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

Purpose: Preferential binding of FP-TZTP at the M2 receptor in vivo led to investigation of [18F]FP-TZTP as a potential PET tracer for Alzheimer's disease, in which a substantial reduction of M2 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 M2 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 Bmax, Ki, and IC50. 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 [18F]FP-TZTP off-rates were determined by measurement of the rate of release into a buffer solution of [18F]FP-TZTP from slide-bound cells that had been preincubated with [18F]FP-TZTP. Results: At equilibrium in vitro, M2 subtype selectivity of [18F]FP- TZTP was not evident. We demonstrated that ATP-dependent mechanisms are not responsible for FP-TZTP M2 selectivity. In vitro off-rate studies from rat brain tissue showed that the off-rate of FP-TZTP varied with the percentage of M2 subtype in the tissue region. Conclusion: The slower dissociation kinetics of FP-TZTP from M2 receptors compared with the four other muscarinic receptor subtypes may be a factor in its M2 selectivity

Prokineticin Receptor 1 Antagonist PC-10 as a Biomarker for Imaging Inflammatory Pain

Prokineticin receptor 1 (PKR1) and its ligand Bv8 were shown to be expressed in inflammation-induced pain and by tumor-supporting fibroblasts. Blocking this receptor might prove useful for reducing pain and for cancer therapy. However, there is no method to quantify the levels of these receptors in vivo. Methods: A nonpeptidic PKR1 antagonist, N-{2-[5-(4-fluorobenzyl)- 1-(4-methoxy-benzyl)-4,6-dioxo-1,4,5,6-tetrahydro-[1,3,5] triazin-2-ylamino]-ethyl}-guanidine, which contains a free guanidine group, was labeled with (18)F by reacting the guanidine function with N-succinimidyl-4-(18)F-fluorobenzoate to give the guanidinyl amide N-(4-(18)F-fluoro-benzoyl)-N9-{2-[5-(4-fluorobenzyl)- 1-(4-methoxy-benzyl)-4,6-dioxo-1,4,5,6-tetrahydro-[1,3,5] triazin-2-ylamino]-ethyl}-guanidine ((18)F-PC-10). Inflammation was induced in C57BL/6 mice by subcutaneous injection of complete Freund adjuvant in the paw. The mice were imaged with (18)F-PC10, (18)F-FDG, and (64)Cu-pyruvaldehyde bis(4-methyl-3-thiosemicarbazone) ((64)Cu-PTSM) at 24 h after complete Freund adjuvant injection using a small-animal PET device. Results: (18)F-PC-10 was synthesized with a radiochemical yield of 16% +/- 3% (decay-corrected). (18)F-PC-10 accumulated specifically in the inflamed paw 4- to 5-fold more than in the control paw. Compared with (18)F-PC-10, (18)F-FDG and (64)Cu-PTSM displayed higher accumulation in the inflamed paw but also had higher accumulation in the control paw, demonstrating a reduced signal-to-background ratio. (18)F-PC-10 also accumulated in PKR1-expressing organs, such as the salivary gland and gastrointestinal tract. Conclusion: (18)F-PC-10 can be used to image PKR1, a biomarker of the inflammation process. However, the high uptake of (18)F-PC-10 in the gastrointestinal tract, due to specific uptake and the metabolic processing of this highly lipophilic molecule, would restrict its utility

Prokineticin Receptor 1 Antagonist PC-10 as a Biomarker for Imaging Inflammatory Pain

