Synthesis and pharmacological characterization of 4-[125I]-N-(N-benzylpiperidin-4-yl)-4-iodobenzamide: a high affinity sigma receptor ligand for potential imaging of breast cancer.

The synthesis of [125I]-N-(N-benzylpiperidin-4-yl)-4-iodobenzamide (4-[125I]BP), a novel radiopharmaceutical that possesses high affinity for both sigma-1 and sigma-2 receptor subtypes, and its binding characteristics to MCF-7 breast cancer cells are described. To obtain high yields (with high specific activity) of radioiodinated ligand, (N-benzylpiperidin-4-yl)-4-tri-butylstannyl benzamide was synthesized. Radiolabeled 4-[125I]BP was prepared from tri-butylstannyl precursor with the use of chloramine-T or hydrogen peroxide as an oxidizing agent in high yields (71-86%). The competition binding studies of 4-[125I]BP in MCF-7 breast tumor cells with haloperidol and DTG (known sigma ligands) showed a dose-dependent displacement and high affinity binding (Ki = 4.6 and 56 nM, respectively), demonstrating that sigma receptors are expressed in MCF-7 breast tumor cells. Scatchard analysis of 4-[125I]BP binding in MCF-7 cells revealed saturable binding, with a Kd = 26 nM and a Bmax = 4000 fmol/mg protein. Furthermore, the Scatchard analysis of [3H]DTG binding in MCF-7 cells gave a Kd of 24.5 nM and a Bmax of 2071 fmol/mg of protein. The biodistribution and clearance of 4-[125I]BP was studied in rats. The radiopharmaceutical cleared quickly from the blood pool but rather slowly from the hepatobiliary system. The in vivo specificity was demonstrated by blocking the receptor binding in the presence of haloperidol. A decrease of 55, 63, 43, and 68% was found at 1 h postinjection in brain, kidney, heart, and lung, respectively. These results demonstrate that a high density of sigma receptors are expressed in MCF-7 cells and that radioiodinated 4-IBP may be useful for imaging breast cancer by targeting sigma sites.

Age-related diminution of dopamine antagonist-stimulated vesamicol receptor binding.

UNLABELLED: Previous studies of radiolabeled vesamicol receptor (VR) ligands suggest that the latter may be used in conjunction with dopamine D2 antagonists to measure changes in striatal cholinergic function. In this study, the effects of aging on vesicular acetylcholine storage/release were investigated with the high-affinity VR ligand (+)-meta-[125I)iodobenzyltrozamicol [(+)-[125I]MIBT]. METHODS: Male Fischer 344 rats (aged 3 and 24 mo) were injected either with a vehicle or a D2 antagonist [haloperidol or S-(-)-eticlopride]. At prescribed intervals thereafter, all animals were intravenously injected with 10 microCi of (+)-[125I]MIBT. Three hours after radiotracer injection, the animals were killed and their brains dissected. The concentration of radiotracer in the striatum, cortex and cerebellum were then determined. RESULTS: In control animals, comparable levels of (+)-[125I]MIBT were observed in corresponding brain regions of young adult and aged Fischer 344 rats. Moreover, in haloperidol- and S-(-)-eticlopride-treated young adult rats, striatal levels of (+)-[125I]MIBT were elevated by 35% and 66%, respectively, relative to controls. In contrast, haloperidol treatment failed to alter the striatal levels of (+)-[125I]MIBT in aged rats while S-(-)-eticlopride displayed a twofold reduction in potency in aged rats. CONCLUSION: Aging is associated with a reduction in striatal cholinergic plasticity or striatal cholinergic reserve and that the D2-stimulated increase in VR ligand binding is a functionally relevant parameter.

Comparative tissue distribution of conformationally restricted radioiodinated vesamicol receptor ligands.

Three conformationally restricted analogs of vesamicol, 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl]-spirol[1H-i nde ne-1,4'- piperidine] (5), 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl]-3,4- dihydrospiro[indene-1,4'-piperidine] (6) and 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl)-3,4- dihydrospiro[naphthalene-1(2H),4'-piperidine] (7), were labelled with iodine-125 and evaluated as potential radioligands for mapping vesamicol receptor (VR) density and cholinergic function in vivo. All compounds showed similar kinetics in most tissues. However, differences were observed in the brain. Although comparable levels of each corresponding enantiomeric pair were obtained initially in the brain, the levels of the dextrorotatory enantiomers (+)-5, (+)-6 and (+)-7 were found to decrease by 72-82% over a period of 3 h. In contrast, the brain levels of the corresponding levorotatory isomers were maintained throughout the duration of the experiment. Among the dextrorotatory isomers, (+)-6 showed the highest brain extraction, while (+)-7 showed the lowest. In tissue dissection experiments, the levels of (+)-5, (+)-6 and (+)-7 were highest in the striatum and moderate to low in the cortex and cerebellum. Co-administration of haloperidol with (+)-6 decreased the levels of the latter in the striatum by 27%, while the levels in the cortex and cerebellum were each reduced by 60%. In addition, haloperidol failed to affect the regional distribution of (+)-7 in the brain. However, both haloperidol and spiperone increased the striatal levels of (+)-5 by 67 and 76%, respectively, suggesting that the binding of this radioligand is related to cholinergic function.(ABSTRACT TRUNCATED AT 250 WORDS)

N-(3-Iodophenyl)trozamicol (IPHT) and related inhibitors of vesicular acetylcholine transport: synthesis and preliminary biological characterization.

