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 experi- function. Therefore, allosteric binding sites are hypothesized ment. Briefly, flasks were rinsed with 10 ml/flask Dulbecco9s phos- to be less evolutionarily and structurally conserved than phate buffered saline solution (magnesium and calcium free, Gibco) orthosteric sites, suggesting that allosteric sites could offer and incubated in 5 ml/flask of 0.05% trypsin-EDTA (Gibco) at 37°C for improved opportunities for receptor subtype selectivity versus 5 minutes. Medium was added to the harvested cells, and then the targeting the traditional orthosteric site of the receptor. This cells were centrifuged in a tabletop centrifuge at 1200 rpm for 10 minutes. The cell pellet was resuspended in 10 ml/flask medium 1 hypothesis has been borne out empirically. For example, small 1% penicillin-streptomycin (Gibco), counted on the Guava Personal molecules targeting allosteric sites of metabotropic glutamate Cell Analysis System (Guava Technologies, Hayward, CA), diluted receptors have been shown to exhibit excellent receptor sub- with medium 1 1% penicillin-streptomycin to a concentration of 1 type selectivity, which had not been achieved by ligands 106 cells/ml, and then 20 ml/well (20,000 cells/well) of cell suspension targeting the highly conserved glutamate binding site (Nickols was added and the plates were incubated at 37°C, 5% CO2 overnight. and Conn 2014). Similarly, allosteric modulators of musca- rinic receptors have been identified that exhibit selectivity 23 Compound Plate Preparation superior to that attained by xanomeline or other orthosteric Compounds were solubilized in dimethylsulfoxide (DMSO) (EMD site M1 receptor activators (Melancon et al., 2013). Chemicals, Gibbstown, NJ) at 10 mM and then serially diluted in half- The current study was designed to test the hypothesis that log increments in 384-well polypropylene REMP microplates (Brooks M1-selective compounds would be free of classic cholinergic Automation, Chelmsford, MA). An intermediate compound plate was side effects in animal models. To test this hypothesis, the prepared by adding 1 ml/well of each DMSO solution to a REMP plate

Fig. 1. Compounds used: 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 (Kuduk et al., 2011), compound B, 1-((4-cyano-4- (pyridin-2-yl)piperidin-1-yl)methyl)-4-oxo-4H-quinolizine- 3-carboxylic acid (Kuduk et al., 2011), and compound C, (R)-ethyl 3-(2-methylbenzamido)-[1,49-bipiperidine]- 19-carboxylate (VU0364572) (Lebois et al., 2011); 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline- 3-carboxylic acid (BQCA) (Ma et al., 2009) was used as a comparator compound for in vitro experiments. Cholinergic Toxicity Produced by Selective M1 Activation 295 followed by 100 ml/well of Hanks’ balanced salt solution/20 mM

HEPES [Hanks’ balanced salt solution (Gibco) supplemented with max E 20 mM HEPES (Gibco), pH 7.4]. 50

Ca21 Flux Assay pEC 4NANA 4NANA 6 6

After overnight incubation, the medium was removed from the max cells by flicking the plates, followed by addition of 20 ml/well of 3 mM E

Fluo4-AM (Invitrogen, Carlsbad, CA), 0.016% pluronic acid (Sigma- to couple activation of these 0.1 100 0.1 98 qi5 Aldrich, Saint Louis, MO), and 2.5 mM probenecid (Sigma-Aldrich) in 50 6 Hanks’ balanced salt solution/20 mM HEPES. Plates were incubated 6 pEC for 1 hour at room temperature prior to assay in the FLIPR Tetra (Molecular Devices, Sunnyvale, CA). For all additions, the FLIPR max

Tetra reads a baseline once a second for 10 seconds prior to compound E

addition, followed by 50 seconds at 1-second intervals and 60 seconds 50

at 3-second intervals. First, the compounds were added from the M). pEC compound plate to assay for agonist activity and 20 ml/well from the m 8NANA6.1 7NANA5.2 6 2 compound plate was added at a height of 10 ml with no mixing. 6 max

Then, the plates were incubated for 20 minutes at room temperature, E Downloaded from and the PAM activity was assayed by adding a 3 solution of receptors also stably expressed G 4 0.1 100 acetylcholine from a bulk reservoir to all wells (20 ml/well at 10 ml 0.2 91 50 ration tested (50 or M 6 height with two 10 ml mixes). The solution was adjusted each day to 6 2 pEC produce an EC10-EC20 response. The ranges of 3 concentrations of acetylcholine used were 15–30 nM for M1,167–250 nM for M2, – – – max 1.67 2nMforM3, 133 400 nM for M4,and15 50 nM for M5. E Raw data files were exported from the FLIPR ScreenWorks 50 jpet.aspetjournals.org software (Molecular Devices). Maximum fold increase in fluorescence levels in Chinese hamster ovary cells recombinantly expressing the various muscarinic 2+ was determined by dividing the maximum value of fluorescence pEC 4NANA5.6 obtained after compound addition by the average of the baseline 3NANA4.7 6 6 max values taken before compound addition. These values were analyzed E using GraphPad PRISM (GraphPad Software, La Jolla, CA). The EC50 values were calculated using nonlinear regression (sigmoidal dose 0.1 100 0.2 102 50 6 response, variable slope). 6 pEC at ASPET Journals on September 28, 2021

In Vitro Selectivity Assays 3NANANANANANANANANANANANA 6 max 5

Selectivity assays for other G protein coupled receptors were E M – performed as standard competitive binding assays using receptor- 1

specific radioligand probes in membrane preparations from cells 0.6 23 50 overexpresssing the various G protein coupled receptors. Phosphodi- 6 pEC

esterase type 4 activity was determined in an electrophoretic mobility 5.6 concentration of acetylcholine (PAM data). Cells expressing M 15 values are relative to a maximal acetylcholine response in the given cell line. assay, using a fluorescently labeled cAMP as a substrate. By the a 13 NA NA 7.3 4 nature of the difference in electrophoretic mobility of the substrate 13 NA NA 6.4 max 6 6 6 max and the hydrolyzed product, the enzymatic activity was determined E 45 upon the electrophoretic separation of the reactions on the caliper (Perkin-Elmer). The activity on GABA (a1b2g2) was measured by 0.3 100 0.3 90 50 a 6 using a cell line expressing a rat GABA (a1b2g2) and a halide-sensing 6 response to an EC pEC

yellow fluorescent protein. Potentiation of GABA activity was de- 2+ termined by the effect of the test compound to enhance the response 1 NANANANANANANANANANANANANANANANA 13 13 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA produced by an EC20 concentration of GABA. Activity on the cardiac 6 NANANANANANANANANANANANANANANANA 6 6 6 6 max

sodium channel was measured using a cell line expressing human E

NaV1.5 and a voltage-sensitive dye on a flurometric plate reader, where the inhibitory activity of the test compounds was assayed in the S.D. from 3 to 5 experiments. E 6 0.1 103 0.2 50 0.2 83 0.2 23 presence of veratridine. 50 6 6 6 6 pEC 5.3

Serum Protein Binding Assays a 2NANA5.3 16 67.4 12 5.9 3NANA4.5 Test compounds were suspended in 100% DMSO as a 10 mM stock. 76.1 max 6 6 6 6 6 6

Five-molar monobasic sodium phosphate was obtained from Sigma- E Aldrich. Pooled, male human, male rat, and male mouse sera were 39

obtained from BioReclamation (Hopewell, NJ). The reusable 96-well values for muscarinic ligands were determined from their ability to directly induce increases in free cytosolic Ca 0.2 100 0.4 24 0.3 44 0.2 99 0.1 70 M1 Agonist M1 PAM M2 Agonist M2 PAM M3 Agonist M3 PAM M4 Agonist M4 PAM M5 Agonist M5 PAM 50 microequilibrium dialysis device was obtained from HTDialysis a 6 6 6 6 6 max

(Gales Ferry, CT). Dialysis membrane strips (10,000-Da mol. wt. pEC cutoff) were also obtained from HTDialysis. Acetonitrile, water, and responses. Data represent mean ) and E 2+ methanol were all obtained from J.T. Baker (Miami, FL). Ammonium 50 acetate, formic acid, and alprenolol were obtained from Sigma- Aldrich. Tolbutamide was obtained from Fluka (Saint Louis, MO). Compound The compound was too weak to develop a full concentration response curve; the percentage of acetylcholine response is provided at the highest concent

Polymerase chain reaction plates were obtained from Axygen (Union BQCA, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid;a NA, no activity. Acetylcholine 6.4 Compound A 5.4 Compound B 4.3 Carbachol 5.4 Compound C 4.4 BQCA subtypes to Ca receptor subtypes (agonist data), or their ability to potentiate the Ca City, CA). TABLE 1 Activity and selectivity profilePotency of (pEC muscarinic ligands for muscarinic receptor subtypes M 296 Alt et al.

