Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2015/11/18/jpet.115.226910.DC1 1521-0103/356/2/293–304$25.00 http://dx.doi.org/10.1124/jpet.115.226910 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 356:293–304, February 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Evidence for Classical Cholinergic Toxicity Associated with s Selective Activation of M1 Muscarinic Receptors Andrew Alt, Annapurna Pendri, Robert L. Bertekap, Jr., Guo Li, Yulia Benitex, Michelle Nophsker, Kristin L. Rockwell, Neil T. Burford, Chi Shing Sum, Jing Chen, John J. Herbst, Meredith Ferrante, Adam Hendricson, Mary Ellen Cvijic, Ryan S. Westphal, Jonathan O’Connell, Martyn Banks, Litao Zhang, Robert G. Gentles, Susan Jenkins, James Loy, and John E. Macor Research and Development/Discovery, Bristol-Myers Squibb Company, Wallingford, Connecticut (A.A., A.P., R.L.B., G.L., Y.B., M.N., K.L.R, N.T.B., J.J.H., M.F., A.H., R.S.W., J.O., M.B., R.G., S.J., J.L.); Research and Development/Discovery, Bristol-Myers Downloaded from Squibb Company, Hopewell, New Jersey (C.S.S., M.E.C.); and Research and Development/Discovery, Bristol-Myers Squibb Company, Lawrence Township, New Jersey (J.C., L.Z., J.E.M.) Received June 19, 2015; accepted November 17, 2015 ABSTRACT jpet.aspetjournals.org The muscarinic acetylcholine receptor subtype 1 (M1) receptors activities that are highly functionally selective for the M1 receptor play an important role in cognition and memory, and are were tested in rats, dogs, and cynomologous monkeys: considered to be attractive targets for the development of novel (3-((1S,2S)-2-hydrocyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl) medications to treat cognitive impairments seen in schizophre- pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one; 1-((4-cyano- nia and Alzheimer’s disease. Indeed, the M1 agonist xanomeline 4-(pyridin-2-yl)piperidin-1-yl)methyl)-4-oxo-4H-quinolizine- has been shown to produce beneficial cognitive effects in both 3-carboxylic acid; and (R)-ethyl 3-(2-methylbenzamido)-[1, ’ 9 9 Alzheimer s disease and schizophrenia patients. Unfortunately, 4 -bipiperidine]-1 -carboxylate). Despite their selectivity for the at ASPET Journals on September 28, 2021 the therapeutic utility of xanomeline was limited by cholinergic M1 receptor, all three compounds elicited cholinergic side effects side effects (sweating, salivation, gastrointestinal distress), such as salivation, diarrhea, and emesis. These effects could not which are believed to result from nonselective activation of other be explained by activity at other muscarinic receptor subtypes, muscarinic receptor subtypes such as M2 and M3. Therefore, or by activity at other receptors tested. Together, these results drug discovery efforts targeting the M1 receptor have focused on suggest that activation of M1 receptors alone is sufficient to the discovery of compounds with improved selectivity profiles. produce unwanted cholinergic side effects such as those seen Recently, allosteric M1 receptor ligands have been described, with xanomeline. This has important implications for the devel- which exhibit excellent selectivity for M1 over other muscarinic opment of M1 receptor–targeted therapeutics since it suggests receptor subtypes. In the current study, the following three that dose-limiting cholinergic side effects still reside in M1 compounds with mixed agonist/positive allosteric modulator receptor selective activators. Introduction subtypes each represent separate gene products and exhibit distinct signaling pathways and tissue distribution, although The neurotransmitter acetylcholine activates two distinct all are expressed within the central nervous system (Ishii and families of receptors: nicotinic and muscarinic acetylcholine Kurachi 2006). receptors, which were initially classified based upon their The M1 muscarinic receptors play an important role in differential activation by the toxins nicotine (Lindstrom 1997) multiple domains of cognitive function (Felder et al., 2000; and muscarine (Wess et al., 1996). Nicotinic acetylcholine Auld et al., 2002), and a significant body of evidence suggests receptors are ligand-gated ion channels; muscarinic acetylcho- that activation of M1 receptors can produce therapeutically line receptors are seven transmembrane guanine nucleotide beneficial effects for the treatment of schizophrenia and binding protein (G protein) coupled receptors. Five subtypes of Alzheimer’s disease (Langmead et al., 2008). Muscarinic – muscarinic acetylcholine receptors exist (M1 M5). These five receptor activation has been shown to reverse cognitive and behavioral deficits in animal models of schizophrenia and ’ dx.doi.org/10.1124/jpet.115.226910. Alzheimer s disease, and