Inositol Phosphate Accumulation in Vivo Provides a Measure of

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Inositol Phosphate Accumulation In Vivo Provides A Measure Of Muscarinic M1 Receptor Activation Michael Popiolek, David Nguyen, Veronica Reinhart Bieber, Jeremy R. Edgerton, John Harms, Susan M. Lotarski, Stefanus J. Steyn, Jennifer Elizabeth Davoren, and Sarah Grimwood Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00688 • Publication Date (Web): 28 Nov 2016 Downloaded from http://pubs.acs.org on November 29, 2016

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1 TITLE: Inositol Phosphate Accumulation In Vivo Provides A Measure Of Muscarinic M1 Receptor Activation. FUNDING SOURCE STATEMENT: At the time that this work was performed all authors were employees of Pfizer Inc. AUTHORS: Michael Popiolek1*, David P. Nguyen2, Veronica Reinhart1, Jeremy R. Edgerton1, John Harms1, Susan M. Lotarski1, Stefanus J. Steyn2, Jennifer E. Davoren3, Sarah Grimwood1 1

Neuroscience and Pain Research Unit, 2Pharmacokinetics, Dynamics and Metabolism, 3Worldwide

Medicinal Chemistry, Pfizer Worldwide Research and Development, Cambridge, Massachusetts 02139.

CORRESPONDING AUTHOR: Michael Popiolek, 610 Main Street, Cambridge, MA 02139 Tel: +1 (617) 395-066 Email: [email protected]

ABBREVIATIONS: ACh, acetylcholine; AD, Alzheimer’s disease; a-LMA, amphetamine-stimulated locomotor activity; amph, amphetamine; CA1, cornus ammonis 1; cAMP, cyclic adenosine monophosphate; Cb,u, unbound (free) brain concentration; CHO, Chinese hamster ovary; CNS, central nervous system; FLIPR, fluorometric imaging plate reader; GPCR, G-protein coupled receptor; HPC, hippocampus; IP, intraperitoneally; IP1, inositol monophosphate; LiCl, lithium chloride; mAChR, muscarinic acetylcholine receptor; mRNA, message ribonucleic acid; PAM, positive allosteric modulator; PD, pharmacodynamics; PDE, phosphodiesterase; PFC, prefrontal cortex; PK, pharmacokinetic; PQCA; 1-((4-cyano-4-(pyridine2-yl)piperidin-1-yl)methyl-4-oxo-4 H-quinolizine-3-carboxylic acid; SEM, standard error of the mean; SC, subcutaneously; STR, striatum; SZ, schizophrenia; Veh, vehicle.

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2 ABSTRACT The rationale for M1-selective muscarinic acetylcholine receptor activators for the treatment of cognitive impairment associated with psychiatric and neurodegenerative disease is well established in the literature.

Here, we investigate measurement of inositol phosphate accumulation, an endpoint

immediately downstream of the M1 muscarinic acetylcholine receptor signaling cascade, as an in vivo biochemical read-out for M1 muscarinic acetylcholine receptor activation. Five brain penetrant M1subtype selective activators from three structurally distinct chemical series were pharmacologically profiled for functional activity in vitro using recombinant cell calcium mobilization and inositol phosphate assays, and a native tissue hippocampal slice electrophysiology assay, to show that all five compounds presented a positive allosteric modulator-agonist profile, within a narrow range of potencies. In vivo characterization using an amphetamine stimulated locomotor activity behavioral assay and the inositol phosphate accumulation biochemical assay demonstrated that the latter has utility for assessing functional potency of M1 activators. Efficacy measured by inositol phosphate accumulation in mouse striatum favorably compared to efficacy in reversing amphetamine induced locomotor activity, suggesting that the inositol phosphate accumulation assay has utility for the evaluation of M1 muscarinic acetylcholine receptor activators in vivo. The benefits of this in vivo biochemical approach include a wide response window, interrogation of specific brain circuit activation, ability to model responses in context of brain exposure, ability to rank order compounds based on in vivo efficacy and minimization of animal use.


