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TM5441 sodium

Alias: TM5441 (sodium); TM-5441 sodium; TM-5441 (sodium);
Cat No.:V84168 Purity: ≥98%
TM5441 is an orally bioavailable plasminogen activator inhibitor-1 (PAI-1) inhibitor
TM5441 sodium
TM5441 sodium Chemical Structure CAS No.: 2319722-53-5
Product category: PAI-1
This product is for research use only, not for human use. We do not sell to patients.
Size Price
500mg
1g
Other Sizes

Other Forms of TM5441 sodium:

  • TM-5441
Official Supplier of:
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Top Publications Citing lnvivochem Products
Product Description
TM5441 is an orally bioavailable plasminogen activator inhibitor-1 (PAI-1) inhibitor with IC50 values ranging from 13.9 to 51.1 μM against multiple cancer cell lines. TM5441 induces intrinsic apoptosis in several human cancer cells. TM5441 attenuates Nω-nitro-1-arginine methyl ester-induced cardiac hypertension and vascular senescence.
Biological Activity I Assay Protocols (From Reference)
Targets
PAI-1/plasminogen activator inhibitor-1/PAI-1
ln Vitro
HT1080, HCT116, Daoy, MDA-MB-231, and Jurkat cells are all dose-dependently decreased by TM-5441, with an IC50 ranging from 13.9 to 51.1 μM[1]. In HT1080 and HCT116 cells, TM5441 increases caspase 3/7 activity in a dose-dependent manner. TM5441 causes HT1080 and HCT116 cells to undergo more apoptosis[1]. Mitochondrial depolarization is induced by TM5441[1]. TM5441 reverses PAI-1-induced inhibition of plasmin activity and effectively blocks fibrosis and inflammation markers' mRNA expression in mouse proximal tubular epithelial cells[2].
TM5275 and v decreased survival of cancer cells [1]
We first screened 17 human cell lines (14 malignant and 3 non-malignant) for their in vitro sensitivity to TM5275 and TM5441 with a cell viability assay. This analysis indicated a significant dose-dependent decrease in cell viability in the presence of TM5275 and TM5441 in several cell lines (HT1080, HCT116, Daoy, MDA-MB-231 and Jurkat) with an IC50 ranging between 9.7 and 60.3 μM (Fig 1 and S1 Table). However many other malignant and non-malignant cell lines were resistant to TM5275 showing an IC50 above 50 μM (S1 Fig). We selected the HT1080 and HCT116 cell lines for further studies because they showed the lowest TM5441 IC50 and because previous work in the laboratory had demonstrated a pro-tumorigenic role of PAI-1 in these cell lines.
TM5275 and TM-5441 decreased proliferation of HT1080 and HCT116 cells [1]
To examine the effect of TM inhibitors on cell proliferation, we used BrdU incorporation to test cell cycle activity. This analysis (Fig 2) revealed a significant decrease in the percentage of BrdU positive cells with both cell lines treated with TM5275 and TM5441 (from 48.5% and 48.7% in DMSO-treated cells to 38.1% and 42.5% in TM5275-treated HT1080 and HCT116 cells and 28.3% and 34.6% for TM5441-treated cells, respectively). These results indicated that TM5275 and TM5441 decreased tumor cell viability in part through diminished proliferation.
TM5275 and TM-5441 increased apoptosis in HT1080 and HCT116 cells [1]
Because previous studies indicated that PAI-1 protects tumor cells from apoptosis [10, 12, 25, 26] we examined the effect of TM5275 and TM5441 on apoptosis in HT1080 and HCT116 cells. Using a caspase 3/7 activity assay, we demonstrated a dose-dependent increase in caspase 3/7 activity for both HT1080 and HCT116 cells exposed to these inhibitors (Fig 3A). The effect on caspase 3/7 activity was statistically much stronger with TM5441 (37-fold and 32-fold, respectively, at 100 μM) than with TM5275 (3-fold and 5-fold; P value = 0.0005 and 0.003, respectively). The effect of the inhibitors on apoptosis was confirmed by analysis of Annexin V and propidium iodide (PI) staining by flow cytometry (Fig 3B). The data indicated a statistically significant dose-dependent increase in early and late apoptosis in HT1080 and HCT116 cells treated with TM5275 or TM5441 inhibitors when compared with DMSO-treated cells. As a readout for PAI-1 activity against uPA, we measured plasmin activity over time. This analysis revealed an increase in cell-associated plasmin activity that peaked between 8 and 24 hours and correlated with an increase in apoptosis (Fig 3C).
We also tested whether TM-5441 at concentrations of 1 μM to 25 μM would affect the sensitivity of HT1080 and HCT116 cells to several chemotherapeutic agents (doxorubicin, etoposide, oxaloplatin, and 5-fluorouracil (5-FU). The data revealed that TM5441 did not potentiate the activity of these chemotherapeutic agents at 1 μM. Even when used at a higher concentration (25 μM), TM5441 did not potentiate the cytotoxic activity of doxorubicin (S2 Fig).
