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1521-0103/356/2/293–304$25.00 http://dx.doi.org/10.1124/jpet.115.226910THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 356:293–304, February 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics
Evidence for Classical Cholinergic Toxicity Associated withSelective Activation of M1 Muscarinic Receptors s
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. MacorResearch 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-MyersSquibb Company, Hopewell, New Jersey (C.S.S., M.E.C.); and Research and Development/Discovery, Bristol-Myers SquibbCompany, Lawrence Township, New Jersey (J.C., L.Z., J.E.M.)
Received June 19, 2015; accepted November 17, 2015
ABSTRACTThe muscarinic acetylcholine receptor subtype 1 (M1) receptorsplay an important role in cognition and memory, and areconsidered to be attractive targets for the development of novelmedications to treat cognitive impairments seen in schizophre-nia and Alzheimer’s disease. Indeed, the M1 agonist xanomelinehas been shown to produce beneficial cognitive effects in bothAlzheimer’s disease and schizophrenia patients. Unfortunately,the therapeutic utility of xanomeline was limited by cholinergicside effects (sweating, salivation, gastrointestinal distress),which are believed to result from nonselective activation of othermuscarinic receptor subtypes such as M2 and M3. Therefore,drug discovery efforts targeting theM1 receptor have focused onthe discovery of compounds with improved selectivity profiles.Recently, allosteric M1 receptor ligands have been described,which exhibit excellent selectivity for M1 over other muscarinicreceptor subtypes. In the current study, the following threecompounds with mixed agonist/positive allosteric modulator
activities that are highly functionally selective for the M1 receptorwere tested in rats, dogs, and cynomologous monkeys:(3-((1S,2S)-2-hydrocyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one; 1-((4-cyano-4-(pyridin-2-yl)piperidin-1-yl)methyl)-4-oxo-4H-quinolizine-3-carboxylic acid; and (R)-ethyl 3-(2-methylbenzamido)-[1,49-bipiperidine]-19-carboxylate). Despite their selectivity for theM1 receptor, all three compounds elicited cholinergic side effectssuch as salivation, diarrhea, and emesis. These effects could notbe explained by activity at other muscarinic receptor subtypes,or by activity at other receptors tested. Together, these resultssuggest that activation of M1 receptors alone is sufficient toproduce unwanted cholinergic side effects such as those seenwith xanomeline. This has important implications for the devel-opment of M1 receptor–targeted therapeutics since it suggeststhat dose-limiting cholinergic side effects still reside in M1receptor selective activators.
IntroductionThe neurotransmitter acetylcholine activates two distinct
families of receptors: nicotinic and muscarinic acetylcholinereceptors, which were initially classified based upon theirdifferential activation by the toxins nicotine (Lindstrom 1997)and muscarine (Wess et al., 1996). Nicotinic acetylcholinereceptors are ligand-gated ion channels; muscarinic acetylcho-line receptors are seven transmembrane guanine nucleotidebinding protein (G protein) coupled receptors. Five subtypes ofmuscarinic acetylcholine receptors exist (M1–M5). These five
subtypes each represent separate gene products and exhibitdistinct signaling pathways and tissue distribution, althoughall are expressed within the central nervous system (Ishii andKurachi 2006).The M1 muscarinic receptors play an important role in
multiple domains of cognitive function (Felder et al., 2000;Auld et al., 2002), and a significant body of evidence suggeststhat activation of M1 receptors can produce therapeuticallybeneficial effects for the treatment of schizophrenia andAlzheimer’s disease (Langmead et al., 2008). Muscarinicreceptor activation has been shown to reverse cognitive andbehavioral deficits in animal models of schizophrenia andAlzheimer’s disease, and the M1 receptor specifically has beenimplicated in mediating these effects (Jones et al., 2005;
dx.doi.org/10.1124/jpet.115.226910.s This article has supplemental material available at jpet.aspetjournals.org.
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 bindingprotein; 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.
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Langmead et al., 2008; Barak and Weiner 2011; Fisher 2008).The most direct evidence for the utility of M1 activators intreating schizophrenia and Alzheimer’s disease comes fromclinical trials using xanomeline, a muscarinic partial agonistwith modest selectivity for M1 and M4 receptors. Xanomelinewas shown to improve psychosis and behavioral disturbancesinAlzheimer’s disease patients (Bodick et al., 1997). Xanomelinewas also found to produce both significant psychiatric im-provements, and improvements in learning and memory inschizophrenia patients (Shekhar et al., 2008). Unfortunately,the clinical utility of xanomeline is limited by its side-effectprofile, which includes salivation, sweating, and gastrointes-tinal distress—all of which are classic cholinergic side effects.These side effects are believed to be due to activity ofxanomeline at M2 and M3 muscarinic receptors (Melanconet al., 2013). Therefore, selectivity has remained the majorfocus for drug discovery efforts targeting the M1 receptor.However, attaining sufficient selectivity for M1 versus othermuscarinic receptors to avoid cholinergic side effects hasproven to be very challenging because the acetylcholinebinding site is highly conserved within all five muscarinicreceptor subtypes (Heinrich et al., 2009), and despite years ofmedicinal chemistry efforts acetylcholine site activators withsufficient M1 selectivity have not been identified.An alternative pharmacological strategy is to modulate the
activity of M1 receptors using molecules that bind to allostericsites that are distinct from the acetylcholine (orthosteric)binding site. Small-molecule positive allosteric modulators(PAMs) have been identified for many G protein coupledreceptors (Wootten et al., 2013), including the M1 receptor(Melancon et al., 2013). Allosteric binding sites do not face thesame evolutionary pressure as the orthosteric binding site,which must bind the endogenous agonist to maintain receptorfunction. Therefore, allosteric binding sites are hypothesizedto be less evolutionarily and structurally conserved thanorthosteric sites, suggesting that allosteric sites could offerimproved opportunities for receptor subtype selectivity versustargeting the traditional orthosteric site of the receptor. Thishypothesis has been borne out empirically. For example, smallmolecules targeting allosteric sites of metabotropic glutamatereceptors have been shown to exhibit excellent receptor sub-type selectivity, which had not been achieved by ligandstargeting the highly conserved glutamate binding site (Nickolsand Conn 2014). Similarly, allosteric modulators of musca-rinic receptors have been identified that exhibit selectivitysuperior to that attained by xanomeline or other orthostericsite M1 receptor activators (Melancon et al., 2013).The current study was designed to test the hypothesis that
M1-selective compounds would be free of classic cholinergicside effects in animal models. To test this hypothesis, the
following three structurally distinct M1 PAMs were chosenfrom the scientific literature (Kuduk et al., 2011; Leboiset al., 2011): 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-carboxylicacid; and compound C, (R)-ethyl 3-(2-methylbenzamido)-[1,49-bipiperidine]-19-carboxylate (Fig. 1). These compounds displaypotency and pharmacokinetic profiles that make them appro-priate for in vivo testing; and, importantly, they show excellentselectivity for M1 over other muscarinic receptor subtypes invitro. In this study, the safety profile of these selective M1
ligands was investigated in rats, dogs, and cynomologousmonkeys. Despite their selectivity for M1, all three compoundswere found to produce effects associatedwith classic cholinergictoxicity such as salivation and diarrhea. Accordingly, theseresults refute the original hypothesis and instead provideevidence to suggest that activation of the M1 receptor alone issufficient to produce cholinergic toxicity in animals.