Four isomeric N-(halophenyl)trozamicol analogues (6a-d) were synthesized and evaluated as potential vesicular acetylcholine transporter (VAChT) ligands. Of the four compounds, N-(3-bromophenyl) trozamicol (6b) and N-(3-iodophenyl)trozamicol (6d) displayed the highest affinity for the VAChT in vitro, whereas the para-substituted compound 6c showed the lowest affinity for this transporter. Tissue distribution studies of N-(3-[125I]iodophenyl)trozamicol ([125I]6d, [125I)IPHT) suggest that the central distribution of the latter is consistent with cholinergic innervation. However, only moderate target-to-background ratios were obtained, suggesting little improvement over the N-(halobenzyl)trozamicols described previously.

Measurement of functional cholinergic innervation in rat heart with a novel vesamicol receptor ligand.

Regional differences in cholinergic activity in the cardiac conduction system have been difficult to study. We tested the utility of (+)-m-[125I]iodobenzyl)trozamicol(+)-[125I]MIBT), a novel radioligand that binds to the vesamicol receptor located on the synaptic vesicle in presynaptic cholinergic neurons, as a functional marker of cholinergic activity in the conduction system. The (+)-[125I]MIBT was injected intravenously into four rats. Three hours later, the rats were killed and their hearts were frozen. Quantitative autoradiography was performed on 20-micron-thick sections that were subsequently stained for acetylcholinesterase to identify specific conduction-system elements. Marked similarities existed between (+)-[125I]MIBT uptake and acetylcholinesterase-positive regions. Optical densitometric analysis of regional (+)-[125I]MIBT uptake revealed significantly greater (+)-[125I]MIBT binding (nCi/mg) in the atrioventricular node (AVN) and His bundle regions compared with other conduction and contractile elements (AVN: 3.43 +/- 0.37; His bundle: 2.16 +/- 0.30; right bundle branch: 0.95 +/- 0.13; right atrium: 0.68 +/- 0.05; right ventricle: 0.57 +/- 0.03; and left ventricle: 0.57 +/- 0.03; p < 0.05 comparing conduction elements with ventricular muscle). This study demonstrates that (+)-[125I]MIBT binds avidly to cholinergic nerve tissue innervating specific conduction-system elements. Thus, (+)-[125I]MIBT may be a useful functional marker in studies on cholinergic innervation in the cardiac conduction system.

The vesamicol receptor ligand (+)-meta-[125I]iodobenzyltrozamicol [(+)-[125I]-MIBT] reveals blunting of the striatal cholinergic response to dopamine D2 receptor blockade in the 6-hydroxydopamine (6-OHDA)-lesioned rat: possible implications for Parkinson's disease.

Previous studies of radiolabelled vesamicol receptor (VR) ligands suggest that the latter may be used, in conjunction with dopamine D2 antagonists, to measure changes in striatal cholinergic function in vivo. In the present study, the radiolabelled VR ligand (+)-meta-[125I]iodobenzyltrozamicol {(+)-[125I]MIBT} was used to assess striatal cholinergic function in the unilateral 6-hydroxydopamine (6-OHDA)-treated rat. In control animals, the levels of this radiotracer monitored at 3 hr post injection displayed bilateral symmetry in the striatum, cerebral cortex and cerebellum. However, in animals pretreated with the dopamine antagonist spiperone (2 mg/kg ip), the radiotracer concentration in the striatal hemisphere ipsilateral to 6-OHDA lesion increased by 23% (p = 0.068) while the concentration in the contralateral striatum was elevated by 87% (p < 0.0001). Since the nigrostriatal dopaminergic system modulates striatal cholinergic function, and dopamine D2 receptor blockade is known to result in increased striatal cholinergic function, the refractoriness of striatal cholinergic neurons following the loss of nigrostriatal dopaminergic innervation confirms the existence of a dopaminergic-cholinergic imbalance in Parkinson's disease. Therefore the combination of a D2 antagonist and radiolabelled VR ligand may provide a potentially useful method for assessing the effects of dopamine depletion in Parkinson's disease.

M1, M3 and M5 muscarinic receptors stimulate mitogen-activated protein kinase.

We report here that the M1, M3 and M5 muscarinic acetylcholine receptor subtypes that have been shown to couple to phosphoinositide hydrolysis also activate the mitogen-activated protein kinase (MAPK). Pharmacological characterization as well as mechanistic details of the activation pathway are presented. Carbachol-induced MAPK activation was time- and concentration-dependent at all subtypes. Pharmacological characterization of the MAPK response revealed that McN-A-343 was a partial agonist at the M1 and M3 subtypes, and that pilocarpine was a partial agonist at the M3 and M5 receptors. Carbachol-mediated MAPK activation at these receptor subtypes was pertussis toxin and wortmannin insensitive. By contrast, both agents significantly inhibited carbachol-induced MAPK activation by the M2 muscarinic receptor subtype. Furthermore, two independent single point mutations in the M1 receptor attenuated carbachol-induced activation of MAPK. Activation of MAPK at the M1, M3 and M5 muscarinic receptor subtypes was not dependent on intracellular or extracellular Ca2+, but was partially dependent upon protein kinase C. These data suggest that activation of MAPK by M1, M3 and M5 muscarinic receptors involves protein kinase C-dependent and independent pathways.