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3 INTRODUCTION The muscarinic acetylcholine receptors (mAChRs) are a family of G- protein-coupled receptors (GPCRs) that share a highly conserved orthosteric binding pocket for acetylcholine (ACh) and mediate wide variety of functions within the central and peripheral nervous systems (Kruse et al., 2014). M1, M3 and M5 mAChRs are excitatory Gq-coupled GPCRs that lead to phospholipase C activation, the subsequent production of inositol phosphate-3 (IP3) and diacylglycerol, and the release of calcium from intracellular stores (Hulme, Birdsall, & Buckley, 1990), while M2 and M4 mAChRs are inhibitory Gi/ocoupled GPCRs that lead to inhibition of adenylyl cyclase, thereby reducing cytoplasmic cyclic adenosine monophosphate (cAMP) levels (Hulme et al., 1990). Several lines of evidence support M1 mAChRs as an attractive target for the treatment of psychosis and cognitive impairment in schizophrenia (SZ) and Alzheimer’s disease (AD) patients (Dean, 2012; Foster, Choi, Conn, & Rook, 2014; Jiang et al., 2014). M1 mAChR mRNA has been demonstrated to be enriched in brain regions implicated in SZ and AD including the striatum (STR), hippocampus (HPC) and prefrontal cortex (PFC) (Buckley, Bonner, & Brann, 1988). This M1 mAChR distribution was confirmed through Western blots and radioligand binding experiments (Bymaster, McKinzie, Felder, & Wess, 2003; Levey, 1993). Consistent with the hypothesis that activation of M1 mAChRs will result in therapeutically beneficial antipsychotic and procognitive efficacy, the reported M1 and M4 agonist xanomeline was demonstrated to have efficacy in both AD and SZ patients (Bodick et al., 1997; Heinrich et al., 2009; Shekhar et al., 2008). However, xanomeline also showed adverse side effects proposed to be mediated by peripheral muscarinic receptor activation (Bodick et al., 1997; Shekhar et al., 2008). In addition, the cholinesterase inhibitor donepezil, which indirectly activates all mAChRs, is a marketed therapeutic for cognitive improvement in AD patients (Evans, Ellis, Watson, & Chowdhury, 2000). Preclinical data obtained for M1 selective mAChR activators have been encouraging and consistent with xanomeline’s beneficial clinical observations, suggesting that selective M1 activators may be therapeutically beneficial. This has been true for both M1 agonists, which directly activate downstream


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4 signaling independently of ACh, and for positive allosteric modulators (PAMs), which bind at a distinct topographical site from ACh and potentiate either the affinity of the receptor for ACh binding or the efficiency of the ACh-bound receptor at driving downstream signaling (for reviews see (Christopoulos, 2014; Kenakin & Miller, 2010). M1 preclinical antipsychotic activity has previously been demonstrated through efficacy in mouse amphetamine-stimulated locomotor activity (a-LMA) with the M1 agonists TBPB and AC-260584, and the M1 PAMs, BQCA, PF-06764427 and PF-06767832(Davoren, Lee, et al., 2016; Davoren, O'Neil, et al., 2016; Jones et al., 2008; Ma et al., 2009; Vanover, Veinbergs, & Davis, 2008). In addition, cognitive enhancement was observed in rodent and non-human primate tests of working and spatial memory with the M1 agonists CDD-0102A and AC-260584, and the M1 selective PAM PQCA (S. D. Kuduk et al., 2011; Lange, Cannon, Drott, Kuduk, & Uslaner, 2015; Ragozzino et al., 2012; Uslaner et al., 2013; Vanover et al., 2008; Vardigan et al., 2015). The present study, using five M1-selective mAChR activators from three structurally distinct chemical series, investigates whether measurement of inositol phosphate levels in vivo can be used to assess levels of M1 mAChR activation. Generation of inositol phosphate 3 (IP3) occurs downstream of M1 mAChR activation. Since IP3 degrades too rapidly for accurate detection, we measured the degradation product, inositol phosphate 1 (IP1), which can be stabilized with lithium chloride (LiCl) treatment and measured through fluorescence-based commercially available kits. EC50 values obtained using the in vivo IP1 assay, along with brain exposures of the test compounds, allowed us to calculate functional potency of compounds.