Materials and MethodsCells
M2, M3, and M4 were recombinantly expressed in a Chinesehamster ovary cell background, with M2 and M4 lines also containinga construct expressing Gqi5 and aequorin. The M1- and M5-expressingcell lines were generated by recombinant expression of M1 or M5 inChinese hamster ovary A12 cells (Perkin-Elmer, Akron, OH).
Cells were cultured in tissue culture–treated T-175 flasks (Corning,Corning, NY) in medium [Dulbecco’s modified Eagle’s medium/F12(Gibco, Waltham, MA), 10% fetal bovine serum (Hyclone, Logan,Utah), and appropriate selection antibiotics] at 37°C, 5% CO2. Cellswere harvested and plated onto 384-well (Corning) tissueculture–treated black/clear plates 16–20 hours prior to the experi-ment. Briefly, flasks were rinsed with 10 ml/flask Dulbecco9s phos-phate buffered saline solution (magnesium and calcium free, Gibco)and incubated in 5 ml/flask of 0.05% trypsin-EDTA (Gibco) at 37°C for5 minutes. Medium was added to the harvested cells, and then thecells were centrifuged in a tabletop centrifuge at 1200 rpm for10 minutes. The cell pellet was resuspended in 10 ml/flask medium 11% penicillin-streptomycin (Gibco), counted on the Guava PersonalCell Analysis System (Guava Technologies, Hayward, CA), dilutedwith medium1 1% penicillin-streptomycin to a concentration of 1 �106 cells/ml, and then 20 ml/well (20,000 cells/well) of cell suspensionwas added and the plates were incubated at 37°C, 5% CO2 overnight.
23 Compound Plate Preparation
Compounds were solubilized in dimethylsulfoxide (DMSO) (EMDChemicals, Gibbstown, NJ) at 10mMand then serially diluted in half-log increments in 384-well polypropylene REMP microplates (BrooksAutomation, Chelmsford, MA). An intermediate compound plate wasprepared 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 compoundC, (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 usedas a comparator compound for in vitro experiments.
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followed by 100 ml/well of Hanks’ balanced salt solution/20 mMHEPES [Hanks’ balanced salt solution (Gibco) supplemented with20 mM HEPES (Gibco), pH 7.4].
Ca21 Flux Assay
After overnight incubation, the medium was removed from thecells by flicking the plates, followed by addition of 20 ml/well of 3 mMFluo4-AM (Invitrogen, Carlsbad, CA), 0.016% pluronic acid (Sigma-Aldrich, Saint Louis, MO), and 2.5 mM probenecid (Sigma-Aldrich) inHanks’ balanced salt solution/20 mM HEPES. Plates were incubatedfor 1 hour at room temperature prior to assay in the FLIPR Tetra(Molecular Devices, Sunnyvale, CA). For all additions, the FLIPRTetra reads a baseline once a second for 10 seconds prior to compoundaddition, followed by 50 seconds at 1-second intervals and 60 secondsat 3-second intervals. First, the compounds were added from thecompound plate to assay for agonist activity and 20 ml/well from the2� compound plate was added at a height of 10 ml with no mixing.Then, the plates were incubated for 20 minutes at room temperature,and the PAM activity was assayed by adding a 3� solution ofacetylcholine from a bulk reservoir to all wells (20 ml/well at 10 mlheight with two 10 ml mixes). The solution was adjusted each day toproduce an EC10-EC20 response. The ranges of 3� concentrations ofacetylcholine used were 15–30 nM for M1, 167–250 nM for M2,1.67–2 nM for M3, 133–400 nM for M4, and 15–50 nM for M5.
Raw data files were exported from the FLIPR ScreenWorkssoftware (Molecular Devices). Maximum fold increase in fluorescencewas determined by dividing the maximum value of fluorescenceobtained after compound addition by the average of the baselinevalues taken before compound addition. These values were analyzedusingGraphPad PRISM (GraphPad Software, La Jolla, CA). The EC50
values were calculated using nonlinear regression (sigmoidal doseresponse, variable slope).
In Vitro Selectivity Assays
Selectivity assays for other G protein coupled receptors wereperformed as standard competitive binding assays using receptor-specific radioligand probes in membrane preparations from cellsoverexpresssing the various G protein coupled receptors. Phosphodi-esterase type 4 activity was determined in an electrophoretic mobilityassay, using a fluorescently labeled cAMP as a substrate. By thenature of the difference in electrophoretic mobility of the substrateand the hydrolyzed product, the enzymatic activity was determinedupon the electrophoretic separation of the reactions on the caliper(Perkin-Elmer). The activity on GABA (a1b2g2) was measured byusing a cell line expressing a rat GABA (a1b2g2) and a halide-sensingyellow fluorescent protein. Potentiation of GABA activity was de-termined by the effect of the test compound to enhance the responseproduced by an EC20 concentration of GABA. Activity on the cardiacsodium channel was measured using a cell line expressing humanNaV1.5 and a voltage-sensitive dye on a flurometric plate reader,where the inhibitory activity of the test compounds was assayed in thepresence of veratridine.
