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 11C-labeled a new VAChT inhibitor, (-)-TZ659. Here we report the in vitro and ex vivo characterization of (-)-TZ659. A stably transfected PC12A123.7 cell line which expresses human VAChT (hVAChT) was used for the in vitro binding characterization of (-)-[3H]TZ659. A saturated binding curve was obtained with Kd=1.97±0.30 nM and Bmax=3240±145.9 fmol/mg protein. In comparison, a PC12A123.7 cell line that expresses mutant hVAChT showed decreased binding affinity (Kd=15.94±0.28 nM). Competitive binding assays using a panel of other CNS ligands showed no inhibition of (-)-[3H]TZ659 binding. On the other hand, binding inhibitions were observed only using VAChT inhibitors (Ki=0.20-31.35 nM). An in vitro assay using rat brain homogenates showed that (-)-[3H]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 (-)-[11C]TZ659. Ex vivo autoradiography of (-)-[11C]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 (-)-[11C]TZ659 may be a promising PET tracer to image VAChT in the brain.
- Binding assay
- Vesicular acetylcholine transporter