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 (EC50 2.4-7.0 nM, Emax 43%-73%), displaying binding selectivity for M1 mAChRs (Kd = 1.5 nM) that was conserved across species (native tissue Kd = 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.

Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Figures

Fig. 1.

Initial identification of LSN3172176. A cassette of nonradiolabeled putative tracer molecules was assessed in filtration binding assays using membranes expressing the human M1 mAChR. The most promising of these compounds was (A) LSN3172176. (B) Single-point binding assay using two different concentrations of LSN3172176 (1 and 10 nM). Nonspecific binding was determined in the presence of 10 μM atropine (C) saturation assay from which specific binding was calculated by subtracting the mean nonspecific binding in the presence of 10 μM atropine from the mean total binding at each ligand concentration performed in triplicate and then expressed as nanograms ligand per gram protein. For both (B and C) after filtration, bound compound was extracted and measured using LC/MS/MS. Data points represent mean ± S.E.M. from three independent experiments.

Fig. 2.

In vitro functional profiling of LSN3172176 and comparators in native cortical tissues. Stimulation of GTPγ[35S] binding to cortical membranes prepared from human, rat, mouse, or mice deficient in the M1 receptor, in response to increasing concentrations of (A) LSN3172176, (B) xanomeline, or (C) GSK1034702. Cortical membranes were incubated at 21°C for 30 minutes with compound. Data shown are increases in GTPγ[35S] binding normalized to the response obtained with a maximally effective concentration of oxotremorine M, or for mouse M1 KO tissue the increase in binding above buffer alone. Data points represent the mean ± S.E.M. of three independent experiments each containing three to six replicates.

Fig. 3.

Recombinant mAChR in vitro binding profile. Displacement of [3H]NMS radioligand binding by increasing concentrations of (A) LSN3172176, (B) xanomeline, and (C) GSK1034702 across human recombinant mAChR subtypes M1–M5. Data points represent mean specific binding ± S.E.M. from three independent experiments each containing three replicates.

Fig. 4.

[3H]LSN3172176 association and dissociation kinetics. For association rate experiments (A) specific binding was calculated by subtracting the mean nonspecific binding in the presence of 10 μM atropine from the mean total binding at each time point performed in quadruplicate. For dissociation rate experiments (B) binding was expressed as a percent of total bound DPM (disintegrations per minute) at 0 minutes. Data points represent mean ± S.E.M. from three independent experiments each containing four replicates.

Fig. 5.

Displacement of [3H]LSN3172176 by known orthosteric mAChR ligands. [3H]LSN3172176 radioligand binding (4 nM) at the human M1 recombinant mAChR was assessed in the presence of increasing concentrations of atropine, NMS, scopolamine, and acetylcholine. Data points represent mean specific binding ± S.E.M. from three independent experiments each containing three replicates.

Fig. 6.

Saturation binding profile of [3H]LSN3172176. Binding of both [3H]NMS and [3H]LSN3172176 was assessed across human M1–M5 recombinant membranes. For both ligands specific binding was calculated by subtracting the mean nonspecific binding in the presence of 10 μM atropine from the mean total binding at each radioligand concentration performed in quadruplicate and then expressed as femtomole [3H]ligand binding per milligram protein. Data points represent mean ± S.E.M. from three independent experiments. HEK, human embryonic kidney cells.

Fig. 7.

Native tissue saturation binding. Saturation binding experiments using [3H]LSN3172176 were conducted using brain membranes (frontal cortex and cerebellum) prepared from wild-type and M1 mAChR KO mouse, rat, rhesus monkey, and human. Specific binding was calculated by subtracting the mean nonspecific binding in the presence of 10 μM atropine from the mean total binding at each radioligand concentration performed in quadruplicate and then expressed as femtomole [3H]3172176 binding per milligram protein. Data points represent mean ± S.E.M. from three independent experiments.

Fig. 8.

Autoradiography with [3H]LSN3172176 in the mouse brain. (A) Binding of [3H]LSN3172176 was compared across serial fresh-frozen slices (20 μm) from wild-type or M1 or M4 KO mice. A final concentration of 5 nM [3H]LSN3172176 was used for all experiments. Representative images from a single brain are shown below. (B) Quantification of regional [3H]LSN3172176 binding was performed using brains from three separate mice from each strain. Bars reflect the average intensity of all pixels in a region of interest and represent the mean ± S.E.M. for each condition.

Fig. 9.

In vivo distribution and occupancy of LSN3172176 in the rat and mouse. (A) Tissue distribution and plasma levels of LSN3172176 in the rat over time. Rats were dosed with 0.3 μg/kg (i.v.) of LSN 3172176 and sacrificed at the indicated intervals. The amount of LSN3172176 in each tissue was quantified by LC/MS as described in Materials andMethods. (B) Scopolamine block of LSN3172176 receptor occupancy in rat frontal cortex. Rats were predosed with 0.01–0.3 mg/kg scopolamine (i.v.) prior to administration of 0.3 μg/kg LSN3172176. After 20 minutes animals were sacrificed and the amount of LSN3172176 in each tissue quantified by LC/MS as described in the Materials andMethods (C) Tissue distribution and plasma levels of LSN3172176 in wild-type and M1–M5 mAChR KO Mice. Mice were dosed with 10 μg/kg LSN3172176. After 20 minutes animals were sacrificed and the amount of LSN3172176 in each tissue quantified by LC/MS as described in the Materials andMethods. (D) Binding potential comparisons for LSN3172176 in wild-type vs. M1–M5 mAChR KO Mice. The in vivo binding potential was calculated by dividing the amount of LSN3172176 in the frontal cortex by the amount determined in the cerebellum by LC/MS. Bars/points represent mean ± S.E.M. from n = 3–5 male mice or rats per treatment group.