Serum Protein Binding Assays
Test compounds were suspended in 100% DMSO as a 10 mM stock.Five-molar monobasic sodium phosphate was obtained from Sigma-Aldrich. Pooled, male human, male rat, and male mouse sera wereobtained from BioReclamation (Hopewell, NJ). The reusable 96-wellmicroequilibrium dialysis device was obtained from HTDialysis(Gales Ferry, CT). Dialysis membrane strips (10,000-Da mol. wt.cutoff) were also obtained from HTDialysis. Acetonitrile, water, andmethanol were all obtained from J.T. Baker (Miami, FL). Ammoniumacetate, formic acid, and alprenolol were obtained from Sigma-Aldrich. Tolbutamide was obtained from Fluka (Saint Louis, MO).Polymerase chain reaction plates were obtained from Axygen (UnionCity, CA). T
ABLE
1Activityan
dselectivitypr
ofileof
mus
carinic
liga
nds
formuscarinic
receptor
subtyp
esM
1–M
5
Poten
cy(pEC50)a
ndEmaxva
lues
formuscarinic
liga
nds
werede
term
ined
from
theirab
ilityto
directly
indu
ceincrea
sesin
free
cytosolicCa2
+leve
lsin
Chineseham
ster
ovarycellsrecombina
ntlyex
pressingtheva
riou
smuscarinic
receptor
subtyp
es(ago
nistda
ta),or
theirab
ilityto
potentiatetheCa2
+resp
onse
toan
EC15concentrationof
acetylch
oline(PAM
data).Cells
expressingM
2or
M4receptorsalso
stab
lyex
pressedG
qi5to
coup
leactiva
tion
ofthese
subtyp
esto
Ca2
+resp
onses.
Datarepr
esen
tmea
n6
S.D
.from
3to
5ex
perimen
ts.E
maxva
lues
arerelative
toamax
imal
acetylch
olineresp
onse
inthegive
ncellline.
Com
pound
M1Ago
nist
M1PAM
M2Ago
nist
M2PAM
M3Ago
nist
M3PAM
M4Ago
nist
M4PAM
M5Ago
nist
M5PAM
pEC50
Emax
pEC50
Emax
pEC50
Emax
pEC50
Emax
pEC50
Emax
pEC50
Emax
pEC50
Emax
pEC50
Emax
pEC50
Emax
pEC50
Emax
Acetylcho
line
6.46
0.2
1006
2NA
NA
5.36
0.3
1006
13NA
NA
7.36
0.1
1006
4NA
NA
5.66
0.1
1006
8NA
NA
6.16
0.1
1006
4NA
NA
Carba
chol
5.46
0.2
996
3NA
NA
4.56
0.3
906
13NA
NA
6.46
0.2
1026
3NA
NA
4.76
0.2
916
7NA
NA
5.26
0.1
986
4NA
NA
BQCA
a39
616
a5.36
0.1
1036
1NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Com
poundA
5.46
0.4
246
67.46
0.2
506
13a
456
4a5.66
0.6
236
3NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Com
poundB
4.36
0.3
446
125.96
0.2
836
13NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Com
poundC
4.46
0.1
706
76.16
0.2
236
6NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
BQCA,1-(4-m
etho
xybe
nzy
l)-4-oxo
-1,4-dihyd
roqu
inoline-3-carbox
ylic
acid;NA,no
activity.
aThecompo
undwas
toowea
kto
deve
lopafullconcentrationresp
onse
curve;
thepe
rcen
tage
ofacetylch
olineresp
onse
ispr
ovided
atthehighestconcentrationtested
(50mM).
Cholinergic Toxicity Produced by Selective M1 Activation 295
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Dialysis membranes were soaked for 15 minutes in distilled waterat room temperature. After the initial soaking, the water was replacedwith 0.133 M sodium phosphate buffer (prepared by dilution of a 5 M
stock with distilled water and adjustment to pH 7.4), and themembranes were soaked for an additional 15 minutes in the buffer.Membrane strips were assembled into the high throughput dialysis
Fig. 3. Activity of muscarinic ligands at receptor subtypes M2–M5. Agonist read (closed symbols): increases in free cytosolic Ca2+ levels were measuredimmediately upon test compound addition. PAM read (open symbols): 20 minutes subsequent to test compound addition cells were challenged with anEC15 concentration of acetylcholine and free cytosolic Ca2+ levels were again measured, with data expressed as fold increase in free intracellular Ca2+
relative to the control EC15 response.
Fig. 2. Activity of muscarinic ligands at receptor subtype M1. Agonist read (closed symbols): increases in free cytosolic Ca2+ levels were measuredimmediately upon test compound addition. PAM read (open symbols): 20 minutes subsequent to test compound addition cells were challenged with anEC15 concentration of acetylcholine and free cytosolic Ca2+ levels were again measured, with data expressed as fold increase in free intracellular Ca2+
relative to the control EC15 response.
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apparatus according to the manufacturer’s instructions, and 150 ml of0.133 M sodium phosphate buffer, pH 7.4, was loaded into one side ofeach dialysis chamber. Test articles and reference compounds (1 mMin 100%DMSO) were added to human, rat, or mouse serum to achieve
a final concentration of 10 mM at 1% DMSO. All sera were adjusted topH 7.4 prior to use.
Test and reference compounds in sera (165 ml) were added to theopposite side of each dialysis chamber from the buffer, and 15 ml of
TABLE 2Activity of test compounds in a broad in vitro selectivity panelPotency (IC50 or EC50) values are reported for those assays in which the maximal observed response produced by the testcompound was greater than or equal to 50% relative to a reference full agonist or inhibitor. Values are otherwise reportedas greater than the top concentration tested or half of the top concentration tested.
TargetIC50 or EC50
Compound A Compound B Compound C
mM mM mM
G-protein receptorsAdenosine A2a .30.0 .30.0 .30.0Adrenergic a 1B .30.0 .30.0 .30.0Adrenergic a 1D ∼4.5 .30.0 .30.0Adrenergic a 2A .30.0 .30.0 .30.0Adrenergic a 2C .30.0 .30.0 .30.0Adrenergic b1 .30.0 .30.0 .30.0Adrenergic b2 .30.0 .30.0 .30.0Cannabinoid CB1 .30.0 .30.0 .30.0Dopamine D1 .30.0 .30.0 .30.0Dopamine D2 .30.0 .30.0 .30.0Histamine H1 .30.0 .30.0 .30.0Histamine H2 .30.0 .30.0 .30.0Muscarinic M2 .30.0 .30.0 .30.0Opioid kappa .30.0 .30.0 .30.0Opioid mu .30.0 .30.0 .30.0Serotonin 5HT1B .30.0 .30.0 .30.0Serotonin 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
TransportersDopamine .30.0 .30.0 .30.0Norepinephrine .30.0 .30.0 .30.0Serotonin .30.0 .30.0 .30.0
Nuclear hormone receptorsAndrogen .150.0 16.1 .150.0Estrogen a .150.0 ∼63.9 ∼114.1Glucocorticoid .150.0 .150.0 .150.0Progesterone .150.0 59.9 .150.0
Ion channel receptorsCalcium channel L-type (Cav1.2) antagonist .25.0 .25.0 .25.0Calcium channel T-type (Cav3.2) activator .25.0 .25.0 .25.0Cardiac sodium channel (hNAV1.5) antagonist 11.5 .30.0 .30.0GABA-A (a1b2g2) antagonist .30.0 .30.0 .30.0GABA-A (a1b2g2) potentiator 1.7 .30.0 .30.0GABA-A (a5b2g2) antagonist .30.0 .30.0 .30.0Nicotinic acetylcholine a1 antagonist .30.0 .30.0 .30.0Nicotinic acetylcholine a4b2 agonist .30.0 .30.0 .30.0Nicotinic acetylcholine a7 antagonist .30.0 .30.0 .30.0NMDA glutamate NR1/2A agonist .25.0 N.D. .30.0NMDA glutamate NR1/2A antagonist .30.0 .30.0 .30.0NMDA glutamate NR1/2B agonist .25.0 .25 .30.0
EnzymesAcetylcholinesterase .60.0 .60 .30.0Monoamine oxidase A 14.3 .30.0 .30.0Monoamine oxidase B .30.0 .30.0 .30.0Phosphodiesterase 3 .30.0 .30.0 .50.0Phosphodiesterase 4 12.6 .30.0 .50.0
N.D., not determined.
