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| ln Vitro |
Treatment with Dofequidar fumarate dose-dependently reduced the proportion of Hoechst33342-effluxing side population (SP) cells, a cancer stem-like cell population, in various cancer cell lines (HeLa, BSY-1, KM12). For example, in HeLa cells, 2.5 µM dofequidar significantly decreased the SP fraction. [4]
In a vesicle transport assay using membrane vesicles overexpressing ABCG2/BCRP, Dofequidar fumarate inhibited the ATP-dependent uptake of [³H]methotrexate ([³H]MTX) in a dose-dependent manner, similar to the known ABCG2 inhibitor fumitremorgin C (FTC). Verapamil (an ABCB1 inhibitor) did not show inhibition. [4] Dofequidar fumarate increased the intracellular accumulation of mitoxantrone (MXR) in K562 cells stably overexpressing ABCG2/BCRP (K562/BCRP), as measured by flow cytometry, indicating inhibition of ABCG2-mediated drug efflux. [4] Dofequidar fumarate sensitized ABCG2/BCRP-overexpressing KB/BCRP cells to mitoxantrone (MXR), reversing their chemoresistance. Treatment with 10 µM dofequidar reduced the GI₅₀ of MXR in KB/BCRP cells from 30.46 nM to 3.84 nM, similar to the effect of 1 µM FTC. [4] Dofequidar fumarate sensitized SP cells isolated from HeLa, HBC-5, and BSY-1 cell lines to chemotherapeutic drugs (topotecan, mitoxantrone). For instance, in HeLa SP cells, dofequidar (3 µM) reduced the GI₅₀ for topotecan from 77.0 nM to 45.6 nM, making it comparable to non-SP (NSP) cells (55.6 nM). [4] |
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| ln Vivo |
In nude mice bearing xenograft tumors derived from HeLa SP cells (which are chemoresistant), oral administration of Dofequidar fumarate (200 mg/kg) prior to intravenous irinotecan (CPT-11, 67 mg/kg) significantly enhanced the antitumor efficacy of CPT-11. The combination therapy (CPT-11 + dofequidar) drastically reduced tumor volume compared to CPT-11 alone, which only arrested tumor growth without reduction. The combination therapy was as effective against SP-derived tumors as CPT-11 alone was against NSP-derived tumors. [4]
Dofequidar fumarate alone (200 mg/kg, orally) had almost no effect on the growth of SP- or NSP-derived tumors in vivo. [4] The combination therapy (CPT-11 + dofequidar) appeared well-tolerated in mice, with very small changes in body weight observed. [4] |
| Enzyme Assay |
An in vitro vesicle transport assay was performed to assess inhibition of ABCG2/BCRP function. Membrane vesicles prepared from insect cells overexpressing human ABCG2/BCRP protein were used. The assay mixture contained 50 mM MOPS-Tris (pH 7.0), 7.5 mM MgCl₂, 70 mM KCl, 160 nM unlabeled methotrexate (MTX), the test inhibitor (dofequidar, FTC, or verapamil), 1 mCi/mL [³H]MTX, and membrane vesicles (25 µg protein) in a 30 µL volume. The mixture was kept on ice for 5 minutes, then the transport reaction was initiated by adding 20 µL of 10 mM ATP (or AMP as a negative control) and incubating at 37°C for 5 minutes. The reaction was terminated by adding an ice-cold stop solution (40 mM MOPS-Tris pH 7.0, 70 mM KCl). The membrane vesicles were collected on a filter plate, washed, and the retained radioactivity was measured with a liquid scintillation counter to quantify ATP-dependent [³H]MTX uptake. [4]
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| Cell Assay |
Side Population (SP) cell analysis and sorting: Cells were trypsinized, resuspended in ice-cold HBSS with 2% FBS, and stained with 2.5-15 µg/mL Hoechst33342 dye for 60 minutes at 37°C in the presence or absence of transporter inhibitors (e.g., dofequidar, reserpine, FTC). After washing, cells were analyzed or sorted using a flow cytometer (FACS Vantage). SP cells were identified as the low-fluorescence population in the Hoechst Blue vs. Hoechst Red dot plot. [4]
Intracellular drug accumulation assay: K562/BCRP cells (5 x 10⁵ cells) were incubated with 3 µM mitoxantrone (MXR) for 30 minutes at 37°C with or without dofequidar or FTC. Cells were then washed with ice-cold PBS and subjected to fluorescence analysis by flow cytometry (excitation 630 nm) to measure intracellular MXR fluorescence. [4] Cell viability assay (MTS assay): Cells (e.g., sorted SP/NSP cells, KB/BCRP cells) were cultured in medium containing serial dilutions of anticancer drugs (e.g., mitoxantrone, topotecan) with or without dofequidar for 3 days. Cell viability was then assessed by adding MTS reagent, incubating for 1 hour, and measuring the absorbance with a microplate spectrophotometer. The 50% growth inhibition (GI₅₀) values were determined. [4] Gene silencing: HeLa cells were transfected with ABCG2-specific siRNA or control siRNA using a lipid-based transfection reagent. After 24-48 hours, cells were harvested for analysis of ABCG2 protein expression by immunoblotting and for SP analysis by Hoechst33342 staining. [4] |