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5 METHODS: Animals were handled and cared for according to the National Institutes for Health Guide for the Care and Use of Laboratory Animals, and all procedures were performed with the approval of the Institutional Animal Care and Use Committee at the Pfizer Global Research and Development site in Cambridge, MA. Human M1, M3, and M5 receptors stably expressed in Chinese hamster ovary (CHO) cells were obtained from Wyeth and HD Biosciences Co. LTD, Shanghai. Muscarinic receptor expression levels were determined using [3H]N-methylscopolamine binding (Smith et al., 2016), resulting in the following saturable binding capacity (Bmax) values: M1 fluorometric imaging plate reader (FLIPR) assay, 384 fmol/mg protein; M1 IP1 assay, 306.9 fmol/mg protein; M3, 976.88 fmol/mg protein; M5, not tested. Calcium mobilization FLIPR assay In-vitro calcium was measured using the Fluo-4 Direct Calcium Assay Kit (Thermo Fisher Scientific, Massachusetts, USA). 10 mL Flou-4-Direct Calcium assay buffer was added to each tube of Flou-4 Direct Calcium assay reagent and stored at -20 oC until use. One day prior to running the assay, M1 cells were plated in a black clear-bottom poly-d-lysine coated 96-well plate (Fisher Scientific, New Hampshire, USA) at a density of 100,000 cells/well in 50 µL media (DMEM supplemented with 10% FBS, 1% penn/sterp, 1 % nonessential amino acids). On the day of the experiment, Fluo-4-Direct Calcium assay buffer was supplemented with prebenecid at 1:50 dilution and 50 µL of this mixture was added to each 96-well for an overall dye to media dilution of 1:1. The plate was incubated at 37 °C for 1 h. The plate was then treated and read for fluorescent Ca2+ using the fluorometric imaging plate reader (FLIPR) Tetra High-Throughput Cellular Screening system (Molecular Devices, California, USA). Compound treatments consisted of half-log concentration responses with a single measurement per concentration. All treatments were prepared in Hanks balanced salt solution without calcium. M1 activators were tested either alone (“agonist mode”) or in the presence of an EC20 (2 nM) of acetylcholine (“PAM mode”). Three individual experiments were conducted for each compound of interest. The data


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6 were analyzed by subtracting the minimum reading from the maximum, and graphed using GraphPad software (GraphPad Software Inc., California, USA). In-vitro IP1 Cell Assay Intracellular IP1 levels were measured using a homogeneous time resolved fluorescence (HTRF) IP-ONE Tb assay kit (CisBio Bioassays, Bedford, Massachusetts). One day prior to running the assay, M1 cells were plated at a density of 20,000 cells per well in white poly-d-lysine coated 384 well plates (Corning Life Sciences, Corning, New York). On the day of the experiment, cell media was removed via aspiration and replaced with 7 µL stimulation buffer with LiCl (vendor supplied) supplemented with M1 activators either alone (“agonist mode”) or with EC20 of acetylcholine (10 nM) (“PAM mode”). Cells were incubated at 37 °C for thirty minutes and then lysed by addition of 3 µL supplied conjugate-lysis buffer containing d2-labeled IP1 and 3 µL supplied conjugate-lysis buffer containing terbium cryptatelabeled anti-IP1 antibody. The plates were briefly spun down and incubated at room temperature for 1 h. Time-resolved fluorescent signals were read using the Envision 2101 Plate Reader (PerkinElmer, Waltham, Massachusetts) at 620 and 665 nm. Results were calculated as a ratio of 665 nm/620 nm and IP1 concentrations were interpolated from a standard curve prepared using the supplied IP1 calibrator, and graphed using GraphPad software (GraphPad Software Inc, California, USA).

Three individual

experiments were conducted for each compound of interest. In order to confirm the M1 specificity of the five tested compounds, intracellular IP1 levels were also measured using the previously described protocol using M3 and M5 overexpressing stable CHO cells in both agonist and PAM mode (in the presence of EC20 concentration of acetylcholine: 20 nM for M3 cells and 63.2 nM for M5 cells). Two individual experiments were conducted for each compound of interest.