TABLE 3Serum protein binding results summary of test compoundsData reported represent mean and S.D. from three determinations; %Rec. denotes percent recovery.
CompoundHuman Rat Mouse
%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 16B 74.8 13.6 118 9 49.2 12.60 87 12 111.5 7.6 70 4C 17.1 2.0 108 9 32.2 7.80 94 19 23.9 1.0 83 6
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compound in serum was immediately removed and added to 135 ml ofmixed matrix solution to create the reference sample (T0). The mixedmatrix solution consisted of 16.7% human serum, 16.7% rat serum,16.7% mouse serum, and 50% 0.133 M sodium phosphate buffer. Theequilibrium dialysis chamber was incubated at 37°C in a 7% CO2
atmosphere for 6.5 hours.Following incubation, 75 ml from the buffer (dialysate) side of the
HTD equilibrium dialysis chamber was added to 75 ml of the mixedserum solution to create the postincubation buffer sample. The mixedmatrix solution consisted of 33% human serum, 33% rat serum, and33% mouse serum. In addition, 15 ml from the serum side of the HTDequilibrium dialysis chamber was added to 135 ml of the appropriatemixed matrix solution to create the postincubation serum sample.
The concentrations of test articles and reference compound sampleswere determined by liquid chromatography–tandem mass spectrom-etry. The analysis system consisted of two sets of binary Shimadzu10ADvp pumps with SCL-10Avp controllers for gradient elution, aLeap HTS Autosampler (Leap Technologies, Carrboro, NC) with twinarms for sample injection, a Thermo Fisher Scientific (Waltham, MA)valve interface module for valve switching and stream selection, andan AB Sciex (Framingham, MA) triple quadrupole mass spectrometeroperated under electrospray ionization mode. To obtain the optimumselected reaction monitoring conditions for sample analysis, tandemmass spectrometry optimization was performed using Discovery-Quant featuring saturation control by AB Sciex (Framingham, MA)with 10 mM standard solutions (in 1:1 methanol/water) prepared fromthe compound stock solutions. The optimization was performed using
a flow injection analysis with an injection volume of 40 ml. The mobilephasewas 25%A:75%Bdelivered by anAgilent (SantaClara, CA) 1100pump at 0.3 ml/min, where A was a mixture of 2 mM ammoniumacetate/acetonitrile/formic acid (980:20:1, v/v/v), and B was a mixtureof acetonitrile/water/formic acid (980:20:1, v/v/v). DiscoveryQuantautomatically determined the optimized ionization polarity (positiveor negative), precursor and product ions, declustering potential, andcollision energy for test and reference compounds.
The optimized selected reaction monitoring tandem mass spec-trometry conditions were used for sample analysis postassay. A5-point calibration curve (5, 50, 500, 1000, and 2000 nM)was preparedfor test and reference compounds in a 1:1 mixture of 133 mM sodiumphosphate buffer and the appropriate mixed serum. A solution ofacetonitrile containing two internal standards (100 nM alprenolol forcompounds requiring positive ionization, 300 nM tolbutamide forcompounds requiring negative ionization) was used for processing theassay samples. Fifty (50) ml of assay samples or calibration standardswere first extracted with 150 ml of acetonitrile containing the internalstandards. After vortexing, centrifugation, and supernatant separa-tion, 15 ml of supernatant was injected onto an Ascentis Express C18high-performance liquid chromatography column (2.7 mm, 2.1 �30 mm; Sigma-Aldrich) at room temperature for analysis. The mobilephase B was the same as that used for optimization, and mobile phaseA was a mixture of 980:20:1 (v/v/v) water/acetonitrile/formic acid. Thepeak area ratios of the test or reference compound to the internalstandard were used for quantification. A linear regression with a1/concentration2 weightingwas applied to the calibration standards toobtain the calibration curve. The concentration of the test or referencecompound was then calculated with the corresponding calibrationcurve. The free fraction (percent free), percent bound, and recoveryvalues were calculated for each test sample and control compounds asfollows:
Percent free5 ðdialysate sample=serum sampleÞ � 100Percent recovery5
�ðdialysate sample1 serum sampleÞ=T0 sample��100
In Vivo Pharmacokinetic Studies
All animal studies were performed under the approval of theBristol-Myers Squibb Animal Care and Use Committee and inaccordance with the Association for Assessment and Accreditation ofLaboratory Animal Care (AAALAC, Frederick, MD).
Rat. Pharmacokinetic studies were conducted in male Sprague-Dawley rats (300–350 g) with cannulae implanted in the jugular veins.After dosing, serial blood samples (0.3 ml) were obtained from theappropriate cannula of each rat by collection into EDTA-containingtubes (Becton Dickinson, Franklin Lakes, NJ), and centrifuged toseparate plasma. Plasma was frozen until analysis. For the i.v.studies, the compound was dissolved (1 mg/ml) in a vehicle of 10%N-methyl pyrrolidinone, 90% polyethylene glycol (PEG) 400 and dosed(1 ml/kg) as a 10-minute constant rate infusion into the jugular vein,
Fig. 4. Plasma exposure of compounds A andB in rats following i.v. (1mg/kg)and oral (5 mg/kg) administration.
