The PET Dept has now performed the seminal experiment to prove that the radioligand F-18 FP-TZTP selectively binds to the M2 subtype muscarinic cholinergic receptor. M2 muscarinic receptors in frontal cortex and hippocampus are reported to decrease in Alzheimer?s disease. Availability of selective M2 receptor ligands with positron-emitting labels would enable PET studies to assess the progression of the disease. (F-18]FP-TZTP) is a muscarinic agonist with reported M2 selectivity. A muscarinic receptor radioligand, 3-(3-(3-fluoropropyl)thio) -1,2,5,thiadiazol-4-yl)-1,2,5,6-tetrahydro- 1-methylpyridine (FP-TZTP) radiolabeled with the positron emitting radionuclide F-18 ([F-18]FP-TZTP) displayed regional brain distribution consistent with M2 receptor densities in rat brain. The purpose of the present study is to further elucidate the subtype selectivity of [F-18]FP-TZTP using genetically engineered mice which lacked functional M1, M2, M3, or M4 muscarinic receptors. Using ex vivo autoradiography, the regional brain localization of [F-18]FP-TZTP in M2 knockout (M2 KO) was significantly decreased (51.3 to 61.4%; P< 0.01) when compared to the wild-type (WT) mice in amygdala, brain stem, caudate putamen, cerebellum, cortex, hippocampus, hypothalamus, superior colliculus, and thalamus. In similar studies with M1KO, M3KO and M4KO compared to their WT mice, [F-18]FP-TZTP uptakes in the same brain regions were not significantly decreased at P < 0.01. However, in amygdala and hippocampus small decreases of 19.5% and 22.7%, respectively, were observed for M1KO vs WT mice at P < 0.05. Given the fact that large decreases in [F-18]FP-TZTP brain uptakes were seen only in M2 KO vs WT mice, our data indicate that [F-18]FP-TZTP selectively labels M2 receptors in vivo. This radiopharmaceutical is currently in clinical trails in collaboration with NIMH scientists. Further clarification of the competitive blocking studies using nonradioactive [F-18]FP-TZTP was accomplished. Muscarinic agonists could alter CBF, but in PET studies [F-18]FP-TZTP is administered in tracer doses. Its specificity, however, was tested by competitive binding inhibition with loading doses of P-TZTP, the non-fluorinated analog of the labeled ligand and an established M2-selective agonist. Effects of loading doses of P-TZTP on CBF were, however, not yet determined. We, therefore, measured CBF with the [C-14]iodoantipyrine method and laser-Doppler flowmetry in rats following administration of either 50 or 500 nmols/rat of P-TZTP, doses used in the saturability studies. Arterial blood pressure (MABP) fell markedly immediately after P-TZTP administration but recovered within one minute, and cortical CBF fell and rose synchronously with MABP. Local CBF decreased significantly immediately after the P-TZTP injection in two cerebral structures but returned to normal by 30 min after administration of either dose. A decrease in CBF may enhance the effects of blocking doses of P-TZTP in saturability studies, thereby overestimating the percentage of specific binding, although this effect will be small. The PET department has also developed a radiolabeled nucleoside that can be used as either a proliferation agent or as a reporter probe. An ester form of the radiotracer has been developed to monitor proliferation in the brain in the presence of a normal blood brain barrier. [Br-76]FBAU is a potential PET tracer for assessing proliferation. This study proposes that [Br-76]FBAU 3',5'-dibenzoate has higher blood-brain-barrier permeability than [Br-76]FBAU itself and thus might be better suited for the application in brain. [Br-76]FBAU 3',5'-dibenzoate was relatively stable in the plasma, gradually being hydrolyzed to [Br-76]FBAU. Biodistribution in rat showed that [Br-76]FBAU 3',5'-dibenzoate had higher brain uptake (0.119?0.023 DUR at 1 h, versus 0.061?0.006 for [Br-76]FBAU, p = 0.003, N = 5). The brain uptake indexes measured after carotid injection (29.6?13.9 for [Br-76]FBAU 