In Vitro Pharmacological Characterization and In Vivo Validation of LSN3172176 a Novel M1 Selective Muscarinic Receptor Agonist Tracer Molecule for Positron Emission Tomography

Abstract

In the search for improved symptomatic treatment options for neurodegenerative and neuropsychiatric diseases, muscarinic acetylcholine M1 receptors (M1 mAChRs) have received significant attention. Drug development efforts have identified a number of novel ligands, some of which have advanced to the clinic. However, a significant issue for progressing these therapeutics is the lack of robust, translatable, and validated biomarkers. One valuable approach to assessing target engagement is to use positron emission tomography (PET) tracers. In this study we describe the pharmacological characterization of a selective M1 agonist amenable for in vivo tracer studies. We used a novel direct binding assay to identify nonradiolabeled ligands, including LSN3172176, with the favorable characteristics required for a PET tracer. In vitro functional and radioligand binding experiments revealed that LSN3172176 was a potent partial agonist (EC 2.4-7.0 nM, E 43%-73%), displaying binding selectivity for M1 mAChRs (K = 1.5 nM) that was conserved across species (native tissue K = 1.02, 2.66, 8, and 1.03 at mouse, rat, monkey, and human, respectively). Overall selectivity of LSN3172176 appeared to be a product of potency and stabilization of the high-affinity state of the M1 receptor, relative to other mAChR subtypes (M1 > M2, M4, M5 > M3). In vivo, use of wild-type and mAChR knockout mice further supported the M1-preferring selectivity profile of LSN3172176 for the M1 receptor (78% reduction in cortical occupancy in M1 KO mice). These findings support the development of LSN3172176 as a potential PET tracer for assessment of M1 mAChR target engagement in the clinic and to further elucidate the function of M1 mAChRs in health and disease.

Identification and pharmacological profile of SPP1, a potent, functionally selective and brain penetrant agonist at muscarinic M1 receptors

“…The affinity and selectivity profile of SPP1 was further assessed in radioligand competition assays by assessing displacement of [ 3 H] NMS by SPP1 from human recombinant M 1 –M 5 receptors. In addition, we employed a tritiated version of a novel M 1 receptor selective ligand [ 3 H]LSN3172176 (Mogg et al ., ) to assess displacement from native M 1 receptors from mouse, rat, monkey and human cortex (Table and Supporting Information Figure S1). SPP1 displayed high binding affinity for human M 1 receptors (K i values of 21.3 ± 1.2 and 14.5 ± 2.5 nM on human recombinant and native M 1 receptors respectively), which was conserved across species (K i values of 2.88 ± 0.36, 8.67 ± 0.41 and 10.7 ± 1.3 nM on mouse, rat and monkey respectively; Table and Supporting Information Figure S1).…”

“…Assessment of central M 1 receptor target engagement (receptor occupancy) was performed by monitoring in vivo block of an M 1 receptor tracer molecule, LSN3172176 (Mogg et al ., ) administered 30 min after p.o. administration of SAR molecules.…”

“…SPP1 displayed modest selectivity for binding to human M 1 receptors over human M 4 > M 2 > M 5 > > M 3 receptor subtypes, as did AZD‐6088. In contrast, HTL‐9936 and xanomeline did not show preferential binding to M 1 receptors (Table and Supporting Information Figure S1; Mogg et al ., ).…”

“…Moving to in vivo studies, assessment of central M 1 receptor target engagement was performed by monitoring in vivo block of a selective receptor occupancy ligand for M 1 receptors (Mogg et al ., ) at various doses or time points after p.o. administration of SPP1 (Figure ).…”

“…Recombinant membranes were incubated with 0.2 nM [ 3 H]‐N‐methylscopolamine ([ 3 H]NMS). Native membranes were incubated with 2 nM [ 3 H]LSN3172176 (Mogg et al ., ). Binding was performed in the presence or absence (total binding) of 11 different concentrations of compound.…”