Plasminogen activator inhibitor-1 (PAI-1) (IC50 = 6.95 μM)

According to docking experiments, TM5275 attaches to PAI-1's strand 4 of the A β-sheet (s4A). TM5275 is a selective PAI-1 that does not interfere with other serpin/serine protease systems at concentrations up to 100 μM[1]. TM5275 inhibits the formation of tPA-GFP-PAI-1 high-molecular-weight complex, hence considerably extending the retention of tPA-GFP on VECs at doses of 20 and 100 μM. TM5275 accelerates the plasminogen's time-dependent buildup and the fibrin clots' disintegration on and around the tPA-GFP-expressing cells[2]. ES-2 and JHOC-9 cells treated with 70-100 μM TM5275 had reduced cell viability after 72 hours. With 100 μM TM5275, cell growth is inhibited for 48–96 hours. When treated with 100 μM TM5275, cells exhibit a substantial decrease in active PAI-1 in cell culture media as compared to the control group. It has been proposed that TM5275 may have anti-proliferative effects on ovarian cancers with elevated PAI-1 expression [3].

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TM5275, 5-chloro-2-[({2-[4-(diphenylmethyl) piperazin-1-yl]-2-oxoethoxy}acetyl)amino]benzoate (Figure 1B), was discovered through an extensive structure-activity relationship study with more than 90 compounds designed and synthesized on the basis of the structure of TM5007 (Figure 1A). TM5275 was eventually selected as the test compound after taking into consideration the in vitro PAI-1 inhibitory activity and pharmacokinetic studies (Tmax, Cmax, T½,) (see below).
The PAI-1 inhibitory activity of TM5275, measured by tPA-dependent hydrolysis of peptide substrate, is comparable to that of TM5007 and PAI-749: Half-maximal inhibition (IC50) values of TM5275, TM5007, and PAI-749 are 6.95, 5.60, and 8.37 μmol/L, respectively.
Docking simulation of the PAI-1 moiety and TM5275 was undertaken to understand the mechanism of TM5275 action. TM5275 binds to strand 4 of the A β-sheet (s4A) position of PAI-1. Although both TM5275 and TM5007 bind within the cleft to the s4A segment of PAI-1, closer inspection reveals that their binding sites differ, as illustrated in Figure 2: the bulky diphenylmethyl group of TM5275 cannot be accommodated to the binding site of TM5007, so that TM5275 is markedly shifted in the s4A cleft.
In vitro, TM5275 (up to 100 μmol/L) does not interfere with other serpin/serine protease systems (that is, alpha1-antitrypsin/trypsin and alpha2-antiplasmin/plasmin). Its PAI-1 inhibitory activity thus appears specific. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the PAI-1 covalent complex formed with tPA is not observed when PAI-1 is preincubated with TM5275 (data not shown). [1]

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TM5275 at concentrations of 20 and 100 μM significantly prolonged the retention of tPA-GFP on VECs by inhibiting tPA-GFP-PAI-1 high-molecular-weight complex formation. TM5275 enhanced the time-dependent accumulation of plasminogen as well as the dissolution of fibrin clots on and around the tPA-GFP-expressing cells.
The profibrinolytic effects of TM5275 were clearly demonstrated by the prolongation of tPA retention and enhancement of plasmin generation on the VEC surface as a result of PAI-1 inhibition. [2]

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TM5275 Inhibited High-molecular-weight Complex Formation Between rtPA and rPAI-1 in a Purified System. TM5275 Prolonged the Retention Time of tPA-GFP on VEC Surface. TM5275 Inhibited High-molecular-weight Complex Formation Between tPA and PAI-1 on the Surface of EA.hy926 Cells, and Decreased the Amounts of tPA-PAI-1 Complex in Supernatants. Effect of TM5275 on Accumulation of Plasminogen on and Around the VECs. Efficacy of TM5275 on Lysis of Fibrin Clot Overlaid on VECs. [2]