3',5'-dibenzoate, versus 10.0?8.7 for [76Br]FBAU, p = 0.012, N = 7) support this claim. The DNA incorporation of [Br-76]FBAU was also confirmed. The results presented support the hypothesis that the dibenzoyl esters will result in higher brain uptake. The PET Dept has also studied radiotracers in the new small animal imaging devices developed in the Dept Nucl Med. In vivo imaging using positron emission tomography (PET) is important in the development of new radiopharmaceuticals in rodent animal models for use as biochemical probes, diagnostic agents, or in drug development. If small animal imaging studies in rodents are to have the same """"""""quality"""""""" as human PET studies, the same number of coincidence events must be detected from a typical rodent imaging """"""""voxel"""""""" as from the human imaging voxel. To achieve this, roughly the same total amount of radiopharmaceutical must be given to the animal as to a human subject. At high specific activities, the mass associated with the human doses may not decrease the uptake of radioactivity at non saturable sites or sites where an enzyme has a high capacity for a substrate. However, in the case of binding sites of low density such as receptors, the increased mass injected could saturate the receptor and lead to physiologic effects and non-linear kinetics. Because of the importance of the mass injected for small animal PET imaging, we experimentally compared (by biodistribution and phosphorimaging) high and low mass preparations of three compounds: 2-fluoro-2-deoxyglucose (FDG), 6-fluoro-L-metatyrosine (FMT) and a receptor-directed compound, the serotonin 5HT1A receptor ligand, trans 4-fluoro-N-{2-[4-(2-methoxylphenyl) piperazino]ethyl}-N-(2-pyridyl) cyclohexanecarboxamide (FCWAY). Changes in the mass injected per rat did not affect the distribution of FDG, FMT, and, in the range of 0.6 nmol to 1.9 nmol, FCWAY. Large changes in the target to nontarget ratio were calculated for injected masses of FCWAY in the range of ~5 nmol per rat. If the specific activity of such compounds and/or the sensitivity of small animal scanners are not increased relative to human studies, small animal PET imaging will not correctly portray the """"""""true"""""""" tracer distribution. These difficulties will only be exacerbated in animals smaller than the rat, e.g. mice. The PET Dept has employed liquid chromatography/ mass spectrometry to develop new analytical techniques that define the chemistry of metabolites formed in vitro and in vivo. The PET Dept has previously reported the development of fluorine-18 radiolabeled FCWAY (trans 4-FCWAY [(N-(2-(1-(4-(2-methoxyphenyl)-1-piperazinyl) ethyl)-N-(2 pyridyl) trans 4-fluorocyclohexanecarboxamide]) as a high affinity ligand for imaging the 5HT-1A receptor in vivo. We have developed three new analogues of FCWAY in a search for radiopharmaceuticals with unique imaging applications using positron emission tomography (PET). Two of the analogues were generated by replacing the fluorocyclohexane carboxylic acid with fluorobenzoic acid (FBWAY) or with 3-methyl-4-fluorobenzoic acid (MeFBWAY). The final analogue was generated by replacing the pyridyl group with a pyrimidyl group and the fluorocyclohexane carboxylate with fluorobenzoic acid (FPWAY). We evaluated the metabolic profile of these compounds using either human or rat hepatocytes to produce metabolites and LC/MS/MS to identify these metabolites. These in vitro metabolism studies indicate that hydrolysis of the amide linkage is the major metabolic pathway for FPWAY and FBWAY in human hepatocytes, whereas aromatic ring-oxidation is the major metabolic pathway for MeFBWAY. Aromatic ring-oxidation is the major metabolic pathway for all three analogs in rat hepatocytes. The value of these in vitro metabolic studies is the ability to demonstrate species differences prior to the acquistion and interpretation of in vivo resul
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