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IntroductionDeficits in cholinergic function have been found in the aged brain and in neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD). The vesicular acetylcholine transporter (VAChT) is a reliable biomarker for the cholinergic system. We previously reported the initial in vitro and ex vivo characterization of (-)-[(11)C]TZ659 as a VAChT specific ligand. Here, we report the in vivo specificity, tracer kinetics, and dose-occupancy studies in the nonhuman primate brain.MethodsMicroPET brain imaging of (-)-[(11)C]TZ659 was performed under baseline conditions in two male macaques. Tracer kinetic modeling was carried out using a two-tissue compartment model (2TCM) and Logan plot with arterial blood input function and using a simplified reference tissue model (SRTM) and Logan plot (LoganREF) without blood input. Specificity for VAChT was demonstrated by pretreatment with (+)-pentazocine, (-)-vesamicol, or S-(-)-eticlopride. Target occupancy (Occ) was calculated following pretreatment with escalating doses of (-)-vesamicol.ResultsBaseline PET imaging revealed selective retention in the striatum with rapid clearance from the cerebellar hemispheres as a reference region. Total volume of distribution (VT) values derived from both 2TCM and Logan analysis with blood input revealed ~3-fold higher levels of (-)-[(11)C]TZ659 in the striatum than the cerebellar hemispheres. Injection of (-)-vesamicol either as a blocking or displacing agent significantly reduced striatal uptake of (-)-[(11)C]TZ659. In contrast, pretreatment with the sigma-1 ligand (+)-pentazocine had no impact. Pretreatment with the S-(-)-eticlopride, a dopamine D2-like receptor antagonist, increased striatal uptake of (-)-[(11)C]TZ659. Striatal binding potential (BPND, range of 0.33-1.6 with cerebellar hemispheres as the reference region) showed good correlation (r(2)=0.97) between SRTM and LoganREF. Occupancy studies found that ~0.0057 mg/kg of (-)-vesamicol produced 50% VAChT occupancy in the striatum.Conclusion(-)-[(11)C]TZ659 demonstrated specific and reversible VAChT binding and favorable pharmacokinetic properties for assessing the density of VAChT in the living brain.

BackgroundThis study aims to further evaluate the specificity and selectivity of [(18)F]FTC-146 and obtain additional data to support its clinical translation.MethodsThe binding of [(19)F]FTC-146 to vesicular acetylcholine transporter (VAChT) was evaluated using [(3)H]vesamicol and PC12(A123.7) cells in an in vitro binding assay. The uptake and kinetics of [(18)F]FTC-146 in S1R-knockout mice (S1R-KO) compared to wild-type (WT) littermates was assessed using dynamic positron emission tomography (PET) imaging. Ex vivo autoradiography and histology were conducted using a separate cohort of S1R-KO/WT mice, and radiation dosimetry was calculated from WT mouse data (extrapolated for human dosing). Toxicity studies in Sprague-Dawley rats were performed with a dose equivalent to 250× the anticipated clinical dose of [(19)F]FTC-146 mass.Results and discussionVAChT binding assay results verified that [(19)F]FTC-146 displays negligible affinity for VAChT (K i = 450 ± 80 nM) compared to S1R. PET images demonstrated significantly higher tracer uptake in WT vs. S1R-KO brain (4.57 ± 1.07 vs. 1.34 ± 0.4 %ID/g at 20-25 min, n = 4, p

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 analogs; the minus enantiomer, (-)-18a displayed high potency (VAChT Ki=0.59 ± 0.06 nM) and high selectivity for VAChT versus σ 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 0.25mg/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.

The loss of cholinergic neurons and synapses relates to the severity of dementia in several neurodegenerative pathologies; and the vesicular acetylcholine transporter (VAChT) provides a reliable biomarker of cholinergic function. We recently characterized and (11)C-labeled a new VAChT inhibitor, (-)-TZ659. Here we report the in vitro and ex vivo characterization of (-)-TZ659. A stably transfected PC12(A123.7) cell line which expresses human VAChT (hVAChT) was used for the in vitro binding characterization of (-)-[(3)H]TZ659. A saturated binding curve was obtained with Kd=1.97±0.30nM and Bmax=3240±145.9fmol/mg protein. In comparison, a PC12(A123.7) cell line that expresses mutant hVAChT showed decreased binding affinity (Kd=15.94±0.28nM). Competitive binding assays using a panel of other CNS ligands showed no inhibition of (-)-[(3)H]TZ659 binding. On the other hand, binding inhibitions were observed only using VAChT inhibitors (Ki=0.20-31.35nM). An in vitro assay using rat brain homogenates showed that (-)-[(3)H]TZ659 had higher binding in striatum than in cerebellum, with a target: non-target ratio>3.46. Even higher ex vivo striatum-to-cerebellum ratios (9.56±1.11) were observed using filtered homogenates of brain tissue after rats were injected intravenously with (-)-[(11)C]TZ659. Ex vivo autoradiography of (-)-[(11)C]TZ659 confirmed high striatal uptake, with a consistently high striatum-to-cerebellum ratio (2.99±0.44). In conclusion, (-)-TZ659 demonstrated high potency and good specificity for VAChT in vitro and in vivo. These data suggest that (-)-[(11)C]TZ659 may be a promising PET tracer to image VAChT in the brain.

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  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.