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7 Hippocampal Slice Electrophysiology Acute hippocampal slices were prepared from 7-10 week old male Sprague-Dawley rats (Charles River Laboratories). Rats were decapitated with a small animal guillotine, and the brain was rapidly removed and immersed in ice-cold cutting solution ((mM): 206 sucrose, 3 KCl, 1.25 NaH2PO4, 7 MgCl2, 26 NaHCO3, 10 D-glucose, 0.5 CaCl2, 1 L-ascorbate, 1 sodium pyruvate). The brain was subsequently cut using a vibratome (Leica VT1000S or 1200S) to 300 micron thick coronal slices containing the dorsal hippocampus. Slices were transferred to an incubation chamber filled with normal recording artificial CSF (aCSF (mM): 124 NaCl2, 3 KCl, 1.25 NaH2PO4, 1.3 MgCl2, 26 NaHCO3, 10 D-glucose, 2 CaCl2, 1 L-ascorbate, 1 sodium pyruvate) in a 32°C water bath for a recovery period of at least 1 h. Both the recording and cutting solutions were bubbled continuously with 95% O2/5% CO2. All recordings were carried out using MED-P515A 64-electrode arrays (Automate Scientific, Inc). After recovery, slices were transferred individually to MED64 recording chambers mounted on dissecting microscopes. The 64 amplifier channels were divided using a 4-way splitter such that 16 channels were active for each slice (2 rows of 8). Slices were positioned with the CA1 stratum pyramidale directly over one row of 8 electrodes, and held down with a U-shaped platinum horseshoe over a square of nylon mesh. Slices were perfused continuously with recirculating aCSF warmed by an inline solution heater (Warner Instruments, Inc), such that the average bath temperature remained about 30-32 °C. Electrophysiological signals were highpass filtered at 0.1 Hz and digitized at 20 kHz using MED64 Mobius acquisition software (WitWerx, Inc). Extracellular action potentials were detected offline using custom-written Matlab scripts. Individual stratum pyramidale electrodes generally detected a mixture of many spike waveforms that we were not able to separate into discrete clusters with confidence; for this reason spiking activity was measured as the multi-unit firing rate for each channel. Spike rates were computed for each stratum pyramidale channel within a slice, and then averaged across the channels to generate a single median activity rate for the slice. Statistical comparisons were performed by considering each slice to be an independent N. For test compound concentration-response curves, compound was added stepwise in serially increasing


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8 concentrations, with each step lasting 20 minutes. The median activity during the last 5 trials of each step was taken as the activity level for that concentration. The effect of the concentration was measured as the median firing rate in the presence of the compound minus the median firing rate under the initial baseline conditions. For assessing PAM activity, compounds were applied either alone (agonist mode) or in the continuous presence of an EC20 of carbachol (100 nM; PAM mode). Mouse in-vivo IP1 Assay Microwave Protocol: Six-week-old CD1 male mice (Charles River Laboratories) were acclimated to the facility for a week prior to the day of the study and to the procedure room for 2 h prior to initiation of the experiment. Mice (n=5 per group) were subcutaneously (SC) administered 100 mg/kg LiCl (Sigma Aldrich #20363710G dissolved in water) administered at 1/100th volume to weight ratio. One hour later, test compounds or vehicle were administered SC. Two hours later, animals were euthanized by brain-focused microwave application (customized instrument from Litton Model 70/50). Hippocampal, striatal and prefrontal cortex tissues were immediately isolated and frozen on dry ice. Samples were then stored at -80oC until subsequent analysis. Tissue preparation and IP1 assay: Frozen tissues were weighed and homogenized with a bead homogenizer using 3.5 mm stainless steel (McMaster-Carr) in 10 µL RIPA buffer (150 nM NaCl, 50 mM Tris, 0.5% deoxycholic acid, 0.1% SDS, 1% NP-40) per mg of tissue for 10 min at 25 hertz. Brain tissue homogenates (~150 µL) were submitted for test compound quantification using LC-MS/MS (see ”Brain Exposure” section below). IP1 was measured using a CisBio IP-One TB kit (62IPAPEC). Brain tissue homogenates (3 µL) were spotted in duplicate into a 384-well solid white low volume plate (Greiner BioOne #784075). Each well was mixed with 8 µL of lysis buffer, 3 µL of IP1-d2 and 3 µL of IP1-cryptate, in that order. The samples were incubated at room temperature for 1 h, before reading the plates on an Envision 2101 Plate Reader (PerkinElmer, Waltham, Massachusetts) at 620 and 665 nm. Results were