2+ Fig. 2. Activity of muscarinic ligands at receptor subtype M1. Agonist read (closed symbols): increases in free cytosolic Ca levels were measured immediately upon test compound addition. PAM read (open symbols): 20 minutes subsequent to test compound addition cells were challenged with an EC concentration of acetylcholine and free cytosolic Ca2+ levels were again measured, with data expressed as fold increase in free intracellular Ca2+

15 Downloaded from relative to the control EC15 response.

Dialysis membranes were soaked for 15 minutes in distilled water stock with distilled water and adjustment to pH 7.4), and the at room temperature. After the initial soaking, the water was replaced membranes were soaked for an additional 15 minutes in the buffer. with 0.133 M sodium phosphate buffer (prepared by dilution of a 5 M Membrane strips were assembled into the high throughput dialysis jpet.aspetjournals.org at ASPET Journals on September 28, 2021

2+ Fig. 3. Activity of muscarinic ligands at receptor subtypes M2–M5. Agonist read (closed symbols): increases in free cytosolic Ca levels were measured immediately upon test compound addition. PAM read (open symbols): 20 minutes subsequent to test compound addition cells were challenged with an 2+ 2+ EC15 concentration of acetylcholine and free cytosolic Ca levels were again measured, with data expressed as fold increase in free intracellular Ca relative to the control EC15 response. Cholinergic Toxicity Produced by Selective M1 Activation 297

TABLE 2 Activity of test compounds in a broad in vitro selectivity panel

Potency (IC50 or EC50) values are reported for those assays in which the maximal observed response produced by the test compound was greater than or equal to 50% relative to a reference full agonist or inhibitor. Values are otherwise reported as greater than the top concentration tested or half of the top concentration tested.

IC50 or EC50 Target Compound A Compound B Compound C

mM mM mM G-protein receptors Adenosine A2a .30.0 .30.0 .30.0 Adrenergic a 1B .30.0 .30.0 .30.0 Adrenergic a 1D ∼4.5 .30.0 .30.0 Adrenergic a 2A .30.0 .30.0 .30.0 Adrenergic a 2C .30.0 .30.0 .30.0 Adrenergic b1 .30.0 .30.0 .30.0 Adrenergic b2 .30.0 .30.0 .30.0 Cannabinoid CB1 .30.0 .30.0 .30.0 Dopamine D1 .30.0 .30.0 .30.0 Dopamine D2 .30.0 .30.0 .30.0

Histamine H1 .30.0 .30.0 .30.0 Downloaded from Histamine H2 .30.0 .30.0 .30.0 Muscarinic M2 .30.0 .30.0 .30.0 Opioid kappa .30.0 .30.0 .30.0 Opioid mu .30.0 .30.0 .30.0 Serotonin 5HT1B .30.0 .30.0 .30.0 Serotonin 5HT2A agonist .10.0 .10.0 N.D. Serotonin 5HT2B agonist .5.0 .10.0 N.D.

Serotonin 5HT4 .30.0 .30.0 .30.0 jpet.aspetjournals.org Transporters Dopamine .30.0 .30.0 .30.0 Norepinephrine .30.0 .30.0 .30.0 Serotonin .30.0 .30.0 .30.0 Nuclear hormone receptors Androgen .150.0 16.1 .150.0 Estrogen a .150.0 ∼63.9 ∼114.1 Glucocorticoid .150.0 .150.0 .150.0

Progesterone .150.0 59.9 .150.0 at ASPET Journals on September 28, 2021 Ion channel receptors Calcium channel L-type (Cav1.2) antagonist .25.0 .25.0 .25.0 Calcium channel T-type (Cav3.2) activator .25.0 .25.0 .25.0 Cardiac sodium channel (hNAV1.5) antagonist 11.5 .30.0 .30.0 GABA-A (a1b2g2) antagonist .30.0 .30.0 .30.0 GABA-A (a1b2g2) potentiator 1.7 .30.0 .30.0 GABA-A (a5b2g2) antagonist .30.0 .30.0 .30.0 Nicotinic acetylcholine a1 antagonist .30.0 .30.0 .30.0 Nicotinic acetylcholine a4b2 agonist .30.0 .30.0 .30.0 Nicotinic acetylcholine a7 antagonist .30.0 .30.0 .30.0 NMDA glutamate NR1/2A agonist .25.0 N.D. .30.0 NMDA glutamate NR1/2A antagonist .30.0 .30.0 .30.0 NMDA glutamate NR1/2B agonist .25.0 .25 .30.0 Enzymes Acetylcholinesterase .60.0 .60 .30.0 Monoamine oxidase A 14.3 .30.0 .30.0 Monoamine oxidase B .30.0 .30.0 .30.0 Phosphodiesterase 3 .30.0 .30.0 .50.0 Phosphodiesterase 4 12.6 .30.0 .50.0

N.D., not determined. apparatus according to the manufacturer’s instructions, and 150 mlof a final concentration of 10 mM at 1% DMSO. All sera were adjusted to 0.133 M sodium phosphate buffer, pH 7.4, was loaded into one side of pH 7.4 prior to use. each dialysis chamber. Test articles and reference compounds (1 mM Test and reference compounds in sera (165 ml) were added to the in 100% DMSO) were added to human, rat, or mouse serum to achieve opposite side of each dialysis chamber from the buffer, and 15 mlof

TABLE 3 Serum protein binding results summary of test compounds Data reported represent mean and S.D. from three determinations; %Rec. denotes percent recovery.

Human Rat Mouse Compound %Free %Free S.D. %Rec. %Rec. S.D. %Free %Free S.D. %Rec. %Rec. S.D. %Free %Free S.D. %Rec. %Rec. S.D. A 0.8 0.4 105 21 0.3 0.10 92 8 0.5 0.1 136 16 B 74.8 13.6 118 9 49.2 12.60 87 12 111.5 7.6 70 4 C 17.1 2.0 108 9 32.2 7.80 94 19 23.9 1.0 83 6 298 Alt et al.

a flow injection analysis with an injection volume of 40 ml. The mobile phase was 25%A:75%B delivered by an Agilent (Santa Clara, CA) 1100 pump at 0.3 ml/min, where A was a mixture of 2 mM ammonium acetate/acetonitrile/formic acid (980:20:1, v/v/v), and B was a mixture of acetonitrile/water/formic acid (980:20:1, v/v/v). DiscoveryQuant automatically determined the optimized ionization polarity (positive or negative), precursor and product ions, declustering potential, and collision energy for test and reference compounds. The optimized selected reaction monitoring tandem mass spec- trometry conditions were used forsampleanalysispostassay.A 5-point calibration curve (5, 50, 500, 1000, and 2000 nM) was prepared for test and reference compounds in a 1:1 mixture of 133 mM sodium phosphate buffer and the appropriate mixed serum. A solution of acetonitrile containing two internal standards (100 nM alprenolol for compounds requiring positive ionization, 300 nM tolbutamide for compounds requiring negative ionization) was used for processing the assay samples. Fifty (50) ml of assay samples or calibration standards were first extracted with 150 ml of acetonitrile containing the internal

standards. After vortexing, centrifugation, and supernatant separa- Downloaded from tion, 15 ml of supernatant was injected onto an Ascentis Express C18 high-performance liquid chromatography column (2.7 mm, 2.1 30 mm; Sigma-Aldrich) at room temperature for analysis. The mobile Fig. 4. Plasma exposure of compounds A and B in rats following i.v. (1 mg/kg) phase B was the same as that used for optimization, and mobile phase and oral (5 mg/kg) administration. A was a mixture of 980:20:1 (v/v/v) water/acetonitrile/formic acid. The peak area ratios of the test or reference compound to the internal compound in serum was immediately removed and added to 135 mlof standard were used for quantification. A linear regression with a jpet.aspetjournals.org 2 mixed matrix solution to create the reference sample (T0). The mixed 1/concentration weighting was applied to the calibration standards to matrix solution consisted of 16.7% human serum, 16.7% rat serum, obtain the calibration curve. The concentration of the test or reference 16.7% mouse serum, and 50% 0.133 M sodium phosphate buffer. The compound was then calculated with the corresponding calibration equilibrium dialysis chamber was incubated at 37°C in a 7% CO2 curve. The free fraction (percent free), percent bound, and recovery atmosphere for 6.5 hours. values were calculated for each test sample and control compounds as Following incubation, 75 ml from the buffer (dialysate) side of the follows: HTD equilibrium dialysis chamber was added to 75 ml of the mixed

5 ð = Þ at ASPET Journals on September 28, 2021 serum solution to create the postincubation buffer sample. The mixed Percent free dialysate sample serum sample 100 à 5 ð 1 Þ= matrix solution consisted of 33% human serum, 33% rat serum, and Percent recovery dialysate sample serum sample T0 sample 100 33% mouse serum. In addition, 15 ml from the serum side of the HTD equilibrium dialysis chamber was added to 135 ml of the appropriate mixed matrix solution to create the postincubation serum sample. In Vivo Pharmacokinetic Studies The concentrations of test articles and reference compound samples were determined by liquid chromatography–tandem mass spectrom- All animal studies were performed under the approval of the etry. The analysis system consisted of two sets of binary Shimadzu Bristol-Myers Squibb Animal Care and Use Committee and in 10ADvp pumps with SCL-10Avp controllers for gradient elution, a accordance with the Association for Assessment and Accreditation of Leap HTS Autosampler (Leap Technologies, Carrboro, NC) with twin Laboratory Animal Care (AAALAC, Frederick, MD). arms for sample injection, a Thermo Fisher Scientific (Waltham, MA) Rat. Pharmacokinetic studies were conducted in male Sprague- valve interface module for valve switching and stream selection, and Dawley rats (300–350 g) with cannulae implanted in the jugular veins. an AB Sciex (Framingham, MA) triple quadrupole mass spectrometer After dosing, serial blood samples (0.3 ml) were obtained from the operated under electrospray ionization mode. To obtain the optimum appropriate cannula of each rat by collection into EDTA-containing selected reaction monitoring conditions for sample analysis, tandem tubes (Becton Dickinson, Franklin Lakes, NJ), and centrifuged to mass spectrometry optimization was performed using Discovery- separate plasma. Plasma was frozen until analysis. For the i.v. Quant featuring saturation control by AB Sciex (Framingham, MA) studies, the compound was dissolved (1 mg/ml) in a vehicle of 10% with 10 mM standard solutions (in 1:1 methanol/water) prepared from N-methyl pyrrolidinone, 90% polyethylene glycol (PEG) 400 and dosed the compound stock solutions. The optimization was performed using (1 ml/kg) as a 10-minute constant rate infusion into the jugular vein,

