Andrei O. Koren

2-[18F]F-A-85380: PET imaging of brain nicotinic acetylcholine receptors and whole body distribution in humans

Noninvasive imaging of nicotinic acetylcholine receptors (nAChRs) in the human brain in vivo is critical for elucidating the role of these receptors in normal brain function and in the pathogenesis of brain disorders. Here we report the first in vivo visualization of human brain areas containing nAChRs by using PET and 2‐[18F]fluoro‐3‐(2(S)azetidinylmethoxy)pyridine (2‐[18F]FA). We acquired scans from six healthy non‐smoking volunteers after i.v. bolus administration of 2‐[18F]FA (1.6 MBq/kg or 0.043 ± 0.002 mCi/kg). This dose was sufficient for visualizing nAChRs in the thalamus up to 5 h after injection. There were no adverse effects associated with administration of no‐carrier‐added 2‐[18F]FA (1.3‐10 pmol/kg). Consistent with the distribution of nAChRs in human brain, accumulated radioactivity was greatest in thalamus, intermediate in the midbrain, pons, cerebellum, and cortex; and least in white matter. As ˜90% of the injected radioactivity was eliminated via the urine (biological half‐life ca. 4 h), the urinary bladder wall received the highest radiation dose. The estimate of radiation dose equivalent to the urinary bladder wall (ca. 180 ± 30 mSv/MBq or 0.7 rem/mCi with a 2.4 h void interval) suggests that multiple studies could be performed in a single subject. The results predict that quantitative PET imaging of nAChRs in human brain with 2‐[18F]FA is feasible.

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 Alzheimers and Parkinsons diseases and other CNS disorders (e.g. Tourettes 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.

2-[18F]fluoro-A-85380, an in vivo tracer for the nicotinic acetylcholine receptors

6-[18F]Fluoro-3-(2(S)-azetidinylmethoxy)pyridine (6-[18F]fluoro-A-85380 or 6-[18F]FA), a new tracer for positron emission tomography, was synthesized by no-carrier-added [18F] fluorination of 6-iodo-3-((1-tert-butoxycarbonyl-2(S)-azetidinyl)methoxy)pyridine followed by acidic deprotection. 6-[18F]FA followed the regional densities of brain nicotinic acetylcholine receptors (nAChRs) reported in the literature. Evidence of binding to nAChRs and high specificity of the binding in vivo was demonstrated by inhibition with nAChR selective ligands as well as with unlabeled 6-FA. A preliminary toxicology study of the 6-FA showed a relatively low biological effect.

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.

Synthesis of a radiotracer for studying nicotinic acetylcholine receptors: 2-[18F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[18F]A-85380)

The radiochemical synthesis of 2-[18F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[18F]A-85380, [18F]1) was accomplished by Kryptofix® 222 assisted nucleophilic no-carrier-added [18F]fluorination of 2-iodo-3((1-tert-butoxycarbonyl-2(S)-azetidinyl)methoxy)pyridine, 2 followed by acidic deprotection. The average radiochemical yield was 10% and the average specific radioactivity was 1050 mCi/μmol, calculated at end-of-synthesis (EOS).

6-[18F]fluoro-A-85380, a novel radioligand for in vivo imaging of central nicotinic acetylcholine receptors.

A novel positron emission tomography (PET) radiotracer, 6-[18F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (6-[18F]fluoro-A-85380, 6-[18F]FA) was synthesized by a no-carrier-added fluorination. In vitro 6-[18F]FA bound to nicotinic acetylcholine receptors (nAChRs), with very high affinity (Kd 28 pM). In PET studies, 6-[18F]FA specifically labeled central nAChRs in the brain of the Rhesus monkey and demonstrated highest levels of accumulation of radioactivity in brain regions enriched with the alpha4beta2 subtype of nAChR. 6-[18F]FA exhibited a target-to-non-target ratio (estimated as radioactivity in the thalamus to that in the cerebellum) of binding in primate brain similar to that previously determined for a labeled analog of epibatidine, [18F]FPH. In contrast to [18F]FPH, the novel tracer is expected to exhibit substantially less toxicity. Thus, the novel radioligand, 6-[18F]FA, appears to be a suitable candidate for imaging nAChRs in human brain.

2-[18F]F: A-85380 : a PET radioligand for α4β2 nicotinic acetylcholine receptors

2-[18F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[18F]F-A-85380), a ligand for nicotinic acetylcholine receptors (nAChRs) was evaluated in an in vitro binding assay with membranes of rat brain and in vivo by PET in Rhesus monkey brain. The ligand has high affinity for alpha4beta2 nAChRs (K(D)=50 pM), crosses the blood-brain barrier, and distributes in the monkey brain in a pattern consistent with that of alpha4beta2 nAChRs. The specific/non-specific binding ratio increased steadily, reaching a value of 3.3 in the thalamus at 4 h. The specific binding of 2-[18F]F-A-85380 was reversed by cytisine. These results, in combination with the data demonstrating low toxicity of 2-[18F]F-A-85380, indicate that this ligand shows promise for use with PET in human subjects.