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Pharmacological inhibition of PAI-1 by TM5275 decreases cell proliferation of ovarian cancer cells [3]
TM5275 is a small molecule inhibitor specific for PAI-1 (Fig. 4A) that has been developed as a therapeutic reagent for PAI-1-associated diseases.17,18 We investigated its potential as a therapeutic reagent for targeting cell proliferation in ovarian cancer. Ovarian cancer cells were treated with various concentrations of TM5275. Cell viability at 72 h treatment was decreased with 70–100 μM TM5275 in ES-2 and JHOC-9 cells, whereas other cell lines were relatively insensitive to TM5275 treatment (Fig. 4B). The IC50 values for TM5275 in ovarian cancer cell lines are shown in Table 1. Further, cell growth was measured at the indicated time points after TM5275 treatment. From 48 h up to 96 h, cell growth was suppressed with 100 μM TM5275 (Fig. 4C). Furthermore, active PAI-1 in cell culture media was significantly decreased in cells treated with 100 μM TM5275 compared to control treatment, confirming TM5275 effectiveness for PAI-1 inhibition (Fig. 4D). These results suggest that ovarian cancer cells with high expression of PAI-1, such as ES-2 and JHOC-9, are prone to growth inhibition by TM5275. Taken together, pharmacological inhibition of PAI-1 by TM5275 is suggested to exert anti-proliferative effects in ovarian cancer with high PAI-1 expression.

To determine the effects of TM5275 on cell cycle progression of ES-2 cells, cells were treated with or without TM5275 for 24 h and cell cycle distribution was analyzed. Compared with vehicle treatment, TM5275 treatment significantly decreased the percentage of cells in G0/G1 (52.0 ± 1.5% to 33.0 ± 4.9%) and increased the percentage in G2/M (18.3 ± 0.8% to 27.7 ± 2.0%) (Fig. 4E). Moreover, cells treated with TM5275 showed a significant increase in the percentage of apoptotic cells (Fig. 4F). These results demonstrated that pharmacological inhibition of PAI-1 induced a G2/M cell cycle arrest and promoted apoptotic cell death in ovarian cancer cells in accordance with the knockdown of PAI-1 by siRNAs.

For mice and rats, TM5275 has a very low toxicity profile and a favorable pharmacokinetics profile. in models of rat thrombosis. Rats given TM5275 at doses of 10 and 50 mg/kg had considerably smaller blood clot weights (60.9±3.0 and 56.8±2.8 mg, respectively) compared to rats given vehicle treatment (72.5±2.0 mg). The antithrombotic efficacy of TM5275 (50 mg/kg) is comparable to that of the reference antithrombotic drug, ticlopidine (500 mg/kg). Following a dosage of 10 mg/kg, the plasma concentration of TM5275 reaches 17.5±5.2 μM. When TM5275 (5 mg/kg) is taken in addition to tPA (0.3 mg/kg), the antithrombotic impact of the tPA (0.3 mg/kg) is greatly increased, yielding benefits comparable to those of a high tPA dose (3 mg/kg)[1].

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Rat Thrombosis Models [1]
The antithrombotic effectiveness of TM5275 in a rat arteriovenous shunt model is described in Table 1. Blood clot weights are significantly lower in rats administered 10 and 50 mg/kg of TM5275 (60.9±3.0 and 56.8±2.8 mg, respectively) than in vehicle-treated rats (72.5±2.0 mg). Up to 300 mg/kg of TM5007 are needed to reach the efficacy of 50 mg/kg of TM5275 in the same model. The antithrombotic effectiveness of TM5275 (50 mg/kg) is equivalent to that of ticlopidine (500 mg/kg), a reference antithrombotic compound. Plasma concentration of TM5275 reaches 17.5±5.2 μmol/L after a dose of 10 mg/kg.

