PAI-1/Plasminogen activator (IC50 = 29 μM)

In Vitro Assessment [1]
We purchased or synthesized 28 of the candidate compounds discovered by virtual screening and tested their biological activities in vitro by three different assays. Inhibition of PAI-1 activity was measured by tPA-dependent hydrolysis of peptide substrate. The 2 most effective candidate compounds, N, N′-bis (3,3′-carboxy-4,4′-phenyl-2,2′-thienyl) hexanedicarboxamide (TM5001) and N, N′-bis [3,3′-carboxy-4,4′-(2,2′-thienyl)-2,2′- thienyl] hexanedicarboxamide (TM5007) had an efficacy comparable to that of tiplaxtinin (IC50 for TM5001, TM5007, and tiplaxtinin 28.6±7.3, 29.2±4.2, and 40±7 μmol/L, respectively). TM5001 and TM5007 share a common binding mode with TM5001 as illustrated in Figure 1. It suggests a similar molecular mechanism for their activity. Neither TM5001 nor TM5007 (up to 250 μmol/L) modified other serpin/serine protease systems (ie, α1-antitrypsin/trypsin and α2-antiplasmin/plasmin): their PAI-1 inhibitory activity appears thus specific (please see supplemental Figure II).
On SDS-PAGE, PAI-1 formed a covalent complex with tPA whereas no PAI-1/tPA complex formation was observed when PAI-1 was preincubated with our compounds (exemplified for TM5007 in Figure 2).
Finally the inhibition of fibrinolysis was tested on a fibrin plate (exemplified for TM5007). The area of tPA-induced fibrinolysis was decreased by PAI-1. Preincubation of PAI-1 with our compounds prevented this effect (please see supplemental Figure III).

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Cytotoxicity of TM5001 and TM5007 was assessed with HeLa cells as the LDH activity released into the culture medium after 24-h incubation. Results are expressed as percentage of the LDH release induced by the lysis of all cells. TM5001 (100 μmol/L) and TM5007 (250 μmol/L) did not significantly raise maximum LDH activity above unstimulated controls (30.8±7.1 and 26.6±2.2%, respectively, versus 23.8±1.6%; P=0.077 and 0.137) [1].

In Vivo Assessment [1]
TM5007 was chosen for further investigations, as it proved more effective than TM5001. Its in vivo anticoagulant effectiveness was assessed by weighing the blood clot obtained in a rat AV shunt model (Table). Blood clot weight was significantly lower in rats given 300 mg/kg of TM5007 (54.8±0.8 mg) than in vehicle-treated rats (74.3±3.2 mg) (P<0.01). This effect, obtained at a plasma concentration of TM5007 of 5.2±0.7 μmol/L, was equivalent to that of warfarin (1.2 mg/kg) and ticlopidine (500 mg/kg), and superior to that of tPA (275000 IU/kg). PAI-1 activity was significantly reduced by TM5007 (0.8±0.1 versus 1.0±0.1 ng/mL in vehicle treated rat) but not by other agents, whereas APTT and PT were not modified (Table).

The anticoagulant effectiveness of TM5007 was also evaluated in FeCl3-treated mouse testicular arteries. Growth of thrombi, followed by 3-dimensional imaging, led to complete arterial occlusion within 12.7±2.7 min (n=29) in vehicle-treated mice versus only 56.3±8.9 min in TM5007-treated mice (n=15, P<0.01 compared to the control group). This effect was obtained at a TM5007 plasma concentration of 4.6±0.6 μmol/L. Tirofiban had a similar effect (58±12.4 min; n=5, P<0.01 compared to the control group).

