| Targets |
IκB kinase β (IKKβ) inhibitor (selective inhibitor; inhibits IκBα phosphorylation and NF-κB p65 nuclear translocation) [1]
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|---|---|
| ln Vitro |
IMD-0560 pretreatment prevents TNFα-induced p65 phosphorylation and IκBα degradation. Moreover, TNFα-induced transcriptional activity is suppressed by IMD-0560. TNFα-induced cell invasion is significantly inhibited by pretreatment with IMD-0560. By preventing MMP-9 expression, pretreatment with IMD-0560 reduces MMP-9 activity[1].
IMD-0560 pretreatment inhibited TNFα-induced p65 phosphorylation (Ser-536) and IκBα degradation in a dose-dependent manner in SCCVII, HSC-2, and Ca9-22 oral squamous cell carcinoma (OSCC) cells. [1] IMD-0560 (1 μM for SCCVII, 10 μM for HSC-2 and Ca9-22) significantly blocked TNFα-induced nuclear translocation of p65 from the cytoplasm to the nucleus in OSCC cells. [1] IMD-0560 suppressed TNFα-induced NF-κB transcriptional activity in SCCVII cells transfected with a luciferase reporter. [1] IMD-0560 strongly inhibited TNFα-induced invasion of SCCVII and HSC-2 cells in a gelatin-coated transwell assay. [1] IMD-0560 suppressed TNFα-induced MMP-9 (92 kDa gelatinase) activity and expression at both protein and mRNA levels in SCCVII and HSC-2 cells, but did not affect MMP-2. [1] IMD-0560 suppressed RANKL expression in osteoblastic cells and RANKL-induced osteoclastogenesis in vitro. [1] IMD-0560 suppressed SCCVII cell proliferation in vitro. [1] IMD-0560 induced apoptosis in SCCVII cells in vitro, as assessed by Annexin V/PI staining. [1] Cell viability was not notably affected by IMD-0560 treatment for up to 24 hours. [1] |
| ln Vivo |
In mice administered with 5 mg/kg IMD-0560, the tumor size decreases in a dose-dependent way, and in certain mice, no tumor is found. When compared to the control groups, the IMD-0560-treated groups exhibited a considerable suppression of zygoma destruction. The 5 mg/kg IMD-0560-treated group had a smaller tumor than the 3 mg/kg IMD-0560-treated group, but both treatments had a similar inhibitory impact on the elimination of zygomas[1].
In a mouse model of jaw bone invasion by SCCVII cells, early local treatment with IMD-0560 (3 or 5 mg/kg, three times per week for 3 weeks, starting 1 week after tumor inoculation) significantly inhibited zygoma and mandible destruction in a dose-dependent manner, reduced tumor volume, and decreased the number of osteoclasts at the tumor-bone interface. [1] Early IMD-0560 treatment reduced the number of Ki-67-positive proliferating SCCVII cells and increased the number of TUNEL-positive apoptotic cells within tumors. [1] Early IMD-0560 treatment reduced the number of MMP-9-positive SCCVII cells and suppressed RANKL expression in both SCCVII cells and adjacent osteoblastic cells. [1] Late local treatment with IMD-0560 (3 or 5 mg/kg, three times per week for 2 weeks, starting 2 weeks after tumor inoculation when tumors were established) also significantly inhibited zygoma destruction, reduced tumor volume, decreased osteoclast numbers, and reduced Ki-67 and MMP-9 positivity in tumor cells, with the 5 mg/kg dose showing stronger effects. [1] IMD-0560 treatment did not significantly affect the body weight of mice compared to the no tumor group. [1] |
| Enzyme Assay |
The study used Western blot analysis to assess the effect of IMD-0560 on IκBα degradation and p65 phosphorylation. Cells were pretreated with various concentrations of IMD-0560 for 120 minutes and then stimulated with TNFα (10 ng/mL) for 15 minutes. Whole-cell lysates were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with specific antibodies against phospho-p65 (Ser536), IκBα, and β-actin. [1]
A luciferase reporter assay was used to measure NF-κB transcriptional activity. SCCVII cells were transiently transfected with a pBIix-luciferase reporter plasmid. After pretreatment with or without IMD-0560 (1 μM) for 120 minutes, cells were treated with or without TNFα (10 ng/mL) for 8 hours. Luciferase activity was measured using a dual-luciferase reporter assay system. [1] |
| Cell Assay |
Cell invasion was assessed using a modified Boyden chamber with a gelatin-coated porous membrane. Cells were suspended in serum-free medium and seeded in the upper chamber. IMD-0560 (1 or 10 μM) was added to the upper chamber 2 hours prior to the addition of TNFα (10 ng/mL) to the lower chamber. After 24 hours of incubation, non-invading cells on the upper surface were scraped off, and cells that migrated to the lower surface were fixed, stained with DAPI, and quantified. [1]
