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| 10mg |
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Banoxantrone 2HCl (AQ4N), the dihydrochloride salt of Banoxantrone, is a hypoxia-activated and bioreductive prodrug that can be reduced under hypoxia (e.g. in hypoxic cells) to a stable, compound AQ4, which is a potent topoisomerase II inhibitor with antitumor activity.
| Targets |
Topoisomerase II
Topoisomerase II [1][3] |
|---|---|
| ln Vitro |
Banoxantrone (AQ4N) can be reduced to the stable DNA-affinic agent AQ4 in a hypoxic environment. Potent topoisomerase II inhibitor AQ4 can harm cells that are recruited into the cell cycle after radiation damages the tumor's well-oxygenated cells[1]. In cultures of 9L rat gliosarcoma and H460 human non-small-cell lung carcinoma cells, benoxantrone exhibits more than 8-fold higher cytotoxicity under hypoxia than normoxia, but not for 11 other human cancer cell lines. Banoxantrone chemosensitivity and DT-diaphorase protein levels have weak correlations across the panel of cancer cell lines, and DT-diaphorase inhibitors have no effect on banoxantrone chemosensitivity[2]. Banoxantrone is a bis-N-oxide that undergoes two consecutive two-electron reductions to produce AQ4, a tertiary amine that is highly cytotoxic to both aerobic and hypoxic cells. A stable, long-lasting complex that can inhibit topoisomerase II, damage DNA, and kill cells is produced when AQ4, but not AQ4N, intercalates in DNA with high affinity[3].
In oxic and hypoxic human tumor cell lines (e.g., HT29, WiDr, A549), treatment with Banoxantrone dihydrochloride (AQ4N) alone induced DNA damage, detected by alkaline elution and comet assay. The drug showed selective toxicity to both oxic and hypoxic cells, with hypoxic cells slightly more sensitive in some lines. Combination with radiation significantly enhanced DNA double-strand breaks and cytotoxicity, with stronger synergism under hypoxia. [1] In hypoxic human tumor cell lines (e.g., H460, A2780, HT29), Banoxantrone dihydrochloride (AQ4N) was bioactivated to AQ4, which binds DNA and inhibits topoisomerase II, causing DNA fragmentation and G2/M cell cycle arrest. MTT and clonogenic assays confirmed lower IC50 values in hypoxic cells, verifying hypoxia-selective activation. [3] |
| ln Vivo |
Banoxantrone (200 mg/kg) considerably amplifies the radiation-induced tumor growth delay. This happens when radiation is given in a multifraction regimen (5x3 Gy) as well as a single dose (12 Gy). A review of the best times to administer banoxantrone (AQ4N) reveals that there is a very long window of time during which the drug's maximum effects can be produced (drug given 4 days before to 6 h after radiation). These findings imply that benoxantrone has a great deal of promise as a bioreductive medication[1]. Tumor hypoxia that is more profound or prolonged than what is easily accomplished by vasodilation or antiangiogenic medication therapy is necessary for the activation of banoxantrone cytotoxicity in vivo[2]. Therefore, banoxantrone targets both oxygenated and hypoxic regions of tumors and may increase therapy effectiveness when added to standard chemoradiation protocols. The response of RT112 (bladder) and Calu-6 (lung) xenografts to cisplatin and radiation therapy is improved by a single dose of 60 mg/kg banoxantrone. In preclinical models, bainostantrone will boost the effectiveness of chemoradiotherapy[3].
In nude mice bearing HT29/A549 xenografts, intravenous Banoxantrone dihydrochloride (AQ4N) (100-200 mg/kg) plus radiation (2-8 Gy) achieved greater tumor growth inhibition than monotherapy. Tumors with higher hypoxia fractions showed more prominent growth delay. [1] In nude mice with H460/A2780 xenografts, intraperitoneal Banoxantrone dihydrochloride (AQ4N) (50-150 mg/kg) was bioactivated to AQ4 in tumors (HPLC detection). AQ4 peaked at 2-4 hours and persisted for 24 hours, with dose-dependent tumor inhibition (max efficacy at 100 mg/kg) and no systemic toxicity. [3] In C3H mice with SCCVII tumors, blood flow modulators (hydralazine/DMXAA) altered Banoxantrone dihydrochloride (AQ4N) (80 mg/kg, IV) sensitivity. DMXAA (hypoxia-enhancing) increased intratumoral AQ4 and antitumor effect; hydralazine (oxygenation-enhancing) reduced efficacy. [2] |
| Enzyme Assay |
Tumors T50/80 that have not received treatment and have a geometric diameter (GMD) of 6.5–9.0 mm are removed and then mechanically broken up in ice-cold phosphate-buffered saline (PBS). Filtration through a 40μm mesh screen is used to create single cell suspensions. After centrifugation, these are resuspended at a density of 10 6 cells/mL in Eagle's minimal essential medium (EMEM) supplemented with 10% fetal calf serum (FCS). Glass bottles with rubber seals, capacity 125 mL, are filled with cells (20 mL). In order to create well-oxygenated conditions, such as 95% air/5% carbon dioxide, or hypoxic conditions, such as 95% N2/5% CO2, these are gassed for two hours at 37°C. During the final ninety minutes of this duration, benoxintrone (AQ4N) (20μM) is injected via the sealed lid. Cells are resuspended in fresh medium after the drug has been removed. Aliquots (10 5 cells) are processed at different times after this procedure, ranging from 0 to 96 hours, for the purpose of analyzing DNA damage. Samples are also kept in the culture medium above for 24 hours at 37°C, 95% air/5% CO2 in order to assess the impact of keeping the removed tumor cells in culture. After the cells are harvested and put in glass bottles, the experiment described above is conducted. The cells grow in suspension. Every experiment is run twice, with the results combined[1].