TM5275 and TM-5441 induced mitochondrial depolarization [1]
To explore the relative contribution of the extrinsic and intrinsic apoptotic pathways in TM inhibitor-induced apoptosis, we examined the effect of these inhibitors on the cleavage (activation) of caspase 3, 8 and 9 by Western blot. The data (Fig 4A) revealed an absence of caspase 8 activation in both cell lines upon treatment with TM5275 or TM5441. In contrast they indicated activation of caspase 9 and 3 and a corresponding cleavage of Poly adipo-ribose polymerase (PARP), a substrate for caspase 3, which was consistent with an activation of the intrinsic apoptotic pathway by TM inhibitors. This was confirmed by the examination of mitochondrial membrane depolarization in HT1080 and HCT116 cells treated with TM inhibitors (50 μM) (Fig 4B). The data indicated an increase in mitochondrial depolarization in cells treated with TM5275 and TM5441 with a much more pronounced effect of TM5441 as previously observed in caspase 3/7 activity assays (Fig 3A). These results indicated that TM5275, and in particular TM5441, are potent stimulators of intrinsic apoptosis in tumor cells.
Inhibited HUVEC branching with TM5275 and TM-5441 [1]
The disruptive effect of TM5441 on the tumor vasculature in vivo was further investigated on EC in vitro (Fig 6). This analysis revealed a dose-dependent inhibition of TM5441 on branching morphogenesis of HUVEC plated in 3D Matrigel cultures (Fig 6A). This effect, however, was not related to a direct effect on EC viability as TM5441 had no effect on HUVEC survival (Fig 6B) and apoptosis (Fig 6C) at a concentration of 50 μM, whereas, it had a significant effect on branching morphogenesis in vitro. A similar effect on EC was observed with TM5275 (S1 and S7 Figs). The data thus indicate that TM inhibitors have a significant vascular disruption activity that is independent of their apoptotic activity.
TM compounds inhibit PAI-1-induced fibrotic and inflammatory responses in vitro [2]
To confirm the efficacy of TM compounds as PAI-1 inhibitors in the kidney, we investigated the effect of TM5275 and TM-5441 on PAI-1-induced markers of both fibrosis and inflammation in mProx cells. PAI-1 treatment significantly increased the mRNA expression of TGF-β, collagen Iα1, collagen Iα2, and MCP-1 (Fig 4A–4D), which suggests that PAI-1 exerted profibrotic and proinflammatory effects; notably, treatment with the TM compounds effectively decreased the PAI-1-induced fibrotic and inflammatory responses (Fig 4A–4D), which confirms the effectiveness of the TM compounds as PAI-1 inhibitors. As expected, PAI-1-induced suppression of plasmin activity was also inhibited following treatment with the TM compounds (Fig 4E). Together, these results suggest that TM compounds can effectively improve PAI-1-induced fibrotic and inflammatory responses in the kidney.
ln Vivo
When TM-5441 (20 mg/kg daily) is given orally to HT1080 and HCT116 xenotransplanted mice, it significantly disrupts the tumor vasculature and increases tumor cell apoptosis, which is linked to a reduction in tumor growth and an increase in survival. One hour after oral administration, the average peak plasma concentration is 11.4 μM, and 23 hours later, it is undetectable[1]. TM-5441 extends life in klotho null mice, reduces the effects of Nω-nitro-l-arginine methyl ester-induced cardiac hypertension and vascular senescence, and has anti-tumorigenic and anti-angiogenic properties in cancer.
TM-5441 has a biological effect in vivo [1]
Due to its higher apoptotic effect in vitro, TM5441 was selected to test its anti-tumor activity in vivo in mice xenotransplanted with HT1080 cells (Fig 5). This experiment revealed that tumor-bearing mice treated with TM5441 (20 mg/kg daily) showed a trend to develop slower growing tumors that reached a volume of 1,500 mm3 at an average of 25.8 (± 3.4) days vs. 21.2 (± 2.5) days for the control group (P value = 0.10) (Fig 5A and 5B). However, there was a statistically significant decrease in bioluminescence activity (relative luciferase units (RLUs) as an indicator of tumor cell viability) over time in the TM5441-treated group (Fig 5C). The effect of TM5441 treatment on survival showed a trend toward an increase in survival in the TM5441-treated group that was, however, not statistically significant (P value = 0.10) (Fig 5D). A histological analysis of these tumors by