TABLE 4 Pharmacokinetic properties of compounds A and B in the rat following i.v. (1 mg/kg) and oral (2.5 mg/kg) administration

Compound A Compound B Parameter Unit Intravenous Oral Intravenous Oral Dose mg/kg 1 2.5 1 2.5 Cmax nM 2840 6 460 1850 6 897 1602 6 285 151 6 69 AUCtot nM·h43576 720 9630 6 4473 727 6 57 714 6 23 t1/2 Hour 3.2 6 1.1 2.5 6 0.3 4.0 6 4.0 3.9 6 2.0 Clearance ml/min/kg 8.4 6 1.5 59 6 4.8 Vss l/kg 1.7 6 0.7 6.1 6 5.1 Bioavaliability %F 86 6 30 39 6 3.6

%F, percentage of absolute oral bioavailability. Cholinergic Toxicity Produced by Selective M1 Activation 299

temperature was taken at t 5 0, 30, and 45 minutes postdose. Plasma prepared from the collected blood samples was stored frozen until analysis.

Pharmacokinetic Data Analysis Pharmacokinetic parameters were obtained by noncompartmental analysis of plasma concentration versus time data (KINETICA software, Version 2.4, InnaPhase Corporation, Philadelphia). The

peak concentration (Cmax) and time for Cmax (tmax) were recorded directly from experimental observations. The area under the curve

(AUC) from time zero to the last sampling time (AUC0–t) and the AUC from time zero to infinity (AUCINF) were calculated using a combina- tion of linear and log trapezoidal summations. The whole body plasma

clearance, steady-state volume of distribution (Vss), apparent terminal half-life (t1/2), and mean residence time were estimated following i.v. administration. The absolute oral bioavailability (F) was estimated as the ratio of the dose-normalized AUC values following oral and i.v.

doses. Downloaded from

Results Fig. 5. Plasma exposure of compound A in monkeys following i.v. Ca21 Flux Assays administration at 0.1 mg/kg, followed by 0.3 mg/kg 1 hour after the first dose. To assess the in vitro activity and selectivity profile of the jpet.aspetjournals.org M1 PAMs used in this study, these compounds were tested using Chinese hamster ovary cells recombinantly expressing with serial blood samples collected before dosing and at 0.17, 0.25, 0.5, the various muscarinic receptor subtypes (M –M ). The well- 0.75, 1, 2, 3, 5, 7, and 24 hours after dosing (n 5 3 rats/dose group). 1 5 characterized M receptor PAM 1-(4-methoxybenzyl)-4-oxo- For solution dosing by mouth, the compound was administered by 1 gastric gavage as an aqueous solution containing 10% N-methyl 1,4-dihydroquinoline-3-carboxylic acid (Ma et al., 2009) was pyrrolidinone, 85% PEG 400, and 5% d-alpha tocopheryl polyethylene also included as a reference for in vitro experiments. The glycol 1000 succinate. Serial blood samples were collected before ability of test compounds to either directly elicit increases in 1 dosing and at 0.25, 0.5, 0.75, 1, 3, 5, 7, and 24 hours after dosing (n 5 intracellular Ca2 levels [agonist (ago) mode], or to increase at ASPET Journals on September 28, 2021 21 3 rats/dose group). Prior to all dosingbymouth,theratswerefasted the Ca response elicited by a low (∼EC15) concentration of overnight with free access to water. acetylcholine (PAM mode) was measured. Potency (EC50) Monkey. Pharmacokinetic and tolerability studies were conducted and Emax values are provided in Table 1. Compounds A, B, in cynomolgous monkeys bearing vascular access ports to facilitate and C, and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline- 5 blood collection in an i.v. rising dose design (n 2). Vehicle (5% 3-carboxylic acid all exhibited mixed ago-PAM activity at the hydroxypropyl beta cyclodextrin; 95% water) was infused at 0.5 ml/kg M1 receptor (Fig. 2). These compounds potentiated the via the venous port over 5 minutes at a constant rate. Animals were 21 observed for 5 minutes. Then, compound (0.1 mg/kg) was infused for cellular Ca response to an EC15 concentration of acetylcholine 5 minutes and animals were observed for clinical signs for 1 hour. Blood at lower concentrations, while at higher concentrations these was taken at the end of infusion (5 minutes) and at 10, 15, 30, and compounds exerted allosteric agonist activity, directly activat- 45 minutes. Body temperature was taken at t 5 0, 30, and 45 minutes ing the receptor even in the absence of orthosteric agonist. In postdose. At the end of the first observation period (1 hour), compound PAM-mode testing, test compounds were preincubated with (0.3 mg/kg) was infused for 5 minutes and animals were observed for cells for 20 minutes prior to addition of acetylcholine and 2 hours for clinical signs. Blood was taken at the end of infusion measurement of the acetylcholine-evoked Ca21 response. Un- (5 minutes) and at 10, 15, 30, and 45 minutes and 1, 2, 3, 5, and 24 hours der these conditions, compounds that exert direct agonist 5 postdose. Body temperature was taken at t 0, 30, and 45 minutes activity (either orthosteric or allosteric) produce desensitization postdose. Plasma prepared from the collected blood samples was stored of the receptor/assay system during this 20-minute preincuba- frozen until analysis. Dog. Pharmacokinetic and tolerability studies were conducted in tion period, inhibiting cellular responses to the subsequent male beagle dogs bearing vascular access ports to facilitate blood acetylcholine challenge and confounding interpretation of collection in an i.v. rising dose design (n 5 2). Vehicle (5% hydroxypropyl beta cyclodextrin; 95% water) was infused at 0.5 ml/kg via the venous TABLE 5 port over 5 minutes at a constant rate. Animals were observed for 5 minutes. Then, compound (low dose) was infused for 5 minutes and Pharmacokinetic parameters of compound A in the monkey following i.v. (0.1 mg/kg) and 0.3 mg/kg 1 hour after the first dose animals were observed for clinical signs for 1 hour. Blood was taken at the end of infusion (5 minutes) and at 15, 30, and 45 minutes. Body Compound A Monkey Dose Escalation (i.v.) temperature was taken at t 5 0, 30, and 45 minutes postdose. At the Parameter Unit end of the first observation period (1 hour), compound (intermediate 0.1 0.3 dose) was infused for 5 minutes and animals were observed for 1 hour mg/kg mg/kg for clinical signs. At the end of the second observation period (1 or Cmax nM 1475 2320 2 hours after the intermediate dose), compound (high dose) was AUCtot nM·h 681 2367 infused for 5 minutes and animals were observed for 2 hours for t1/2 Hour 0.9 3.2 clinical signs. Blood was taken at the end of infusion (5 minutes) and at Clearance ml/min 5.4 4.6 15, 30, and 45 minutes and 1, 2, 3, 5, 7, and 24 hours postdose. Body Vss Liter 0.3 0.9 300 Alt et al.

A was found to be highly (.99%) protein bound in human and rodent serum, while compounds B and C exhibited high free fractions in serum (Table 3).

Pharmacokinetic and Tolerability Studies Rat. Compound A was dosed i.v. (1 mg/kg) as a clear colorless solution in N-methyl pyrrolidinone:PEG 400 (10:90) and orally (2.5 mg/kg) in N-methyl pyrrolidinone:PEG 400: d-alpha tocopheryl polyethylene glycol 1000 succinate (10:85: 5). Plasma was sampled out to 24 hours (Fig. 4; Table 4). Following i.v. administration of compound A at 1 mg/kg in the rat, a low clearance (8.4 ml/min/kg) and a high volume of distribution (1.7 l/kg) were observed. The resulting half-life was moderate at 3.2 hours. Following oral administration (2.5 mg/kg) a Cmax of 1.9 mM was observed 2.7 hours postdose. The AUC (0–24 hours) was 9.6 mM·h. Bioavailability of

