Ravasi L, Kiesewetter D O, Shimoji K, Lucignani G, Eckelman W C
PET Radiochemistry Group, National Institute for Biomedical Imaging and Bioengineering, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892, USA.
Eur J Nucl Med Mol Imaging. 2006 Mar;33(3):292-300. doi: 10.1007/s00259-005-1966-x. Epub 2005 Dec 7.
Preferential binding of FP-TZTP at the M(2) receptor in vivo led to investigation of [(18)F]FP-TZTP as a potential PET tracer for Alzheimer's disease, in which a substantial reduction of M(2) receptors has been observed in autopsy studies. We hereby investigated in vitro the FP-TZTP behavior to further elucidate the properties of FP-TZTP that lead to its M(2) selectivity.
Chinese hamster ovarian cells expressing the five subtypes of human muscarinic receptor as well as the wild type were harvested in culture to assess equilibrium binding. Specific binding was calculated by subtraction of non-specific binding from total binding. Internal specific binding was calculated by subtraction of external specific binding from the total specific binding. Saturation assays were also performed to calculate B(max), K(i), and IC(50). In addition, equilibrium binding and dissociation kinetic studies were performed on rat brain tissue. Selected regions of interest were drawn on the digital autoradiograms and [(18)F]FP-TZTP off-rates were determined by measurement of the rate of release into a buffer solution of [(18)F]FP-TZTP from slide-bound cells that had been preincubated with [(18)F]FP-TZTP.
At equilibrium in vitro, M(2) subtype selectivity of [(18)F]FP-TZTP was not evident. We demonstrated that ATP-dependent mechanisms are not responsible for FP-TZTP M(2) selectivity. In vitro off-rate studies from rat brain tissue showed that the off-rate of FP-TZTP varied with the percentage of M(2) subtype in the tissue region.
The slower dissociation kinetics of FP-TZTP from M(2) receptors compared with the four other muscarinic receptor subtypes may be a factor in its M(2) selectivity.
FP-TZTP在体内对M(2)受体的优先结合促使人们研究[(18)F]FP-TZTP作为阿尔茨海默病潜在正电子发射断层显像（PET）示踪剂的可能性，在尸检研究中已观察到M(2)受体显著减少。我们在此进行了体外FP-TZTP行为研究，以进一步阐明导致其M(2)选择性的FP-TZTP特性。
收获培养的表达人毒蕈碱受体五种亚型以及野生型的中国仓鼠卵巢细胞，以评估平衡结合。通过从总结合中减去非特异性结合来计算特异性结合。通过从总特异性结合中减去外部特异性结合来计算内部特异性结合。还进行了饱和试验以计算B(max)、K(i)和IC(50)。此外，对大鼠脑组织进行了平衡结合和解离动力学研究。在数字放射自显影片上绘制选定的感兴趣区域，并通过测量预先用[(18)F]FP-TZTP预孵育的载玻片结合细胞中[(18)F]FP-TZTP释放到缓冲溶液中的速率来确定[(18)F]FP-TZTP的解离速率。
在体外平衡时，[(18)F]FP-TZTP的M(2)亚型选择性不明显。我们证明ATP依赖性机制与FP-TZTP的M(2)选择性无关。大鼠脑组织的体外解离速率研究表明，FP-TZTP的解离速率随组织区域中M(2)亚型的百分比而变化。
与其他四种毒蕈碱受体亚型相比，FP-TZTP从M(2)受体解离的动力学较慢可能是其M(2)选择性的一个因素。