hematoxylin and eosin (H&E) indicated the presence of larger hemorrhagic areas with disrupted vasculature in tumors from TM5441-treated mice. This disruption of the tumor vasculature was confirmed by staining for CD31 (PECAM) which revealed multiple areas of discontinuity in EC comprising blood vessels and a statistically significant decrease in blood vessel density in the TM5441-treated group (P value = 0.002) (Fig 5F). There was also an increased amount of TUNEL positive staining (a marker of tumor cell apoptosis) in tumors from TM5441-treated mice (P value = 0.05) (Fig 5G). Taken together, the data demonstrate that TM5441 has a biological activity on the tumor vasculature and on tumor cells in vivo that, however, was not sufficient to significantly affect tumor growth. Similar results were seen in our in vivo HCT116 tumor model (S3 Fig). Pharmacokinetics studies revealed in mice treated with 20 mg/kg an average peak concentration of 11.4 μM (only slightly lower than the IC50 of 13.9 μM for HT1080) and undetectable trough levels (S4 Fig). Administration of 50 mg/kg and 100 mg/kg of TM5441, although increasing the peak plasma concentration levels to 15.0 μM and 35.6 μM, respectively, did not significantly increase the trough levels and did not affect tumor growth (S5 Fig). Interestingly, the administration of TM5441 had no systemic effect on bleeding time in mice treated with 50 or 100 mg/kg (S6A Fig). In an in vitro human plasma clot lysis assay TM5275 effectively inhibited PAI-1 from maintaining the clot and allowed lysis to occur similarly to the addition of tPA (S6B Fig).
Diabetic nephropathy is the leading cause of end-stage renal disease worldwide, but no effective therapeutic strategy is available. Because plasminogen activator inhibitor-1 (PAI-1) is increasingly recognized as a key factor in extracellular matrix (ECM) accumulation in diabetic nephropathy, this study examined the renoprotective effects of TM5275 and TM-5441, two novel orally active PAI-1 inhibitors that do not trigger bleeding episodes, in streptozotocin (STZ)-induced diabetic mice. TM5275 (50 mg/kg) and TM5441 (10 mg/kg) were administered orally for 16 weeks to STZ-induced diabetic and age-matched control mice. Relative to the control mice, the diabetic mice showed significantly increased (p < 0.05) plasma glucose and creatinine levels, urinary albumin excretion, kidney-to-bodyweight ratios, glomerular volume, and fractional mesangial area. Markers of fibrosis and inflammation along with PAI-1 were also upregulated in the kidney of diabetic mice, and treatment with TM5275 and TM5441 effectively inhibited albuminuria, mesangial expansion, ECM accumulation, and macrophage infiltration in diabetic kidneys. Furthermore, in mouse proximal tubular epithelial (mProx24) cells, both TM5275 and TM5441 effectively inhibited PAI-1-induced mRNA expression of fibrosis and inflammation markers and also reversed PAI-1-induced inhibition of plasmin activity, which confirmed the efficacy of the TM compounds as PAI-1 inhibitors. These data suggest that TM compounds could be used to prevent diabetic kidney injury. [2]
TM compounds improve kidney function and morphology in STZ-induced diabetic mice [1]
At 16 weeks after STZ injection, mice showed lower body weight gain and increased plasma glucose level as compared to age-matched control mice. The plasma glucose level in diabetic mice was not affected by treatment with either TM5275 (50 mg/kg) or TM-5441 (10 mg/kg) (Table 1). The diabetic mice also showed increased kidney-to-bodyweight ratio (Table 1) and plasma creatinine level (Fig 1A), and these alterations were again not markedly affected by the TM compounds (Fig 1A). Furthermore, STZ-induced diabetic mice showed a significant increase in urinary albumin excretion, glomerular volume, and FMA. Intriguingly, the TM compounds effectively reduced albuminuria and FMA in the diabetic mice (Fig 1B, 1C and 1E), although neither inhibitor exerted a large effect on STZ-induced glomerular hypertrophy (Fig 1C and 1D). Together, these data demonstrate that the TM compounds TM5275 and TM5441 protect mice against diabetes-induced albuminuria and mesangial expansion without affecting hyperglycemia.
Cell Assay
Flow cytometry [1]