The antithrombotic effectiveness of TM5275 in a rat FeCl3 carotid artery thrombosis model is illustrated in Figure 3. TM5275 and clopidogrel, another standard antithrombotic drug, prove antithrombotic in a dose-dependent manner (Figure 3). The minimum effective doses of TM5275 and clopidogrel are 1 and 3 mg/kg, respectively. It corresponds to a TM5275 plasma concentration of 4.9±3.6 μmol/L. As expected, clopidogrel prolongs bleeding time in a dose-dependent manner (Figure 3). By contrast, TM5275 does not affect bleeding time, a potential benefit as an antithrombotic agent. Two hours after oral administration, TM5275 (10 mg/kg) does not affect platelet aggregation induced by ADP and collagen (data not shown). Its antithrombotic effect is thus independent of any effect on platelets.

TM5275 has been further combined with tPA in the same model, as illustrated in Figure 4. tPA (0.3 mg/kg) alone does not provide a significant antithrombotic effect. However, TM5275 (5 mg/kg) combined with tPA (0.3 mg/kg) significantly enhances the antithrombotic effect of tPA (0.3 mg/kg) alone and provides a benefit similar to that of a high tPA dose (3 mg/kg). The bleeding time of the combination therapy is similar to that of a low tPA dose (0.3 mg/kg) alone.

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Monkey Thrombosis Model [1]
The antithrombotic effect of TM5275 has been evaluated in a cynomolgus monkey model of photochemically induced thrombosis (Table 2). Total occlusion time is significantly reduced in both the TM5275 (53.9±19.9 mins) and clopidogrel (39.4±25.8 mins) groups (10 mg/kg, each) in comparison with the vehicle group (119.0±17.4 mins). Plasma concentration of TM5275 reaches 18.9±3.7 μmol/L in the TM5275 group.

The benefits of TM5275, as an antithrombotic agent devoid of effects on bleeding time, have been confirmed in nonhuman primates (Table 3). Two hours after administration of 50 mg/kg (five times higher than the effective dose) of TM5275, the bleeding time is only slightly longer (146.7±3.3 secs) than before administration (83.3±6.7 secs), whereas it is markedly extended in the 10 mg/kg clopidogrel group (>600 secs) above that observed before administration (113.3±8.8 secs).

In Vitro PAI-1 Activity Assay [1]
PAI-1 inhibitory activity was assessed by a previously described chromogenic assay (Izuhara et al, 2008). The composition of the incubation medium was adapted to increase the assay's sensitivity: 0.15 mol/L NaCl, 50 mmol/L Tris-HCl pH8, 0.2 mmol/L CHAPS, 0.1% PEG-6000, 1% dimethylsulfoxide, 5 nmol/L human active PAI-1, 2 nmol/L human 2-chain tPA and 0.2 mmol/L Spectrozyme tPA at final concentration. Tested compounds were added at various concentrations and the IC50 was calculated by logit-log analysis.

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PAI-1/tPA Complex on Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis [1]
The effect of the tested compound on the formation of a PAI-1/tPA complex was estimated in an incubation medium mixing PAI-1, tPA, and the compound. The eventual composition included 100 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 0.05% Tween 20, 0.8% dimethylsulfoxide, 0.875 μmol/L PAI-1, 0.7 μmol/L tPA, and compound (160 μmol/L). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by Coomassie staining.

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SDS-polyacrylamide Gel Electrophoresis (SDS-PAGE) [2]
The effects of TM5275 on the formation of a tPA-PAI-1 complex were evaluated in a purified system using SDS-PAGE. After incubation with TM5275 at concentrations of 0 (solvent alone), 20, and 100 μM in HBS for 10 min at 37 °C, rPAI-1 (final concentration, 250 nM) was incubated with rtPA (final concentration, 270 nM) for 30 min at 37 °C. After mixing with sample buffer (non-reducing), the mixture was subjected to 10% SDS-PAGE, and the protein bands were stained with Coomassie Brilliant Blue.