Prokineticin receptor 1 (PKR1) and its ligand Bv8 were shown to be expressed in inflammation-induced pain and by tumor-supporting fibroblasts. Blocking this receptor might prove useful for reducing pain and for cancer therapy. However, there is no method to quantify the levels of these receptors in vivo. Methods: A nonpeptidic PKR1 antagonist, N-{2-[5-(4-fluoro-benzyl)-1-(4-methoxy-benzyl)-4,6-dioxo-1,4,5,6-tetrahydro-[1,3,5]triazin-2-ylamino]-ethyl}-guanidine, which contains a free guanidine group, was labeled with 18F by reacting the guanidine function with N-succinimidyl-4-18F-fluorobenzoate to give the guanidinyl amide N-(4-18F-fluoro-benzoyl)-N′-{2-[5-(4-fluoro-benzyl)-1-(4-methoxy-benzyl)-4,6-dioxo-1,4,5,6-tetrahydro-[1,3,5]triazin-2-ylamino]-ethyl}-guanidine (18F-PC-10). Inflammation was induced in C57BL/6 mice by subcutaneous injection of complete Freund adjuvant in the paw. The mice were imaged with 18F-PC-10, 18F-FDG, and 64Cu-pyruvaldehyde bis(4-methyl-3-thiosemicarbazone) (64Cu-PTSM) at 24 h after complete Freund adjuvant injection using a small-animal PET device. Results: 18F-PC-10 was synthesized with a radiochemical yield of 16% ± 3% (decay-corrected). 18F-PC-10 accumulated specifically in the inflamed paw 4- to 5-fold more than in the control paw. Compared with 18F-PC-10, 18F-FDG and 64Cu-PTSM displayed higher accumulation in the inflamed paw but also had higher accumulation in the control paw, demonstrating a reduced signal-to-background ratio. 18F-PC-10 also accumulated in PKR1-expressing organs, such as the salivary gland and gastrointestinal tract. Conclusion: 18F-PC-10 can be used to image PKR1, a biomarker of the inflammation process. However, the high uptake of 18F-PC-10 in the gastrointestinal tract, due to specific uptake and the metabolic processing of this highly lipophilic molecule, would restrict its utility

Usefulness of [18F]-DA and [18F]-DOPA for PET imaging in a mouse model of pheochromocytoma

Purpose: To evaluate the usefulness of [ 18F]-6-fluorodopamine ([ 18F]-DA) and [ 18F]-L-6-fluoro-3,4-dihydroxyphenylalanine ([ 18F]-DOPA) positron emission tomography (PET) in the detection of subcutaneous (s.c.) and metastatic pheochromocytoma in mice; to assess the expression of the norepinephrine transporter (NET) and vesicular monoamine transporters 1 and 2 (VMAT1 and VMAT2), all important for [ 18F]-DA and [ 18F]-DOPA uptake. Furthermore, to compare tumor detection by micro-computed tomography (microCT) to magnetic resonance imaging (MRI) in individual mouse. Methods: SUV max values were calculated from [ 18F]-DA and [ 18F]-DOPA PET, tumor-to-liver ratios (TLR) were obtained and expression of NET, VMAT1 and VMAT2 was evaluated. Results: [ 18F]-DA detected less metastatic lesions compared to [ 18F]-DOPA. TLR values for liver metastases were 2.26-2.71 for [ 18F]-DOPA and 1.83-2.83 for [ 18F]-DA. A limited uptake of [ 18F]-DA was found in s.c. tumors (TLR=0.22-0.27) compared to [ 18F]-DOPA (TLR=1.56-2.24). Overall, NET and VMAT2 were expressed in all organ and s.c. tumors. However, s.c. tumors lacked expression of VMAT1. We confirmed [ 18F]-DA's high affinity for the NET for its uptake and VMAT1 and VMAT2 for its storage and retention in pheochromocytoma cell vesicles. In contrast, [ 18F]-DOPA was found to utilize only VMAT2. Conclusion: MRI was superior in the detection of all organ tumors compared to microCT and PET. [ 18F]-DOPA had overall better sensitivity than [ 18F]-DA for the detection of metastases. Subcutaneous tumors were localized only with [ 18F]-DOPA, a finding that may reflect differences in expression of VMAT1 and VMAT2, perhaps similar to some patients with pheochromocytoma where [ 18F]-DOPA provides better visualization of lesions than [ 18F]-DA