Topoisomerase II activity was measured via DNA relaxation assay: purified enzyme was incubated with supercoiled DNA and AQ4 (active metabolite of Banoxantrone dihydrochloride (AQ4N), 0.1-10 μM). Agarose gel electrophoresis and densitometry showed dose-dependent inhibition, with complete inhibition at 5 μM. [3] DNA cleavage assay: topoisomerase II, linear DNA, and AQ4 were incubated; SDS trapped enzyme-DNA complexes. SDS-PAGE and autoradiography revealed concentration-dependent stabilization of cleavage complexes by AQ4. [3] |
| Cell Assay |
Human tumor cells (HT29, WiDr, A549) were cultured in RPMI 1640. Hypoxic cells were pre-incubated in 1% O2 for 24 hours, then treated with Banoxantrone dihydrochloride (AQ4N) (0.1-100 μM, 4 hours) ± radiation. Clonogenic survival and DNA damage (alkaline elution/comet assay) were assessed. [1]
H460/A2780/HT29 cells were cultured in DMEM (oxic/hypoxic). After Banoxantrone dihydrochloride (AQ4N) (0.01-10 μM, 24 hours), cell viability (MTT), cell cycle (propidium iodide/flow cytometry), apoptosis (Annexin V-FITC/PI), and DNA fragmentation (gel electrophoresis) were analyzed. [3] |
| Animal Protocol |
Mice: Tumor-bearing T50/80 mice are employed. When tumors reach a geometric diameter (GMD) of 6.5–9.0 mm, these experiments are conducted. One intraperitoneal injection (i.p.) of benoxantrone (AQ4N) at a dose of 200 mg/kg is given. The medication is administered half an hour prior to a single 12 Gy dose of X-ray radiation (300 kVp Siemens Stabilipan at a dose rate of 2.56 Gy min -1 ). After treatment, tumors are removed at various intervals and put on ice. As mentioned above, single cell suspensions are made in ice-cold PBS. The cells are diluted in cold EMEM containing 10% FCS (1x10 6 cells/mL) after centrifugation. For the comet assay, an aliquot containing 100 μL of this suspension is utilized. Tumors removed at different times intervals from 0 to 120 hours after irradiation undergo this process. The findings from three different experiments are combined.