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Fibrin Autography [2]
To evaluate the effects of TM5275 on the ability of PAI-1 to form high-molecular-weight complexes with tPA either on cultured EA.hy926 cells or in the supernatant, the amounts of tPA-PAI-1 complex and free tPA were semi-quantitated by fibrin autography. Culture media from tPA-GFP-expressing or non-expressing EA.hy926 cells were collected after 3 h of incubation in the presence of 0 (solvent alone), 20, and 100 μM TM5275 at 37 °C, centrifuged at 3,000 ×g for 10 min to remove cell debris, mixed with SDS sample buffer, and subjected to 10% SDS-PAGE. tPA-dependent activities were then detected by plasminogen-rich fibrin indicator gels after separation of the protein bands as previously reported [17].

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Plasminogen-accumulation Analysis [2]
After treatment with 100 μM TM5275 or solvent in HBS/3% BSA for 30 min at 37 °C, tPA-GFP-expressing cells were incubated with human plasminogen (0.5 μM) containing plg-568 (20 nM). The accumulation of plg-568 on or around the cell surface was then analyzed every 10 min with a confocal laser scanning microscope equipped with a 60X oil-immersion objective lens that captured the fluorescence of plg-568 at wavelengths from 570 nm to 670 nm. Just before the end of each experiment, we added 2.5 μg/mL Cell Mask Deep Red plasma membrane stain to identify the localization of tPA-GFP-expressing cells. We created a region of interest (ROI) around a single cell, including the pericellular area at the most basal focal plane, and measured the fluorescence intensity using FV10-ASW software (Olympus). Because the mean fluorescence intensity within the ROI increased linearly for 10 min, we calculated the slope of the fluorescence increase over time, representing a time-dependent accumulation of plg-568, and referred to this as dF-plg.

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Fibrin Clot Lysis Imaging [2]
tPA-GFP-transfected cells were preincubated with 100 μM TM5275 or solvent in HBS/3%BSA for 30 min at 37 °C. Fibrin clots were then formed over the cells by mixing 0.5 μM human plasminogen, containing 20 nM plg-568, 2 U/mL thrombin, and 1 mg/mL human fibrinogen containing 10 μg/mL fbg-647, in HBS/3%BSA. After fibrin clots were formed on the VECs, we started to collect images every 10 min through an automatically selected dichroic mirror and an appropriate range of wavelengths for each fluorescent dye using FV1000. We then calculated the lysis area originated from single randomly chosen tPA-GFP expressing cell at a focal plane approximately 3 μm above the bottom of the dish using FV10-ASW software.

Cell viability assay [3]
Cell viability was assessed with the CellTiter-Glo Luminescent Cell Viability Assay. Cells were seeded on 96-well plates at a density of 1–2 × 103/well. After treatment with siRNA TM5275, 80 μl CellTiter-Glo reagent was added to each well, and then plate contents were mixed on an orbital shaker. Luminescence was quantified on a standard luminometer.

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Cell cycle analysis [3]
ES-2 cells (1 × 106/100-mm dish) were subjected to the appropriate treatment. The cells were trypsinized and fixed with ice-cold 70% ethanol overnight. Fixed cells were washed with PBS and stained with 50 μg/ml propidium iodide (PI) in the presence of 100 μg/ml RNase. Cellular fluorescence was quantitated using the FL3 channel of a flow cytometer. The cell cycle distribution was determined using FlowJo software.

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Annexin V staining [3]
ES-2 cells (1 × 106/100-mm dish) were subjected to the appropriate treatment. The cells were trypsinized, washed with PBS, and stained with AlexaFluor-488-conjugated Annexin V and PI from the Dead Cell Apoptosis Kit. The stained cells were analyzed using a flow cytometer.

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Caspase assay [3]
Caspase activation was determined by Caspase-Glo 3/7 or Caspase-Glo 8 assay kits.42 Cells, seeded on 96-well plates at a density of 2 × 103/well, were transfected with siRNAs. After 72 h, 80 μl of individual Caspase-Glo reagent was added. Following 0.5 h incubation at room temperature, samples were read on a microplate luminometer.