MMP-9 activity was assessed by gelatin zymography. Cells were incubated in serum-free medium for 24 hours in the presence or absence of TNFα and IMD-0560. Conditioned media were collected, resolved by 10% SDS-PAGE containing gelatin, and processed to visualize gelatinolytic activity as clear bands against a Coomassie blue-stained background. [1] MMP-9 mRNA expression was analyzed by real-time RT-PCR. Total RNA was extracted from cells using TRIzol reagent, reverse-transcribed into cDNA, and amplified using specific primers for mmp-9 and β-actin. [1] Immunofluorescence microscopy was used to visualize p65 nuclear translocation. Cells grown on coverslips were pretreated with or without IMD-0560 for 120 minutes, treated with TNFα (10 ng/mL) for 30 minutes, fixed, permeabilized, and incubated with an anti-p65 antibody followed by an Alexa Fluor 430-conjugated secondary antibody. Nuclei were counterstained with DAPI, and localization was observed by fluorescence microscopy. [1] Apoptosis was assessed in vitro by Annexin V and propidium iodide (PI) staining. [1] Cell proliferation was assessed in vitro. [1] |
| Animal Protocol |
For the early treatment model, 8- to 10-week-old male C3H/HeN mice were injected with SCCVII cells (1.0 x 10^5 cells in 0.1 mL DMEM) into the left masseter region. One week after inoculation, mice were locally injected between the masseter region and the mandibular bone surface with either vehicle (50 μL of 0.5% carboxymethyl-cellulose, CMC) or IMD-0560 (3 or 5 mg/kg in 50 μL CMC). Injections were administered three times per week for a total of 3 weeks. Mice were euthanized 28 days after tumor inoculation for analysis. [1]
For the late treatment model, the same tumor inoculation procedure was used. Two weeks after inoculation (when tumors were established), mice began receiving local injections of vehicle or IMD-0560 (3 or 5 mg/kg in CMC) three times per week for 2 weeks. Analysis was performed 28 days post-inoculation. [1] Tumor volume was measured using calipers and calculated with the formula: (width)^2 x length x 0.52. [1] Bone destruction was assessed by micro-computed tomography (μ-CT) imaging of the skulls, and a clinical scoring system (0-4) was applied to evaluate zygoma destruction. [1] Tissues were fixed, decalcified, sectioned, and stained with H&E, TRAP (for osteoclasts), and immunohistochemistry for RANKL, Ki-67, and MMP-9. [1] |
| ADME/Pharmacokinetics |
The prodrug of IMD-0560, IMD-2560, is undergoing clinical trials for the treatment of rheumatoid arthritis and inflammation-related cardiovascular disease. [1]
Intraperitoneal injection of IMD-0560 (1 or 3 mg/kg every 48 hours) showed efficacy in a mouse model of collagen-induced arthritis. [1] This study did not describe in detail the specific ADME/PK parameters (absorption, distribution, metabolism, excretion, half-life, bioavailability) of IMD-0560. [1] |
| Toxicity/Toxicokinetics |
In vitro experiments showed that IMD-0560 treatment for up to 24 hours did not significantly affect the viability of oral squamous cell carcinoma (OSCC) cells. [1] In vivo experiments showed that IMD-0560 treatment (3 or 5 mg/kg) did not result in a significant decrease in body weight in mice compared with the non-tumor control group. [1] No serious side effects of IMD-0560 treatment were reported in the completed Phase I clinical trials. [1] IMD-0560 treatment did not affect the proliferation of basal and parabasal cells in normal tongue tissue, suggesting that it has a certain specificity for tumor cells. [1]
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| References | |
| Additional Infomation |
IMD-0560 (N-[2,5-bis(trifluoromethyl)phenyl]-5-bromo-2-hydroxybenzamide) is a novel synthetic small molecule IKKβ inhibitor. [1]
It is designed based on the binding mode of aspirin to IKKβ. [1] It blocks the phosphorylation of IκBα and inhibits the nuclear translocation of NF-κB p65. [1] Studies have shown that it can inhibit TNFα-induced IL-6 production in human fibroblast-like synovial cells of patients with rheumatoid arthritis. [1] It has also shown efficacy in preclinical models of collagen-induced arthritis and myocardial ischemia. [1] This study suggests that IMD-0560 may be a novel potential therapeutic for preventing bone invasion of oral squamous cell carcinoma (OSCC). [1] A local administration strategy is recommended for systemic side effects caused by OSCC invasion of the mandible. [1] |
| Exact Mass |
426.964
|
|---|---|
| CAS # |
439144-66-8
|
| PubChem CID |
11464503
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| Appearance |
White to off-white solid powder
|
| LogP |
5.828
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| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
8
|
| Rotatable Bond Count |
2
|
| Heavy Atom Count |
25
|
| Complexity |
484
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
SVGRIJCSKWXOPA-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C15H8BrF6NO2/c16-8-2-4-12(24)9(6-8)13(25)23-11-5-7(14(17,18)19)1-3-10(11)15(20,21)22/h1-6,24H,(H,23,25)
|
| Chemical Name |
N-[2,5-Bis(trifluoromethyl)phenyl]-5-bromo-2-hydroxybenzamide
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| Synonyms |
IMD0560; IMD 0560; IMD-0560
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
Ethanol : ~50 mg/mL (~116.79 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.84 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.84 mM) (saturation unknown) in 10% EtOH + 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 EtOH stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
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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