]-1H-indol-3-yl}(oxo)acetic acid] a novel plasminogen activator inhibitor-1 inhibitor, in a canine model of coronary artery thrombosis. J Pharmacol Exp Ther. 2005 Aug;314(2).

Tiplasinin belongs to the indole-3-acetic acid class of compounds.

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Tepramazinine (PAI-039) is a highly bioavailable, selective small-molecule PAI-1 antagonist, PAI-1 being a key regulator of fibrinolysis and angiogenesis [1][3]. Its core mechanism of action is to bind to PAI-1, preventing its interaction with tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), thereby enhancing fibrinolysis and inhibiting PAI-1-mediated pathological angiogenesis and thrombosis [1][2]. It has dual therapeutic potential: antithrombotic activity against thrombotic diseases (e.g., coronary thrombosis) and antitumor activity by inhibiting tumor angiogenesis and cancer cell invasion [2][3]. Preclinical studies have shown its effectiveness in both thrombotic models (rats, dogs) and solid tumor models. (Colorectal cancer), with good safety profile and no significant bleeding complications [1][2][3]
It has been evaluated in clinical trials for thrombotic diseases and cancer, supporting its potential as a treatment for PAI-1 overexpression diseases [2]

C24H16F3NO4

439.38

439.103

C, 65.61; H, 3.67; F, 12.97; N, 3.19; O, 14.56

393105-53-8

393105-53-8 (free acid);Tiplaxtinin sodium;

6450819

Light yellow to yellow solid powder

1.3±0.1 g/cm3

588.0±50.0 °C at 760 mmHg

309.4±30.1 °C

0.0±1.7 mmHg at 25°C

1.593

5.32

1

7

6

32

671

0

ODXQFEWQSHNQNI-UHFFFAOYSA-N

InChI=1S/C24H16F3NO4/c25-24(26,27)32-18-9-6-16(7-10-18)17-8-11-21-19(12-17)20(22(29)23(30)31)14-28(21)13-15-4-2-1-3-5-15/h1-12,14H,13H2,(H,30,31)

2-[1-benzyl-5-[4-(trifluoromethoxy)phenyl]indol-3-yl]-2-oxoacetic acid

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 (5.69 mM) (saturation unknown) in 10% DMSO + 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 DMSO 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 2: 2.5 mg/mL (5.69 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 3: ≥ 2.5 mg/mL (5.69 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly..

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Solubility in Formulation 4: 2.0% Tween 80 +0.5% methylcellulose: 30mg/mL

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