compound A was 86% following oral dosing. It should be noted Downloaded from Fig. 6. Plasma exposure in dogs following i.v. administration of that some variation was observed in the individual rats after compound B at 1 mg/kg in dogs A and B, followed by 1.5 mg/kg (dog B) or oral dosing. Following oral and i.v. administration, all rats had 3 mg/kg (dog A) 1 hour after the first dose. diarrhea between 45 minutes and 1 hour postdose. No other clinical observations were noted for the remainder of the PAM-mode results. Therefore, for compounds A, B, and C, and study, except for bright yellow urine 24 hours postdose in the cages of animals that were dosed orally. 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic jpet.aspetjournals.org acid, the PAM-mode curves have been truncated to exclude Compound B was dosed in rat using the same study design results at concentrations of test compound that elicit direct as compound A. Following i.v. administration of compound allosteric agonist activity (Fig. 2). With the exception of B at 1 mg/kg in the rat as a clear yellow solution, a high compound A, which exhibited very weak but measurable clearance (59 ml/min/kg) and a high volume of distribution (6.1 l/kg) were observed (Fig. 4; Table 4). The resulting half-life activity at M2 (Fig. 3), none of the M1 PAMs tested had any measurable agonist or PAM activity at any of the other was moderate at 4 hours (individual rat half-life varied from 1.6 to 8.6 hours). Clearance (13.5 ml/min/kg) was much muscarinic receptor subtypes, M2–M5. at ASPET Journals on September 28, 2021 higher compared with previously reported values (Kuduk et al., 2011). Following oral administration of compound B at Selectivity Profiling 2.5 mg/kg, the Cmax, tmax,andAUC0-tot values were 0.2 mM, We evaluated compounds A, B, and C in concentration- 1.0 hour, and 0.7 mM·h, respectively. The bioavailability of response (30 mM top concentration) in a broad panel of compound B was 39%, which is lower compared with pre- selectivity assays comprising G protein coupled receptors, viously reported values (Kuduk et al., 2011). No clinical biogenic amine transporters, ion channels, enzymes, and observations were noted other than bright yellow urine found nuclear hormone receptors (Table 2; Supplemental Tables in the bedding from all animals postdose. 1–3). This panel consists of targets that have been implicated Monkey. Compound A was dosed i.v. in a dose escalation in various safety/liability concerns within central nervous, study at 0.1 and 0.3 mg/kg as described in Materials and cardiac, pulmonary, renal, immune, gastrointestinal, and Methods. Plasma concentration measurements are shown in reproductive systems. Compound A was noted to have weak Fig. 5, and pharmaokinetic properties are summarized in inhibitory activity at cardiac sodium channel NaV1.5 (IC50 5 Table 5. Pharmacokinetic parameters were not calculated 11 mM) and phosphodiesterase type 4 (IC50 5 13 mM). It also for the 0.1 mg/kg dose group due to the short time interval was found to bind to the adrenergic a1D receptor, based upon (,1 hour). Although the plasma levels of compound A were competition in a radioligand binding assay (IC50 5 4 mM). somewhat higher for the second dose as a result of the first Furthermore, in an assay that measures potentiation of GABA dose, the expected contribution was calculated to be minimal receptor GABAA (a1b2g2) activity, compound A was observed (, 10%). Following i.v. administration at 0.3 mg/kg, compound to potentiate GABA-evoked responses with EC505 2 mM, with a maximal response that reached a plateau at ∼36% of the maximal GABA-stimulated response. Compound B exhibited TABLE 6 5 Pharmacokinetic properties of compound B in the dog following i.v. very weak binding to androgen (IC50 16 mM), estrogen administration at 1 mg/kg in dogs A and B and 1.5 mg/kg (dog B) or 3 mg/ (IC50 5 64 mM), and progesterone (IC50 5 60 mM) receptors. kg (dog A) 1 hour after the first dose Compound C did not display any measurable activity at any of Compound B Dog Dose Escalation (i.v.) the 43 targets in this selectivity panel at the concentrations Parameter Unit tested. Dogs A and B Dog B Dog A Dose mg/kg 1 1.5 3 Serum Protein Binding Studies Cmax nM 2815 5240 16,000 AUCtot nM·h 1456 3844 13,183 Compounds A, B, and C were tested in a serum protein t1/2 Hour 0.5 5.0 6.4 binding panel using human, rat, and mouse serum. These Clearance ml/min·kg 30 17 10 V Liter 1.1 3.6 3.6 compounds exhibited a wide range of protein binding. Compound ss Cholinergic Toxicity Produced by Selective M1 Activation 301

TABLE 7 Pharmacokinetic properties of compound C in the dog following i.v. administration at 0.5 and 1 mg/kg in dogs A and B 1 hour after the first dose and 5 mg/kg (dog A) 2 hours after the first dose

Compound C Dog Dose Escalation (i.v.)

Parameter Unit 0.5 mg/kg 1 mg/kg 5 mg/kg

Dog A Dog B Dog A Dog B Dog A

Cmax nM 2560 4080 7290 17,300 41,300 AUCtot nM·h 639 722 2301 3712 13,213 t1/2 Hour 0.2 0.2 0.2 0.9 4.2 Clearance ml/min 35 31 N.D. N.D. N.D. Vss l/kg 0.7 0.5 N.D. N.D. N.D.

N.D., not determined.

point. Due to the severe effects in the first dog, the infusion for

the second dog was stopped after 2.5 minutes, resulting in a Downloaded from lower dose of 1.5 mg/kg. Mild salivation and licking was observed after 2 minutes of infusion. No other adverse events were noted. Exposure at the end of infusion was 5.2 mM. Fig. 7. Plasma exposure of compound C in dogs following i.v. adminis- Pharmacokinetic parameters for this dog were similar to the tration at 0.5 mg/kg, followed by 1 mg/kg 1 hour postdose (dogs A and B) and 5 mg/kg 2 hour postdose (dog A). first dog with a Vss of 3.6 l/kg and a t1/2 of 5.0 hours, but clearance was somewhat higher (17 ml/min/kg). jpet.aspetjournals.org Compound C was dosed in the same manner as compound B A had low clearance (4.6 ml/min), moderate V (0.9 l), and ss with a 5-minute infusion of 0.5, 1, and 5 mg/kg with 1 hour moderate t (3.2 hours). Plasma C (5 minutes postdose) 1/2 max observation between doses. Plasma concentration measure- was 2.3 mM and declined to 1.0 mM by 15 minutes postdose. No ments are shown in Fig. 7, and pharmaokinetic parameters effects were observed in the monkeys following administration are summarized in Table 7. Following the vehicle and 0.5 mg/kg of vehicle and the 0.1 mg/kg dose. At 0.3 mg/kg, nasal dose, no adverse events were noted. Exposures at the end discharge and hypersalivation were observed in one monkey of the infusion were 2.6 and 4.1 mM. Clearance was high at from approximately 5 to 15 minutes postdose. In the other at ASPET Journals on September 28, 2021 31–35 ml/min/kg, V was moderate (0.5–0.7 l/kg), and t was animal, hypersalivation, emesis (single episode), urination ss 1/2 short (0.2 hours). Following administration at 1 mg/kg, slight (single episode), and miosis were observed from approxi- clear nasal discharge was observed in both animals approxi- mately 5 to 20 minutes postinfusion. mately 3 to 4 minutes into the infusion. One animal also Dog. Compound B was dosed i.v. in a dose escalation study experienced hypersalivation and decreased activity. Expo- at 1 and 3 mg/kg as described in the Materials and Methods. sures at the end of the infusion were 7.3 and 17.3 mM. Both Plasma concentration measurements are shown in Fig. 6, and dogs had no other symptoms by 5 minutes postdose. Only 1 dog pharmacokinetic parameters are summarized in Table 6. was administered the 5 mg/kg dose. Decreased activity, Pharmacokinetic parameters were not calculated for the licking, and excessive hypersalivation were noted 2.5 minutes 1 mg/kg dose, due to the short time interval (,1 hour). after the start of the infusion with moderate nasal discharge. Following administration at 1 mg/kg, both dogs had slight By the end of the infusion, dilated pupils, clear ocular drooling approximately 4 minutes into the infusion; no other discharge, and slight ataxia with a wide stance were observed symptoms were observed. Exposures at the end of infusion in addition to hypersalivation and licking. Symptoms abated were 2.6 and 3.1 mM. Although the plasma levels of compound rapidly, with no further observations by 20 minutes postdose. B were somewhat higher for the second dose as a result of the A summary of the observed in vivo effects in all species is first dose, the expected contribution was calculated to be provided in Table 8. minimal (,10%). Following i.v. administration in one dog at 3 mg/kg, compound B had low clearance (10 ml/min), high Vss (3.6 l), and long t1/2 (6.4 hours). Three minutes into the Discussion infusion, nasal discharge, licking lips, and salivation were visible. Severe salivation was noted at end of the infusion A significant body of research exists to support the potential (5 minutes). Exposure at the end of administration was 16 mM. therapeutic efficacy of M1 agonists for treating cognitive Vomiting was observed between 7 and 9 minutes postdose. disorders such as schizophrenia and Alzheimer’s disease. M1 Severe diarrhea was observed between 8 and 17 minutes agonists have been shown to reverse cognitive deficits in postdose. Ataxia was observed from 11 to 50 minutes postdose, animal models (Jones et al., 2005; Langmead et al., 2008; starting with abnormal stance with front legs straight and Barak and Weiner 2011), and more importantly the M1 back legs splayed behind. Ataxia was severe by 20 minutes agonist xanomeline has been shown to produce beneficial postdose, with complete loss of hind end control and knuckling psychiatric and cognitive effects in human patients (Bodick on front feet. Ataxia lessened by 29 minutes postdose, with the et al., 1997; Shekhar et al., 2008). Unfortunately, xanomeline dog walking in circles. The dog was alert and exploring by also produces serious side effects including salivation, sweat- 50 minutes postdose, with no other adverse events noted after ing, and gastrointestinal distress. Although xanomeline had 1 hour postdose. Exposure was 2.2 mM at the 1 hour time originally been reported to be highly selective for M1 over 302 Alt et al.

