Sivapackiam Jothilingam, Harpstrite Scott E, Prior Julie L, Mattingly Stephen, Sharma Vijay
ICCE Institute, Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
ICCE Institute, Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA; Students and Teachers As Research Scientists (STARS), Washington University School of Medicine, St. Louis, MO 63110, USA.
Nucl Med Biol. 2016 Mar;43(3):191-7. doi: 10.1016/j.nucmedbio.2015.12.001. Epub 2015 Dec 8.
For stratification of chemotherapeutic choices, radiopharmaceuticals capable of imaging breast cancer resistance protein (BCRP/ABCG2)-mediated functional transport are desired. To accomplish this objective, Galmydar, a fluorescent and moderately hydrophobic Ga(III) cationic complex and its (67/68)Ga-radiolabeled counterparts were interrogated in HEK293 cells stably transfected with BCRP and their WT counterparts transfected with empty vector. Additionally, the sensitivity and specificity of (68)Ga-Galmydar to evaluate functional expression of BCRP at the blood-brain barrier (BBB) was investigated in gene-knockout mdr1a/1b(-/-) (double knockout, dKO) and mdr1a/1b(-/-)ABCG2(-/-) (triple knockout, tKO) mouse models.
For radiotracer uptake assays and live cell fluorescence imaging, either (67)Ga-Galmydar or its unlabeled counterpart was incubated in HEK293 cells transfected with BCRP (HEK293/BCRP) and their WT counterparts at 37°C under a continuous flux of CO2 (5%) in the presence or absence of Ko143, a potent BCRP antagonist, and cellular uptake was measured to assess the sensitivity of Galmydar to probe BCRP-mediated functional transport activity in cellulo. For assessing the potential of Galmydar to enable diagnostic imaging of targeted tissues in vivo, the (67)Ga-radiolabeled counterpart was incubated in either human serum albumin or human serum at 37°C and the percentage of unbound (67)Ga-Galmydar was determined. To evaluate the sensitivity of (68)Ga-Galmydar for molecular imaging of BCRP-mediated efflux activity in vivo, microPET/CT brain imaging was performed in dKO and tKO mice and their age-matched WT counterparts, 60min post-intravenous injection.
(67)Ga-Galmydar shows uptake profiles in HEK293 cells inversely proportional to BCRP expression, and antagonist (Ko143) induced accumulation in HEK293/BCRP cells, thus indicating target sensitivity and specificity. Furthermore, employing the fluorescent characteristics of Galmydar, optical imaging in HEK293/BCRP cells shows an excellent correlation with the radiotracer cellular accumulation data. (67)Ga-Galmydar shows > 85% unbound fraction and presence of parental compound in human serum. Finally, microPET/CT imaging shows higher retention of (68)Ga-Galmydar in brains of dKO and tKO mice compared to their age-matched WT counterparts, 60min post-intravenous tail-vein injection.
Combined data indicate that Galmydar could provide a template scaffold for development of a PET tracer for imaging BCRP-mediated functional transport activity in vivo.
为了对化疗选择进行分层，需要能够对乳腺癌耐药蛋白（BCRP/ABCG2）介导的功能性转运进行成像的放射性药物。为实现这一目标，在稳定转染了BCRP的HEK293细胞及其转染空载体的野生型对应细胞中研究了Galmydar（一种荧光且具有适度疏水性的Ga(III)阳离子复合物）及其（67/68）Ga放射性标记的类似物。此外，在基因敲除mdr1a/1b(-/-)（双敲除，dKO）和mdr1a/1b(-/-)ABCG2(-/-)（三敲除，tKO）小鼠模型中研究了（68）Ga-Galmydar评估血脑屏障（BBB）处BCRP功能表达的敏感性和特异性。
对于放射性示踪剂摄取测定和活细胞荧光成像，将（67）Ga-Galmydar或其未标记的类似物在37°C、5% CO2连续气流存在或不存在强效BCRP拮抗剂Ko143的条件下，与转染了BCRP的HEK293细胞（HEK293/BCRP）及其野生型对应细胞一起孵育，并测量细胞摄取以评估Galmydar在细胞内探测BCRP介导的功能性转运活性的敏感性。为了评估Galmydar在体内对靶向组织进行诊断成像的潜力，将（67）Ga放射性标记的类似物在37°C下与人血清白蛋白或人血清一起孵育，并测定未结合的（67）Ga-Galmydar的百分比。为了评估（68）Ga-Galmydar在体内对BCRP介导的外排活性进行分子成像的敏感性，在静脉注射后60分钟，对dKO和tKO小鼠及其年龄匹配的野生型对应小鼠进行了microPET/CT脑成像。
（67）Ga-Galmydar在HEK293细胞中的摄取情况与BCRP表达呈反比，拮抗剂（Ko143）诱导其在HEK293/BCRP细胞中积累，从而表明其对靶点的敏感性和特异性。此外，利用Galmydar的荧光特性，HEK293/BCRP细胞中的光学成像与放射性示踪剂细胞积累数据显示出极好的相关性。（67）Ga-Galmydar在人血清中的未结合分数>85%，且存在亲本化合物。最后，microPET/CT成像显示，在尾静脉注射后60分钟，与年龄匹配的野生型对应小鼠相比，dKO和tKO小鼠脑中（68）Ga-Galmydar的滞留更高。
综合数据表明，Galmydar可为开发用于体内成像BCRP介导的功能性转运活性的PET示踪剂提供模板支架。