Pharmacokinetic Studies [1]
TM5275, suspended in 0.5% carboxymethyl cellulose sodium salt (CMC) solution, was administered orally by gavage to male ICR mice (50 mg/kg), male Wistar rats (50 mg/kg), and male cynomolgus monkeys (1 mg/kg). Heparinized blood samples were collected from the vein before (0 h) and 1, 2, 6, and 24 h after oral drug administration. Plasma drug concentration was determined on a reverse-phase high-performance liquid chromatography. Maximum drug concentration time (Tmax), maximum drug concentration (Cmax), and drug half-life (T½) were then calculated. For the bioavailability (BA) study in monkeys, heparinized blood samples were collected from the vein before (0 h) and 0.5, 1, 2, 4, 6, 8, 24, 48, 72, 120, and 168 h after oral drug administration, and before (0 h) and 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, 24, 48, 72, 120, and 168 h after intravenous drug injection. BA was calculated by noncompartment model analysis using WinNonlin Professional Software, version 5.01 (Pharsight Co., NC, USA).

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Toxicity [1]
For the evaluation of acute toxicity, TM5275 (1000 mg/kg for mice and 2000 mg/kg for rats and monkeys), suspended in 0.5% CMC solution, was administered orally by gavage to male (n=5) and female (n=5) ICR mice (CLEA Japan Inc.), male (n=5) and female (n=5) Sprague–Dawley rats (Charles River Japan Inc., Kanagawa, Japan), and male cynomolgus monkeys (n=2) (Japan SLC). The animal's body weight was monitored once a week. Various organs underwent histological studies 2 weeks (mice) and 1 week (rats) after drug administration.

For the evaluation of the subacute toxicity, three different doses of TM5275 (200, 600, and 2000 mg/kg/day) were administered for 2 weeks by gavage to male (n=5) and female (n=5) Sprague–Dawley rats and male cynomolgus monkeys (n=2) (Japan SLC). At the end of the study, blood glucose, total cholesterol, triglyceride, aspartate aminotransferase, alanine aminotransferase, creatinine, urea nitrogen, total protein, albumin, hemoglobin, red blood cells, and hematocrit levels as well as activated partial thromboplastin time and prothrombin time were assessed. Body weight was measured and urinary analysis performed.

The following safety pharmacology core battery was used: a modified Irwin's test for the central nervous system in Sprague–Dawley rats dosing TM5275 up to 2 g/kg per os, and three cardiovascular tests. (1) QT interval in telemetry electrocardiogram recording in beagle dogs administered an oral dose of 2 g/kg of TM5275; (2) action potentials of guinea-pig right ventricular papillary muscles at a dose of 5 μmol/L of TM5275; and (3) hERGIkr current measured in stably transfected human embryonic kidney (HEK) 293 cells at a dose of 5 μmol/L of TM5275.

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Arteriovenous Shunt Thrombosis Rat Model [1]
Thrombus formation in arteriovenous shunts was achieved in male CD rats by a previously described method (Morishima et al, 1997). Either TM5275 (10 and 50 mg/kg, n=9) or ticlopidine (500 mg/kg, n=6), suspended in 0.5% CMC solution, was administered orally by gavage 90 mins before the study. Control rats were administered only a 0.5% CMC solution (n=10). Blood was allowed to circulate through the shunt for 30 mins. The wet weight of the thrombus covering the silk thread was eventually measured.

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Ferric Chloride-Treated Carotid Artery Thrombosis Rat Model [1]
Male Sprague–Dawley rats weighing 280 to 310 g were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally) and fixed on a heating pad. During the experiment, rectal temperature was maintained at 38°C. The left common carotid artery was exposed, and a piece of filter paper (2.5 × 4.2 mm) was folded around it. The probe of a pulsed Doppler flowmeter was placed to measure the arterial blood flow. After obtention of a steady baseline flow, 2 μL of ferric chloride (FeCl3) saline solution (35% (w/w)) was added to the filter paper. Five minutes later, the filter paper was removed and the artery washed with saline. Blood flow in the common carotid artery was continuously monitored for 30 mins after FeCl3 saline exposure. Time to primary occlusion was calculated.

