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]

• ATPase 분석

  P-gp ATPase 활성은 ATP로부터 무기 인산염이 유리되는 것으로 측정된다. 이 분석은 96-웰 플레이트에서 37°C로 90분간 수행된다. 막(8 μg-10 μg 단백질)은 5 mM 아지드화 나트륨, 1 mM 우아바인, 1 mM EGTA, 3 mM ATP, 5 mM 포스포엔올피루브산과 3.6 units/mL 피루브산 키나아제로 구성된 ATP 재생 시스템을 포함하는 버퍼 A 100 μL 총 부피 내에서 1 mM 바나듐산 나트륨 존재 및 부재 하에 배양된다. P-gp ATPase 활성은 전체 ATPase 활성 중 바나듐산염에 민감한 부분으로 정의된다. 플레이트는 검출 용액 첨가 3분 후 판독된다. 흡광도는 마이크로타이터 디쉬 리더를 사용하여 690 nm에서 측정된다. 인산염 표준 곡선을 사용하여 형성된 인산염의 μmol을 계산한다. 샘플은 3회 반복 측정된다.

• 세포주

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

• 농도

  0.05 μM to 5 μM

• 배양 시간

  72 hours

• 방법

  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.

• 동물 모델

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

• 용량

  ≤30 mg/kg

• 투여

  Administered via i.p. and i.v.