PurposeThe vesicular acetylcholine transporter (VAChT) is a specific biomarker for imaging presynaptic cholinergic neurons. The syntheses and C-11 labeling of two potent enantiopure VAChT inhibitors are reported here.ProceduresTwo VAChT inhibitors, (±)-2 and (±)-6, were successfully synthesized. A chiral HPLC column was used to resolve the enantiomers from each corresponding racemic mixture for in vitro characterization. The radiosyntheses of (-)-[(11)C]2 and (-)-[(11)C]6 from the corresponding desmethyl phenol precursor was accomplished using [(11)C]methyl iodide or [(11)C]methyl triflate, respectively.ResultsThe synthesis of (-)-[(11)C]2 was accomplished with 40-50 % radiochemical yield (decay-corrected), SA > 480 GBq/μmol (EOB), and radiochemical purity >99 %. Synthesis of (-)-[(11)C]6 was accomplished with 5-10 % yield, SA > 140 GBq/μmol (EOB), and radiochemical purity >97 %. The radiosynthesis and dose formulation of each tracer was completed in 55-60 min.ConclusionsTwo potent enantiopure VAChT ligands were synthesized and (11)C-labeled with good radiochemical yield and specific activity.

The σ1 receptor is an important target for CNS disorders. We previously identified a σ1 ligand TZ3108 having highly potent (Ki-σ1 = 0.48 nM) and selective affinity for σ1 versus σ2 receptors. TZ3108 was 18F-labeled with F-18 for in vivo evaluation. Biodistribution and blocking studies of [18F]TZ3108 in male Sprague-Dawley rats demonstrated high brain uptake, which was σ1-specific with no in vivo defluorination. MicroPET studies in cynomolgus macaques showed high brain penetration of [18F]TZ3108; the regional brain distribution was consistent with that of the σ1 receptor. Pseudo-equilibrium in the brain was reached ~ 45 min post-injection. Metabolite analysis of [18F]TZ3108 in NHP blood and rodent blood and brain revealed that ~ 70% parent remained in the plasma of NHPs 60 min post-injection and the major radiometabolite did not cross the blood-brain barrier in rats. In summary, the potent, selective and metabolically stable σ1 specific radioligand [18F]TZ3108 represents a potentially useful PET radioligand for quantifying the σ1 receptor in the brain.

The roles of 7 conserved ionic residues in the 12 putative transmembrane domains (TMDs) of vesicular acetylcholine transporter (VAChT) were investigated, and transport and ligand binding were characterized at different pH values using filter assays. The pH effects indicate that sites that must be unprotonated for transport and vesamicol binding remain unidentified.

Tests to determine whether transport-dependent changes in exposure of the acetylcholine(ACh) binding site can be detected based on the ability of external ACh to compete with [(super 3)H] vasamicol for binding is reported. It was found that a molecule of vesicular acetylcholine transporter (VAChT) not bound to vasamicol binds and transports a molecule of ACh in the presence of proton-motive force and trace [H](super 3) vasamicol.

The vesicular acetylcholine transporter (V AchT) catalyzes ACh storage in synaptic vesicles by exchanging cytoplasmic ACh with vesicular protons. The stoichiometry of exchange was determined by the examination of the ligand binding and transport kinetics. Findings are in agreement with a two-proton model. The transport cycle starts with binding of external ACh to externally oriented site 2 and protonation of internally oriented site 1.

The synthesis of a tritiated arylazido ligand (azido ABV) of the vesamicol receptor (VR) in cholinergic synaptic vesicles was conducted on the basis of the high-affinity vesamicol analog 4-aminobenzovesamicol (ABV). Acetylcholine (AcCh) active transport was repressed by azido ABV and bound to VR with great affinity. The equilibrium dissociation constant and the rate of dissociation of synaptic vessels was lower than those of vesamicol. Autofluorography was followed by sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis, which showed specific labeling over a wide molecular weight range.

A study was done on the linkage of the acetylcholine transporter-vesamicol receptor to proteoglycan in synaptic vesicles. Results show a strong linkage between the AcChT-VR and proteoglycan which is controlled by the glycosaminoglycan-like polysaccharide. These findings suggest that the AcChT-VR in intact terminals may be able to communicate with extracellular matrix and participate in stabilization and operation of the synapse.

A study was done on vesamicol receptor (VR) purified by 7% yield in hydroxylapatite. It was shown that purified VR has variable electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and very diffuse protein staining at 240 kDa. Substantially purified VR retained both enantioselectivity for (-)-vesamicol and a linked AcCh-binding site which confirms the allosteric model for the VR in the AcCh transporter.

A study was done on the photoaffinity labeling of the acetylcholine transporter. It was shown that the high-affinity analogue of AcCh containing an aryl azido group known as cyclohexylmethyl cis-N-(4-azidophenacyl)-N-methylisonipecotate bromide was able to specifically label the material and exhibits a continuous substitution from 50 to 250 kDa. This finding suggests that the AcChT has structure which closely resembles the synaptic vesicle proteoglycan.

A kinetic and allosteric model was used to study the acetylcholine transporter(AcChT)-vesamicol receptor in synaptic vesicles. It was shown that the vesamicol family of compounds binds to an allosteric site in the AcChT and the initiation of transport is not affected by the affinities of vesamicol and AcCh analogues. This finding suggests that most of the AcChT-VR in purified vesicles is transport incompetent.