TABLE 4Pharmacokinetic properties of compounds A and B in the rat following i.v. (1 mg/kg) and oral (2.5 mg/kg)administration
Parameter UnitCompound A Compound B
Intravenous Oral Intravenous Oral
Dose mg/kg 1 2.5 1 2.5Cmax nM 2840 6 460 1850 6 897 1602 6 285 151 6 69AUCtot nM·h 4357 6 720 9630 6 4473 727 6 57 714 6 23t1/2 Hour 3.2 6 1.1 2.5 6 0.3 4.0 6 4.0 3.9 6 2.0Clearance ml/min/kg 8.4 6 1.5 59 6 4.8Vss l/kg 1.7 6 0.7 6.1 6 5.1Bioavaliability %F 86 6 30 39 6 3.6
%F, percentage of absolute oral bioavailability.
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with serial blood samples collected before dosing and at 0.17, 0.25, 0.5,0.75, 1, 2, 3, 5, 7, and 24 hours after dosing (n 5 3 rats/dose group).
For solution dosing by mouth, the compound was administered bygastric gavage as an aqueous solution containing 10% N-methylpyrrolidinone, 85% PEG 400, and 5% d-alpha tocopheryl polyethyleneglycol 1000 succinate. Serial blood samples were collected beforedosing and at 0.25, 0.5, 0.75, 1, 3, 5, 7, and 24 hours after dosing (n53 rats/dose group). Prior to all dosing by mouth, the rats were fastedovernight with free access to water.
Monkey. Pharmacokinetic and tolerability studies were conductedin cynomolgous monkeys bearing vascular access ports to facilitateblood collection in an i.v. rising dose design (n 5 2). Vehicle (5%hydroxypropyl beta cyclodextrin; 95% water) was infused at 0.5 ml/kgvia the venous port over 5 minutes at a constant rate. Animals wereobserved for 5 minutes. Then, compound (0.1 mg/kg) was infused for5 minutes and animals were observed for clinical signs for 1 hour. Bloodwas taken at the end of infusion (5 minutes) and at 10, 15, 30, and45 minutes. Body temperature was taken at t 5 0, 30, and 45 minutespostdose. At the end of the first observation period (1 hour), compound(0.3 mg/kg) was infused for 5 minutes and animals were observed for2 hours for clinical signs. Blood was taken at the end of infusion(5minutes) and at 10, 15, 30, and 45minutes and 1, 2, 3, 5, and 24 hourspostdose. Body temperature was taken at t 5 0, 30, and 45 minutespostdose. Plasma prepared from the collected blood samples was storedfrozen until analysis.
Dog. Pharmacokinetic and tolerability studies were conducted inmale beagle dogs bearing vascular access ports to facilitate bloodcollection in an i.v. rising dose design (n5 2). Vehicle (5%hydroxypropylbeta cyclodextrin; 95% water) was infused at 0.5 ml/kg via the venousport over 5 minutes at a constant rate. Animals were observed for5 minutes. Then, compound (low dose) was infused for 5 minutes andanimals were observed for clinical signs for 1 hour. Blood was taken atthe end of infusion (5 minutes) and at 15, 30, and 45 minutes. Bodytemperature was taken at t 5 0, 30, and 45 minutes postdose. At theend of the first observation period (1 hour), compound (intermediatedose) was infused for 5 minutes and animals were observed for 1 hourfor clinical signs. At the end of the second observation period (1 or2 hours after the intermediate dose), compound (high dose) wasinfused for 5 minutes and animals were observed for 2 hours forclinical signs. Bloodwas taken at the end of infusion (5minutes) and at15, 30, and 45 minutes and 1, 2, 3, 5, 7, and 24 hours postdose. Body
temperature was taken at t5 0, 30, and 45 minutes postdose. Plasmaprepared from the collected blood samples was stored frozen untilanalysis.
Pharmacokinetic Data Analysis
Pharmacokinetic parameters were obtained by noncompartmentalanalysis of plasma concentration versus time data (KINETICAsoftware, Version 2.4, InnaPhase Corporation, Philadelphia). Thepeak concentration (Cmax) and time for Cmax (tmax) were recordeddirectly from experimental observations. The area under the curve(AUC) from time zero to the last sampling time (AUC0–t) and the AUCfrom time zero to infinity (AUCINF) were calculated using a combina-tion of linear and log trapezoidal summations. The whole body plasmaclearance, steady-state volume of distribution (Vss), apparent terminalhalf-life (t1/2), and mean residence time were estimated following i.v.administration. The absolute oral bioavailability (F) was estimated asthe ratio of the dose-normalized AUC values following oral and i.v.doses.
ResultsCa21 Flux Assays
To assess the in vitro activity and selectivity profile of theM1 PAMs used in this study, these compounds were testedusing Chinese hamster ovary cells recombinantly expressingthe various muscarinic receptor subtypes (M1–M5). The well-characterized M1 receptor PAM 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (Ma et al., 2009) wasalso included as a reference for in vitro experiments. Theability of test compounds to either directly elicit increases inintracellular Ca21 levels [agonist (ago) mode], or to increasethe Ca21 response elicited by a low (∼EC15) concentration ofacetylcholine (PAM mode) was measured. Potency (EC50)and Emax values are provided in Table 1. Compounds A, B,and C, and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid all exhibited mixed ago-PAM activity at theM1 receptor (Fig. 2). These compounds potentiated thecellularCa21 response toanEC15 concentration of acetylcholineat lower concentrations, while at higher concentrations thesecompounds exerted allosteric agonist activity, directly activat-ing the receptor even in the absence of orthosteric agonist. InPAM-mode testing, test compounds were preincubated withcells for 20 minutes prior to addition of acetylcholine andmeasurement of the acetylcholine-evoked Ca21 response. Un-der these conditions, compounds that exert direct agonistactivity (either orthosteric or allosteric) produce desensitizationof the receptor/assay system during this 20-minute preincuba-tion period, inhibiting cellular responses to the subsequentacetylcholine challenge and confounding interpretation of
Fig. 5. Plasma exposure of compound A in monkeys following i.v.administration at 0.1 mg/kg, followed by 0.3 mg/kg 1 hour after the firstdose.
TABLE 5Pharmacokinetic 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
Parameter UnitCompound A Monkey Dose Escalation (i.v.)
0.1 0.3
mg/kg mg/kg
Cmax nM 1475 2320AUCtot nM·h 681 2367t1/2 Hour 0.9 3.2Clearance ml/min 5.4 4.6Vss Liter 0.3 0.9
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PAM-mode results. Therefore, for compounds A, B, and C, and1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylicacid, the PAM-mode curves have been truncated to excluderesults at concentrations of test compound that elicit directallosteric agonist activity (Fig. 2). With the exception ofcompound A, which exhibited very weak but measurableactivity at M2 (Fig. 3), none of the M1 PAMs tested had anymeasurable agonist or PAM activity at any of the othermuscarinic receptor subtypes, M2–M5.