The in vivo antifibrotic effect of TM5007 was tested in a mouse model of bleomycin induced pulmonary fibrosis. Bleomycin increased significantly the lung hydroxyproline content above that of control mice (232.9±8.5 versus 140.2±4.8 μg/lung, P<0.001). TM5007 significantly lowered the bleomycin-induced lung hydroxyproline content (204.2±9.5 μg/lung, P<0.05). Bleomycin raised plasma PAI-1 activity above that of control mice (1.7±0.2 versus 0.8±0.1 ng/mL, P<0.001). TM5007, at a plasma concentration of 9.2±0.2 μmol/L, lowered significantly the bleomycin-induced PAI-1 rise (1.2±0.1 ng/mL, P<0.05). Histological evidence of pulmonary fibrosis was markedly improved by TM5007 (Figure 3A through 3C). Azan staining disclosed the accumulation of collagen in bleomycin-treated lungs (Figure 3D through 3F). Bleomycin raised the fibrosis score above control (4.7±0.37 versus 0.5±0.17, P<0.001), a rise that was partially prevented by TM5007 (2.9±0.38, P<0.01), in good agreement with the results of plasma PAI-1 activity.

PAI-1 activity assays [1]
PAI-1 activity was evaluated by two different methods. For the screening of our chemical libraries for their PAI-1 inhibitory activities, we used a biological assay utilizing a synthetic substrate for t-PA. In brief, human PAI-1 was incubated at 37 °C for 15 min in the reaction buffer containing 100 mM Tris HCl, pH 8, 0.1 % Tween 80 in the presence or absence of the tested compounds in a 96-well polystyrene plate. The mixture was subsequently incubated for 15 min with human tPA, and eventually fortified with a chromogenic substrate, S-2288 (Chromogenix, Milano, Italy). The final mixture contained 100 mM Tris-HCl, pH 8, 30 mM NaCl, 1 % DMSO, 0.1 % Tween 80, 67 nM PAI-1, 9.8 nM tPA, 1 mM S-2288, and tested compounds at various concentrations (20, 35, 50, and 100 μM). Kinetics of p-nitroanilide release during peptide cleavage was monitored with a spectrophotometer at 405 nm. The residual inhibitory activity of PAI-1 was expressed as the percentage of the initial activity. The inhibitory activity of the tested compounds was also evaluated in other serpin/serine protease systems (i.e., α1-antitrypsin/trypsin and α2-antiplasmin/plasmin) by a chromogenic assay with synthetic substrates.
PAI-1 activity was determined in citrated blood samples of both rat and mouse by, ELISA kits: functionally active PAI-1 molecule present in plasma reacts with tPA (for rat) or uPA (for mouse) coated and dried on a microtiter plate. In these ELISAs, non-active PAI-1 molecules, such as latent or complexed PAI-1, do not bind to the plate and remain therefore undetected. PAI-1 activity is expressed as a mass of active material.

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Fibrinolytic activity assay [1]
The effect of the tested compound on fibrinolysis was evaluated according to the method of Matsuo et al. In brief, thrombin (10 NIH U/ml) dissolved in 0.2 ml of saline was mixed in a 9 cm plate with 9 ml of a fibrinogen (1.5 mg/ml) solution containing 25 mM barbital sodium, 50 mM NaCl, and 25 mM CaCl2. The plate was kept for 2 hr at room temperature and used for the fibrinolytic assay. The solution containing 100 mM Tris-HCl, pH 8, 30 mM NaCl, 1 % DMSO, 0.1 % Tween 80, 87 nM PAI-1, 18 nM tPA, and the tested compound (4, 16.5, 65, and 260 μM) was spotted on the plate very gently. After an 18-hr reaction at room temperature, the fibrinolysed area was measured.

Cellular toxicity assay [1]
HeLa cells were cultured for 24 hrs in the Dulbecco’s MEM in the presence of the tested compound (50-250 μM). Cytotoxicity was determined by the release of lactate dehydrogenase (LDH) measured using a kit (Promega, Madison, WI) and expressed as percentage of the total LDH released after freeze and thaw lysis of all culture cells.

Arteriovenous Shunt Model [1]
Thrombus formation in arteriovenous (AV) shunts was achieved in male CD rats by a previously described method.23 Before the study, TM5007 (300 mg/kg), warfarin (1.2 mg/kg), or ticlopidine (500 mg/kg), suspended in 0.5% carboxymethyl cellulose sodium salt (CMC) solution, was given by gavage, or tPA (275000 IU/kg) was administered intravenously by a bolus injection (n=7 for each group). Control rats were given 0.5% CMC solution only (n=7). Blood was allowed to circulate through the shunt for 30 min. The wet weight of the thrombus covering the silk thread was eventually measured.

