Zosuquidar trihydrochloride (LY-335979 trihydrochloride) is a potent modulator of P-glycoprotein-mediated multi-drug resistance with Ki of 60 nM. Phase 3.

P-gp:60 nM(Ki)

Zosuquidar通过阻断[3H]azidopine光亲和标记CEM/VLB100质膜中的Pgp，竞争性抑制[3H]vinblastine与Pgp的平衡结合。[1] Zosuquidar单独对药物敏感和多药抗性（MDR）细胞系表现出具有IC50在6 μM至16 μM范围内的细胞毒性，并且能够以0.1及0.5 μM浓度完全逆转MDR细胞系P388/ADR、MCF7/ADR、2780AD或UCLA-P3.003VLB对化疗化合物（vinblastine、doxorubicin或etoposide）的抗性。[1] Zosuquidar显著恢复表达P-gp的白血病细胞系（包括K562/HHT40、K562/HHT90、K562/DOX和HL60/DNR）对药物的敏感性，并增强含有活跃P-gp的初级AML爆发细胞对蒽环类抗生素（daunorubicin、idarubicin、mitoxantrone）和吉木替尼（Mylotarg）的细胞毒性。[2] 最新的研究表明，Zosuquidar完全抑制在ABCB1转导细胞中(Z)-endoxifen的顶向转运。[3]

Zosuquidar trihydrochloride 在血脑屏障作为P-gp的抑制剂仅表现出适度活性。口服25 mg/kg的Zosuquidar trihydrochloride能在给予紫杉醇后1小时内使大脑浓度约增加2.5倍，在24小时后增加5倍。Zosuquidar 以剂量依赖的方式增强了奈非那韦进入大脑的吸收。在没有Zosuquidar给药的情况下，大脑组织/血浆中奈非那韦的浓度比为0.06±0.03，而在给予2 mg/kg静脉注射Zosuquidar后2至6小时之间增至0.09±0.02，6小时后达到0.85±0.19，20 mg/kg Zosuquidar后增至1.58±0.67。

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.

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 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 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.(Only for Reference)