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9 calculated as a ratio of 665nm/620nm and IP1 concentrations were interpolated off of a standard curve prepared using the supplied IP1 calibrator, graphed using GraphPad software (GraphPad Software Inc, California, USA), and analyzed using one-way ANOVA with multiple comparisons. Mouse locomotor activity (LMA) assays Male C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were received at 7-8 weeks of age and group housed (4/cage) upon arrival. Animals were acclimated to the facility for a minimum of one week prior to testing. Versamax Animal Activity Chambers (Accuscan Instruments, Inc., Columbus, OH) were enclosed in sound attenuating chambers and used to record locomotor activity. Each chamber measured 16” x 16” and had infra-red photobeams along the x, y and z axis to capture movement of the animal. Each chamber also has a fan for ventilation. Lights (~60 lux) remained on for testing and sound attenuation chambers were closed. Spontaneous LMA: Following a one hour acclimation to the test room mice (N=8-10) were administered vehicle or test compound and returned to the home cage for a 30 min pretreatment time. Each animal was then placed into a test box for a 90 min test session. Amphetamine-stimulated LMA: Following a one hour acclimation to the test room, mice (n=10/group) were placed in the test box for a 60 min habituation period, followed by dosing with vehicle or test compound. Thirty minutes later animals are then administered d-amphetamine and returned to the chamber for a 90 min test session. Lights remained on during testing. Statistical analysis (R stats): Data were recorded in 10 min bins and analyzed using R 3.0.1 statistical software (Team, 2014). The effects of treatment and time and their interaction on total distance were assessed using a two-way repeated measures ANOVA using generalized least squares methods from NLME library (Pinheiro J, 2014). To account for correlations within subjects a first order autoregressive


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10 scheme was employed, which assumed that correlations decay exponentially with the lag between measurements. We also allowed for time dependence of the variance. The model was fitted using the method of restricted maximum likelihood. Significant ANOVA results were followed by post-hoc pairwise comparisons of least squared means (Lenth, 2014) across treatment arms, separately at each time point. In order to adjust for multiple hypothesis testing we used a false discovery rate (FDR) method which controls the expected proportion of false discoveries amongst the rejected hypotheses (Benjamini, Drai, Elmer, Kafkafi, & Golani, 2001). Data were considered statistically significant at a level of p 10-fold higher potency in PAM mode (175 – 285 nM), compared to agonist mode (2313 – 5080), again demonstrating a PAM-agonist profile (Figure 3; Table 1). Figure 3: M1 PAM-agonists induce IP1 accumulation in stably expressed M1 cells

Figure 3 legend: IP1 functional responses shown are the mean ± SEM of at least 3 experiments using human M1 cells. Filled circles indicate the response to the test compound alone (agonist mode), while open circles indicate data obtained in the presence of an EC20 of acetylcholine (PAM mode). IP1 experiments performed for the five test compounds using M3 and M5 mAChR cells in agonist and PAM modes resulted in no detectable increase in IP1 for concentrations tested (up to 10 µM ; Supplementary Table 1), suggesting functional selectivity for the M1 mAChR over M3 and M5 mAChRs.


