Striatal, Hippocampal, and Cortical Networks Are Differentially

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Striatal, hippocampal and cortical networks are differentially responsive to the M4- and M1-muscarinic acetylcholine receptor mediated effects of xanomeline Catherine A Thorn, Joshua Moon, Clinton A Bourbonais, John Harms, Jeremy R Edgerton, Eda Stark, Stefanus J. Steyn, Christopher R. Butler, John T. Lazzaro, Rebecca E O'Connor, and Michael Popiolek ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00625 • Publication Date (Web): 27 Nov 2018 Downloaded from http://pubs.acs.org on November 28, 2018

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ACS Chemical Neuroscience

Manuscript Title: Striatal, hippocampal and cortical networks are differentially responsive to the M4and M1-muscarinic acetylcholine receptor mediated effects of xanomeline Authors: Catherine A. Thorn1, Joshua Moon1, Clinton A. Bourbonais1, John Harms1, Jeremy R. Edgerton1, Eda Stark1, Stefanus J. Steyn2, Christopher R. Butter3, John T. Lazzaro4, Rebecca E. O’Connor4, Michael Popiolek1,5* 1Internal

Medicine Research Unit, 2Pharmacokinetics, Dynamics and Metabolism, 3Medicine Design, Pfizer Worldwide Research and Development, Cambridge, Massachusetts 02139. 4Primary Pharmacology Group, Pfizer Worldwide Research and Development, Groton, Connecticut 06340, United States 5Current address: Sage Therapeutics, 215 First St Suite 220, Cambridge, MA 02142 *corresponding author: [email protected] Funding source statement: At the time that this work was performed all authors were employees of

Pfizer Inc. Authors do not declare any conflict of interest. PT-3763, PT-8658, MK-7622, PF-96827443, Merck compound 1, xanomeline and oxotremorine were synthesized by Pfizer. Abbreviations: ACSF, artificial cerebrospinal fluid; AD, Alzheimer’s disease; CA1, Cornu Ammonis 1; cAMP, cyclic adenosine monophosphate; CREB, 3'-5'-cyclic adenosine monophosphate-response element binding protein; FLIPR, fluorescent imaging plate reader; fSPs, synaptically evoked extracellular field potentials; GPCR, g-protein coupled receptor; h, human; IP1, inositol phosphate-1; IP3, inositol 1,4,5-triphosphate; mAChR, muscarinic acetylcholine receptor; nPO, nucleus pontis oralis; PAM, positive allosteric modulator; pCREB, phosphorylated CREB; r, rat; SC, Schaffer collateral; SZ, schizophrenia Author contributions: CAT, MP wrote the article; CAT, JM, CB, JH, JRE, ES, SJS, JTL, REO, MP ran studies supporting this article; CB designed compounds used in this article; MP designed the research; CAT, JM, CB, JH, JRE, ES, JTL, REO, MP analyzed the data

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Abstract Preclinical and clinical data suggest that muscarinic acetylcholine receptor activation may be therapeutically beneficial for the treatment of schizophrenia and Alzheimer’s diseases. This is best exemplified by clinical observations with xanomeline, whose efficacy is thought to be mediated through co-activation of the M1and the M4 muscarinic acetylcholine receptors (mAChRs). Here we examined the impact of treatment with xanomeline and compared it to selective M1 and M4 mAChR activators on in vivo intracellular signaling cascades in mice, including 3'-5'-cyclic adenosine monophosphate-response element binding protein (CREB) phosphorylation and inositol phosphate-1 (IP1) accumulation, in striatum, hippocampus and prefrontal cortex. We additionally assessed the effects of xanomeline on hippocampal electrophysiological signatures in rats, using ex vivo recordings from CA1 as well as in vivo hippocampal theta. As expected, xanomeline’s effects across these readouts were consistent with activation of both M1 and M4 mAChRs, however, differences were observed across different brain regions suggesting non-uniform activation of these receptor subtypes in the central nervous system. Interestingly, despite near equal in vitro potency at the M1 and the M4 mAChRs, during in vivo assays xanomeline produced M4-like effects at significantly lower brain exposures than those at which M1-like effects were observed. Our results raise the possibility that clinical efficacy observed with xanomeline was, in part, driven through its non-uniform activation of mAChR subtypes in the central nervous system and, at lower doses, through preferential agonism of the M4 mAChR.