the M1 receptor specifically has been s This article has supplemental material available at jpet.aspetjournals.org. implicated in mediating these effects (Jones et al., 2005; ABBREVIATIONS: ago, agonist; AUC, area under the curve; compound A, 3-((1S,2S)-2-hydrocyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin- 3-yl)methyl)benzo[h]quinazolin-4(3H)-one; compound B, 1-((4-cyano-4-(pyridin-2-yl)piperidin-1-yl)methyl)-4-oxo-4H-quinolizine-3-carboxylic acid; compound C, (R)-ethyl 3-(2-methylbenzamido)-[1,49-bipiperidine]-19-carboxylate; DMSO, dimethylsulfoxide; G protein, guanine nucleotide binding protein; M1–M5, muscarinic acetylcholine receptor subtypes 1–5; PAM, positive allosteric modulator; PEG, polyethylene glycol; t1/2, half-life; Vss, steady-state volume of distribution. 293 294 Alt et al. Langmead et al., 2008; Barak and Weiner 2011; Fisher 2008). following three structurally distinct M1 PAMs were chosen The most direct evidence for the utility of M1 activators in from the scientific literature (Kuduk et al., 2011; Lebois treating schizophrenia and Alzheimer’s disease comes from et al., 2011): compound A, (3-((1S,2S)-2-hydrocyclohexyl)-6- clinical trials using xanomeline, a muscarinic partial agonist ((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h] with modest selectivity for M1 and M4 receptors. Xanomeline quinazolin-4(3H)-one; compound B, 1-((4-cyano-4-(pyridin- was shown to improve psychosis and behavioral disturbances 2-yl)piperidin-1-yl)methyl)-4-oxo-4H-quinolizine-3-carboxylic in Alzheimer’s disease patients (Bodick et al., 1997). Xanomeline acid; and compound C, (R)-ethyl 3-(2-methylbenzamido)-[1,49- was also found to produce both significant psychiatric im- bipiperidine]-19-carboxylate (Fig. 1). These compounds display provements, and improvements in learning and memory in potency and pharmacokinetic profiles that make them appro- schizophrenia patients (Shekhar et al., 2008). Unfortunately, priate for in vivo testing; and, importantly, they show excellent the clinical utility of xanomeline is limited by its side-effect selectivity for M1 over other muscarinic receptor subtypes in profile, which includes salivation, sweating, and gastrointes- vitro. In this study, the safety profile of these selective M1 tinal distress—all of which are classic cholinergic side effects. ligands was investigated in rats, dogs, and cynomologous Thesesideeffectsarebelievedtobeduetoactivityof monkeys. Despite their selectivity for M1, all three compounds were found to produce effects associated with classic cholinergic xanomeline at M2 and M3 muscarinic receptors (Melancon et al., 2013). Therefore, selectivity has remained the major toxicity such as salivation and diarrhea. Accordingly, these focus for drug discovery efforts targeting the M receptor. results refute the original hypothesis and instead provide 1 Downloaded from evidence to suggest that activation of the M receptor alone is However, attaining sufficient selectivity for M1 versus other 1 muscarinic receptors to avoid cholinergic side effects has sufficient to produce cholinergic toxicity in animals. proven to be very challenging because the acetylcholine binding site is highly conserved within all five muscarinic Materials and Methods receptor subtypes (Heinrich et al., 2009), and despite years of medicinal chemistry efforts acetylcholine site activators with Cells jpet.aspetjournals.org sufficient M1 selectivity have not been identified. M2,M3, and M4 were recombinantly expressed in a Chinese An alternative pharmacological strategy is to modulate the hamster ovary cell background, with M2 and M4 lines also containing activity of M1 receptors using molecules that bind to allosteric a construct expressing Gqi5 and aequorin. The M1- and M5-expressing sites that are distinct from the acetylcholine (orthosteric) cell lines were generated by recombinant expression of M1 or M5 in binding site. Small-molecule positive allosteric modulators Chinese hamster ovary A12 cells (Perkin-Elmer, Akron, OH). – (PAMs) have been identified for many G protein coupled Cells were cultured in tissue culture treated T-175 flasks (Corning, Corning, NY) in medium [Dulbecco’s modified Eagle’s medium/F12 receptors (Wootten et al., 2013), including the M1 receptor (Gibco, Waltham, MA), 10% fetal bovine serum (Hyclone, Logan, at ASPET Journals on September 28, 2021 (Melancon et al., 2013). Allosteric binding sites do not face the Utah), and appropriate selection antibiotics] at 37°C, 5% CO2. Cells same evolutionary pressure as the orthosteric binding site, were harvested and plated onto 384-well (Corning) tissue which must bind the endogenous agonist to maintain receptor culture–treated black/clear plates 16–20 hours prior to the