| Animal Protocol |
Tumor xenograft model: Sorted HeLa SP or NSP cells (100 to 10,000 cells) were suspended in DMEM growth medium, mixed 1:1 with Matrigel, and subcutaneously injected into 5-6 week-old female BALB/c nude mice. Tumor formation was monitored. For secondary transplantation, established tumors were resected, diced into pieces, and transplanted into new mice. [4]
Drug treatment: When secondary tumor volume reached approximately 100 mm³, mice were sorted into groups. Dofequidar fumarate was administered orally at a dose of 200 mg/kg (vehicle/control not specified in method details, but water was used as control in results) 30 minutes before the intravenous administration of irinotecan (CPT-11) at 67 mg/kg. This treatment schedule (dofequidar + CPT-11) was repeated on days 0, 4, and 8. Tumor size and body weight were measured every 2-3 days. [4] |
| References |
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| Additional Infomation |
See also: dofquinda fumarate (note moved to).
Dofquinda fumarate (MS-209) is an orally effective quinoline derivative that was initially developed as a multidrug resistance (MDR) reversal agent targeting ABCB1/P-gp and ABCC1/MRP1. [4] The evidence provided in this study suggests that dofquinda fumarate can also effectively inhibit the ABCG2/BCRP transporter, which is highly expressed in cancer stem cell-like side population (SP) cells. By inhibiting ABCG2, it can sensitize these resistant cells to chemotherapeutic agents such as mitoxantrone, topotecan, and irinotecan (via its active metabolite SN-38). [4] Its mechanism of action involves inhibiting the drug efflux function of the ABC transporter, thereby increasing the intracellular concentration of chemotherapeutic agents and overcoming chemotherapeutic resistance, especially in stem cell-like cancer cells. [4] Studies have shown that combining dofequinda fumarate with conventional chemotherapy (such as CPT-11) may be a strategy to target chemotherapy-resistant cancer stem cells, potentially improving treatment outcomes and reducing recurrence rates. [4] Phase III clinical trials for breast cancer have shown that dofequinda in combination with the CAF regimen (cyclophosphamide, doxorubicin, fluorouracil) can improve progression-free survival and overall survival in previously untreated patients, which aligns with the concept of targeting pre-existing drug-resistant stem cell-like cells. [4] |
| Molecular Formula |
C30H31N3O3
|
|---|---|
| Molecular Weight |
481.5854
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| Exact Mass |
597.247
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| CAS # |
153653-30-6
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| Related CAS # |
Dofequidar;129716-58-1
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| PubChem CID |
9960287
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
10
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| Heavy Atom Count |
44
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| Complexity |
771
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1CN(CCN1CC(COC2=CC=CC3=C2C=CC=N3)O)C(=O)C(C4=CC=CC=C4)C5=CC=CC=C5.C(=C/C(=O)O)\C(=O)O
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| InChi Key |
QIAVTDQTRFYXSD-WLHGVMLRSA-N
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| InChi Code |
InChI=1S/C30H31N3O3.C4H4O4/c34-25(22-36-28-15-7-14-27-26(28)13-8-16-31-27)21-32-17-19-33(20-18-32)30(35)29(23-9-3-1-4-10-23)24-11-5-2-6-12-24;5-3(6)1-2-4(7)8/h1-16,25,29,34H,17-22H2;1-2H,(H,5,6)(H,7,8)/b;2-1+
|
| Chemical Name |
(E)-but-2-enedioic acid;1-[4-(2-hydroxy-3-quinolin-5-yloxypropyl)piperazin-1-yl]-2,2-diphenylethanone
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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, 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 : ~100 mg/mL (~167.32 mM)
H2O : ~1 mg/mL (~1.67 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.18 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 25.0 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: ≥ 2.5 mg/mL (4.18 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 25.0 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: ≥ 2.5 mg/mL (4.18 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0765 mL | 10.3823 mL | 20.7646 mL | |
| 5 mM | 0.4153 mL | 2.0765 mL | 4.1529 mL | |
| 10 mM | 0.2076 mL | 1.0382 mL | 2.0765 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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