Key words: Xanomeline, muscarinic acetylcholine receptor, in vivo potency, striatum, hippocampus, biochemistry, electrophysiology


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Introduction Disrupted cholinergic signaling in frontal circuits is thought to contribute to the development of cognitive impairments and psychotic symptoms in diseases as diverse as Alzheimer’s disease (AD) and schizophrenia (SZ). Deficits in memory and attention, associated with cholinergic degeneration in AD, are present in almost all SZ patients1, are a major predictor of poor social and vocational outcome2, and are resistant to treatment with traditional antipsychotic medications 3. Similarly, psychosis, a defining symptom of SZ, is present in as many as 50% of AD patients 4, and is associated with greater rates of functional decline in these patients5 as well as increased burden on caregivers6. Thus, pharmacological interventions that improve cognitive and psychotic symptoms hold great promise for improving quality of life for patients and their families. AD and SZ have been proposed to share dysregulated cholinergic signaling, which has been implicated in psychosis and cognition1,7. Preclinical and clinical data suggest that muscarinic acetylcholine receptor activation may be therapeutically beneficial for the treatment of these symptoms in AD and SZ8. M1 and M4 mAChRs, in particular, have long been hypothesized to be viable therapeutic targets for SZ and AD on the basis of expression in key brain regions including striatum and hippocampus9,10, as well as mechanistic11–13 and behavioral studies14–16. Research efforts focused on the M1 and M4 mAChRs culminated in development of xanomeline, a reported M1, M4 and M5 preferring mAChR agonist17,18, which displayed robust antipsychotic and modest cognitive improvements in both SZ and AD patients19,20. Due to a significant side effect profile, however, xanomeline failed to advance to clinical use, but its efficacy profile raised the possibility that compounds with greater selectivity for M1 and/or M4 receptors might better address disease symptoms while producing fewer side effects. The mAChRs are seven membrane spanning g-protein coupled receptors (GPCRs) consisting of five family members (M1-M5) which share a highly conserved binding pocket for acetylcholine, posing a significant challenge for the development of selective compounds. Recently, the targeting of subtypespecific allosteric binding sites has made it possible to develop compounds that selectively modulate the activity of M1, M4, and other mAChRs15,16,21. In this study, to determine whether the in vivo and ex vivo



actions of xanomeline were more consistent with M1 or M4 mAChR activation, we compared the biochemical and electrophysiological effects of xanomeline in striatum, hippocampus and prefrontal cortex to the actions of several more recently developed, highly specific M1 and M4 activators. Biochemical readouts can be used to differentiate M1 and M4 mAChR mediated activation because the two receptors are differentially coupled. M1, M3 and M5 mAChRs are Gq-coupled excitatory GPCRs which are linked to phospholipase C activation, resulting in inositol phosphate 3 production and subsequent Ca+2 release from intracellular compartments22. M2 and M4 mAChRs are Gi/o-coupled inhibitory GPCRs whose activation leads to inhibition of adenyl cyclase, reduction in cytoplasmic cyclic adenosine monophosphate (cAMP), inhibition of protein kinase A and decrease in CREB phosphorylation (pCREB)22. We found that xanomeline, which demonstrated equal potency at M1 and M4 receptors in vitro, produced effects consistent with activation of both M1 and M4 mAChRs in vivo and ex vivo. However, we noticed brain region selectivity exemplified by dual M1 and M4 mAChR activation in hippocampal and prefrontal networks, and preferential M4 mAChR activation in the striatum. Furthermore, in vivo, we observed that xanomeline produced functional effects in M4-related readouts at brain exposures approximately 10-fold lower than the exposures required to produce effects in M1-related readouts, raising the possibility that in vivo xanomeline may be more potent at the M4 than the M1 mAChR. Taken together, our results suggest that xanomeline exhibits a differential profile of mAChR activation across different brain regions that may underlie its unique clinical efficacy.