In vivo studies with [125I]5-I-A-85380, a nicotinic acetylcholine receptor radioligand.

5-[125I]iodo-3-(2(S)-azetidinylmethoxy)pyridine ([125I]5-I-A-85380) was evaluated in the mouse as a potential in vivo imaging ligand for central nicotinic acetylcholine receptors (nAChRs). After i.v. administration of [125I]5-I-A-85380, peak brain levels of radioactivity were measured within 1 h and declined slowly over 4 h. [125I]5-I-A-85380 binding was saturable, and both its pharmacology, based upon inhibition studies, and its pattern of accumulation in brain regions having high nAChR densities were consistent with an interaction at α4β2 nAChR agonist binding sites. The thalamus:cerebellum radioactivity ratio, a measure of specific labeling, reached 37. Therefore, radiolabeled 5-I-A-85380 has excellent potential as an imaging radiotracer for nAChRs, particularly with single photon emission computed tomography, when 123I is incorporated into the molecule.

Synthesis and characterization of binding of 5-[76Br]bromo-3-[[2(S)-azetidinyl]methoxy]pyridine, a novel nicotinic acetylcholine receptor ligand, in rat brain.

Abstract : 5‐[76Br]Bromo‐3‐[[2(S)‐azetidinyl]methoxy]‐pyridine ([76Br]BAP), a novel nicotinic acetylcholine receptor ligand, was synthesized using [76Br]bromide in an oxidative bromodestannylation of the corresponding trimethylstannyl compound. The radiochemical yield was 25%, and the specific radioactivity was on the order of 1 Ci/μmol. The binding properties of [76Br]BAP were characterized in vitro and in vivo in rat brain, and positron emission tomography (PET) experiments were performed in two rhesus monkeys. In association experiments on membranes of the cortex and thalamus, >90% of maximal specific [76Br]BAP binding was obtained after 60 min. The dissociation half‐life of [76Br]BAP was 51 ± 6 min in cortical membranes and 56 ± 3 min in thalamic membranes. Saturation experiments with [76Br]BAP revealed one population of binding sites with dissociation constant (KD) values of 36 ± 9 and 30 ± 9 pM in membranes of cortex and thalamus, respectively. The maximal binding site density (Bmax) values were 90 ± 17 and 207 ± 33 fmol/mg in membranes of cortex and thalamus, respectively. Scatchard plots were nonlinear, and the Hill coefficients were <1, suggesting the presence of a lower‐affinity binding site. In vitro autoradiography studies showed that binding of [76Br]BAP was high in the thalamus and presubiculum, moderate in the cortex and striatum, and low in the cerebellum and hippocampus. A similar pattern of [76Br]BAP accumulation was observed by ex vivo autoradiography. In vivo, binding of [76Br]BAP in whole rat brain was blocked by preinjection of (S)(‐)‐nicotine (0.3 mg/kg) by 27, 52, 68, and 91% at survival times of 10, 25, 40, 120, and 300 min, respectively. In a preliminary PET study in rhesus monkeys, the highest [76Br]BAP uptake was found in the thalamus, and radioactivity was displaceable by ~60% with cytisine and by 50% with (S)(‐)‐nicotine. The data of this study indicate that [76Br]BAP is a promising radioligand for the characterization of nicotinic acetylcholine receptors in vivo.

Synthesis and initial in vitro characterization of 6-[18F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine, a high-affinity radioligand for central nicotinic acetylcholine receptors

6-[18F]Fluoro-3-(2(S)-azetidinylmethoxy)pyridine ([18F]1), a novel analogue of the high-affinity nicotinic acetylcholine receptor ligand, A-85380, was prepared by a two-step procedure from an appropriate nitro precursor. In the first step, a Kryptofix 222-assisted 18F nucleophilic heteroaromatic substitution in 6-nitro-3-((1-tert-butoxycarbonyl-2(S)-azetidinyl)methoxy)pyridine provided a radio-fluorinated Boc-protected intermediate. Subsequent acidic deprotection of the intermediate gave [18F]1 with an overall radiochemical yield of 8 to 12% (non-decay-corrected). The typical synthesis time was ca. Specific radioactivity of the final product ranged from 1000 to 4500 mCi/μmol, as calculated at the end-of-synthesis. In vitro studies demonstrated that the novel radioligand bound to a single population of sites in rat brain membranes, presumably, to the α4β2 subtype of nicotinic acetylcholine receptor. This binding was characterized by a Kd value of 28 pM, consistent with the Ki value of 25 pM, derived previously for unlabeled 1 in competition assays with (±)-[3H]epibatidine Copyright