Selectivity Profiling
We evaluated compounds A, B, and C in concentration-response (30 mM top concentration) in a broad panel ofselectivity assays comprising G protein coupled receptors,biogenic amine transporters, ion channels, enzymes, andnuclear hormone receptors (Table 2; Supplemental Tables1–3). This panel consists of targets that have been implicatedin various safety/liability concerns within central nervous,cardiac, pulmonary, renal, immune, gastrointestinal, andreproductive systems. Compound A was noted to have weakinhibitory activity at cardiac sodium channel NaV1.5 (IC50 511 mM) and phosphodiesterase type 4 (IC50 5 13 mM). It alsowas found to bind to the adrenergic a1D receptor, based uponcompetition in a radioligand binding assay (IC50 5 4 mM).Furthermore, in an assay thatmeasures potentiation of GABAreceptor GABAA (a1b2g2) activity, compound A was observedto potentiate GABA-evoked responses with EC505 2 mM, witha maximal response that reached a plateau at ∼36% of themaximal GABA-stimulated response. Compound B exhibitedvery weak binding to androgen (IC50 5 16 mM), estrogen(IC50 5 64 mM), and progesterone (IC50 5 60 mM) receptors.Compound C did not display anymeasurable activity at any ofthe 43 targets in this selectivity panel at the concentrationstested.
Serum Protein Binding Studies
Compounds A, B, and C were tested in a serum proteinbinding panel using human, rat, and mouse serum. Thesecompounds exhibited awide range of protein binding.Compound
A was found to be highly (.99%) protein bound in human androdent serum, while compounds B and C exhibited high freefractions in serum (Table 3).
Pharmacokinetic and Tolerability Studies
Rat. Compound A was dosed i.v. (1 mg/kg) as a clearcolorless 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 therat, a low clearance (8.4 ml/min/kg) and a high volume ofdistribution (1.7 l/kg) were observed. The resulting half-lifewas moderate at 3.2 hours. Following oral administration(2.5 mg/kg) a Cmax of 1.9 mMwas observed 2.7 hours postdose.The AUC (0–24 hours) was 9.6 mM·h. Bioavailability ofcompound A was 86% following oral dosing. It should be notedthat some variation was observed in the individual rats afteroral dosing. Following oral and i.v. administration, all rats haddiarrhea between 45 minutes and 1 hour postdose. No otherclinical observations were noted for the remainder of thestudy, except for bright yellow urine 24 hours postdose in thecages of animals that were dosed orally.Compound B was dosed in rat using the same study design
as compound A. Following i.v. administration of compoundB at 1 mg/kg in the rat as a clear yellow solution, a highclearance (59 ml/min/kg) and a high volume of distribution(6.1 l/kg) were observed (Fig. 4; Table 4). The resulting half-lifewas moderate at 4 hours (individual rat half-life varied from1.6 to 8.6 hours). Clearance (13.5 ml/min/kg) was muchhigher compared with previously reported values (Kuduket al., 2011). Following oral administration of compound B at2.5 mg/kg, the Cmax, tmax, and AUC0-tot values were 0.2 mM,1.0 hour, and 0.7 mM·h, respectively. The bioavailability ofcompound B was 39%, which is lower compared with pre-viously reported values (Kuduk et al., 2011). No clinicalobservations were noted other than bright yellow urine foundin the bedding from all animals postdose.Monkey. Compound A was dosed i.v. in a dose escalation
study at 0.1 and 0.3 mg/kg as described in Materials andMethods. Plasma concentration measurements are shown inFig. 5, and pharmaokinetic properties are summarized inTable 5. Pharmacokinetic parameters were not calculatedfor the 0.1 mg/kg dose group due to the short time interval(,1 hour). Although the plasma levels of compound A weresomewhat higher for the second dose as a result of the firstdose, the expected contribution was calculated to be minimal(, 10%). Following i.v. administration at 0.3mg/kg, compound
Fig. 6. Plasma exposure in dogs following i.v. administration ofcompound B at 1 mg/kg in dogs A and B, followed by 1.5 mg/kg (dog B) or3 mg/kg (dog A) 1 hour after the first dose.
TABLE 6Pharmacokinetic properties of compound B in the dog following i.v.administration at 1 mg/kg in dogs A and B and 1.5 mg/kg (dog B) or 3 mg/kg (dog A) 1 hour after the first dose
Parameter UnitCompound B Dog Dose Escalation (i.v.)
Dogs A and B Dog B Dog A
Dose mg/kg 1 1.5 3Cmax nM 2815 5240 16,000AUCtot nM·h 1456 3844 13,183t1/2 Hour 0.5 5.0 6.4Clearance ml/min·kg 30 17 10Vss Liter 1.1 3.6 3.6
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A had low clearance (4.6 ml/min), moderate Vss (0.9 l), andmoderate t1/2 (3.2 hours). Plasma Cmax (5 minutes postdose)was 2.3mMand declined to 1.0 mMby 15minutes postdose. Noeffects were observed in themonkeys following administrationof vehicle and the 0.1 mg/kg dose. At 0.3 mg/kg, nasaldischarge and hypersalivation were observed in one monkeyfrom approximately 5 to 15 minutes postdose. In the otheranimal, hypersalivation, emesis (single episode), urination(single episode), and miosis were observed from approxi-mately 5 to 20 minutes postinfusion.Dog. Compound B was dosed i.v. in a dose escalation study
at 1 and 3 mg/kg as described in the Materials and Methods.Plasma concentration measurements are shown in Fig. 6, andpharmacokinetic parameters are summarized in Table 6.Pharmacokinetic parameters were not calculated for the1 mg/kg dose, due to the short time interval (,1 hour).Following administration at 1 mg/kg, both dogs had slightdrooling approximately 4 minutes into the infusion; no othersymptoms were observed. Exposures at the end of infusionwere 2.6 and 3.1 mM. Although the plasma levels of compoundB were somewhat higher for the second dose as a result of thefirst dose, the expected contribution was calculated to beminimal (,10%). Following i.v. administration in one dog at3 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 theinfusion, nasal discharge, licking lips, and salivation werevisible. Severe salivation was noted at end of the infusion(5minutes). Exposure at the end of administrationwas 16mM.Vomiting was observed between 7 and 9 minutes postdose.Severe diarrhea was observed between 8 and 17 minutespostdose. Ataxia was observed from 11 to 50minutes postdose,starting with abnormal stance with front legs straight andback legs splayed behind. Ataxia was severe by 20 minutespostdose, with complete loss of hind end control and knucklingon front feet. Ataxia lessened by 29minutes postdose, with thedog walking in circles. The dog was alert and exploring by50 minutes postdose, with no other adverse events noted after1 hour postdose. Exposure was 2.2 mM at the 1 hour time
