Zosuquidar 3HCl

LY335979 competitively inhibits equilibrium binding of 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 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 and 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]

• Saggio dell'ATPasi

  L'attività ATPasica della P-glicoproteina viene misurata tramite la liberazione di fosfato inorganico dall'ATP. Il saggio viene eseguito in una piastra a 96 pozzetti per 90 minuti a 37 °C. Le membrane (8 μg-10 μg di proteine) vengono incubate in un volume totale di 100 μL di tampone A contenente 5 mM di azoturo di sodio, 1 mM di ouabaina, 1 mM di EGTA, 3 mM di ATP, un sistema di rigenerazione dell'ATP composto da 5 mM di fosfoenolpiruvato e 3,6 unità/mL di piruvato chinasi in presenza e assenza di 1 mM di vanadato di sodio. L'attività ATPasica della Pgp è definita come la porzione dell'attività ATPasica totale sensibile al vanadato. Le piastre vengono lette 3 minuti dopo l'aggiunta della soluzione di rilevamento. L'assorbanza viene misurata a 690 nm mediante un lettore di micropiastre. Una curva standard di fosfato viene utilizzata per calcolare i μmol di fosfato formato. I campioni vengono misurati in triplicato.

• Linee cellulari

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

• Concentrazioni

  0.05 μM to 5 μM

• Tempo di incubazione

  72 hours

• Metodo

  Cell viability is determined using a modified the MTT 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 mM 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 MTT(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.

• Modelli animali

  P388 or P388/ADR cells are implanted by i.p. injection into female BDF₁ mice.

• Dosaggi

  ≤30 mg/kg

• Somministrazione

  Administered via i.p. and i.v.