Liow Jeih-San, Zoghbi Sami S, Hu Shuo, Hall Matthew D, Hines Christina S, Shetty H Umesha, Araneta Maria D, Page Emily M, Pike Victor W, Kreisl William C, Herscovitch Peter, Gottesman Michael M, Theodore William H, Innis Robert B
Molecular Imaging Branch, NIMH/NIH, Bethesda, MD, USA.
Molecular Imaging Branch, NIMH/NIH, Bethesda, MD, USA.
Neuroimage. 2016 Sep;138:134-140. doi: 10.1016/j.neuroimage.2016.05.045. Epub 2016 May 19.
Efflux transporters at the blood-brain barrier can decrease the entry of drugs and increase the removal of those molecules able to bypass the transporter. We previously hypothesized that (18)F-FCWAY, a radioligand for the serotonin 5-HT1A receptor, is a weak substrate for permeability glycoprotein (P-gp) based on its very early peak and rapid washout from human brain. To determine whether (18)F-FCWAY is a substrate for P-gp, breast cancer resistance protein (BCRP), and multidrug resistance protein (MRP1) - the three most prevalent efflux transporters at the blood-brain barrier - we performed three sets of experiments. In vitro, we conducted fluorescence-activated cell sorting (FACS) flow cytometry studies in cells over-expressing P-gp, BCRP, and MRP1 treated with inhibitors specific to each transporter and with FCWAY. Ex vivo, we measured (18)F-FCWAY concentration in plasma and brain homogenate of transporter knockout mice using γ-counter and radio-HPLC. In vivo, we conducted positron emission tomography (PET) studies to assess changes in humans who received (18)F-FCWAY during an infusion of tariquidar (2-4mg/kg iv), a potent and selective P-gp inhibitor. In vitro studies showed that FCWAY allowed fluorescent substrates to get into the cell by competitive inhibition of all three transporters at the cell membrane. Ex vivo measurements in knockout mice indicate that (18)F-FCWAY is a substrate only for P-gp and not BCRP. In vivo, tariquidar increased (18)F-FCWAY brain uptake in seven of eight subjects by 60-100% compared to each person's baseline. Tariquidar did not increase brain uptake via some peripheral mechanism, given that it did not significantly alter concentrations in plasma of the parent radioligand (18)F-FCWAY or its brain-penetrant radiometabolite (18)F-FC. These results show that (18)F-FCWAY is a weak substrate for efflux transport at the blood-brain barrier; some radioligand can enter brain, but its removal is hastened by P-gp. Although (18)F-FCWAY is not ideal for measuring 5-HT1A receptors, it demonstrates that weak substrate radioligands can be useful for measuring both increased and decreased function of efflux transporters, which is not possible with currently available radioligands such as (11)C-loperamide and (11)C-verapamil that are avid substrates for transporters.
血脑屏障处的外排转运体可减少药物的进入，并增加那些能够绕过转运体的分子的清除。我们之前推测，基于其在人脑中的极早峰值和快速洗脱，用于5-羟色胺5-HT1A受体的放射性配体（18）F-FCWAY是通透性糖蛋白（P-糖蛋白）的弱底物。为了确定（18）F-FCWAY是否是P-糖蛋白、乳腺癌耐药蛋白（BCRP）和多药耐药蛋白（MRP1）（血脑屏障处三种最普遍的外排转运体）的底物，我们进行了三组实验。在体外，我们在用每种转运体特异性抑制剂和FCWAY处理过的过表达P-糖蛋白、BCRP和MRP1的细胞中进行了荧光激活细胞分选（FACS）流式细胞术研究。在离体实验中，我们使用γ计数器和放射性高效液相色谱法测量了转运体基因敲除小鼠血浆和脑匀浆中的（18）F-FCWAY浓度。在体内，我们进行了正电子发射断层扫描（PET）研究，以评估在输注强效选择性P-糖蛋白抑制剂 tariquidar（2-4mg/kg静脉注射）期间接受（18）F-FCWAY的人体的变化。体外研究表明，FCWAY通过竞争性抑制细胞膜上的所有三种转运体，使荧光底物进入细胞。基因敲除小鼠的离体测量表明，（18）F-FCWAY仅是P-糖蛋白的底物，而非BCRP的底物。在体内，与每个受试者的基线相比，tariquidar使8名受试者中的7名受试者的（18）F-FCWAY脑摄取增加了60-100%。鉴于tariquidar并未显著改变母体放射性配体（18）F-FCWAY或其脑渗透性放射性代谢物（18）F-FC的血浆浓度，因此它不是通过某些外周机制增加脑摄取的。这些结果表明，（18）F-FCWAY是血脑屏障处外排转运的弱底物；一些放射性配体可进入脑内，但P-糖蛋白会加速其清除。虽然（18）F-FCWAY对于测量5-HT1A受体并不理想，但它表明弱底物放射性配体可用于测量外排转运体功能的增加和减少，而这对于目前可用的放射性配体如（11）C-洛哌丁胺和（11）C-维拉帕米（它们是转运体的avid底物）来说是不可能的。
