Evaluation of newly synthesized TZTP-derivatives as potential SPECT tracers for imaging muscarinic M2 receptors

Aim: A loss of presynaptic muscarinic receptors of the M2 subtype accompanies the cholinergic defect in Alzheimer's disease. Imaging of the M2 receptor may therefore be of value to detect loss of cholinergic neurons in this form of dementia. 3-(3-alkylthio-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (TZTP)-derivatives, are known to bind to muscarinic receptors and many derivatives have a higher affinity for the M2 receptor than for the other muscarinic receptor subtypes. The goal of the current study was to evaluate the in vitro and in vivo binding characteristics of five newly synthesized TZTP-derivatives as potential SPECT tracers. Materials and Methods: Binding affinity and in vitro selectivity were determined by competitive binding assays with human muscarinic receptor subtypes that were cloned in Chinese hamster ovary (CHO) cells. The in vivo binding characteristics of the [123I]-radiolabeled analogue of one these derivatives were evaluated by performing biodistribution and blocking experiments in rats. Results: Four out of five tested TZTP derivatives showed a nanomolar binding affinity for cloned human muscarinic receptors (M1-M3), of which the iodo-pentenyl-TZTP-derivative had the most favorable affinity for the M2 receptor (Ki = 4.9 nM), compared to M1 and M3 receptors (ratios of 2 and 10 for M2 to M1 and M2 to M3, respectively). Intravenous injection in rats of the [123I]-radiolabeled analogue of this tracer resulted in a distribution pattern in the brain that was similar to the distribution of M2 receptors in the first 30 min post-injection, while measurements after this time point resembled non-specific uptake. Only blocking of the muscarinic receptor system resulted in a decrease of radioactive uptake in all brain areas at 15 min post-injection, but not at 90 min post-injection. Apart from in vivo binding to M2 receptors, this radioligand showed also intense uptake in liver and kidneys. Conclusion: We synthesized a series of TZTP-derivatives with high affinity for the muscarinic M2 receptor. One of these compounds, an iodo-pentenyl-TZTP-derivative, has high in vitro affinity and moderate selectivity for the muscarinic M2 receptor subtype. Although this tracer labels muscarinic receptors in vivo, possible fast metabolism may limit its value for SPECT imaging in the human brain