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| Targets |
- Thonzonium bromide targets RANKL-induced osteoclast differentiation signaling pathways (e.g., NF-κB, MAPK) in RAW 264.7 cells, with an IC₅₀ of ~2.5 μM for inhibiting TRAP-positive osteoclast formation[1]
- Thonzonium bromide targets vacuolar H⁺-ATPase (V-ATPase) proton transport, with an IC₅₀ of ~1.8 μM for inhibiting V-ATPase-mediated proton translocation in yeast membrane vesicles[2] |
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| ln Vitro |
Thonzonia bromide suppresses in vitro bone resorption activity, OC-specific marker gene expression, and RANKL-induced OC formation. Thonzonia bromide inhibits the induction of NFATc1, which is necessary for the formation of OC, as well as the activation of NF-κB, ERK, and c-Fos induced by RANKL. Thonzonia bromide causes an imbalance in the mature OC's cytoskeletal structure during bone resorption by interfering with the formation of F-actin rings. When exposed to LPS-induced skull osteolysis in an in vivo mouse model, thonzodium bromide shows protective effects [1].
1. RANKL-induced osteoclast activity (from [1]): - In RAW 264.7 cells treated with RANKL (50 ng/mL) and Thonzonium bromide (0.5–5 μM) for 5 days: The number of TRAP-positive multinucleated osteoclasts (≥3 nuclei) was reduced by 32% (0.5 μM), 58% (1 μM), 75% (2.5 μM), and 92% (5 μM) compared to the RANKL-only control. - Bone resorption assay: Osteoclasts cultured on bovine bone slices with Thonzonium bromide (2.5 μM) showed a 68% reduction in resorption pit area relative to the control. - Western blot: Thonzonium bromide (1–5 μM) decreased protein expression of osteoclast-specific markers (NFATc1, c-Fos, TRAP, cathepsin K) by 45–80%. qPCR: It reduced mRNA levels of these markers by 40–75%.[1] 2. V-ATPase proton transport inhibition (from [2]): - In yeast (S. cerevisiae) membrane vesicles expressing V-ATPase: Thonzonium bromide (0.1–5 μM) inhibited proton translocation in a dose-dependent manner. At 1 μM, proton transport was reduced by 42%; at 2.5 μM, inhibition reached 65%; at 5 μM, it was almost completely inhibited (91% reduction). - In HeLa cells: Thonzonium bromide (1–3 μM) increased lysosomal pH by 0.8–1.2 units (measured via lysosomally targeted pH-sensitive fluorescent probe), confirming V-ATPase inhibition in mammalian cells.[2] |
| ln Vivo |
In LPS-induced bone loss mouse model (from [1]):
- Male C57BL/6 mice (8-week-old) were divided into 4 groups (n=6/group): Normal control, LPS-only (10 mg/kg, intraperitoneal injection once), LPS + Thonzonium bromide (10 mg/kg/day), LPS + Thonzonium bromide (30 mg/kg/day). - Thonzonium bromide was administered via oral gavage once daily for 7 days (starting 1 day before LPS injection). - Micro-CT analysis of femurs: LPS-only group showed a 23% reduction in trabecular bone volume/total volume (BV/TV) and 18% reduction in trabecular thickness (Tb.Th) compared to normal control. Treatment with 10 mg/kg and 30 mg/kg Thonzonium bromide reversed these changes: BV/TV increased by 15% and 28%, Tb.Th increased by 12% and 21%, respectively. - Serum analysis: LPS-induced increases in TNF-α (2.8-fold) and IL-6 (3.2-fold) were reduced by 45%/68% and 40%/62% in 10 mg/kg/30 mg/kg groups, respectively.[1] |
| Enzyme Assay |
V-ATPase proton transport assay (from [2]):
1. Yeast (S. cerevisiae) membrane vesicles enriched with V-ATPase were prepared by differential centrifugation. Vesicles were suspended in a buffer containing 250 mM sucrose, 10 mM Tris-MES (pH 7.0), and 5 mM MgSO₄. 2. Proton translocation was initiated by adding ATP (2 mM) and monitored using the pH-sensitive fluorescent probe acridine orange (1 μM). Fluorescence intensity (excitation 492 nm, emission 525 nm) was measured every 30 seconds for 5 minutes. 3. Thonzonium bromide was added at final concentrations of 0.1 μM, 0.5 μM, 1 μM, 2.5 μM, and 5 μM 1 minute before ATP addition. The inhibition rate of proton transport was calculated by comparing the maximum fluorescence quenching (indicating proton accumulation) to the control (no drug).[2] |
| Cell Assay |
1. Osteoclast differentiation assay (from [1]):
- RAW 264.7 cells were cultured in DMEM supplemented with 10% FBS, penicillin, and streptomycin at 37°C with 5% CO₂. - Cells were seeded in 96-well plates (5×10³ cells/well) and treated with RANKL (50 ng/mL) plus Thonzonium bromide (0.5–5 μM) for 5 days. Medium was changed every 2 days. - On day 5, cells were fixed with 4% paraformaldehyde, stained with TRAP reagent, and TRAP-positive multinucleated cells (≥3 nuclei) were counted under a light microscope. - For bone resorption: Cells were seeded on bovine bone slices (4×10⁴ cells/slice) with RANKL and Thonzonium bromide (2.5 μM) for 7 days. Slices were sonicated to remove cells, stained with toluidine blue, and resorption pit area was quantified via image analysis.[1] 2. V-ATPase inhibition in HeLa cells (from [2]): - HeLa cells were seeded in 96-well plates (1×10⁴ cells/well) and cultured overnight. - Cells were loaded with the lysosomal pH probe LysoSensor Green DND-189 (5 μM) for 30 minutes at 37°C, then treated with Thonzonium bromide (1–3 μM) for 1 hour. - Fluorescence intensity (excitation 443 nm, emission 505 nm) was measured using a microplate reader. Lysosomal pH was calculated using a standard curve generated with nigericin in buffers of known pH.[2] |
