Characterizing the binding of TC-5619 and encenicline on the alpha7 nicotinic acetylcholine receptor using PET imaging in the pig.

The alpha7 nicotinic acetylcholine receptor (α7-nAChR) has has long been considered a promising therapeutic target for addressing cognitive impairments associated with a spectrum of neurological and psychiatric disorders, including Alzheimer's disease and schizophrenia. However, despite this potential, clinical trials employing α7-nAChR (partial) agonists such as TC-5619 and encenicline (EVP-6124) have fallen short in demonstrating sufficient efficacy. We here investigate the target engagement of TC-5619 and encenicline in the pig brain by use of the α7-nAChR radioligand 11C-NS14492 to characterize binding both with in vitro autoradiography and in vivo occupancy using positron emission tomography (PET). In vitro autoradiography demonstrates significant concentration-dependent binding of 11C-NS14492, and both TC-5619 and encenicline can block this binding. Of particular significance, our in vivo investigations demonstrate that TC-5619 achieves substantial α7-nAChR occupancy, effectively blocking approximately 40% of α7-nAChR binding, whereas encenicline exhibits more limited α7-nAChR occupancy. This study underscores the importance of preclinical PET imaging and target engagement analysis in informing clinical trial strategies, including dosing decisions.

Evaluation of [18 F]2FP3 in pigs and non-human primates.

So far, no suitable 5-HT7 R radioligand exists for clinical positron emission tomography (PET) imaging. [18 F]2FP3 was first tested in vivo in cats, and the results were promising for further evaluations. Here, we evaluate the radioligand in pigs and non-human primates (NHPs). Furthermore, we investigate species differences in 5-HT7 R binding with [3 H]SB-269970 autoradiography in post-mortem pig, NHP, and human brain tissue. Specific binding of [18 F]2FP3 was investigated by intravenous administration of the 5-HT7 R specific antagonist SB-269970. [3 H]SB-269970 autoradiography was performed as previously described. [18 F]2FP3 was synthesized in an overall yield of 35% to 45%. High brain uptake of the tracer was found in both pigs and NHPs; however, pretreatment with SB-269970 only resulted in decreased binding of 20% in the thalamus, a 5-HT7 R-rich region. Autoradiography on post-mortem pig, NHP, and human tissues revealed that specific binding of [3 H]SB-269970 was comparable in the thalamus of pig and NHP. Despite the high uptake of [18 F]2FP3 in both species, the binding could only be blocked to a limited degree with the 5-HT7 R antagonists. We speculate that the affinity of the radioligand is too low for imaging the 5-HT7 Rs in vivo and that part of the PET signal arises from targets other than the 5-HT7 R.

Radiosynthesis and in vitro validation of (3)H-NS14492 as a novel high affinity alpha7 nicotinic receptor radioligand.

The neuronal α7 nicotinic acetylcholine receptor is a homo-pentameric ligand-gated ion channel that is a promising drug target for cognitive deficits in Alzheimer׳s disease and schizophrenia. We have previously described (11)C-NS14492 as a suitable agonist radioligand for in vivo positron emission tomography (PET) occupancy studies of the α7 nicotinic receptor in the pig brain. In order to investigate the utility of the same compound for in vitro studies, (3)H-NS14492 was synthesized and its binding properties were characterized using in vitro autoradiography and homogenate binding assays in pig frontal cortex. (3)H-NS14492 showed specific binding to α7 nicotinic receptors in autoradiography, revealing a dissociation constant (Kd) of 2.1±0.7nM and a maximum number of binding sites (Bmax) of 15.7±2.0fmol/mg tissue equivalent. Binding distribution was similar to that of another selective ligand (125)I-α-bungarotoxin ((125)I-BTX) in autoradiography, and unlabeled NS14492 displaced (125)I-BTX with an inhibition constant (Ki) of 23nM. (3)H-NS14492 bound to α7 nicotinic receptors in homogenized pig frontal cortex with a Kd of 0.8±0.3nM and a Bmax of 30.2±11.6fmol/mg protein. This binding assay further revealed the Ki rank order for a number of α7 nicotinic receptor agonists, and positive allosteric modulators (PAMs). Further, we saw increased binding of (3)H-NS14492 to pig frontal cortex membranes when co-incubated with PNU-120596, a type II PAM. Taken together, these findings show that (3)H-NS14492 is a useful new in vitro radioligand for the pig α7 nicotinic receptor.

Labeling and preliminary in vivo evaluation of the 5-HT(7) receptor selective agonist [(11)C]E-55888.

E-55888 has been identified as a selective serotonin 7 (5-HT7) receptor agonist. In this study, we describe the synthesis, radiolabeling and in vivo evaluation of [(11)C]E-55888 as a radioligand for positron emission tomography (PET) imaging. [(11)C]E-55888 was obtained by N-methylation of an appropriate precursor using [(11)C]MeOTf in acetone at 60 °C giving isolated quantities in the range of 1.7-2.4 GBq. Studies in Danish Landrace pigs demonstrated that [(11)C]E-55888 has good brain uptake, however, the distribution in the brain tissue was dominated by non-specific binding, as binding could neither be displaced by the structurally different 5-HT7 receptor ligand SB-269970 nor by self-block with unlabeled E-55888. Based on these data, [(11)C]E-55888 does not show promise as a PET radioligand for imaging the 5-HT7 receptor in vivo.