Cells were plated in triplicate in 6-well plates at a density of 120,000 cells per well and treated with 50 μM TM5275 or TM-5441 the next day for eight hours (BromodeoxyUridine (BrdU) incorporation) or 24 and 48 hours (mitochondrial depolarization). For Annexin V, cells were treated with the indicated doses for 48 hours. For BrdU incorporation, cells were pulsed with 10 μM BrdU for 20 minutes before being harvested using the fluorescein isothiocyanate (FITC) BrdU Flow kit (BD) according to the manufacturer’s recommendations. Mitochondrial depolarization was assessed using the MitoProbe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) assay kit according to the manufacturer’s recommendations. Apoptotic cells (early apoptotic Annexin V+/PI- cells and late apoptotic Annexin V+/PI+ cells) were evaluated using the Annexin V FITC apoptosis detection kit I (BD) according to the manufacturer’s recommendations. The cells were analyzed by flow cytometry in a BD LSR II system (BD) with DiVA software (version 6.0, BD).
Caspase 3/7 activity assay [1]
Cells were plated as described for cell viability and treated with increasing concentrations of TM5275 or TM-5441 for 48 hours. The ApoLive-Glo kit was used to measure cell viability with a fluorescent dye followed by the measurement of caspase 3/7 activity with luminescence activity according to the manufacturer’s recommendations at room temperature. Caspase 3/7 activity was normalized to cell viability and plotted as fold change compared to DMSO control cells.
Cell culture [2]
Mouse proximal tubular epithelial (mProx24) cells were cultured in DMEM containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin at 37°C in a humidified 5% CO2 atmosphere. Near-confluent cells were incubated with serum-free media for 24 h and pretreated with TM5275 at 50 μM or TM-5441 at 10 μM for 4 h before stimulation with recombinant PAI-1 (approximately 90% biological activity).
Animal Protocol
20 mg/kg; oral
Mice bearing HT1080 and HCT116 xenotransplanted tumors [1]
For in vivo experiments, TM-5441 (20, 50 or 100 mg/kg) was dissolved in DMSO and incorporated into individual servings of peanut butter and honey. Controls were given equal amounts of vehicle (equal volumes of DMSO mixed in peanut butter and honey). Each mouse was then administered the inhibitor or vehicle mixture until it had eaten the entire dose.[1]
HT1080 cells were transduced with a firefly luciferase lentiviral vector and selected against 100 μg/ mL geneticin for two weeks. Cells were then maintained with geneticin. Five-week-old nu/nu female mice were injected with 5x106 HT1080-luciferase cells subcutaneously into the right flank as described previously. Palpable tumors and mouse weight were measured every 2–3 days. Mice were sacrificed when tumor volume reached a maximum of 1,500 mm3 calculated with the modified ellipsoid formula: tumor volume (mm3) = (width in mm)2 X (length in mm) X π/6. For bioluminescence imaging, mice were injected intravenously (i.v.) with D-luciferin (0.3 mL) at a dose of 5 mg/kg and luciferase activity was determined after 15 minutes with a two-second exposure time using the Xenogen IVIS system. Bioluminescence emitted from the HT1080 tumor cells (total flux) was measured and plotted as relative luciferase units. For bleeding time testing, the dorsal tail vein was cut using an automated Surgicutt Newborn device 0.5 mm deep and 2.5 mm long. Filter paper was carefully blotted against the bleeding wound every 15 seconds until the incision stopped bleeding. Bleeding time was performed on the last day one hour after administration of the last dose of TM-5441 and before the mice were sacrificed (at day 16–30). Measurements were recorded to the nearest 15 seconds.[1]
We used 6-week-old male C57BL/6 mice, which were divided into 6 groups. Diabetes was induced by intraperitoneally injecting the mice with 150 mg/kg streptozotocin (STZ). Age-matched control mice were injected with an equivalent volume of sodium citrate buffer (100 mM sodium citrate, 100 mM citric acid, pH 4.5). TM5275 at 50 mg/kg/day and TM-5441 at 10 mg/kg/day were orally administered in control and diabetic mice for 16 weeks. The effective doses of TM5275 and TM-5441 were determined based on previous studies and our preliminary studies (data not shown). Mice that were not administered the TM compounds were injected with an equivalent volume of 0.5% carboxymethyl cellulose, the vehicle for TM5275 and TM-5441. Mice were monitored at least once a day, and no deaths occurred during the experimental period. All mice were sacrificed at 16 weeks after STZ injection via anesthesia with 16.5% urethane (10 mL/kg). Blood was collected in a heparinized syringe. We collected blood for measurement of plasma glucose and creatinine, urine for protein measurement, and kidneys for immunohistochemical analysis. [2]
References

