In Vitro and In Vivo Characterization of Two C-11-Labeled PET Tracers for Vesicular Acetylcholine Transporter

PurposeThe vesicular acetylcholine transporter (VAChT) is a specific biomarker for imaging presynaptic cholinergic neurons. Herein, two potent and selective (11)C-labeled VAChT inhibitors were evaluated in rodents and nonhuman primates for imaging VAChT in vivo.ProceduresFor both (-)-[(11)C]2 and (-)-[(11)C]6, biodistribution, autoradiography, and metabolism studies were performed in male Sprague Dawley rats. Positron emission tomography (PET) brain studies with (-)-[(11)C]2 were performed in adult male cynomolgus macaques; 2 h dynamic data was acquired, and the regions of interest were drawn by co-registration of the PET images with the MRI.ResultsThe resolved enantiomers (-)-2 and (-)-6 were very potent and selective for VAChT in vitro (K i  < 5 nM for VAChT with >35-fold selectivity for VAChT vs. σ receptors); both radioligands, (-)-[(11)C]2 and (-)-[(11)C]6, demonstrated high accumulation in the VAChT-enriched striatum of rats. (-)-[(11)C]2 had a higher striatum to cerebellum ratio of 2.4-fold at 60 min; at 30 min, striatal uptake reached 0.550 ± 0.086 %ID/g. Uptake was also specific and selective; following pretreatment with (±)-2, striatal uptake of (-)-[(11)C]2 in rats at 30 min decreased by 50 %, while pretreatment with a potent sigma ligand had no significant effect on striatal uptake in rats. In addition, (-)-[(11)C]2 displayed favorable in vivo stability in rat blood and brain. PET studies of (-)-[(11)C]2 in nonhuman primates indicate that it readily crosses the blood-brain barrier (BBB) and provides clear visualization of the striatum; striatal uptake reaches the maximum at 60 min, at which time the target to nontarget ratio reached ~2-fold.ConclusionsThe radioligand (-)-[(11)C]2 has high potential to be a suitable PET radioligand for imaging VAChT in the brain of living subjects

Synthesis and biological characterization of a promising F-18 PET tracer for vesicular acetylcholine transporter

Nine fluorine-containing vesicular acetylcholine transporter (VAChT) inhibitors were synthesized and screened as potential PET tracers for imaging the VAChT. Compound 18a was one of the most promising carbonyl-containing benzovesamicol analogues; the minus enantiomer, (-)-18a displayed high potency (VAChT K(i) = 0.59 ± 0.06 nM) and high selectivity for VAChT versus receptors (> 10,000-fold). The radiosynthesis of (-)-[(18)F]18a was accomplished by a two-step procedure with 30 – 40% radiochemical yield. Preliminary biodistribution studies of (-)-[(18)F]18a in adult male Sprague–Dawley rats at 5, 30, 60 and 120 min post-injection (p.i.) were promising. The total brain uptake of (-)-[(18)F]18a was 0.684 ID%/g at 5 min p.i. and by 120 min p.i. slowly washed out to 0.409 %ID/g.; evaluation of regional brain uptake showed stable levels of ~0.800 %ID/g from 5 to 120 min p.i in the VAChT-enriched striatal tissue of rats, indicating the tracer had crossed the blood brain barrier and was retained in the striatum. Subsequent microPET brain imaging studies of (-)-[(18)F]18a in nonhuman primates (NHPs) showed high striatal accumulation in the NHP brain; the standardized uptake value (SUV) for striatum reached a maximum value of 5.1 at 15 min p.i. The time-activity curve for the target striatal region displayed a slow and gradual decreasing trend 15 min after injection, while clearance of the radioactivity from the cerebellar reference region was much more rapid. Pretreatment of NHPs with 0.25 mg/kg of the VAChT inhibitor (-)-vesamicol resulted in a ~90% decrease of striatal uptake compared to baseline studies. HPLC metabolite analysis of NHP plasma revealed that (-)-[(18)F]18a had a good in vivo stability. Together, these preliminary results suggest (-)-[(18)F]18a is a promising PET tracer candidate for imaging VAChT in the brain of living subjects

Heteroaromatic and Aniline Derivatives of Piperidines As Potent Ligands for Vesicular Acetylcholine Transporter

To identify suitable lipophilic compounds having high potency and selectivity for vesicular acetylcholine transporter (VAChT), a heteroaromatic ring or a phenyl group was introduced into the carbonyl-containing scaffold for VAChT ligands. Twenty new compounds with ALogD values between 0.53 and 3.2 were synthesized, and their in vitro binding affinities were assayed. Six of them (<b>19a</b>, <b>19e</b>, <b>19g</b>, <b>19k</b>, and <b>24a</b>–<b>b</b>) displayed high affinity for VAChT (<i>K</i>_i = 0.93–18 nM for racemates) and moderate to high selectivity for VAChT over σ₁ and σ₂ receptors (<i>K</i>_i = 44–4400-fold). These compounds have a methyl or a fluoro substitution that provides the position for incorporating PET radioisotopes C-11 or F-18. Compound (−)-[¹¹C]<b>24b</b> (<i>K</i>_<i>i</i> = 0.78 nM for VAChT, 1200-fold over σ receptors) was successfully synthesized and evaluated in vivo in rats and nonhuman primates. The data revealed that (−)-[¹¹C]<b>24b</b> has highest binding in striatum and has favorable pharmacokinetics in the brain