title: Inositol Phosphate Accumulation in Vivo Provides a Measure of Muscarinic M1Receptor Activation.
abstract: The rationale for using M1selective muscarinic acetylcholine receptor activators for the treatment of cognitive impairment associated with psychiatric and neurodegenerative disease is well-established in the literature. Here, we investigate measurement of inositol phosphate accumulation, an end point immediately downstream of the M1muscarinic acetylcholine receptor signaling cascade, as an in vivo biochemical readout for M1muscarinic acetylcholine receptor activation. Five brain penetrant M1-subtype selective activators from three structurally distinct chemical series were pharmacologically profiled for functional activity in vitro using recombinant cell calcium mobilization and inositol phosphate assays, and a native tissue hippocampal slice electrophysiology assay, to show that all five compounds presented a positive allosteric modulator agonist profile, within a narrow range of potencies. In vivo characterization using an amphetamine-stimulated locomotor activity behavioral assay and the inositol phosphate accumulation biochemical assay demonstrated that the latter has utility for assessing functional potency of M1activators. Efficacy measured by inositol phosphate accumulation in mouse striatum compared favorably to efficacy in reversing amphetamine-induced locomotor activity, suggesting that the inositol phosphate accumulation assay has utility for the evaluation of M1muscarinic acetylcholine receptor activators in vivo. The benefits of this in vivo biochemical approach include a wide response window, interrogation of specific brain circuit activation, an ability to model responses in the context of brain exposure, an ability to rank order compounds based on in vivo efficacy, and minimization of animal use.
authors.affiliation: Neuroscience and Pain Research Unit, ‡Pharmacokinetics, Dynamics and Metabolism, and §Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
authors.forename: Michael
authors.identifier: http://orcid.org/0000-0001-7372-1982
authors.initials: M
authors.lastname: Popiolek
authors.affiliation: Neuroscience and Pain Research Unit, ‡Pharmacokinetics, Dynamics and Metabolism, and §Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
authors.forename: David P
authors.initials: DP
authors.lastname: Nguyen
authors.affiliation: Neuroscience and Pain Research Unit, ‡Pharmacokinetics, Dynamics and Metabolism, and §Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
authors.forename: Veronica
authors.initials: V
authors.lastname: Reinhart
authors.affiliation: Neuroscience and Pain Research Unit, ‡Pharmacokinetics, Dynamics and Metabolism, and §Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
authors.forename: Jeremy R
authors.initials: JR
authors.lastname: Edgerton
authors.affiliation: Neuroscience and Pain Research Unit, ‡Pharmacokinetics, Dynamics and Metabolism, and §Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
authors.forename: John
authors.initials: J
authors.lastname: Harms
authors.affiliation: Neuroscience and Pain Research Unit, ‡Pharmacokinetics, Dynamics and Metabolism, and §Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Lotarski
authors.affiliation: Neuroscience and Pain Research Unit, ‡Pharmacokinetics, Dynamics and Metabolism, and §Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
authors.forename: Stefanus J
authors.initials: SJ
authors.lastname: Steyn
authors.affiliation: Neuroscience and Pain Research Unit, ‡Pharmacokinetics, Dynamics and Metabolism, and §Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Davoren
authors.affiliation: Neuroscience and Pain Research Unit, ‡Pharmacokinetics, Dynamics and Metabolism, and §Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.
authors.forename: Sarah
authors.initials: S
authors.lastname: Grimwood
mesh_terms: D000661:Amphetamine / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D003342:Corpus Striatum / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D015259:Dopamine Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D055724:Electrophysiological Phenomena / Q000187:drug effects; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D008297:Male; D051379:Mice; D009043:Motor Activity / Q000187:drug effects; D018721:Muscarinic Agonists / Q000145:classification / Q000494:pharmacology*; D017207:Rats, Sprague-Dawley; D043643:Receptor, Muscarinic M1 / Q000819:agonists* / Q000378:metabolism
chemical_list: D015259:Dopamine Agents; D007295:Inositol Phosphates; D018721:Muscarinic Agonists; D043643:Receptor, Muscarinic M1; D000661:Amphetamine; D002118:Calcium