The role of species-dependent metabolism in the regional brain retention of 18F-labeled muscarinic acetylcholine receptor ligands.

We have developed PET radioligands for the muscarinic acetylcholine receptor designed to be sensitive to endogenous acetylcholine changes. These radioligands were based on the piperidyl and pyrrolidyl benzilate scaffold and include (R)-N-(2-[18F]fluoroethyl)-3-piperidyl benzilate (1b), (R)-N-(2-[18F]fluoroethyl)-3-pyrrolidyl benzilate (2b), and N-(2-[18F]fluoroethyl)-4-piperidyl benzilate (3b). In the mouse, intravenous injection of 2b produced a heterogeneous receptor-mediated regional retention of radioactivity, whereas in the rat a homogeneous brain distribution was observed. Analyses of blood and brain extracts showed a radiolabeled metabolite for 2b which was formed to a much greater extent in mice than rats. This metabolite may have a higher receptor binding affinity than authentic 2b, and thus be responsible for the apparent receptor-mediated binding in the mouse brain. Our findings emphasize the importance of metabolite analysis in multiple species when developing novel radiopharmaceuticals for in vivo use.

Acetylcholinesterase inhibition increases in vivo N-(2-[18F]fluoroethyl)-4-piperidyl benzilate binding to muscarinic acetylcholine receptors.

Although the inhibition of acetylcholinesterase remains the primary treatment of Alzheimer's disease, little is known of the results of increased acetylcholine levels on muscarinic receptor occupancy or function. Using N-(2-[18F]fluoroethyl)-4-piperidyl benzilate ([18F]FEPB), a moderate affinity (Ki = 1.7 nmol/L) nonsubtype-selective muscarinic receptor antagonist, the authors examined the sensitivity of equilibrium in vivo radioligand binding in rat brain with changes in endogenous acetylcholine levels produced by treatments with acetylcholinesterase inhibitors. Phenserine administration 30 minutes before resulted in a dose-dependent into muscarinic cholinergic receptors, reaching a maximum increase of 90% in the striatum at a dose of 5 mg/kg intraperitoneally. Constant infusion of physostigmine at a dosage of 250 microg/kg/min produced an identical increase in radioligand binding. This agonist-induced increase of in vivo mAChR radioligand binding offers a new method for monitoring of the efficacy of acetylcholinesterase inhibitors or other drugs to enhance acetylcholine actions at the muscarinic receptors.

Radiolabeled cholinesterase substrates: in vitro methods for determining structure-activity relationships and identification of a positron emission tomography radiopharmaceutical for in vivo measurement of butyrylcholinesterase activity.

There is currently great interest in developing radiolabeled substrates for acetylcholinesterase and butyrylcholinesterase that would be useful in the in vivo imaging of patients with Alzheimer's disease. Using a simple in vitro spectrophotometric assay for determination of enzymatic cleavage rates, the structure-activity relationship for a short series of 1-methyl-4-piperidinyl esters was investigated. Relative enzymatic hydrolysis rates for the well-characterized 1-methyl-4-piperidinyl acetate, propionate, and i-butyrate esters were in agreement with literature values. The 4 and 5 carbon esters of 1-methyl-4-piperidinol were specific for butyrylcholinesterase and cleaved in the rank order n-valerate > n-butyrate >> 2-methylbutyrate, iso-valerate. These spectrophotometric results were also in agreement with in vitro hydrolysis rates in mouse blood and with in vivo regional retention of radioactivity in mouse brain of 11C-labeled analogs. Brain uptake and apparent enzymatic rate constants for 1-[11C]methyl-4-piperidinyl n-butyrate and n-valerate were calculated from in vivo measurements in M. nemistrina using positron emission tomography. Based on higher brain uptake of radioactivity and superior pharmacokinetics, 1-[11C]methyl-4-piperidinyl n-butyrate was identified as a new radiopharmaceutical for the in vivo measurement of butyrylcholinesterase activity.

Synthesis, (18)F-labeling, and biological evaluation of piperidyl and pyrrolidyl benzilates as in vivo ligands for muscarinic acetylcholine receptors.

