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Research Area
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Description
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Cancer |
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Biological Activity
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Description
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LY335979 (Zosuquidar) is a potent modulator of P-glycoprotein-mediated multidrug resistance with Ki of 60 nM. |
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Targets
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P-glycoprotein |
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IC50 |
60 nM (Ki) [1] |
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In Vitro
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LY335979 competitively inhibits equilibrium binding of [3H]vinblastine to Pgp by blocking [3H]azidopine photoaffinity labeling of the Pgp in CEM/VLB100 plasma membranes. [1] LY335979 alone shows the cytotoxicity to drug-sensitive and MDR cell lines with IC50 ranging from 6 μM-16 μM and produces its ability to completely reverse the resistance of the oncolytics (vinblastine, doxorubicin, or etoposide) to the MDR cell lines P388/ADR, MCF7/ADR, 2780AD, or UCLA-P3.003VLB at concentration of 0.1 and 0.5 μM. [1] LY335979 significantly restores drug sensitivity in P-gp-expressing leukemia cell lines including K562/HHT40, K562/HHT90, K562/DOX and HL60/DNR, and enhances the cytotoxicity of anthracyclines (daunorubicin, idarubicin, mitoxantrone) and gemtuzumab ozogamicin (Mylotarg) in primary AML blasts with active P-gp. [2] A latest paper indicates that LY335979 completely inhibits apically directed transport of (Z)-endoxifen in the ABCB1-transduced cells. [3] |
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In Vivo
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Clinical Trials
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Features
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Combination Therapy
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Description
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In the MDR P388/ADR mouse model, combined treatment of LY335979 (30 mg/kg) and doxorubicin (1 mg/kg) exhibits a significant antitumor activity than doxorubicin alone. In UCLA-P3.003VLB human non-small cell lung carcinoma xenografts model, combined treatment of Taxol (20 mg/kg) and LY335979 (30 mg/kg) leads to a significant suppression of solid tumor growth compared to either treatment given alone. [1] |
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Protocol
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Kinase Assay
[1]
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| ATPase Assay |
P-Glycoprotein ATPase activity is measured by the liberation of inorganic phosphate from ATP. The assay is measured in a 96-well plate for 90 min at 37 °C. Membranes (8 μg-10 μg protein) are incubated in a total volume of 100 μL of buffer A containing 5 mM sodium azide, 1 mM ouabain, 1 mM EGTA, 3 mM ATP, an ATP regenerating system composed of 5 mM phosphoenolpyruvate, and 3.6 units/mL pyruvate kinase in the presence and absence of 1 mM sodium vanadate. Pgp-ATPase activity is defined as the vanadate-sensitive portion of the total ATPase activity. Plates are read 3 minutes after the addition of the detection solution. The absorbance is measured at 690 nm by a microtiter dish reader. A phosphate standard curve is used to calculate the μmol of phosphate formed. Samples are measured in triplicate. |
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Cell Assay
[1]
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| Cell Lines |
CEM/VLB100, P388/ADR, MCF7/ADR, 2780AD, and UCLA-P3.OO3VLB cells |
| Concentrations |
0.05 μM to 5 μM |
| Incubation Time |
72 hours |
| Methods |
Cell viability is determined using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction method. Cells are harvested during logarithmic growth phase, and seeded in 96-well plates. The cells are then cultured for 72 hours in the presence of oncolytics with or without modulators. MCF-7 and MCF-7/ADR cells are incubated 24 hours before the addition of the drug with and without the LY335979. LY335979 is prepared as 2 nM DMSO stocks and added to wells to give final concentrations ranging from 0.05 to 5 μM. After 72 hours, 20 μL of freshly prepared 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (5 mg/mL in Dulbecco's PBS) is added to each well and incubated for 4 hours in a 37 °C incubator containing 5% CO2. Cells are pelleted in a Sorvall RT6000B centrifuge, 70 μL of medium is carefully removed from each well, and 100 μL of 2-propanol/0.04 N HC1 is added. Cells are resuspended 5-10 times with a Multipipettor or until no particulate matter is visible. Plates are immediately read on a Titertek Multiskan MCC/340 microplate reader Flow Laboratories with a test wavelength of 570 nm and a reference wavelength of 630 nm. Controls are measured in quadruplicate and modulators are measured in duplicate. Cytotoxicity analyses are also performed using the CeliTiter 96 AQueous assay kit. |
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Animal Study
[1]
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| Animal Models |
P388 or P388/ADR cells are implanted by i.p. injection into female BDF1 mice. |
| Formulation |
LY335979 is dissolved in 5% mannitol. |
| Doses |
≤30 mg/kg |
| Administration |
Administered via i.p. and i.v. |
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