Zosuquidar 3HCl

2 years -80 in solvent

Administered via i.p. and i.v.

CEM/VLB100, P388/ADR, MCF7/ADR, 2780AD, and UCLA-P3.OO3VLB cells

<=30 mg/kg

60 nM (Ki), [1], 60 nM (Ki), [1], 60 nM (Ki), [1], 60 nM (Ki), [1], 60 nM (Ki), [1], 60 nM (Ki), [1]

72 hours

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 uM. After 72 hours, 20 uL 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 uL of medium is carefully removed from each well, and 100 uL 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.

Zosuquidar 3HCl is a potent modulator of P-glycoprotein-mediated multi-drug resistance with Ki of 60 nM in a cell-free assay. Phase 3.