Several concentrations of TM5275 (0.3, 1, 5 mg/kg) and clopidogrel (1, 3, 10 mg/kg), suspended in 0.5% CMC solution, were administered orally by gavage (n=8, each group) 2 h before FeCl3 exposure. After a 30 mins blood flow monitoring, a sphygmomanometer cuff was placed on the tail and inflated to 40 mm Hg. An incision was made with an animal lancet (Goldenrod, Medipoint Inc., Mineola, NY, USA) and, every 30 secs, a wick of filtration paper was inserted on the wound until no further staining was observed. Bleeding time was determined to the nearest 30 secs. If it lasted more than 10 mins, the experiment was discontinued.

The benefits of combining TM5275 with tPA were further ascertained. Either TM5275 (5 mg/kg, n=10), tPA (0.3 or 3 mg/kg, n=10 each), or TM5275 (5 mg/kg) plus tPA (0.3 mg/kg) (n=10) were administered orally (TM5275) or intravenously (tPA). The experiments were performed along the conditions described above.

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Photochemically Induced Arterial Thrombosis Monkey Model [1]
Three- to 4-year-old male cynomolgus monkeys weighing 2.8 to 3.5 kg underwent anesthesia by an intramuscular injection of 10 mg/kg ketamine hydrochloride followed by the intravenous injection of 25 mg/kg pentobarbital sodium. Animals were fixed on a heating pad, and rectal temperature was maintained at 36.5 to 37.5°C. The saphenous artery was exposed by a 2 cm incision and thrombosis was induced by a photochemical reaction according to the modified method of Umemura et al (1993). Briefly, the saphenous artery was irradiated with green light (wave length 540 nm, 900,000 lx) generated by a xenon lamp with a heat-absorbing filter and a green filter. Irradiation was directed by a 3-mm diameter optic fiber mounted on a micromanipulator. The probe of a pulsed Doppler flowmeter was placed on the saphenous artery to measure arterial blood flow. Once the baseline flow was steady, a 20 mins photo-irradiation was undertaken and a 6 mins intravenous rose bengal (20 mg/kg) injection initiated. TM5275 (10 mg/kg) or clopidogrel (10 mg/kg) suspended in 0.5% CMC solution were administered by gavage (n=6, each group) 2 h before photochemical thrombosis. Blood flow of the saphenous artery was monitored for 3 h after the start of photo-irradiation. In monkeys, in contrast with rodents, photochemically induced arterial thrombosis progressively reduced cerebral blood flow, and was followed by recanalization and eventually by rethrombosis, a sequence observed in stroke patients and called cyclical flow reduction (Maeda et al, 2005a, 2005b). Therefore, total occlusion time was calculated during the experiment.

Bleeding time was measured in monkeys previously acclimated to chair restraint during repeated training sessions several times before the experiment. A sphygmomanometer cuff, placed on the upper leg of conscious monkeys fixed to a monkey chair, was inflated to 40 mm Hg. TM5275 (50 mg/kg) or clopidogrel (10 mg/kg) suspended in 0.5% CMC solution were administered orally by gavage (n=3, each group) 2 h before the study. Bleeding was produced inside the lower leg by a Micro Lancet with a 21-gauge needle. Every 10 sec, a wick of filtration paper was placed on the wound until no further staining was observed. Bleeding time was determined to the nearest 10 secs. Maximum observation period was 10 mins.

Pharmacokinetics [1]
The pharmacokinetics of TM5275 improved significantly when compared with that of TM5007. An oral dose of 50 mg/kg of TM5275, administered in rats, yields calculated plasma Tmax, Cmax, and T½ of 2 h, 34 μmol/L, and 2.5 h, respectively, versus 18 h, 8.8 μmol/L, and 124 h, respectively, in rats administered the same dose of TM5007. TM5275 thus increases Cmax fourfold, and markedly shortens both Tmax and T½. In mice, an oral dose of 50 mg/kg of TM5275 yields the following values for these parameters: 1 h, 6.9 μmol/L, and 6.5 h, respectively. In monkeys, an oral dose of 1 mg/kg of TM5275 yields Tmax, Cmax, and T½ values of 6 h, 10.5 μmol/L, and 114.7 h, respectively. Bioavailability of TM5275 reaches 96% in monkeys.

