Kinetics modeling and occupancy studies of a novel C-11 PET tracer for VAChT in nonhuman primates

Abstract

Introduction: Deficits 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.

Methods: MicroPET 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.

Results: Baseline 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.

Keywords: (−)-[(11)C]TZ659; Binding potential; Occupancy; Tracer kinetics; Vesicular acetylcholine transporter.

Figure 1. Specificity of (−)-[¹¹C]TZ659 in vivo binding to VAChT

Representative coronal images of PET (left) and the coregistered MRI/PET (right) in a male macaque (Subject A). The baseline scan (A) of (−)-[¹¹C]TZ659 clearly demonstrates striatal accumulation (caudate and putamen). B–D show (−)-[¹¹C]TZ659 striatal activity after pretreatment with (B) 0.25 mg/kg of the allosteric antagonist (−)-vesamicol; (C) 1 mg/kg of the sigma-1 receptor ligand (+)-pentazocine or (D) 0.025 mg/kg of the dopamine D₂-like receptor antagonist S-(−)-eticlopride. Striatal accumulation of (−)-[¹¹C]TZ659 was significantly reduced in response to (−)-vesamicol pretreatment, while (+)-pentazocine pretreatment showed no significant change in striatal activity. D₂–like receptor antagonism with S-(−)-eticlopride clearly increased the striatal uptake of the radiotracer.

Figure 2. Cerebellar and striatal time-activity curves (TACs) from baseline and (−)-vesamicol pretreatment (0.25 and 0.05 mg/kg)

Cerebellar TACs at baseline (filled circles) and after (−)-vesamicol treatment (open circles) reached a similar steady state (by 70 min p.i.) although the initial uptake differed; this suggests the cerebellar hemispheres are a suitable non-target reference region. Striatal uptake (combination of caudate and putamen) after pretreatment using 0.05 mg/kg (−)-vesamicol (half-filled triangles) and 0.25 mg/kg (−)-vesamicol (open triangles), was significantly lower than baseline striatal uptake (filled triangles). A greater reduction in striatal uptake during steady state was seen with the higher blocking dose. Baseline data represents the average of all four scans for Subject A.

Figure 3. Logan graphic analysis of brain regions

Time activity data for different brain regions (striatum “×”, thalamus “○”, occipital cortex “–”, temporal cortex “◊”, frontal cortex “□”, midbrain “●” and hippocampus “Δ”) for the graphic Logan plots for baseline (A) and experiments with pretreatment by (−)-vesamicol 0.25 mg/kg (B).

Figure 4. Reversible tracer binding

(−)-Vesamicol (0.3 mg/kg i.v.) was injected 20 min p.i., during striatal equilibrium for displacement studies. Striatal uptake of (−)-[¹¹C]TZ659 significantly decreased after injection of (−)-vesamicol under displacement conditions (gray triangles) compared to baseline uptake (black triangles). Cerebellar uptake of (−)-[¹¹C]TZ659 after injection of (−)-vesamicol (grey circles) was almost identical to baseline cerebellar uptake (black circles). Baseline data are the average of all four scans for Subject A.

Figure 5. (−)-Vesamicol dose and VAChT occupancy relationship

A) Scatter plot of BP_ND estimates with SRTM versus BP_ND estimates with LoganREF modeling. Data are for striatal regions under either baseline (circles) or after pretreatment (triangles). All six baseline, four (−)-vesamicol blocking, one (+)-pentazocine blocking, and one S-(−)-eticlopride blocking studies are shown. B). (−)-Vesamicol dose and VAChT occupancy relationship. The occupancy percentages were calculated from BPND. The fitted ED₅₀ is 0.0057 ± 0.0017 mg/kg.

Figure 6. Simplified circuitry cartoon showing the interaction between cholinergic and dopaminergic system in the striatum

A). Acetylcholine (ACh) activation of nicotinic receptors (nAChRs) facilitates dopamine release in the striatum. In contrast, the predominant effect of dopamine is suppression of ACh release, mediated by D₂-like receptors. B). S-(−)-eticlopride is an antagonist for D₂-like receptors and therefore down-regulates the consequences for activation of dopaminergic output and enhances cholinergic activity.