Identification and Preclinical Development of a Radiofluorinated Probe for Imaging the Muscarinic Acetylcholine Receptor M4 by Positron Emission Tomography

Introduction: An accumulating body of evidence suggests that the muscarinic acetylcholine receptor 4 (M₄) is implicated in schizophrenia and dementia with Lewy body. Of note, previous studies revealed that M₄ is markedly expressed in the brain, with high abundancies in the striatum and thalamus. Strenuous research efforts prompted the discovery of several M₄ agonists, however, they have not yet reached advanced clinical stages, at least in part, due to the lack of a clinically validated M₄-targeted probe. As such, we envisioned the development of a suitable M₄ PET ligand that allows the visualization of M₄ in vitro and in vivo.

Methods: Structure-activity relationship studies of pyrazol-4-yl-pyridine derivates prompted the discovery of target compound 2, a positive allosteric modulator (PAM) of M₄. The radiofluorinated analogue, [¹⁸F]2, was challenged by in vitro autoradiography on mouse, rat, non-human primate (NHP) and human brain sections. To assess the effect of orthosteric ligand binding on the binding attributes of [¹⁸F]2, we performed all autoradiographic studies either in the presence or in the absence of the commercially available orthosteric agonist, carbachol. PET imaging was conducted in NHPs.

Results: Initial in vitro autoradiograms on rodent brain sections were performed in the absence of carbachol and showed only marginal specificity and a low selectivity of [¹⁸F]2 for the M₄-expressing striatum. Nonetheless, upon addition of carbachol, a substantially higher tracer binding was observed in the rat striatum, which was significantly reduced under blocking conditions (Figure 1), suggesting that orthosteric ligand interaction is essential for efficient binding of [¹⁸F]2 to the allosteric binding site. Similar observations were made on autoradiograms of mouse brain sections. Notably, the presence of carbachol did not further improve the specificity and selectivity of [¹⁸F]2 in brain sections of NHPs and humans. In conclusion, these findings revealed significant species-differences with regard to the effects of orthosteric ligand binding on the tracer performance characteristics and paved the way for a preliminary PET study in NHPs, where brain uptake of [¹⁸F]2 was highest in the putamen and in cortical regions with known M₄ expression.

Conclusions: We report on the identification and preclinical development of the first radiofluorinated M₄ PET radioligand with promising in vitro and in vivo performance characteristics. Indeed, specificity and selectivity were demonstrated across different species by in vitro autoradiography on mouse, rat, NHP and human brain sections. PET imaging in NHP showed highest tracer uptake in brain regions with known M₄ expression. The availability of a clinically validated M₄ PET radioligand holds promise to provide a useful diagnostic tool for non-invasive imaging, thereby facilitating the clinical development of M4-targeted drugs in the pipeline.

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