A series of 31 compounds based on the piperidyl or pyrrolidyl benzilate scaffold were prepared from methyl benzilate and 4-piperidinol, (R)-(+)-3-piperidinol, or (R)-(+)-3-pyrrolidinol. Amine substituents included alkyl and aralkyl groups. In vitro K(i) values ranged from 0.05 nM to >100 nM. (R)-N-(2-Fluoroethyl)-3-piperidyl benzilate (3-FEPB, 22, K(i) = 12.1 nM) and N-(2-fluoroethyl)-4-piperidyl benzilate (4-FEPB, 8, K(i) = 1. 83 nM) were selected for radiolabeling with fluorine-18. Using alkylation with 2-[(18)F]fluoroethyl triflate, 3-[(18)F]FEPB (42) and 4-[(18)F]FEPB (43) were produced in 7-9% radiochemical yield and >97% radiochemical purity. For in vivo studies, retention was moderate in mouse brain for 42; however, blocking with scopolamine showed that uptake was not muscarinic cholinergic receptor-mediated. Conversely, 43 exhibited high, receptor-mediated retention in mouse brain, with significant clearance after 1 h. These results suggest that 43 could have applications as an in vivo probe for measuring endogenous acetylcholine levels.

Radiochemical synthesis and tissue distribution of Tc-99m-labeled 7alpha-substituted estradiol complexes.

The diagnosis and staging of breast cancer could be improved by the development of radiopharmaceutical imaging agents that provide a noninvasive determination of the estrogen receptor (ER) status of tumor cells. Agents labeled with (99m)Tc would be especially valuable in this regard. In attempting to achieve this goal, we synthesized four (99m)Tc-labeled 7alpha-substituted estradiol complexes. One complex utilizes the "3+1" mixed ligand design to introduce the Tc metal, whereas the other three took advantage of the cyclopentadienyltricarbonylmetal (CpTM) design. The Tc moieties were attached to the 7alpha position of estradiol with a hexyl tether, a monoether tether, or a polyether tether. The corresponding rhenium compounds have binding affinities for the ER of 20-45% compared with estradiol. Radiochemical yields of the (99m)Tc-labeled compounds ranged from approximately 15% for the CpT-Tc complexes to 95% for the 3 + 1 inorganic complex. Tissue distribution studies in immature female rats showed low nonreceptor-mediated uptake in the target organs and high uptake in nontarget organs such as the liver and fat. These complexes represent the first time that estradiol has been labeled at the 7alpha position with (99m)Tc and provide a further refinement of our understanding of ligand structure-binding affinity correlations for the ER.

Synthesis of novel progestin-rhenium conjugates as potential ligands for the progesterone receptor.

To assist in the development of technetium-based radiopharmaceuticals that are useful for the diagnostic imaging of steroid receptor-positive breast tumors, we have synthesized a series of small-sized metal chelates according to 'n + 1' mixed-ligand, thioether-carbonyl and organometallic designs. In these preliminary investigations, rhenium was used as a model for the radioactive technetium. The metal chelates contain the rhenium metal in several oxidation states, being + 5, + 3, and + 1, and they were attached to 21-substituted progesterone derivatives. A competitive receptor-binding assay (rat uterine cytosol, 0 degrees C) was used to determine the binding affinity of these conjugates for the progesterone receptor. The highest affinity of 9% (RU5020 = 100%) was obtained with a '3 + 1' mixed-ligand complex, containing a NMe group as the central donor atom in the tridentate ligand part. This value reflects a relative binding affinity of 75% compared with the parent steroid progesterone.

Integrated "3+1" oxorhenium(V) complexes as estrogen mimics.

The diagnosis and staging of breast cancer could be improved by the development of imaging radiopharmaceuticals that provide a noninvasive determination of the estrogen receptor (ER) status of tumor cells. Toward this goal, we have synthesized a number of integrated "3+1" oxorhenium(V) complexes designed to mimic estradiol and a class of nonsteroidal estrogens, the tetrahydrochrysenes (THC). The monodentate component of the estradiol mimic is a p-hydroxyphenethyl thiol ligand with ethyl substituents at the benzylic and homobenzylic positions. Model complexes of this ligand were easily made, but steric hindrance of the secondary thiol prevented the formation of the complex with the disubstituted ligand. The three "3+1" oxorhenium(V) complexes prepared to mimic the THC class mimics represent the first pyridinedithiol rhenium complexes of their kind to be made. These complexes are quite stable to air and moisture. The target tridentate ligand was prepared from chelidamic acid, and the VT NMR of the rhenium complex displays interesting fluxional behavior. The binding affinities of these complexes for the estrogen receptor are low, and their lipophilicities are rather high. Nevertheless, our findings provide a further refinement of our understanding of ligand structure-binding affinity correlations for the estrogen receptor.