TABLE 8 Summary of doses, dose routes, and observed effects for all species used in the evaluation of compounds A, B, and C in tolerability studies

Compound Species Route Dose Observed Effect

mg/Kg A Rat i.v. 1 No effects A Rat By mouth 2.5 Diarrhea, bright yellow urine A Cyno i.v. 0.3 Hypersalivation, licking, emesis, urination, miosis A Dog i.v. 1.5 Hypersalivation, licking A Dog i.v. 3 Hypersalivation, licking, emesis B Rat i.v. 1 No effects B Rat By mouth 2.5 Bright yellow urine B Dog i.v. 1 Slight drooling, runny nose B Dog i.v. 1.5 Mild salvation, licking lips B Dog i.v. 3 Nose running, licking lips, salvation, vomiting, diarrhea, ataxia C Dog i.v. 1 Nasal discharge, hypersalivation, licking, decreased activity C Dog i.v. 5 Nasal discharge, hypersalivation, licking, decreased activity, ataxia

Cyno, cynomologous monkey. Downloaded from other muscarinic receptor subtypes based upon ex vivo studies The M1 PAMs used in this study were profiled in an in vitro (Sauerberg et al., 1992; Shannon et al., 1994), subsequent safety panel measuring activity at 43 G protein coupled studies using cloned human muscarinic receptors suggested receptors, ion channels, transporters, nuclear hormone recep- that xanomeline exhibits very little selectivity among the five tors, and enzymes (Table 2). Compound A showed some muscarinic receptor subtypes (Watson et al., 1998; Wood et al., measurable activity at several of the receptor targets in this jpet.aspetjournals.org 1999). Therefore, the untoward side-effect profile of xanomeline panel: the a1D-adrenergic receptor, the GABAA receptor, has been attributed to nonselective activity at other muscarinic phosphodiesterase 4, and the cardiac sodium channel NaV1.5. receptor subtypes, in particular M2 and M3 (Mirza et al., 2003). However, compound A exhibited only very low potency Recently, the identification of small molecules that bind to (2–13 mM) at each of these targets. Compound B showed some allosteric binding sites that are distinct from the (orthosteric) binding activity at androgen, estrogen, and progesterone endogenous agonist binding site has emerged as a strategy for receptors, but again with very low (16–64 mM) potency. targeting G protein coupled receptors (Wootten et al., 2013). Compound C showed no significant activity at any of the

Allosteric modulators have several potential advantages over targets in this panel. It is important to note that many of these at ASPET Journals on September 28, 2021 traditional orthosteric ligands (Wootten et al., 2013), includ- selectivity assays (e.g., radioligand binding–based assays) ing the potential for improved receptor subtype selectivity. would not be expected to detect potential allosteric modulation Indeed, the M1 PAMs used in the current study show exquisite of the receptor tested. However, with this caveat, these results functional selectivity for M1 over other muscarinic receptor —combined with the similarity of the in vivo toxicity profile subtypes in vitro (Figs. 2 and 3; Table 1). Therefore, it was observed with compounds A, B, and C—again support the surprising that all three of the M1 PAMs tested produced in conclusion that the toxic effects observed in the current study vivo effects consistent with cholinergic toxicity, such as were mediated by M1 receptors rather than by some off-target hypersalivation, vomiting, and severe diarrhea. These effects activity of the compounds. are reminiscent of the side effects seen in humans during It is common for PAMs to also exhibit direct agonist activity clinical trials with xanomeline (Bodick et al., 1997; Shekhar at higher concentrations (Schwartz and Holst 2006, 2007; et al., 2008), suggesting that some or all of xanomeline’s Bridges and Lindsley 2008; Burford et al., 2011). Such adverse effects may in fact be mediated by activation of the M1 compounds are commonly referred to as ago-PAMs (Noetzel receptor itself, rather than by other muscarinic receptor et al., 2012). Indeed, all three of the M1 PAMs used in the subtypes, as had been previously assumed. current study were found to exhibit direct agonist activity at It is important to note that, in this study, the in vitro concentrations approximately 50- to 100-fold higher than assays used to assess selectivity of the test compounds for M1 those required for PAM activity in vitro (Fig. 2; Table 1). versus the other muscarinic receptor subtypes used cells Ago-PAMs and pure PAMs (those with no direct agonist recombinantly expressing each of the muscarinic receptors. activity) can show differential activity in vivo (Bridges et al., Furthermore, in the case of the M2-andM4-expressing cell 2013; Rook et al., 2013). This may in part reflect the ability of lines, the mutant G protein Gqi5 was coexpressed to force PAMs to maintain the temporal and spatial fidelity of native 21 coupling of the M2 and M4 receptors to a Ca mobilization signaling because pure PAMs do not activate the receptor on pathway instead of their natural Gi-mediated coupling to their own. They exert an effect only when and where the native inhibition of adenylyl cyclase. Therefore, the cellular models agonist is present, thereby preserving some aspects of native used for selectivity assessment represent highly artificial receptor signaling and its physiologic regulation, and thus systems. While these recombinant systems afford excellent might be expected to avoid some side effects associated with sensitivity and reproducibility of data generated, it is direct activation of the receptor (for reviews, see Wootten possible that artificial G protein coupling or other differences et al., 2013; Burford et al., 2015). Within this context, it is from the receptors’ native neuronal environment could lead to interesting to note that the doses in this study, which pro- results that do not reflect the true activity of these compounds duced adverse events in vivo, generally attained relatively in vivo, and this caveat must be kept in mind when interpreting high (i.e., micromolar) plasma exposure levels in test animals. these results. At these concentrations, the M1 PAMs tested may be producing Cholinergic Toxicity Produced by Selective M1 Activation 303 direct agonism at the receptor in vivo, based upon their in vitro Acknowledgments profiles (Fig. 2; Table 1). Additionally, the fact that the side The authors thank Reshma Panemangalore, Rudy Krause, Jeremy effects were transient further suggests that these side effects Stewart, Lizbeth Gallagher, Glen Farr, Michele Matchett, and Ron resulted from high concentrations of the compounds. There- Knox for the execution of various in vitro selectivity assays. fore, this raises the intriguing possibility that direct activation Authorship Contributions of M1 is required to produce the unwanted cholinergic side effects observed, and that pure M1 PAMs, or ago-PAMs Participated in research design: Alt, Pendri, Cvijic, Westphal, administered at doses below those required for direct receptor O’Connell, Zhang, Gentles, Jenkins, Loy, Macor. agonism, could still be safe, and therefore the identification of Conducted experiments: Bertekap, Benitex, Nophsker, Rockwell, M PAMs with acceptable safety margins may be possible. Burford, Sum, Chen, Herbst, Ferrante. 1 Contributed new reagents or analytic tools: Pendri, Li, Gentles. Within this context, it should be noted that compound B Performed data analysis: Alt, Bertekap, Benitex, Nophsker, Rock- from this study has also been tested in vivo by Kuduk et al. well, Burford, Sum, Chen, Herbst, Ferrante, Hendricson, Jenkins, (2011), who also reported that the compound is selective Loy. against other muscarinic receptor subtypes, in addition to Wrote or contributed to the writing of the manuscript: Alt, Pendri, being selective against a wide panel of other targets, in Sum, Herbst, Banks, Jenkins, Loy, Macor. agreement with the current study. This group has further reported that compound B did not produce significant gastro- References intestinal or salivation disturbances at doses up to five times Auld DS, Kornecook TJ, Bastianetto S, and Quirion R (2002) Alzheimer’s disease and Downloaded from the basal forebrain cholinergic system: relations to b-amyloid peptides, cognition, greater than those that produced procognitive effects in and treatment strategies. Prog Neurobiol 68:209–245. rhesus monkeys, suggesting that a therapeutic window be- Barak S and Weiner I (2011) The M₁/M₄ preferring agonist xanomeline reverses amphetamine-, MK801- and scopolamine-induced abnormalities of latent in- tween beneficial and undesired effects can be achieved hibition: putative efficacy against positive, negative and cognitive symptoms in (Vardigan et al., 2015). Furthermore, compound B was shown schizophrenia. Int J Neuropsychopharmacol 14:1233–1246. Bodick NC, Offen WW, Levey AI, Cutler NR, Gauthier SG, Satlin A, Shannon HE, to produce synergistic procognitive effects when combined Tollefson GD, Rasmussen K, and Bymaster FP, et al. (1997) Effects of xanomeline, with either the muscarinic agonist xanomeline, or the acetyl- a selective muscarinic receptor agonist, on cognitive function and behavioral jpet.aspetjournals.org symptoms in Alzheimer disease. Arch Neurol 54:465–473. cholinesterase inhibitor donepezil, suggesting combination Bridges TM and Lindsley CW (2008) G-protein-coupled receptors: from classical therapy as another potential strategy for achieving acceptable modes of modulation to allosteric mechanisms. ACS Chem Biol 3:530–541. Bridges TM, Rook JM, Noetzel MJ, Morrison RD, Zhou Y, Gogliotti RD, Vinson PN, safety margins with M1-targeted therapeutics. Xiang Z, Jones CK, and Niswender CM, et al. (2013) Biotransformation of a novel The current study does not include testing in cognition positive allosteric modulator of metabotropic glutamate receptor subtype 5 con- tributes to seizure-like adverse events in rats involving a receptor agonism- models, and was not designed to gauge safety margins. dependent mechanism. Drug Metab Dispos 41:1703–1714. Instead, the current study was designed to test the hypothesis Burford NT, Traynor JR, and Alt A (2015) Positive allosteric modulators of the