point. Due to the severe effects in the first dog, the infusion forthe second dog was stopped after 2.5 minutes, resulting in alower dose of 1.5 mg/kg. Mild salivation and licking wasobserved after 2 minutes of infusion. No other adverse eventswere noted. Exposure at the end of infusion was 5.2 mM.Pharmacokinetic parameters for this dog were similar to thefirst dog with a Vss of 3.6 l/kg and a t1/2 of 5.0 hours, butclearance was somewhat higher (17 ml/min/kg).Compound C was dosed in the samemanner as compound B
with a 5-minute infusion of 0.5, 1, and 5 mg/kg with 1 hourobservation between doses. Plasma concentration measure-ments are shown in Fig. 7, and pharmaokinetic parametersare summarized in Table 7. Following the vehicle and 0.5mg/kgdose, no adverse events were noted. Exposures at the endof the infusion were 2.6 and 4.1 mM. Clearance was high at31–35 ml/min/kg, Vss was moderate (0.5–0.7 l/kg), and t1/2 wasshort (0.2 hours). Following administration at 1 mg/kg, slightclear nasal discharge was observed in both animals approxi-mately 3 to 4 minutes into the infusion. One animal alsoexperienced hypersalivation and decreased activity. Expo-sures at the end of the infusion were 7.3 and 17.3 mM. Bothdogs had no other symptoms by 5minutes postdose. Only 1 dogwas administered the 5 mg/kg dose. Decreased activity,licking, and excessive hypersalivation were noted 2.5 minutesafter the start of the infusion with moderate nasal discharge.By the end of the infusion, dilated pupils, clear oculardischarge, and slight ataxia with a wide stance were observedin addition to hypersalivation and licking. Symptoms abatedrapidly, with no further observations by 20 minutes postdose.A summary of the observed in vivo effects in all species isprovided in Table 8.
DiscussionA significant body of research exists to support the potential
therapeutic efficacy of M1 agonists for treating cognitivedisorders such as schizophrenia and Alzheimer’s disease. M1
agonists have been shown to reverse cognitive deficits inanimal models (Jones et al., 2005; Langmead et al., 2008;Barak and Weiner 2011), and more importantly the M1
agonist xanomeline has been shown to produce beneficialpsychiatric and cognitive effects in human patients (Bodicket al., 1997; Shekhar et al., 2008). Unfortunately, xanomelinealso produces serious side effects including salivation, sweat-ing, and gastrointestinal distress. Although xanomeline hadoriginally been reported to be highly selective for M1 over
TABLE 7Pharmacokinetic 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 firstdose and 5 mg/kg (dog A) 2 hours after the first dose
Parameter Unit
Compound C Dog Dose Escalation (i.v.)
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 41,300AUCtot nM·h 639 722 2301 3712 13,213t1/2 Hour 0.2 0.2 0.2 0.9 4.2Clearance 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.
Fig. 7. Plasma exposure of compound C in dogs following i.v. adminis-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).
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othermuscarinic receptor subtypes based upon ex vivo studies(Sauerberg et al., 1992; Shannon et al., 1994), subsequentstudies using cloned human muscarinic receptors suggestedthat xanomeline exhibits very little selectivity among the fivemuscarinic receptor subtypes (Watson et al., 1998;Wood et al.,1999). Therefore, the untoward side-effect profile of xanomelinehas been attributed to nonselective activity at other muscarinicreceptor subtypes, in particular M2 andM3 (Mirza et al., 2003).Recently, the identification of small molecules that bind to
allosteric binding sites that are distinct from the (orthosteric)endogenous agonist binding site has emerged as a strategy fortargeting G protein coupled receptors (Wootten et al., 2013).Allosteric modulators have several potential advantages overtraditional orthosteric ligands (Wootten et al., 2013), includ-ing the potential for improved receptor subtype selectivity.Indeed, theM1 PAMs used in the current study show exquisitefunctional selectivity for M1 over other muscarinic receptorsubtypes in vitro (Figs. 2 and 3; Table 1). Therefore, it wassurprising that all three of the M1 PAMs tested produced invivo effects consistent with cholinergic toxicity, such ashypersalivation, vomiting, and severe diarrhea. These effectsare reminiscent of the side effects seen in humans duringclinical trials with xanomeline (Bodick et al., 1997; Shekharet al., 2008), suggesting that some or all of xanomeline’sadverse effects may in fact be mediated by activation of theM1
receptor itself, rather than by other muscarinic receptorsubtypes, as had been previously assumed.It is important to note that, in this study, the in vitro
assays used to assess selectivity of the test compounds for M1
versus the other muscarinic receptor subtypes used cellsrecombinantly expressing each of the muscarinic receptors.Furthermore, in the case of the M2- and M4-expressing celllines, the mutant G protein Gqi5 was coexpressed to forcecoupling of the M2 and M4 receptors to a Ca21 mobilizationpathway instead of their natural Gi-mediated coupling toinhibition of adenylyl cyclase. Therefore, the cellular modelsused for selectivity assessment represent highly artificialsystems. While these recombinant systems afford excellentsensitivity and reproducibility of data generated, it ispossible that artificial G protein coupling or other differencesfrom the receptors’ native neuronal environment could lead toresults that do not reflect the true activity of these compoundsin vivo, and this caveatmust be kept inmindwhen interpretingthese results.
