Why does the agonist [(18)F]FP-TZTP bind preferentially to the M(2) muscarinic receptor?
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Ravasi
L
L
PET Radiochemistry Group, National Institute for Biomedical Imaging and Bioengineering, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892, USA.
Kiesewetter
D O
DO
Shimoji
K
K
Lucignani
G
G
Eckelman
W C
WC
D000818:Animals; D001921:Brain / Q000000981:diagnostic imaging / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005462:Fluorine Radioisotopes / Q000493:pharmacokinetics*; D008657:Metabolic Clearance Rate; D009928:Organ Specificity; D011485:Protein Binding; D011725:Pyridines / Q000493:pharmacokinetics*; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D043585:Receptor, Muscarinic M2 / Q000819:agonists* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013844:Thiazoles / Q000493:pharmacokinetics*; D014018:Tissue Distribution
D005462:Fluorine Radioisotopes; D011725:Pyridines; D019275:Radiopharmaceuticals; D043585:Receptor, Muscarinic M2; D013844:Thiazoles; C115177:FP-TZTP