Toxicity [1]
Acute toxicity has been evaluated in vivo. A single dose of TM5275 of 1000 mg/kg in mice and 2000 mg/kg in rats and monkeys elicited no symptoms after 2 weeks in the former group and after 1 week in the latter two groups. Body weight and the histology of various organs are not modified.

Subacute toxicity has been assessed in rats and monkeys administered daily three different doses of TM5275 (200, 600, and 2000 mg/kg/day) for 2 weeks. Body weight and the histology of various organs are not modified. No abnormality is noted in the biochemistry of plasma and urine, including activated partial thromboplastin time, prothrombin time, and red blood cell count.

In safety pharmacology studies, TM5275 does not modify tests of the central nervous system (a modified Irwin's test in rats) or of the cardiovascular system: (1) QT interval in electrocardiogram recording in dogs; (2) action potentials of guinea-pig right ventricular papillary muscles; and (3) hERGIkr current in HEK293 cells.

[1]. A novel inhibitor of plasminogen activator inhibitor-1 provides antithrombotic benefits devoid of bleeding effect in nonhuman primates. J Cereb Blood Flow Metab. 2010 May;30(5):904-12.

[2]. TM5275 prolongs secreted tissue plasminogen activator retention and enhances fibrinolysis on vascular endothelial cells. Thromb Res. 2013 Jul;132(1):100-5.

[3]. Inhibition of plasminogen activator inhibitor-1 is a potential therapeutic strategy in ovarian cancer. Cancer Biol Ther. 2015;16(2):253-60.

Inhibition of plasminogen activator inhibitor (PAI)-1 is useful to treat several disorders including thrombosis. An inhibitor of PAI-1 (TM5275) was newly identified by an extensive study of structure-activity relationship based on a lead compound (TM5007) which was obtained through virtual screening by docking simulations. Its antithrombotic efficacy and adverse effects were tested in vivo in rats and nonhuman primates (cynomolgus monkey). TM5275, administered orally in rats (1 to 10 mg/kg), has an antithrombotic effect equivalent to that of ticlopidine (500 mg/kg) in an arterial venous shunt thrombosis model and to that of clopidogrel (3 mg/kg) in a ferric chloride-treated carotid artery thrombosis model. TM5275 does not modify activated partial thromboplastin time and prothrombin time or platelet activity and does not prolong bleeding time. Combined with tissue plasminogen activator, TM5275 improves the latter's therapeutic efficacy and reduces its adverse effect. Administered to a monkey model of photochemical induced arterial thrombosis, TM5275 (10 mg/kg) has the same antithrombotic effect as clopidogrel (10 mg/kg), without enhanced bleeding. This study documents the antithrombotic benefits of a novel, more powerful, PAI-1 inhibitor in rats and, for the first time, in nonhuman primates. These effects are obtained without adverse effect on bleeding time. [1]

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Introduction: Elevated plasminogen activator inhibitor-1 (PAI-1) reduces fibrinolytic potential in plasma, contributing to thrombotic disease. Thus, inhibiting PAI-1 activity is clinically desirable. We recently demonstrated that tissue plasminogen activator (tPA) remains on the surface of vascular endothelial cells (VECs) after secretion in a heavy-chain dependent manner, which is essential for high fibrinolytic activity on the surface of VECs, and that PAI-1 dissociates retained tPA from the cell surface as a result of high-molecular weight complex formation. Based on the model whereby amounts of tPA and its equilibrium with PAI-1 dynamically change after exocytosis, we examined how TM5275, a newly synthesized small molecule PAI-1 inhibitor, modulated tPA retention and VEC surface-derived fibrinolytic activity using microscopic techniques.