m-opioid receptor: a novel approach for future pain medications. Br J Pharmacol at ASPET Journals on September 28, 2021 that selective activation of the M1 receptor alone would be 172:277–286. insufficient to produce classic cholinergic toxicity. Our results Burford NT, Watson J, Bertekap R, and Alt A (2011) Strategies for the identification refute this hypothesis. Toxic effects were observed in rats, of allosteric modulators of G-protein-coupled receptors. Biochem Pharmacol 81: 691–702. dogs, and cynomologous monkeys. These effects were similar Felder CC, Bymaster FP, Ward J, and DeLapp N (2000) Therapeutic opportunities across the species tested, and were produced by three different for muscarinic receptors in the central nervous system. J Med Chem 43:4333–4353. Fisher A (2008) M1 muscarinic agonists target major hallmarks of Alzheimer’s dis- M1 receptor PAMs. Furthermore, these effects are similar to ease–the pivotal role of brain M1 receptors. Neurodegener Dis 5:237–240. Heinrich JN, Butera JA, Carrick T, Kramer A, Kowal D, Lock T, Marquis KL, Pausch those seen in clinical trials using the M1 agonist xanomeline. MH, Popiolek M, and Sun SC, et al. (2009) Pharmacological comparison of mus- All three of the M1 PAMs used in this study exhibited excellent carinic ligands: historical versus more recent muscarinic M1-preferring receptor agonists. Eur J Pharmacol 605:53–56. selectivity for M1 over other muscarinic receptor subtypes in Ishii M and Kurachi Y (2006) Muscarinic acetylcholine receptors. Curr Pharm Des 12: vitro, and in a safety panel no off-target activity was detected 3573–3581. for these compounds, which would explain the toxic effects Jones CK, Eberle EL, Shaw DB, McKinzie DL, and Shannon HE (2005) Pharmaco- logic interactions between the muscarinic cholinergic and dopaminergic systems in observed. Based upon these combined results, we now hypoth- the modulation of prepulse inhibition in rats. J Pharmacol Exp Ther 312: – esize that activation of the M1 receptor alone is sufficient to 1055 1063. Kuduk SD, Chang RK, Di Marco CN, Pitts DR, Greshock TJ, Ma L, Wittmann M, produce a cholinergic toxicity syndrome. This has important Seager MA, Koeplinger KA, and Thompson CD, et al. (2011) Discovery of a selec- implications for the development of M1-targeted therapeutics tive allosteric M1 receptor modulator with suitable development properties based on a quinolizidinone carboxylic acid scaffold. J Med Chem 54:4773–4780. since it would suggest that dose-limiting cholinergic side effects Langmead CJ, Watson J, and Reavill C (2008) Muscarinic acetylcholine receptors as such as those observed in clinical trials with xanomeline cannot CNS drug targets. Pharmacol Ther 117:232–243. be completely avoided simply by the use of drugs that exhibit Lebois EP, Digby GJ, Sheffler DJ, Melancon BJ, Tarr JC, Cho HP, Miller NR, Morrison R, Bridges TM, and Xiang Z, et al. (2011) Development of a highly se- greater selectivity for the M1 receptor versus other muscarinic lective, orally bioavailable and CNS penetrant M1 agonist derived from the receptor subtypes. MLPCN probe ML071. Bioorg Med Chem Lett 21:6451–6455. Lindstrom J (1997) Nicotinic acetylcholine receptors in health and disease. Mol Finally, it should be noted that a novel allosteric M1 agonist, Neurobiol 15:193–222. HTL9936, has been investigated in a phase 1 clinical trial as Ma L, Seager MA, Wittmann M, Jacobson M, Bickel D, Burno M, Jones K, Graufelds VK, Xu G, and Pearson M, et al. (2009) Selective activation of the M1 muscarinic a potential treatment of cognitive impairment associated acetylcholine receptor achieved by allosteric potentiation. Proc Natl Acad Sci USA with Alzheimer’s disease (http://clinicaltrials.gov/ct2/show/ 106:15950–15955. Melancon BJ, Tarr JC, Panarese JD, Wood MR, and Lindsley CW (2013) Allosteric NCT02291783), and was reported to be well tolerated in this modulation of the M1 muscarinic acetylcholine receptor: improving cognition and a study. It will be of great interest to see whether this initial potential treatment for schizophrenia and Alzheimer’s disease. Drug Discov Today 18:1185–1199. report is replicated, and whether the central nervous system Mirza NR, Peters D, and Sparks RG (2003) Xanomeline and the antipsychotic po- exposure levels achieved in this safety study are sufficient to tential of muscarinic receptor subtype selective agonists. CNS Drug Rev 9: 159–186. produce therapeutic effects. Therefore, future clinical stud- Nickols HH and Conn PJ (2014) Development of allosteric modulators of GPCRs for ies using HTL9936 are expected to provide additional treatment of CNS disorders. Neurobiol Dis 61:55–71. Noetzel MJ, Rook JM, Vinson PN, Cho HP, Days E, Zhou Y, Rodriguez AL, Lavreysen insight into the side-effect liabilities associated with M1 H, Stauffer SR, and Niswender CM, et al. (2012) Functional impact of allosteric receptor activation in humans. agonist activity of selective positive allosteric modulators of metabotropic 304 Alt et al.

glutamate receptor subtype 5 in regulating central nervous system function. Mol xanomeline as a novel treatment approach for schizophrenia. Am J Psychiatry 165: Pharmacol 81:120–133. 1033–1039. Rook JM, Noetzel MJ, Pouliot WA, Bridges TM, Vinson PN, Cho HP, Zhou Y, Vardigan JD, Cannon CE, Puri V, Dancho M, Koser A, Wittmann M, Kuduk SD, Gogliotti RD, Manka JT, and Gregory KJ, et al. (2013) Unique signaling profiles of Renger JJ, and Uslaner JM (2015) Improved cognition without adverse effects: positive allosteric modulators of metabotropic glutamate receptor subtype 5 de- novel M1 muscarinic potentiator compares favorably to donepezil and xanomeline termine differences in in vivo activity. Biol Psychiatry 73:501–509. in rhesus monkey. Psychopharmacology (Berl) 232:1859–1866. Sauerberg P, Olesen PH, Nielsen S, Treppendahl S, Sheardown MJ, Honoré T, Mitch Watson J, Brough S, Coldwell MC, Gager T, Ho M, Hunter AJ, Jerman J, Middlemiss CH, Ward JS, Pike AJ, and Bymaster FP, et al. (1992) Novel functional M1 DN, Riley GJ, and Brown AM (1998) Functional effects of the muscarinic receptor selective muscarinic agonists. Synthesis and structure-activity relationships of agonist, xanomeline, at 5-HT1 and 5-HT2 receptors. Br J Pharmacol 125:1413–1420. 3-(1,2,5-thiadiazolyl)-1,2,5,6-tetrahydro-1-methylpyridines. J Med Chem 35: Wess J, Blin N, Yun J, Schöneberg T, and Liu J (1996) Molecular aspects of mus- 2274–2283. carinic receptor assembly and function. Prog Brain Res 109:153–162. Schwartz TW and Holst B (2006) Ago-allosteric modulation and other types of allo- Wood MD, Murkitt KL, Ho M, Watson JM, Brown F, Hunter AJ, and Middlemiss DN stery in dimeric 7TM receptors. J Recept Signal Transduct Res 26:107–128. (1999) Functional comparison of muscarinic partial agonists at muscarinic receptor Schwartz TW and Holst B (2007) Allosteric enhancers, allosteric agonists and ago- subtypes hM1,hM2,hM3,hM4 and hM5 using microphysiometry. Br J Pharmacol – allosteric modulators: where do they bind and how do they act? Trends Pharmacol 126:1620 1624. Sci 28:366–373. Wootten D, Christopoulos A, and Sexton PM (2013) Emerging paradigms in GPCR – Shannon HE, Bymaster FP, Calligaro DO, Greenwood B, Mitch CH, Sawyer BD, allostery: implications for drug discovery. Nat Rev Drug Discov 12:630 644. Ward JS, Wong DT, Olesen PH, and Sheardown MJ, et al. (1994) Xanomeline: a novel muscarinic receptor agonist with functional selectivity for M1 receptors. J Address correspondence to: Andrew Alt, Leads Discovery and Optimiza- Pharmacol Exp Ther 269:271–281. tion, Bristol-Myers Squibb, 5 Research Parkway, Wallingford, CT 06492. Shekhar A, Potter WZ, Lightfoot J, Lienemann J, Dubé S, Mallinckrodt C, Bymaster E-mail: [email protected] FP, McKinzie DL, and Felder CC (2008) Selective muscarinic receptor agonist Downloaded from jpet.aspetjournals.org at ASPET Journals on September 28, 2021 Supplemental data for JPET #226910

Journal of Pharmacology and Experimental Therapeutics #226910

Evidence for classical cholinergic toxicity associated with 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 Westphal, Jonathan O’Connell, Martyn Banks, Litao Zhang, Robert

Gentles, Susan Jenkins, James Loy, and John E. Macor

Supplemental Data

Table S1. Activity of Compound A in a broad in vitro selectivity panel. Potency (IC50 or EC50) values are reported for those assays in which the maximal observed response produced by the test compound (Ymaxobs) was greater

than or equal to 50% relative a reference full agonist or inhibitor. Values are otherwise reported as greater than

the top concentration tested or half of the top concentration tested.