The M1 PAMs used in this study were profiled in an in vitrosafety panel measuring activity at 43 G protein coupledreceptors, ion channels, transporters, nuclear hormone recep-tors, and enzymes (Table 2). Compound A showed somemeasurable activity at several of the receptor targets in thispanel: the a1D-adrenergic receptor, the GABAA receptor,phosphodiesterase 4, and the cardiac sodium channel NaV1.5.However, compound A exhibited only very low potency(2–13 mM) at each of these targets. Compound B showed somebinding activity at androgen, estrogen, and progesteronereceptors, but again with very low (16–64 mM) potency.Compound C showed no significant activity at any of thetargets in this panel. It is important to note thatmany of theseselectivity assays (e.g., radioligand binding–based assays)would not be expected to detect potential allostericmodulationof the receptor tested. However, with this caveat, these results—combined with the similarity of the in vivo toxicity profileobserved with compounds A, B, and C—again support theconclusion that the toxic effects observed in the current studywere mediated by M1 receptors rather than by some off-targetactivity of the compounds.It is common for PAMs to also exhibit direct agonist activity
at higher concentrations (Schwartz and Holst 2006, 2007;Bridges and Lindsley 2008; Burford et al., 2011). Suchcompounds are commonly referred to as ago-PAMs (Noetzelet al., 2012). Indeed, all three of the M1 PAMs used in thecurrent study were found to exhibit direct agonist activity atconcentrations approximately 50- to 100-fold higher thanthose required for PAM activity in vitro (Fig. 2; Table 1).Ago-PAMs and pure PAMs (those with no direct agonistactivity) can show differential activity in vivo (Bridges et al.,2013; Rook et al., 2013). This may in part reflect the ability ofPAMs to maintain the temporal and spatial fidelity of nativesignaling because pure PAMs do not activate the receptor ontheir own. They exert an effect onlywhen andwhere the nativeagonist is present, thereby preserving some aspects of nativereceptor signaling and its physiologic regulation, and thusmight be expected to avoid some side effects associated withdirect activation of the receptor (for reviews, see Woottenet al., 2013; Burford et al., 2015). Within this context, it isinteresting to note that the doses in this study, which pro-duced adverse events in vivo, generally attained relativelyhigh (i.e., micromolar) plasma exposure levels in test animals.At these concentrations, the M1 PAMs tested may be producing
TABLE 8Summary of doses, dose routes, and observed effects for all species used in the evaluation of compounds A, B, and C intolerability studies
Compound Species Route Dose Observed Effect
mg/Kg
A Rat i.v. 1 No effectsA Rat By mouth 2.5 Diarrhea, bright yellow urineA Cyno i.v. 0.3 Hypersalivation, licking, emesis, urination, miosisA Dog i.v. 1.5 Hypersalivation, lickingA Dog i.v. 3 Hypersalivation, licking, emesisB Rat i.v. 1 No effectsB Rat By mouth 2.5 Bright yellow urineB Dog i.v. 1 Slight drooling, runny noseB Dog i.v. 1.5 Mild salvation, licking lipsB Dog i.v. 3 Nose running, licking lips, salvation, vomiting, diarrhea, ataxiaC Dog i.v. 1 Nasal discharge, hypersalivation, licking, decreased activityC Dog i.v. 5 Nasal discharge, hypersalivation, licking, decreased activity, ataxia
Cyno, cynomologous monkey.
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direct agonism at the receptor in vivo, based upon their in vitroprofiles (Fig. 2; Table 1). Additionally, the fact that the sideeffects were transient further suggests that these side effectsresulted from high concentrations of the compounds. There-fore, this raises the intriguing possibility that direct activationof M1 is required to produce the unwanted cholinergic sideeffects observed, and that pure M1 PAMs, or ago-PAMsadministered at doses below those required for direct receptoragonism, could still be safe, and therefore the identification ofM1 PAMs with acceptable safety margins may be possible.Within this context, it should be noted that compound B
from this study has also been tested in vivo by Kuduk et al.(2011), who also reported that the compound is selectiveagainst other muscarinic receptor subtypes, in addition tobeing selective against a wide panel of other targets, inagreement with the current study. This group has furtherreported that compound B did not produce significant gastro-intestinal or salivation disturbances at doses up to five timesgreater than those that produced procognitive effects inrhesus monkeys, suggesting that a therapeutic window be-tween beneficial and undesired effects can be achieved(Vardigan et al., 2015). Furthermore, compound B was shownto produce synergistic procognitive effects when combinedwith either the muscarinic agonist xanomeline, or the acetyl-cholinesterase inhibitor donepezil, suggesting combinationtherapy as another potential strategy for achieving acceptablesafety margins with M1-targeted therapeutics.The current study does not include testing in cognition
models, and was not designed to gauge safety margins.Instead, the current study was designed to test the hypothesisthat selective activation of the M1 receptor alone would beinsufficient to produce classic cholinergic toxicity. Our resultsrefute this hypothesis. Toxic effects were observed in rats,dogs, and cynomologous monkeys. These effects were similaracross the species tested, and were produced by three differentM1 receptor PAMs. Furthermore, these effects are similar tothose seen in clinical trials using the M1 agonist xanomeline.All three of theM1 PAMs used in this study exhibited excellentselectivity for M1 over other muscarinic receptor subtypes invitro, and in a safety panel no off-target activity was detectedfor these compounds, which would explain the toxic effectsobserved. Based upon these combined results, we now hypoth-esize that activation of the M1 receptor alone is sufficient toproduce a cholinergic toxicity syndrome. This has importantimplications for the development of M1-targeted therapeuticssince it would suggest that dose-limiting cholinergic side effectssuch as those observed in clinical trials with xanomeline cannotbe completely avoided simply by the use of drugs that exhibitgreater selectivity for the M1 receptor versus other muscarinicreceptor subtypes.Finally, it should be noted that a novel allostericM1 agonist,
HTL9936, has been investigated in a phase 1 clinical trial asa potential treatment of cognitive impairment associatedwith Alzheimer’s disease (http://clinicaltrials.gov/ct2/show/NCT02291783), and was reported to be well tolerated in thisstudy. It will be of great interest to see whether this initialreport is replicated, and whether the central nervous systemexposure levels achieved in this safety study are sufficient toproduce therapeutic effects. Therefore, future clinical stud-ies using HTL9936 are expected to provide additionalinsight into the side-effect liabilities associated with M1
receptor activation in humans.
Acknowledgments
The authors thank Reshma Panemangalore, Rudy Krause, JeremyStewart, Lizbeth Gallagher, Glen Farr, Michele Matchett, and RonKnox for the execution of various in vitro selectivity assays.
Authorship Contributions
Participated in research design: Alt, Pendri, Cvijic, Westphal,O’Connell, Zhang, Gentles, Jenkins, Loy, Macor.
Conducted experiments: Bertekap, Benitex, Nophsker, Rockwell,Burford, Sum, Chen, Herbst, Ferrante.
Contributed new reagents or analytic tools: Pendri, Li, Gentles.Performed data analysis: Alt, Bertekap, Benitex, Nophsker, Rock-
well, Burford, Sum, Chen, Herbst, Ferrante, Hendricson, Jenkins,Loy.
Wrote or contributed to the writing of the manuscript: Alt, Pendri,Sum, Herbst, Banks, Jenkins, Loy, Macor.
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Address correspondence to: Andrew Alt, Leads Discovery and Optimiza-tion, Bristol-Myers Squibb, 5 Research Parkway, Wallingford, CT 06492.E-mail: [email protected]
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