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16 Hippocampal slice electrophysiology: M1 mAChR activation is known to produce direct excitatory effects on hippocampal pyramidal neurons (Dasari & Gulledge, 2011; Langmead et al., 2008). This excitation results in increased spontaneous firing rates of CA1 neurons in hippocampal slices, which can be measured extracellularly with multi-electrode arrays. We used this assay to determine whether the PAM-agonist profile observed with the test compounds using M1 cell line assays would also be portrayed using a native cell system. We first measured CA1 neural firing rates while applying increasing concentrations of the non-selective cholinergic agonist carbachol, allowing for the approximation of an EC20 which was applied in subsequent potentiation studies (Figure 4). When applied per se M1 activators elicited a response which is likely to be the direct agonist response observed in-vitro (although some contribution of endogenous ACh released within the slices cannot be ruled out), with EC50 values ranging from 514 – 3869 nM. When the M1 activators were co-applied with an EC20 of carbachol the potency of the M1 activators was enhanced, ranging from 115 – 321 nM, suggesting a dual PAM-agonist profile, consistent with the pharmacology previously observed using the M1 stable cell line (Figure 4 and Table 1). Figure 4: Hippocampal slice electrophysiology.


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17 Figure 4 legend: Hippocampal slices were prepared from rats and bath incubated with increasing concentrations of test compounds as indicated. The observed concentration-dependent increases in spike rates suggest functional efficacy of carbachol and the five M1 selective activators. Filled circles indicate the response to the test compound alone (agonist mode), while open circles indicate data obtained in the presence of an EC20 of carbachol (PAM mode). Data shown are the mean ± SEM of at least 3 slices. Table 1 summarizes EC50 values obtained for M1 mAChR activators in vitro. Potency values obtained across compounds were similar within each experimental format, with PAM mode EC50 values varying from 1.6 to 4 fold across experimental formats, and agonist mode EC50 values varying from 2 – 7 fold across experimental formats (Table 1). The observed variability, particularly for agonist mode, meant that potency values for the five M1 mAChR activators did not rank in exactly the same order for each assay format, however, in all cases, for both recombinant cell and native brain slice assays, all compounds displayed a PAM-agonist profile in vitro, i.e. with higher potency values observed in PAM mode. Table 1: Summary of in-vitro characterization of M1 activators.

Assay mode

PT-4186

PT-1631

PT-8345

PF-06764427

PT-3214

PAM

25 ± 18

63 ± 35

102 ± 16

55 ± 13a

63 ± 13

Agonist

607 ± 257

1982 ± 8997

2154 ± 1530

1193 ± 556

3659 ± 2464

In-vitro IP1

PAM

175 ± 225

280 ± 14

211 ± 162

187 ± 194

285 ± 213

(EC50, nM)

Agonist

3415 ± 597

4851 ± 696

2765 ± 1567

2313 ± 1430

5080 ± 4259

HPC spike rate

PAM

256 ± 221

115 ± 464

118 ± 62

161 ± 53

321 ± 197

(EC50, nM)

Agonist

514 ± 200

3869 ± 1320

1749 ± 775

2788 ± 739

3580 ± 245

FLIPR (EC50, nM)


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18 Table 1 legend: Compiled FLIPR (from Figure 2) and IP1 (from Figure 3) data from M1 stable cell line experiments, and rat hippocampal (HPC) spike rate (from Figure 4). Experiments were performed in either agonist (test compound alone) or positive allosteric modulator (PAM; tested in the presence of EC20 of ACh or carbachol) modes. aData previously reported by Davoren and colleagues (Davoren, O'Neil, et al., 2016). Data sets are complimentary and indicate that M1 mAChR activators have PAM-agonist profiles, with higher potencies observed in PAM mode. Selectivity of M1 mAChR activators: Compounds were tested for selectivity in functional assays using both agonist and PAM modes against both M2 and M4 receptors, demonstrating no observable activity (Supplementary Table 1). Functional selectivity beyond muscarinic receptors was investigated against twenty other GPCRs, ion channels, and phosphodiesterases (Supplementary Table 1). No significant activity was observed at these targets, suggesting that these compounds are highly selective for M1 mAChR. 3.7 In-vivo biochemical efficacy as measured by IP1 In order to measure IP1 accumulation in vivo it is necessary to prevent the endogenous degradation of IP1, which can be achieved with LiCl (Bymaster et al., 1998). We therefore performed preliminary experiments to investigate the effect of different LiCl doses on IP1 degradation in the striatum, hippocampus and pre-frontal cortex in vivo. 50 mg/kg LiCl did not alter IP1 levels in the three brain regions relative to vehicle treated mice (Figure 5A). At 250 mg/kg LiCl we observed very robust IP1 increases in the three brain regions, potentially leaving a very small window for further IP1 increase (Figure 5A). At the mid dose of 100 mg/kg LiCl a modest IP1 increase was observed suggesting sufficient brain bioavailability to enhance IP1 levels, but not high enough for saturation (Figure 5A), therefore 100 mg/kg LiCl was selected for testing compounds. In addition, we determined that LiCl had no effect on the activity of the M1 mAChR in our recombinant cell line (Supplementary Figure 1). With 100 mg/kg LiCl treatment we consistently observed lowest to highest baseline IP1 in STR, HPC and PFC