Materials and methods: The effects of TM5275 on the kinetics of the secretion and retention of green fluorescent protein (GFP)-tagged tPA (tPA-GFP) on VECs were analyzed using total internal reflection fluorescence microscopy. The effects of TM5275 on the generation of plasmin activity were evaluated by both plasminogen accumulation and fibrin clot lysis on tPA-GFP-expressing VECs using confocal laser scanning microscopy.

Results: TM5275 at concentrations of 20 and 100 μM significantly prolonged the retention of tPA-GFP on VECs by inhibiting tPA-GFP-PAI-1 high-molecular-weight complex formation. TM5275 enhanced the time-dependent accumulation of plasminogen as well as the dissolution of fibrin clots on and around the tPA-GFP-expressing cells.

Conclusions: The profibrinolytic effects of TM5275 were clearly demonstrated by the prolongation of tPA retention and enhancement of plasmin generation on the VEC surface as a result of PAI-1 inhibition.

Keywords: CLSM; HBS; HEPES-buffered solution; PAI-1; TIRF; TM5275; VEC; confocal laser scanning microscope; fibrinolysis; plasminogen activator inhibitor-1; plasminogen activator inhibitor-1 (PAI-1); plasminogen activator inhibitor-1 (PAI-1) inhibitor; tPA; tissue plasminogen activator; total internal reflection fluorescence; total internal reflection fluorescence (TIRF) microscopy; vascular endothelial cell.[2]

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Plasminogen activator inhibitor (PAI)-1 is predictive of poor outcome in several types of cancer. The present study investigated the biological role for PAI-1 in ovarian cancer and potential of targeted pharmacotherapeutics. In patients with ovarian cancer, PAI-1 mRNA expression in tumor tissues was positively correlated with poor prognosis. To determine the role of PAI-1 in cell proliferation in ovarian cancer, the effects of PAI-1 inhibition were examined in PAI-1-expressing ovarian cancer cells. PAI-1 knockdown by small interfering RNA resulted in significant suppression of cell growth accompanied with G2/M cell cycle arrest and intrinsic apoptosis. Similarly, treatment with the small molecule PAI-1 inhibitor TM5275 effectively blocked cell proliferation of ovarian cancer cells that highly express PAI-1. Together these results suggest that PAI-1 promotes cell growth in ovarian cancer. Interestingly, expression of PAI-1 was increased in ovarian clear cell carcinoma compared with that in serous tumors. Our results suggest that PAI-1 inhibition promotes cell cycle arrest and apoptosis in ovarian cancer and that PAI-1 inhibitors potentially represent a novel class of anti-tumor agents.[3]

C28H27CLN3NAO5

543.98

543.153

C, 61.82; H, 5.00; Cl, 6.52; N, 7.72; Na, 4.23; O, 14.71

1103926-82-4

1103928-13-7 (free acid);1103926-82-4 (sodium);

53240409

White to off-white solid powder

2.541

1

6

9

38

752

0

C1CN(CCN1C(C2=CC=CC=C2)C3=CC=CC=C3)C(=O)COCC(=O)NC4=C(C=C(C=C4)Cl)C(=O)[O-].[Na+]

JSHSGBIWNPQCQZ-UHFFFAOYSA-M

InChI=1S/C28H28ClN3O5.Na/c29-22-11-12-24(23(17-22)28(35)36)30-25(33)18-37-19-26(34)31-13-15-32(16-14-31)27(20-7-3-1-4-8-20)21-9-5-2-6-10-21;/h1-12,17,27H,13-16,18-19H2,(H,30,33)(H,35,36);/q;+1/p-1

sodium;2-[[2-[2-(4-benzhydrylpiperazin-1-yl)-2-oxoethoxy]acetyl]amino]-5-chlorobenzoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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 (4.60 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 (4.60 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 3: ≥ 2.5 mg/mL (4.60 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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 (Please use freshly prepared in vivo formulations for optimal results.)