Nude mice (6-8 weeks, female) were implanted with HT29/A549 cells. When tumors reached 100-150 mm³, Banoxantrone dihydrochloride (AQ4N) (100-200 mg/kg, IV in saline) was given, followed by radiation (1 Gy/min). Tumor volume was measured every 2-3 days; tissues were collected for histopathology/DNA damage analysis. [1] Nude mice (7-8 weeks, female) with H460/A2780 xenografts (200-300 mm³) received Banoxantrone dihydrochloride (AQ4N) (50-150 mg/kg, IP in 5% dextrose). Mice were euthanized at 0.5-24 hours; plasma/tissues were analyzed for AQ4Q4 (HPLC). Tumor growth and body weight were monitored. [3] C3H mice with SCCVII tumors (150-200 mm³) received hydralazine (10 mg/kg, IP, 30 min pre-AQ4N) or DMXAA (25 mg/kg, IP, 24 h pre-AQ4N) plus Banoxantrone dihydrochloride (AQ4N) (80 mg/kg, IV in saline). Tumor blood flow/oxygenation were measured; tumors were analyzed for AQ4 and apoptosis. [2] |
| ADME/Pharmacokinetics |
In the H460 xenograft nude mouse model, plasma concentrations peaked at 1 hour (8.6 ± 1.2 μg/mL, t1/2 = 3.2 ± 0.5 h) after intraperitoneal injection of baronone hydrochloride (AQ4N) (100 mg/kg). AQ4 peaked at 2 hours (2.1 ± 0.3 μg/mL, t1/2 = 4.8 ± 0.7 h) and accumulated in the tumor (peak at 4 hours: 3.8 ± 0.6 μg/g). [3] In C3H mice, DMXAA pretreatment increased tumor AQ4 concentration by 2.3-fold; hydralazine reduced it by 45%. Flow modifiers had no effect on plasma AQ4N/AQ4 concentrations. [2]
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| Toxicity/Toxicokinetics |
In nude mice (intravenous injection of up to 200 mg/kg of baronone hydrochloride (AQ4N)), no significant changes in body weight, hematology, or liver and kidney function were observed. Histopathological examination showed no obvious toxicity in normal tissues. [1][3] In C3H mice (intravenous injection of 80 mg/kg of baronone hydrochloride (AQ4N), with or without a modulator), no acute toxicity or organ weight abnormalities were observed. [2] Plasma protein binding rates: 78 ± 4% for baronone hydrochloride (AQ4N) and 92 ± 3% for AQ4 (human plasma, in vitro). [3]
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| References |
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| Additional Infomation |
Bananthrone hydrochloride (AQ4N) is a hypoxia-activated prodrug; nitro reduction generates AQ4, which inhibits topoisomerase II and induces DNA damage. Tumor hypoxia confers its selective toxicity relative to normal tissues. [1][2][3]
When used in combination with radiotherapy, it can simultaneously target both aerobic and hypoxic tumor cells. By modulating tumor blood flow, altering oxygenation and drug activation, the efficacy is optimized. [1][2] |
| Molecular Formula |
C₂₂H₃₀CL₂N₄O₆
|
|---|---|
| Molecular Weight |
517.402803897858
|
| Exact Mass |
516.154
|
| Elemental Analysis |
C, 51.07; H, 5.84; Cl, 13.70; N, 10.83; O, 18.55
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| CAS # |
252979-56-9
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| Related CAS # |
136470-65-0 (Free base); 70476-63-0 (AQ4); 252979-56-9 (2HCl)
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| PubChem CID |
72941779
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| Appearance |
Blue to dark blue solid powder
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| LogP |
3.637
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| Hydrogen Bond Donor Count |
6
|
| Hydrogen Bond Acceptor Count |
8
|
| Rotatable Bond Count |
8
|
| Heavy Atom Count |
34
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| Complexity |
644
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| Defined Atom Stereocenter Count |
0
|
| SMILES |
Cl.Cl.[O-][N+](C)(C)CCNC1C=CC(=C2C(C3C(=CC=C(C=3C(C2=1)=O)O)O)=O)NCC[N+](C)(C)[O-]
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| InChi Key |
SBWCPHUXRZRTDP-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C22H28N4O6.2ClH/c1-25(2,31)11-9-23-13-5-6-14(24-10-12-26(3,4)32)18-17(13)21(29)19-15(27)7-8-16(28)20(19)22(18)30;;/h5-8,23-24,27-28H,9-12H2,1-4H3;2*1H
|
| Chemical Name |
2-[[4-[2-[dimethyl(oxido)azaniumyl]ethylamino]-5,8-dihydroxy-9,10-dioxoanthracen-1-yl]amino]-N,N-dimethylethanamine oxide;dihydrochloride
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| Synonyms |
AQ-4N; AZD-1689; AQ4N; AZD1689; AQ 4N; AZD 1689
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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: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). 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) |
DMSO: ~25 mg/mL (~48.3 mM)
H2O: ~20 mg/mL (~38.7 mM) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 7.5 mg/mL (14.50 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 75.0 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. Solubility in Formulation 2: ≥ 7.5 mg/mL (14.50 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 75.0 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. View More
Solubility in Formulation 3: 8.33 mg/mL (16.10 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C). |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9327 mL | 9.6637 mL | 19.3274 mL | |
| 5 mM | 0.3865 mL | 1.9327 mL | 3.8655 mL | |
| 10 mM | 0.1933 mL | 0.9664 mL | 1.9327 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT00109356 | Unknown | Drug: AQ4N (Chemotherapy) |
Non-Hodgkin's Lymphoma Small Lymphocytic Leukemia |
Novacea | March 2005 | Phase 1 Phase 2 |
| NCT00394628 | Unknown | Drug: AQ4N Drug: Temozolomide |
Glioblastoma Multiforme | Novacea | October 2006 | Phase 1 Phase 2 |
| NCT00090727 | Unknown | Drug: AQ4N | Solid Malignancies Non-Hodgkin's Lymphoma |
Novacea | August 2004 | Phase 1 |
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