[1]. Small Molecule Inhibitors of Plasminogen Activator Inhibitor-1 Elicit Anti-Tumorigenic and Anti-Angiogenic Activity. PLoS One. 2015 Jul 24;10(7):e0133786.

[2]. Novel Plasminogen Activator Inhibitor-1 Inhibitors Prevent Diabetic Kidney Injury in a Mouse Model. PLoS One. 2016 Jun 3;11(6):e0157012.

Additional Infomation
Numerous studies have shown a paradoxical positive correlation between elevated levels of plasminogen activator inhibitior-1 (PAI-1) in tumors and blood of cancer patients with poor clinical outcome, suggesting that PAI-1 could be a therapeutic target. Here we tested two orally bioavailable small molecule inhibitors of PAI-1 (TM5275 and TM-5441) for their efficacy in pre-clinical models of cancer. We demonstrated that these inhibitors decreased cell viability in several human cancer cell lines with an IC50 in the 9.7 to 60.3 μM range and induced intrinsic apoptosis at concentrations of 50 μM. In vivo, oral administration of TM5441 (20 mg/kg daily) to HT1080 and HCT116 xenotransplanted mice increased tumor cell apoptosis and had a significant disruptive effect on the tumor vasculature that was associated with a decrease in tumor growth and an increase in survival that, however, were not statistically significant. Pharmacokinetics studies indicated an average peak plasma concentration of 11.4 μM one hour after oral administration and undetectable levels 23 hours after administration. The effect on tumor vasculature in vivo was further examined in endothelial cells (EC) in vitro and this analysis indicated that both TM5275 and TM5441 inhibited EC branching in a 3D Matrigel assay at concentrations where they had little effect on EC apoptosis. These studies bring novel insight on the activity of PAI-1 inhibitors and provide important information for the future design of inhibitors targeting PAI-1 as therapeutic agents in cancer. [1]
This data represents a first in vivo analysis of TM-5441 PAI-1 inhibitor activity in cancer. TM5275 and TM5441 induced intrinsic apoptosis in several human cancer cell lines and inhibited EC branching in a manner that was independent from their apoptotic activity on EC in vitro. These in vivo results in HT1080 and HCT116 xenograft models showed that although TM5441 had a vascular disruptive effect (and a trend of decreased tumor growth and increased survival in the HT1080 model), these effects were not sufficient to affect tumor growth even as we documented a significant decrease in TUNEL staining in vivo.

As a basis for comparison, the IC50 of TM5275 and TM-5441 treatment is similar to the IC50 of PAI-039, another previously reported PAI-1 inhibitor. The IC50 measured by tPA-dependent hydrolysis for the compounds were 8.37 μM for PAI-749, which is a more potent derivative of PAI-039, and 6.95 μM for TM5275. When defined on the basis of cell viability, the IC50 of PAI-039 was calculated from previous data to be 29 μM and 32 μM for HT1080 and HCT116 cells, respectively, which is in the range of the IC50 found for TM5275 and TM5441. There was no correlation between the IC50 of the TM compounds and the total PAI-1 levels measured in the cell lysates. This suggested that other factors besides PAI-1 played a role, which may include membrane-associated plasmin, uPA, or sensitivity to apoptosis that all contributed to the control of cell viability. [1]
This study was conducted to provide experimental evidence that two novel orally active PAI-1 inhibitors, TM5275 and TM-5441, can prevent the development and progression of diabetic kidney injury, and to suggest the use of TM compounds as a new strategy for preventing diabetic nephropathy. Accordingly, at 16 weeks after injection of STZ, the diabetic mice showed an increase (relative to control mice) in various parameters of kidney injury, such as plasma creatinine level, urinary albumin excretion, kidney-to-body weight ratio, glomerular volume, and FMA. Notably, treatment with TM5275 and TM5441 effectively reduced urinary albumin excretion and FMA. Consistent with our results, another compound of TM series significantly reduced proteinuria in NEP25/LMB2 podocyte injury mouse model. With regard to the mechanic explanation for the role of PAI-1 on urine albumin levels, PAI-1/uPA complex-mediated uPAR-dependent podocyte β1-integrin endocytosis has been proposed in progressive podocyte injury leading to proteinuria. However, inhibition of PAI-1 did not affect plasma glucose levels in STZ-induced diabetic mice, which agreed with the results of a previous study [26]. These data indicate that TM compounds improve kidney function and morphology in diabetic mice.