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Ferric Chloride–Induced Thrombosis Model and Visualization of Thrombi [1]
Male mice were anesthetized with an intraperitoneal injection of 12 mL/kg ketamine-xylazine. Rhodamine 6G (0.1 mL of 0.1%) was injected intravenously. A testicular artery (100 to 150 μm in diameter) was carefully exposed for ferric chloride (FeCl3) treatment. A cotton thread (0.2 mm in diameter) saturated with 0.25 mol/L FeCl3 was applied to the adventitial surface of the testicular artery. After 5 minutes, the cotton thread was replaced by a saline solution in the wound. Thrombus formation in the testicular artery was subsequently monitored through 3-dimensional imaging using an ultrafast laser confocal microscope equipped with a piezo-electric motor control unit as previously reported.24 The time from endothelial damage by FeCl3 to occlusion of testicular arteries by large thrombi was measured. Mice were pretreated either by gavage of TM5007 (200 mg/kg), twice a day for 4 days. The antiplatelet glycoprotein (GP) IIb/IIIa agent, tirofiban (0.13 mg/kg, Wako), was single administered in a single intravenous injection before the injury.

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Bleomycin-Induced Pulmonary Fibrosis [1]
Male C57BL/6J (CLEA Japan Inc.) mice weighing 19 to 21 g were anesthetized with intraperitoneal pentobarbital and their trachea exposed by a cervical incision. Ten animals served as controls. Ten animals received an intratracheal instillation of bleomycin dissolved in saline (1.5 U/kg), and 10 animals received in addition by gavage TM5007 (200 mg/kg) suspended in 0.5% CMC, twice a day for 14 days. Lung tissue was obtained for histological analysis and measurement of hydroxyproline content. Hydroxyproline was measured in tissue hydrolysates by the method of Kivirikko et al.25 Tissue sections were stained with hematoxylin and eosin and pulmonary fibrosis was scored on a scale of 0 to 8 using a previously described method.26 Azan stain for collagen was also used.

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Toxicity in rodents [1]
For the evaluation of acute toxicity, TM5001 and TM5007 (500, 1000, 1500, and 2000 mg/kg) were given once by gavage to ICR mice. Two weeks later, each mouse was autopsied and various organs evaluated. For the evaluation of sub-acute toxicity, two different doses of TM5007 (300 mg/kg/day for 1 week or 2000 mg/kg/day for two week) were given daily by gavage to male Wistar rats. At the end of the study, bleeding test, activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombo test (TT) were assessed as well as blood glucose, total cholesterol, triglyceride, AST, ALT, creatinine, urea nitrogen, total protein, albumin, hemoglobin, red blood cells, and hematocrit levels. Blood pressure and body weight were also measured and urinary analysis was performed.

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Pharmacokinetics [1]
TM5001 and TM5007 (50 mg/kg) were given by gavage to male Wistar rats. Heparinized blood samples were collected from the vein before (0 h) and at 1, 2, 6, 18, 24 h, and 7 day after administration. Drug concentration was determined in the plasma by reverse-phase HPLC. Maximum drug concentration time (Tmax), maximum drug concentration (Cmax), and drug half-life (T1/2) were calculated.

Rats were orally administered 50 mg/kg of each of the two compounds, and their plasma Tmax, Cmax, and T1/2 were calculated. The Tmax, Cmax, and T1/2 of TM5001 were 18 hours, 32 μmol/L, and 54 hours, respectively, while those of TM5007 were 18 hours, 8.8 μmol/L, and 124 hours, respectively. [1]

Acute toxicity of TM5001 and TM5007 was evaluated in mice. No symptoms were observed within two weeks of a single dose of up to 2000 mg/kg for either compound. Subacute toxicity of TM5007 was evaluated in rats by administering two different daily doses (300 mg/kg/day for 1 week or 2000 mg/kg/day for 2 weeks). No changes were observed in blood pressure or body weight. No abnormalities were observed in plasma and urine biochemistry, including bleeding time, APTT, PT, TT, and red blood cell count (see supplementary table for details). [1]

[1]. Inhibition of plasminogen activator inhibitor-1: its mechanism and effectiveness on coagulation and fibrosis. Arterioscler Thromb Vasc Biol. 2008 Apr;28(4):672-7.