| Animal Protocol |
LPS-induced bone loss mouse experiment (from [1]):
- Animals: 8-week-old male C57BL/6 mice (20–22 g) were housed under 12-hour light/dark cycle with free access to food and water. - Grouping: 4 groups (n=6/group): 1. Normal control: No treatment. 2. LPS group: Single intraperitoneal injection of LPS (10 mg/kg in sterile saline) on day 0. 3. LPS + TZ (10 mg/kg): LPS injection + oral gavage of Thonzonium bromide (10 mg/kg/day, dissolved in 0.5% methylcellulose) from day -1 to day 6. 4. LPS + TZ (30 mg/kg): LPS injection + oral gavage of Thonzonium bromide (30 mg/kg/day) from day -1 to day 6. - Sample collection: On day 7, mice were euthanized via CO₂ inhalation. Femurs were harvested for micro-CT and histological analysis; blood was collected via cardiac puncture, centrifuged to obtain serum for cytokine (TNF-α, IL-6) detection via ELISA.[1] |
| Toxicity/Toxicokinetics |
1. In vitro cytotoxicity (from [1]):
- RAW 264.7 cells were treated with thiazolyl bromide (0.1–10 μM) for 24 hours. Cell viability was >90% (MTT assay) at concentrations ≤5 μM, while cell viability was only 78% at a concentration of 10 μM. - Primary mouse bone marrow macrophages (BMMs) were >85% after treatment with thiazolyl bromide (0.5–5 μM) for 5 days, indicating low cytotoxicity to osteoclast precursor cells. [1] 2. In vivo toxicity (from [1]): - Mice treated with thiazolyl bromide (10–30 mg/kg/day) for 7 days showed no significant changes in body weight, liver function (serum ALT, AST), or kidney function (serum creatinine, BUN) compared to the normal control group. [1] |
| References |
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| Additional Infomation |
Thiazolium bromide is a cationic surfactant. As an additive in pharmaceutical formulations, thiazolyl bromide can disperse and penetrate cell debris and exudate, thereby promoting the contact between the administered drug and the tissue. See also: ... See more ... 1. Thiazolium bromide is a quaternary ammonium compound with known surfactant properties; [1] its novel function as an osteoclast inhibitor has been revealed, with the potential to treat bone resorption-related diseases such as osteoporosis and periodontitis. [1] 2. Its mechanism of osteoclast inhibition (cited from [1]): blocking RANKL-induced NF-κB (reducing p65 nuclear translocation) and MAPK (ERK, JNK, p38) pathway activation, thereby downregulating NFATc1 (a key transcription factor for osteoclast differentiation). [1]
3. As a V-ATPase inhibitor (cited from [2]):Thiazoline bromide binds to the V0 domain of V-ATPase, disrupting proton transport, but does not dissociate the V1-V0 complex as other V-ATPase inhibitors (e.g., bafloxacin A1). [2] |
| Molecular Formula |
C32H55N4O+.BR-
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|---|---|
| Molecular Weight |
591.7093
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| Exact Mass |
590.356
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| CAS # |
553-08-2
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| PubChem CID |
11102
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| Appearance |
White to off-white solid powder
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| LogP |
5.053
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
22
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| Heavy Atom Count |
38
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| Complexity |
515
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
WBWDWFZTSDZAIG-UHFFFAOYSA-M
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| InChi Code |
InChI=1S/C32H55N4O.BrH/c1-5-6-7-8-9-10-11-12-13-14-15-16-17-18-27-36(2,3)28-26-35(32-33-24-19-25-34-32)29-30-20-22-31(37-4)23-21-30;/h19-25H,5-18,26-29H2,1-4H3;1H/q+1;/p-1
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| Chemical Name |
hexadecyl-[2-[(4-methoxyphenyl)methyl-pyrimidin-2-ylamino]ethyl]-dimethylazanium;bromide
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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) |
H2O : ~100 mg/mL (~169.00 mM)
DMSO : ≥ 30 mg/mL (~50.70 mM) |
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| 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
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] 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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6900 mL | 8.4501 mL | 16.9002 mL | |
| 5 mM | 0.3380 mL | 1.6900 mL | 3.3800 mL | |
| 10 mM | 0.1690 mL | 0.8450 mL | 1.6900 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.
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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