Development of ligands for in vivo imaging of cerebral nicotinic receptors.

Nicotinic acetylcholine receptors (nAChRs) mediate a variety of brain functions. Findings from postmortem studies and clinical investigations have implicated them in the pathophysiology and treatment of Alzheimer's and Parkinson's diseases and other CNS disorders (e.g. Tourette's syndrome, epilepsy, nicotine dependence). Therefore, it ultimately might be useful to image nAChRs noninvasively for diagnosis, for studies on how changes in nAChRs might contribute to cerebral disorders, for development of therapies targeted at nAChRs, and to monitor the effects of such treatments. To date, only (S)-(-)-nicotine, radiolabeled with 11C, has been used for external imaging of nAChRs in human subjects. Since this radiotracer presents drawbacks, new ligands, with more favorable properties, have been synthesized and tested. Three general classes of compounds, namely, nicotine and its analogs, epibatidine and related compounds, and 3-pyridyl ether compounds, including A-85380, have been evaluated. Analogs of A-85380 appear to be the most promising candidates because of their low toxicity and high selectivity for the alpha4beta2 subtype of nAChRs.

5-Iodo-A-85380, an alpha4beta2 subtype-selective ligand for nicotinic acetylcholine receptors.

In an effort to develop selective radioligands for in vivo imaging of neuronal nicotinic acetylcholine receptors (nAChRs), we synthesized 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-iodo-A-85380) and labeled it with (125)I and (123)I. Here we present the results of experiments characterizing this radioiodinated ligand in vitro. The affinity of 5-[(125)I]iodo-A-85380 for alpha4beta2 nAChRs in rat and human brain is defined by K(d) values of 10 and 12 pM, respectively, similar to that of epibatidine (8 pM). In contrast to epibatidine, however, 5-iodo-A-85380 is more selective in binding to the alpha4beta2 subtype than to other nAChR subtypes. In rat adrenal glands, 5-iodo-A-85380 binds to nAChRs containing alpha3 and beta4 subunits with 1/1000th the affinity of epibatidine, and exhibits 1/60th and 1/190th the affinity of epibatidine at alpha7 and muscle-type nAChRs, respectively. Moreover, unlike epibatidine and cytisine, 5-[(125)I]iodo-A-85380 shows no binding in any brain regions in mice homozygous for a mutation in the beta2 subunit of nAChRs. Binding of 5-[(125)I]iodo-A-85380 in rat brain is reversible, and is characterized by high specificity and a slow rate of dissociation of the receptor-ligand complex (t(1/2) for dissociation approximately 2 h). These properties, along with other features observed previously in in vivo experiments (low toxicity, rapid penetration of the blood-brain barrier, and a high ratio of specific to nonspecific binding), suggest that this compound, labeled with (125)I or (123)I, is superior to other radioligands available for in vitro and in vivo studies of alpha4beta2 nAChRs, respectively.

Radiosynthesis and preliminary evaluation of 5-[123/125I]iodo-3-(2(S)-azetidinylmethoxy)pyridine: a radioligand for nicotinic acetylcholine receptors.

The radiochemical syntheses of 5-[125I]iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-[125I]-iodo-A-85380, [125I]1) and 5-[123I]-iodo-A-85380, [123I]1, were accomplished by radioiodination of 5-trimethylstannyl-3-((1-tert-butoxycarbonyl-2(S)-azetidinyl)metho xy)pyridine, 2, followed by acidic deprotection. Average radiochemical yields of [125I]1 and [123I]1 were 40-55%; and the average specific radioactivities were 1,700 and 7,000 mCi/mumol, respectively. Binding affinities of [125I]1 and [123I]1 in vitro (rat brain membranes) were each characterized by a Kd value of 11 pM. Preliminary in vivo assay and ex vivo autoradiography of mouse brain indicated that [125I]1 selectively labels nicotinic acetylcholine receptors (nAChRs) with very high affinity and specificity. These studies suggest that [123I]1 may be useful as a radioligand for single photon emission computed tomography (SPECT) imaging of nAChRs.

In vivo imaging of brain nicotinic acetylcholine receptors with 5-[123I]iodo-A-85380 using single photon emission computed tomography.

The distribution and kinetics of 5-[123I]iodo-A-85380, a novel ligand for brain nicotinic acetylcholine receptors (nAChRs), were evaluated in the Rhesus monkey using single photon emission computed tomography (SPECT). Peak levels of radioactivity were measured in brain at 90 min after injection of the tracer. Accumulation of radioactivity was highest in the thalamus, intermediate in the frontal cortex and basal ganglia, and lowest in the cerebellum. The ratio of specific to nonspecific binding (V3") in the thalamus, estimated from the (thalamic-cerebellar)/cerebellar radioactivity ratio, reached a value of 6 at 4 h post-injection. Specific binding was reduced by subcutaneous injection of 1 mg/kg cytisine at 2.25 h after injection of radiotracer. At 2.5 h after cytisine administration, radioactivity in the thalamus was reduced by 84%, in the frontal cortex, by 76%, and in the basal ganglia, by 57% of the level measured at the time of cytisine administration, demonstrating that the binding was reversible. On the basis of these findings, together with other data indicating high affinity, receptor subtype selectivity, low nonspecific binding and lack of toxicity in animals, 5-[123I]iodo-A-85380 appears to be a promising ligand for SPECT imaging of nAChRs in the human brain.