Piwnica-Worms D, Chiu M L, Budding M, Kronauge J F, Kramer R A, Croop J M
Department of Radiology, Brigham and Women's Hospital, Boston, Massachusetts 02115.
Cancer Res. 1993 Mar 1;53(5):977-84.
The multidrug-resistant P-glycoprotein (Pgp), a M(r) 170,000 plasma membrane protein encoded by the mammalian multidrug resistance gene (MDR1), appears to function as an energy-dependent efflux pump. Many of the drugs that interact with Pgp are lipophilic and cationic at physiological pH. We tested the hypothesis that the synthetic gamma-emitting organotechnetium complex, hexakis(2-methoxyisobutylisonitrile)technetium(I) ([99mTc]SESTAMIBI), a lipophilic cationic radiopharmaceutical, could be a suitable Pgp transport substrate capable of functional imaging of the MDR phenotype. The cellular pharmacological profile of [99mTc]SESTAMIBI transport was examined in Chinese hamster V79 lung fibroblasts and the 77A and LZ derivative cell lines which express modestly low, intermediate, and very high levels of Pgp, respectively. Steady-state contents of [99mTc]SESTAMIBI in V79, 77A, and LZ cells were 10.0 +/- 0.5 (SEM) (n = 9), 3.6 +/- 0.5 (n = 8), and 0.4 +/- 0.02 (n = 9) fmol.(mg protein)-1 (nMo)-1, respectively, consistent with enhanced extrusion of the imaging agent by Pgp-enriched cells. Maximal doses (> 100 microM) of the multidrug-resistant reversal agents verapamil and cyclosporin A enhanced [99mTc]SESTAMIBI accumulation in V79, 77A, and LZ cells by approximately 10-, 25-, and 200-fold, respectively. The median effective concentration values for tracer accumulation in the presence of verapamil in V79, 77A, and LZ cells were 4, 100, and 200 microM, and those for cyclosporin A were 0.9, 3, and > 25 microM, respectively. Pgp-mediated [99mTc]SESTAMIBI transport occurred against its electrochemical gradient and was found to be ATP dependent displaying an apparent Km of 50 microM. Carrier-added [99Tc]SESTAMIBI was 11- to 13-fold less toxic in multidrug-resistant cells, and inhibited photolabeling of Pgp by [125I]iodoaryl azidoprazosin in a concentration-dependent manner; half-maximal displacement was observed at approximately 100- to 1000-fold molar excess [99Tc]SESTAMIBI. Exploiting the favorable gamma emission properties of 99mTc, functional expression of Pgp was successfully imaged in human tumor xenographs in nude mice with pharmacologically inert tracer quantities of [99mTc]SESTAMIBI. Functional imaging with these organotechnetium complexes may provide a novel mechanism to rapidly characterize Pgp expression in human tumors in vivo, target reversal agents in vivo, and ultimately provide a means to direct patients to specific cancer therapies.
多药耐药性P-糖蛋白（Pgp）是一种分子量为170,000的质膜蛋白，由哺乳动物多药耐药基因（MDR1）编码，似乎起着能量依赖性外排泵的作用。许多与Pgp相互作用的药物在生理pH值下是亲脂性和阳离子性的。我们检验了这样一个假设，即合成的发射γ射线的有机锝络合物六（2-甲氧基异丁基异腈）锝（I）（[99mTc]SESTAMIBI），一种亲脂性阳离子放射性药物，可能是一种合适的Pgp转运底物，能够对MDR表型进行功能成像。在中国仓鼠V79肺成纤维细胞以及分别表达适度低、中、高水平Pgp的77A和LZ衍生细胞系中研究了[99mTc]SESTAMIBI转运的细胞药理学特征。V79、77A和LZ细胞中[99mTc]SESTAMIBI的稳态含量分别为10.0±0.5（SEM）（n = 9）、3.6±0.5（n = 8）和0.4±0.02（n = 9）fmol·（mg蛋白）-1（nMo）-1，这与富含Pgp的细胞对成像剂的增强外排一致。多药耐药逆转剂维拉帕米和环孢素A的最大剂量（>100μM）分别使V79、77A和LZ细胞中[99mTc]SESTAMIBI的积累增加了约10倍、25倍和200倍。在维拉帕米存在下，V79、77A和LZ细胞中示踪剂积累的半数有效浓度值分别为4μM、100μM和200μM，环孢素A的分别为0.9μM、3μM和>25μM。Pgp介导的[99mTc]SESTAMIBI转运是逆其电化学梯度进行的，并且发现是ATP依赖性的，表观Km为50μM。添加载体的[99Tc]SESTAMIBI在多药耐药细胞中的毒性低11至13倍，并以浓度依赖性方式抑制[125I]碘芳基叠氮哌唑对Pgp的光标记；在大约100至1000倍摩尔过量的[99Tc]SESTAMIBI时观察到半数最大置换。利用99mTc良好的γ发射特性，用药理学惰性示踪量的[99mTc]SESTAMIBI成功地在裸鼠体内的人肿瘤异种移植模型中对Pgp的功能表达进行了成像。用这些有机锝络合物进行功能成像可能提供一种新机制，以快速在体内表征人肿瘤中Pgp的表达、在体内靶向逆转剂，并最终提供一种将患者导向特定癌症治疗的方法。