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19 respectively. The CisBio IP1 kit includes a standard IP1 allowing for back calculation of IP1 in the tissue. Applying the standard we calculated baseline in STR at 754 ± 81nM, in HPC at 1402 ± 141 nM and in PFC at 3672 ± 432 nM (± SEM). Addition of the five test compounds dose-dependently increased IP1 levels in striatal, hippocampal and pre-frontal cortex tissue (Figure 5B-F; Table 2) beyond levels observed with LiCl alone. Figure 5: Brain tissue IP1 levels after treatment with M1 mAChR activators.

A

B

C

D

E

F

Figure 5 legend: In order to select an appropriate dose of LiCl mice were administered three doses of LiCl and levels of IP1 were examined relative to vehicle in the striatum (STR), hippocampus (HPC) and prefrontal cortex (PFC) (A). 100 mg/kg of LiCl was selected for subsequent compound studies. Each compound resulted in dose-dependent statistically significant IP1 increases in all three brain regions examined (B-F). Compounds under investigation resulted in different IP1 fold changes hence the y-axis is different for every compound. One-way ANOVA with multiple comparisons in GraphPad was used to asses statistical significance outcome of which is indicated with asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.


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20 Table 2: IP1 fold change relative to 100 mg/kg LiCl per compound per dose in three distinct brain regions

Compound PT-4186

PT-1631

PT-8345

PF-06764427

PT-3214

Dose (mg/kg) 1 3.2 10 3.2 10 32 0.32 1 3.2 10 1 3.2 10 3.2 10 32

STR 2.3 ±0.5 13.2 ± 4.1 47.3 ± 6.4 **** 2.8 ± 0.6 14.4 ± 3.5 * 30.6 ± 4.4 **** 1.1 ± 0.2 1.2 ± 0.3 1.7 ± 0.3 8.8 ± 1.9 **** 1.2 ± 0.3 2.1 ± 0.4 8.5 ± 1.9 *** 1.2 ± 0.2 3.8 ± 1.1 5.7 ± 2.4

HPC 2.2 ± 0.7 6.1 ± 0.6 38.3 ± 13.5 ** 2.6 ± 0.6 5.1 ± 1.0 17.0 ± 1.8 **** 1.2 ± 0.3 1.3 ± 0.2 2.4 ± 0.3 6.5 ± 1.1 **** 0.7 ± 0.2 0.7 ± 0.2 4.1 ± 1.0 ** 1.5 ± 0.3 3.0 ± 0.3 **** 1.9 ± 0.2 *

PFC 3.2 ± 0.7 7.1 ± 1.9 20.3 ± 7.0 ** 2.2 ± 0.8 3.4 ± 0.8 7.2 ± 1.7 ** 1.0 ± 0.1 1.0 ± 0.1 1.9 ± 0.1 *** 2.5 ± 0.2 **** 1.2 ± 0.3 2.2 ± 0.4 4.3 ±0.9 ** 1.0 ± 0.3 3.3 ± 1.3 1.3 ± 0.7

Table 2 legend: Data shown are the mean ± SEM IP1 fold changes measured relative to the vehicle treated mice (n of at least 4 per brain region). Dose-dependent IP1 accumulation was observed for each compound within the brain regions examined: HPC, hippocampus; STR, striatum and PFC, prefrontal cortex. One-way ANOVA with multiple comparisons in GraphPad was used to asses statistical significance outcome of which is indicated with asterisks: * p

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