1

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurement

G-Protein Displacement of 3H-CGS- Coupled Adenosine A2A >30 40.6 21680, 25 nM Receptors

G-Protein Displacement of Prazosin, [7- Coupled Adrenergic α1B >30 4.8 Methoxy-3H], 1 nM Receptors

G-Protein Displacement of Prazosin, [7- Coupled Adrenergic α1D ~4.5 0.4 68.0 Methoxy-3H], 0.5 nM Receptors

G-Protein Displacement of 3H-MK912, 1 Coupled Adrenergic α2A >30 31.6 nM Receptors

G-Protein Displacement of Rauwolsine, [7- Coupled Adrenergic α2C >30 9.3 Methoxy-3H], 1nM Receptors

G-Protein Displacement of Coupled Adrenergic β1 >30 19.5 Dihydroalprenolol hydrochloride, Receptors levo-[ring, propyl-3H(N)], 1 nM

2

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurement

G-Protein Displacement of Coupled Adrenergic β2 >30 -2.0 Dihydroalprenolol hydrochloride, Receptors levo-[ring, propyl-3H(N)], 0.5 nM

G-Protein Displacement of [3H]- CP- Coupled Cannabinoid CB1 >30 7.1 55,940, 2.0 nM Receptors

G-Protein Displacement of SCH-23390 [N- Coupled Dopamine D1 >30 24.0 Methyl-3H], 1nM Receptors

G-Protein Displacement of 3H- Coupled Dopamine D2 >30 28.3 Methylspiperone, 0.6 nM Receptors

G-Protein Displacement of [Pyridinyl-5-3H]- Coupled Histamine H1 >30 pyrilamine, 2.5 nM Receptors

G-Protein Displacement of [3H]-Tiotidine, Coupled Histamine H2 >30 18.6 3.0 nM Receptors

3

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurement

G-Protein Displacement of [3H]-N-Methyl- Coupled Muscarinic M2 >30 9.1 Scopolamine Methyl Chloride, Receptors 0.5 nM

G-Protein Displacement of Naloxone-[N- Coupled Opioid kappa >30 0.8 41.9 Allyl-2,3-3H], 3 nM Receptors

G-Protein Displacement of Naloxone-[N- Coupled Opioid mu >30 12.6 Allyl-2,3-3H], 1.1 nM Receptors

G-Protein Displacement of [3H]- Coupled Serotonin 5HT1B >30 19.9 GP125743, 3.0 nM Receptors

G-Protein Serotonin 5HT2A Coupled >10 6.7 Stimulation of calcium flux agonist Receptors

G-Protein Serotonin 5HT2B Coupled >5 16.1 Stimulation of calcium flux agonist Receptors

4

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurement

G-Protein Displacement of 3H-GR113808, Coupled Serotonin 5HT4 >30 3.4 0.6 nM Receptors

Displacement of 125I RTI-55, Transporters Dopamine >30 0.125 nM

Displacement of 125I Nisoxetine, Transporters Norepinephrine >30 0.55 nM

Displacement of 125I RTI-55, Transporters Serotonin >30 0.26 nM

Nuclear Fluorescent polarization ligand Hormone Androgen >150 17.8 binding competition with Receptors Fluormone™ AL Red, 2 nM

Nuclear Fluorescent polarization ligand Hormone Estrogen A >150 32.2 binding competition with Receptors Fluormone™ EL Red, 1 nM

5

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurement

Nuclear Fluorescent polarization ligand Hormone Glucocorticoid >150 5.27 binding competition with Receptors Fluormone™ GS Red, 1 nM

Nuclear Fluorescent polarization ligand Hormone Progesterone >150 30.6 binding competition with Receptors Fluormone™ PL Red, 2 nM

Calcium Channel L- Ion Channels type (Cav1.2) >25 Inhibition of calcium flux Antagonist

Calcium Channel T- Ion Channels >25 11.3 Stimulation of calcium flux type (Cav3.2) Activator

Cardiac Sodium Inhibition of membrane potential Ion Channels Channel (hNAV1.5) 11.5 0.6 change triggered by 40 uM Antagonist veratridine

Inhibition of channel activity GABA-A (α1β2γ2) Ion Channels >30 triggered by GABA at EC95 Antagonist concentration

6

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurement

Potentiation of channel activity GABA-A (α1β2γ2) Ion Channels 1.7 1.6 triggered by GABA at EC20 Potentiator concentration

Inhibition of channel activity GABA-A (α5β2γ2) Ion Channels >30 triggered by GABA at EC95 Antagonist concentration

Inhibition of calcium flux Nicotinic Acetylcholine Ion Channels >30 stimulated with 10 µM α1 Antagonist acetylcholine

Nicotinic Acetylcholine Ion Channels >30 Stimulation of calcium flux α4β2 Agonist

Inhibition of calcium flux Nicotinic Acetylcholine Ion Channels >30 stimulated with 15 µM α7 Antagonist acetylcholine

NMDA Glutamate Ion Channels >25 -9.48 Stimulation of calcium flux NR1/2A Agonist

7

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurement

Inhibition of calcium flux NMDA Glutamate Ion Channels >30 stimulated with 100 µM NR1/2A Antagonist glutamate

NMDA Glutamate Ion Channels >25 31.1 Stimulation of calcium flux NR1/2B Agonist

Inhibiiton of enzyme activity on Enzymes Acetylcholinesterase >60 acetylcholine substrate, 50 µM

Inhibition of enzyme activity on Enzymes Monoamine Oxidase A 14.3 0.6 kynuramine substrate, 40 µM

Inhibition of enzyme activity on Enzymes Monoamine Oxidase B >30 1.2 kynuramine substrate, 20 µM

SPA-based enzyme inhibition Enzymes Phosphodiesterase 3 >30 assay with [3H]cAMP, 100 nM

8

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurement

SPA-based enzyme inhibition Enzymes Phosphodiesterase 4 12.6 assay with [3H]cAMP, 300 nM

9

Supplemental data for JPET #226910

Table S2. Activity of Compound B in a broad in vitro selectivity panel. Potency (IC50 or EC50) values are reported for those assays in which the maximal observed response produced by the test compound (Ymaxobs) was greater than or equal to 50% relative a reference full agonist or inhibitor. Values are otherwise reported as greater than the top concentration tested or half of the top concentration tested.

10

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurements

G-Protein Displacement of 3H-CGS- Adenosine A2A >30 -11.44 Receptors 21680, 25 nM

G-Protein Displacement of Prazosin, [7- Adrenergic A1B >30 -11.94 Receptors Methoxy-3H], 1 nM

G-Protein Displacement of Prazosin, [7- Adrenergic A1D >30 17.76 Receptors Methoxy-3H], 0.5 nM

G-Protein Displacement of 3H-MK912, 1 Adrenergic A2A >30 -6.44 Receptors nM

G-Protein Displacement of Rauwolsine, [7- Adrenergic A2C >30 23.65 Receptors Methoxy-3H], 1nM

Displacement of G-Protein Adrenergic B1 >30 -10.23 Dihydroalprenolol hydrochloride, Receptors levo-[ring, propyl-3H(N)], 1 nM

11

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurements

Displacement of G-Protein Adrenergic B2 >30 9.45 Dihydroalprenolol hydrochloride, Receptors levo-[ring, propyl-3H(N)], 0.5 nM

G-Protein Displacement of [3H]- CP- Cannabinoid CB1 >30 13.50 Receptors 55,940, 2.0 nM

G-Protein Displacement of SCH-23390 [N- Dopamine D1 >30 8.41 Receptors Methyl-3H], 1nM

G-Protein Displacement of 3H- Dopamine D2 >30 -8.20 Receptors Methylspiperone, 0.6 nM

G-Protein Displacement of [Pyridinyl-5-3H]- Histamine H1 >30 -2.91 Receptors pyrilamine, 2.5 nM

G-Protein Displacement of [3H]-Tiotidine, Histamine H2 >30 -11.01 Receptors 3.0 nM

12

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurements

Displacement of [3H]-N-Methyl- G-Protein Muscarinic M2 >30 28.10 Scopolamine Methyl Chloride, Receptors 0.5 nM

G-Protein Displacement of Naloxone-[N- Opioid kappa >30 -11.65 Receptors Allyl-2,3-3H], 3 nM

G-Protein Displacement of Naloxone-[N- Opioid mu >30 -7.17 Receptors Allyl-2,3-3H], 1.1 nM

G-Protein Displacement of [3H]- Serotonin 5HT1B >30 10.14 Receptors GP125743, 3.0 nM

G-Protein Serotonin 5HT2A >10 Stimulation of calcium flux Receptors

G-Protein Serotonin 5HT2B >10 8.62 Stimulation of calcium flux Receptors

13

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurements

G-Protein Displacement of 3H-GR113808, Serotonin 5HT4 >30 -5.13 Receptors 0.6 nM

Displacement of 125I RTI-55, Transporters Dopamine >30 0.125 nM

Displacement of 125I Nisoxetine, Transporters Norepinephrine >30 0.55 nM

Displacement of 125I RTI-55, Transporters Serotonin >30 0.26 nM

Nuclear Fluorescent polarization ligand Hormone Androgen 16.13446 11.0 103.91 binding competition with Receptors Fluormone™ AL Red, 2 nM