Objective: Serine protease inhibitors (serpins) play a central role in various pathological processes, including coagulation, fibrinolysis, malignancy, and inflammation. Inhibition of serine protease inhibitors may hold therapeutic potential. However, to date, very few small-molecule serine protease inhibitors have been reported. We are the first to use a novel virtual screening method to discover novel orally active small-molecule serine protease inhibitors and report their efficacy. Methods and Results: We focused on a clinically significant serine protease inhibitor—plasminogen activator inhibitor-1 (PAI-1)—whose crystal structure has been resolved. We screened for several novel orally active molecules that could act as mimics to enter the fourth chain position (s4A) of the PAI-1 Aβ fold. In vitro experiments showed that these molecules specifically inhibited PAI-1 activity and enhanced fibrinolytic activity. In vivo, the most effective molecule (TM5007) inhibited coagulation in two models: a rat arteriovenous (AV) shunt model and a ferric chloride-induced mouse testicular artery thrombosis model. It also inhibits the fibrotic process induced by bleomycin in the lungs of mice. Conclusion: This study confirms the beneficial effects of novel PAI-1 inhibitors in vitro and in vivo. Our method has proven to be an effective tool for obtaining effective serine protease inhibitors. [1] Based on virtual screening and the three-dimensional structure of PAI-1 and its inhibitory peptide complex, we identified two novel, orally bioavailable small molecule PAI-1 inhibitors, TM5001 and TM5007. In vitro experiments showed that both inhibitors were stable, non-toxic, and non-cytotoxic because they did not increase LDH levels in cultured HeLa cells. No acute or subacute toxicity was confirmed in mice after a single dose of up to 2000 mg/kg, or in rats after 300 mg/kg for one week or 2000 mg/kg for two weeks. TM5007 demonstrated in vivo efficacy in animal models of acute vascular thrombosis and chronic pulmonary fibrosis without adverse effects on blood pressure or bleeding, highly consistent with previous findings in PAI-1 deficient mice and humans. The specificity of TM5007 for PAI-1 action was further confirmed by a colorimetric assay using the synthetic substrate, a finding also validated against other serine protease inhibitor/serine protease systems (e.g., α1-antitrypsin/trypsin and α2-antiplasmin/plasmin). At a concentration of 250 μmol/L, TM5007 showed no inhibitory activity against any closely related serine protease inhibitors or serine proteases. This concentration is approximately 10 times the IC50 value of TM5007 against PAI-1 in in vitro studies (29 μmol/L) and approximately 25 to 50 times the peak plasma concentrations of this compound in mice and rats (5 to 10 μmol/L). Therefore, the activity of TM5007 appears to be specific to PAI-I. All known PAI-I inhibitors have been shown to inhibit thrombosis, both in vitro and in acute thrombosis models. In contrast, tepraxetine only showed a delayed effect on subsequent tissue remodeling in angiotensin II-induced hypertensive mouse model. We demonstrated that TM5007 is a potent antithrombotic agent that does not prolong bleeding time, prothrombin time (PT), or activated partial thromboplastin time (APTT). Its efficacy at a dose of 300 mg/kg is comparable to warfarin (1.2 mg/kg) and ticlopidine (500 mg/kg), and superior to tPA (275,000 IU/kg). The antithrombotic effect of TM5007 was also demonstrated in a FeCl₃-induced testicular artery thrombosis mouse model. Pretreatment with oral TM5007 (200 mg/kg, twice daily for 4 days) was comparable in efficacy to a single intravenous injection of the antiplatelet drug tirofiban (0.13 mg/kg). Notably, the in vitro calculated IC50 value of TM5007 against PAI-1 (29.2 μmol/L) was higher than the peak plasma concentrations of this compound observed in mice and rats (5 to 10 μmol/L). This difference may reflect variations in experimental systems. In vitro, PAI-1 inhibition was directly measured. In contrast, its effect on thrombosis in vivo is more complex, as it involves multiple factors beyond PAI-1/tPA. In this study, we demonstrate for the first time that PAI-1 inhibition can prevent bleomycin-induced fibrosis in the lungs. Eitzman et al. investigated mice with PAI-1 gene overexpression or deletion, confirming a strong correlation between PAI-1 expression and collagen accumulation in lung tissue. ⁹ The inhibitory effect of TM5007 on pulmonary fibrosis suggests that PAI-1 is not a surrogate marker of fibrosis, but rather its primary cause. This finding has potentially significant implications. Fibrotic changes are indeed associated with the failure of multiple organ functions, including the heart, blood vessels, liver, and kidneys. Prevention of fibrotic changes may alter the prognosis of various diseases, such as cardiovascular disease, cirrhosis, kidney disease, and radiation injury.
Some small-molecule PAI-I inhibitors were discovered through low-efficiency high-throughput screening (HTS) of compound libraries. ^3-5 Gerlatova et al. demonstrated that the binding epitope of tipalaxtinin is adjacent to a previously identified polinecin interaction site, suggesting that the drug's antiserine protease inhibitory activity is mediated by the interaction between PAI-I and polinecin. In contrast, we focused on the s4A site as a target site for PAI-I inhibition. Our molecular docking simulations showed that TM5001 and TM5007 preferentially bind to this site, suggesting that our compounds exert inhibitory activity by blocking the s4A site. SDS-PAGE electrophoresis results showed that no PAI-1/tPA complex formation was observed after pre-incubation of PAI-1 with our compound (Figure 2). Therefore, the results indicate that the inhibitory mechanism of our compound is different from that of tipaxtinin. Interestingly, although tipaxtinin and ZK4044 have completely different chemical structures, they both appeared to bind to the s4A site in our molecular docking simulations, which is consistent with the previous hypothesis of Gerlatova et al.³⁰ (tiplaxtinin inhibits PAI-1 through multiple mechanisms). In summary, these findings confirm the crucial role of the s4A site as a target site for PAI-1 inhibition.
Utilizing the three-dimensional structure of PAI-1 not only helps in understanding the molecular events leading to PAI-1 inhibition but also facilitates virtual screening of novel inhibitors for clinical use. Potential applications include thrombotic diseases (arterial and venous), fibrotic diseases, amyloidosis, obesity, and type 2 diabetes. The emergence of specific inhibitors will also provide a potentially important pharmacological tool for studying the role of PAI-1 in these processes. Finally, elucidating the three-dimensional structures of other serine protease inhibitors and combining them with virtual screening will help to discover other small molecule serine protease inhibitors, thereby mitigating their harmful effects. [1]