TM5007, the parent compound of TM5275 and TM-5441, prevents bleomycin-induced lung fibrosis in mice, and tiplaxtinin (an indole oxoacetic acid derivative) attenuates angiotensin II-induced aortic remodeling in mice. These two previous studies suggest that the best-in-class PAI-1 inhibitors could be effective antifibrotic agents. Here, our study demonstrating the antifibrotic effect of TM5275 and TM5441 in diabetic kidney injury is consistent with previous studies conducted using PAI-1 null mice. PAI-1 deficiency reduces ECM accumulation and tubulointerstitial or glomerular fibrosis in STZ-induced diabetic mice and db/db diabetic mice.

In summary, the TM compounds improved kidney function, fibrosis, and inflammation in STZ-induced diabetic mice. Therefore, oral administration of TM5275 and TM-5441, two novel PAI-1 inhibitors that do not induce bleeding episodes, could emerge as an effective measure for treating diabetic nephropathy.
These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C21H18CLN2NAO6
Molecular Weight
452.820195674896
Elemental Analysis
C, 55.95; H, 3.58; Cl, 7.86; N, 6.21; Na, 5.10; O, 21.29
CAS #
2319722-53-5
Related CAS #
1190221-43-2
PubChem CID
129316186
Appearance
Typically exists as solid at room temperature
Hydrogen Bond Donor Count
2
Hydrogen Bond Acceptor Count
6
Rotatable Bond Count
8
Heavy Atom Count
31
Complexity
625
Defined Atom Stereocenter Count
0
SMILES
N(C1=CC=CC(C2=COC=C2)=C1)C(=O)COCC(=O)NC1C=CC(Cl)=CC=1C(=O)O.[NaH]
InChi Key
RDKPAESSEMYFJL-UHFFFAOYSA-M
InChi Code
InChI=1S/C21H17ClN2O6.Na/c22-15-4-5-18(17(9-15)21(27)28)24-20(26)12-30-11-19(25)23-16-3-1-2-13(8-16)14-6-7-29-10-14;/h1-10H,11-12H2,(H,23,25)(H,24,26)(H,27,28);/q;+1/p-1
Chemical Name
sodium;5-chloro-2-[[2-[2-[3-(furan-3-yl)anilino]-2-oxoethoxy]acetyl]amino]benzoate
Synonyms
TM5441 (sodium); TM-5441 sodium; TM-5441 (sodium);
HS Tariff Code
2934.99.9001
Storage

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Shipping Condition
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
Solubility Data
Solubility (In Vitro)
Typically soluble in DMSO (e.g. 10 mM)
Solubility (In Vivo)
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.

Injection Formulations
(e.g. IP/IV/IM/SC)
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 : PEG300Tween 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 : PEG300Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH 400 μLPEG300 50 μL Tween 80 450 μL Saline)


Oral Formulations
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


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.

 (Please use freshly prepared in vivo formulations for optimal results.)
Preparing Stock Solutions 1 mg 5 mg 10 mg
1 mM 2.2084 mL 11.0419 mL 22.0838 mL
5 mM 0.4417 mL 2.2084 mL 4.4168 mL
10 mM 0.2208 mL 1.1042 mL 2.2084 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

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  • Molar mass (molar weight) is the mass of one mole of a substance and is expressed in g/mol.
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Reconstitution Calculator allows you to calculate the volume of solvent required to reconstitute your vial.

  • Enter the mass of the reagent and the desired reconstitution concentration as well as the correct units
  • Click the “Calculate” button
  • The answer appears in the Volume (to add to vial) box
In vivo Formulation Calculator (Clear solution)
Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)
Step 2: Enter in vivo formulation (This is only a calculator, not the exact formulation for a specific product. Please contact us first if there is no in vivo formulation in the solubility section.)
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Calculation results

Working concentration mg/mL;

Method for preparing DMSO stock solution mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.

(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
             (2) Be sure to add the solvent(s) in order.

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