Nuclear Fluorescent polarization ligand Hormone Estrogen A ~63.92667 2.4 54.04 binding competition with Receptors Fluormone™ EL Red, 1 nM

14

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurements

Nuclear Fluorescent polarization ligand Hormone Glucocorticoid >150 0.7 26.54 binding competition with Receptors Fluormone™ GS Red, 1 nM

Nuclear Fluorescent polarization ligand Hormone Progesterone 59.8759 1.8 84.68 binding competition with Receptors Fluormone™ PL Red, 2 nM

Calcium Channel L- Ion Channel type (Cav1.2) >25 8.39 Inhibition of calcium flux Receptors Antagonist

Ion Channel Calcium Channel T- >25 2.48 Stimulation of calcium flux Receptors type (Cav3.2) Activator

Cardiac Sodium Inhibition of membrane potential Ion Channel Channel (hNAV1.5) >30 0.4 13.92 change triggered by 40 uM Receptors Antagonist veratridine

Inhibition of channel activity Ion Channel GABA-A (A1B2G2) >30 129.27 triggered by GABA at EC95 Receptors Antagonist concentration

15

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurements

Potentiation of channel activity Ion Channel GABA-A (A1B2G2) >30 -2.7 6.56 triggered by GABA at EC20 Receptors Potentiator concentration

Inhibition of channel activity Ion Channel GABA-A (A5B2G2) >30 -8.6 109.68 triggered by GABA at EC95 Receptors Antagonist concentration

Inhibition of calcium flux Ion Channel Nicotinic Acetylcholine >30 stimulated with 10 µM Receptors A1 Antagonist acetylcholine

Ion Channel Nicotinic Acetylcholine >30 1.38 Stimulation of calcium flux Receptors A4B2 Agonist

Inhibition of calcium flux Ion Channel Nicotinic Acetylcholine >30 stimulated with 15 µM Receptors A7 Antagonist acetylcholine

Ion Channel NMDA Glutamate n.d. Stimulation of calcium flux Receptors NR1/2A Agonist

16

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurements

Inhibition of calcium flux Ion Channel NMDA Glutamate >30 stimulated with 100 µM Receptors NR1/2A Antagonist glutamate

Ion Channel NMDA Glutamate >25 16.46 Stimulation of calcium flux Receptors NR1/2B Agonist

Inhibiiton of enzyme activity on Enzymes Acetylcholinesterase >60 1.62 acetylcholine substrate, 50 µM

Inhibition of enzyme activity on Enzymes Monoamine Oxidase A >30 1.1 27.83 kynuramine substrate, 40 µM

Inhibition of enzyme activity on Enzymes Monoamine Oxidase B >30 27.89 kynuramine substrate, 20 µM

SPA-based enzyme inhibition Enzymes Phosphodiesterase 3 >30 assay with [3H]cAMP, 100 nM

17

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurements

SPA-based enzyme inhibition Enzymes Phosphodiesterase 4 >30 assay with [3H]cAMP, 300 nM

18

Supplemental data for JPET #226910

Table S3. Activity of Compound C in a broad in vitro selectivity panel. Potency (IC50 or EC50) values are reported for those assays in which the maximal observed response produced by the test compound (Ymaxobs) was greater than or equal to 50% relative a reference full agonist or inhibitor. Values are otherwise reported as greater than the top concentration tested or half of the top concentration tested.

19

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurements

G-Protein Displacement of 3H-CGS- Adenosine A2A >30 30.02 Receptors 21680, 25 nM

G-Protein Displacement of Prazosin, [7- Adrenergic A1B >30 5.81 Receptors Methoxy-3H], 1 nM

G-Protein Displacement of Prazosin, [7- Adrenergic A1D >30 18.88 Receptors Methoxy-3H], 0.5 nM

G-Protein Displacement of 3H-MK912, 1 Adrenergic A2A >30 1.3 49.68 Receptors nM

G-Protein Displacement of Rauwolsine, [7- Adrenergic A2C >30 8.43 Receptors Methoxy-3H], 1nM

Displacement of G-Protein Adrenergic B1 >30 3.82 Dihydroalprenolol hydrochloride, Receptors levo-[ring, propyl-3H(N)], 1 nM

20

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurements

Displacement of G-Protein Adrenergic B2 >30 -6.55 Dihydroalprenolol hydrochloride, Receptors levo-[ring, propyl-3H(N)], 0.5 nM

G-Protein Displacement of [3H]- CP- Cannabinoid CB1 >30 27.87 Receptors 55,940, 2.0 nM

G-Protein Displacement of SCH-23390 [N- Dopamine D1 >30 -5.75 Receptors Methyl-3H], 1nM

G-Protein Displacement of 3H- Dopamine D2 >30 17.48 Receptors Methylspiperone, 0.6 nM

G-Protein Displacement of [Pyridinyl-5-3H]- Histamine H1 >30 33.71 Receptors pyrilamine, 2.5 nM

G-Protein Displacement of [3H]-Tiotidine, Histamine H2 >30 11.64 Receptors 3.0 nM

21

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurements

Displacement of [3H]-N-Methyl- G-Protein Muscarinic M2 >30 21.44 Scopolamine Methyl Chloride, Receptors 0.5 nM

G-Protein Displacement of Naloxone-[N- Opioid kappa >30 10.60 Receptors Allyl-2,3-3H], 3 nM

G-Protein Displacement of Naloxone-[N- Opioid mu >30 16.24 Receptors Allyl-2,3-3H], 1.1 nM

G-Protein Displacement of [3H]- Serotonin 5HT1B >30 14.57 Receptors GP125743, 3.0 nM

G-Protein Serotonin 5HT2A n.d. Stimulation of calcium flux Receptors

G-Protein Serotonin 5HT2B n.d. Stimulation of calcium flux Receptors

22

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurements

G-Protein Displacement of 3H-GR113808, Serotonin 5HT4 >30 -1.68 Receptors 0.6 nM

Displacement of 125I RTI-55, Transporters Dopamine >30 0.0 23.83 0.125 nM

Displacement of 125I Nisoxetine, Transporters Norepinephrine >30 16.58 0.55 nM

Displacement of 125I RTI-55, Transporters Serotonin >30 8.10 0.26 nM

Nuclear Fluorescent polarization ligand Hormone Androgen >150 37.87 binding competition with Receptors Fluormone™ AL Red, 2 nM

Nuclear Fluorescent polarization ligand Hormone Estrogen A ~114.1183 0.7 61.92 binding competition with Receptors Fluormone™ EL Red, 1 nM

23

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurements

Nuclear Fluorescent polarization ligand Hormone Glucocorticoid >150 11.63 binding competition with Receptors Fluormone™ GS Red, 1 nM

Nuclear Fluorescent polarization ligand Hormone Progesterone >150 0.6 16.20 binding competition with Receptors Fluormone™ PL Red, 2 nM

Calcium Channel L- Ion Channel type (Cav1.2) >25 Inhibition of calcium flux Receptors Antagonist

Ion Channel Calcium Channel T- >25 0.04 Stimulation of calcium flux Receptors type (Cav3.2) Activator

Cardiac Sodium Inhibition of membrane potential Ion Channel Channel (hNAV1.5) >30 12.58 change triggered by 40 uM Receptors Antagonist veratridine

Inhibition of channel activity Ion Channel GABA-A (A1B2G2) >30 106.50 triggered by GABA at EC95 Receptors Antagonist concentration

24

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurements

Potentiation of channel activity Ion Channel GABA-A (A1B2G2) >30 -2.58 triggered by GABA at EC20 Receptors Potentiator concentration

Inhibition of channel activity Ion Channel GABA-A (A5B2G2) >30 110.22 triggered by GABA at EC95 Receptors Antagonist concentration

Inhibition of calcium flux Ion Channel Nicotinic Acetylcholine >30 -0.8 5.28 stimulated with 10 µM Receptors A1 Antagonist acetylcholine

Ion Channel Nicotinic Acetylcholine >30 -0.2 1.12 Stimulation of calcium flux Receptors A4B2 Agonist

Inhibition of calcium flux Ion Channel Nicotinic Acetylcholine >30 -28.95 stimulated with 15 µM Receptors A7 Antagonist acetylcholine

Ion Channel NMDA Glutamate >30 3.11 Stimulation of calcium flux Receptors NR1/2A Agonist

25

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurements

Inhibition of calcium flux Ion Channel NMDA Glutamate >30 8.97 stimulated with 100 µM Receptors NR1/2A Antagonist glutamate

Ion Channel NMDA Glutamate >30 2.17 Stimulation of calcium flux Receptors NR1/2B Agonist

Inhibiiton of enzyme activity on Enzymes Acetylcholinesterase >30 9.1 7.59 acetylcholine substrate, 50 µM

Inhibition of enzyme activity on Enzymes Monoamine Oxidase A >30 -3.21 kynuramine substrate, 40 µM

Inhibition of enzyme activity on Enzymes Monoamine Oxidase B >30 8.16 kynuramine substrate, 20 µM

SPA-based enzyme inhibition Enzymes Phosphodiesterase 3 >50 12.72 assay with [3H]cAMP, 100 nM

26

Supplemental data for JPET #226910

Class Assay IC50/EC50 SLOPE Ymaxobs Graph Measurements

SPA-based enzyme inhibition Enzymes Phosphodiesterase 4 >50 -1.32 assay with [3H]cAMP, 300 nM

27