C24H20N2O6S4

560.68540096283

560.02

C, 51.41; H, 3.60; N, 5.00; O, 17.12; S, 22.87

342595-05-5

4199057

Typically exists as solid at room temperature

1.5±0.1 g/cm3

800.4±65.0 °C at 760 mmHg

437.8±34.3 °C

0.0±3.0 mmHg at 25°C

1.730

4.96

4

10

11

36

766

0

C(NC1SC=C(C2SC=CC=2)C=1C(O)=O)(=O)CCCCC(NC1SC=C(C2SC=CC=2)C=1C(O)=O)=O

USLUSWIVVBUXLU-UHFFFAOYSA-N

InChI=1S/C24H20N2O6S4/c27-17(25-21-19(23(29)30)13(11-35-21)15-5-3-9-33-15)7-1-2-8-18(28)26-22-20(24(31)32)14(12-36-22)16-6-4-10-34-16/h3-6,9-12H,1-2,7-8H2,(H,25,27)(H,26,28)(H,29,30)(H,31,32)

2-[[6-[(3-carboxy-4-thiophen-2-ylthiophen-2-yl)amino]-6-oxohexanoyl]amino]-4-thiophen-2-ylthiophene-3-carboxylic acid

TM5007; 342595-05-5; 2-[[6-[(3-carboxy-4-thiophen-2-ylthiophen-2-yl)amino]-6-oxohexanoyl]amino]-4-thiophen-2-ylthiophene-3-carboxylic acid; SCHEMBL1798341; EX-A4825; SNA59505; STK400044;

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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