Results and Discussion Xanomeline is a potent activator of M1, M4 and M5 receptors Using in vitro GloSensor and fluorescent imaging plate reader (FLIPR) assays, we verified the functional activity of the compounds used in this study at the five muscarinic receptor subtypes (Table 1); whose chemical structures for the compounds used can be found in Figure 1. As other groups have reported, we found that xanomeline was a potent activator of human (h) M1, M4 and M5 receptors, as well


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as rat (r) M4 receptor17,18, exhibiting EC50 values that were more than 10-fold lower for these receptor subtypes compared to hM2 and hM3 mAChRs. We also assessed xanomeline’s selectivity beyond mAChRs. Potential for an interaction with other targets exists as observed by functional and binding assays at multiple GPCRs, ion channels, transporters, kinases and enzymes (Table 2). We observed functional interactions by xanomeline at a number of non-muscarinic targets including serotonergic, adrenergic and opioid receptors. In particular, xanomeline exhibited high in vitro potency at the delta opioid receptor (EC50 of 79 nM), suggesting significant potential for this compound to impact cognitive and emotional processing via non-muscarinic mechanisms23. The pan-muscarinic agonist oxotremorine displayed similar potency across various mAChR cell lines. In line with a previous study from Vanderbilt University24 and our own group25, we observed that the M4 PAM PT-3763 selectively activated hM4 and rM4 receptors with EC50 values that were more than 20-fold (hM4) and more than 150-fold (rM4) lower than for hM2 mAChR, with no appreciable activation at other muscarinic subtypes. We additionally found that a second M4 PAM, PT-8658, similar in structure to VU046715426,27, exhibited approximately 15-fold lower, and 750-fold lower, EC50 values for hM4 and rM4 receptors, respectively, over hM2 receptors. PT-8658 also did not exhibit significant activation of other mAChR subtypes. The previously published M1 PAMs, MK-7622 and PF-0682744315,16,28, were both found to selectively activate hM1 receptors at low nanomolar concentrations, without any significant activation of other muscarinic receptor subtypes. Likewise, Merck compound 1, an M1 PAM, selectively activated hM1 but not any of the other mAChRs.



Figure 1: Structures of the muscarinic compounds used in this article.


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Table 1. Functional activity of test compounds at mAChRs. Xanomeline agonizes M1, M4, and M5 receptors at low nM concentrations. M4 PAMs PT-3763 and PT-8658 are more than 50-fold selective for rM4 receptors over other subtypes. M1 PAMs MK-7622, PF-06827443 and Merck compound 1 are highly selective for hM1 mAChR over other subtypes. All PAM EC50s are reported for “PAM mode” in which an EC20 of acetylcholine was additionally included to orthosterically activate mAChRs (hM1: 5 nM; hM2: 3 nM; hM3: 0.8 nM; hM4: 15 nM; hM5: 2 nM; rM4: 0.8 nM). Xanomeline and Oxotremorine were tested in “agonist mode” without the addition of acetylcholine. All EC50 values are reported in nM. * indicates average values obtained from experiments with fewer than 3 replicates otherwise average of at least N = 3 presented. ND = not determined. hM1

hM2

hM3

hM4

rM4

hM5

Xanomeline

14

6,845

595

20

6*

56

Oxotremorine

12

40

ND

14

10

7

PT-3763 (M4 PAM)

>10,000

954

>10,000

46

12

>10,000

PT-8658 (M4 PAM)

>7,500*

563*

>7,500*

197

4

>7,500*

MK-7622 (M1 PAM)

63

1,161

>10,000

>10,000

>10,000

>10,000

PF-96827443 (M1 PAM)

47

>10,000

>10,000

>10,000

>10,000

>10,000

Merck compound 1 (M1

102

>10,000

>10,000

>10,000

ND

9,914

PAM)



Table 2: Functional and binding selectivity of xanomeline at non-muscarinic GPCRs, ion channels, transporters, enzymes and kinases tested at 10 M concentration. If an effect over 50% was observed at any one target, then a full dose response of xanomeline was applied to find potency at that target. Color coding: green indicates no significant effect, red indicates maximum effect, gradients of colors in between indicate magnitude of intermediate effects. Note that xanomeline-mediated agonism of ∂-opioid receptors was observed at low concentrations (79 nM), and that additional non-muscarinic effects were observed at monoaminergic receptors and transporters as well as at calcium channels.


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Xanomeline produces differential effects on mouse CREB phosphorylation in the striatum, hippocampus, and prefrontal cortex. To examine the functional effects of xanomeline on striatal, hippocampal and cortical circuits in vivo, we compared pCREB changes induced by systemic administration of xanomeline in mice to those induced by more selective M1 or M4 activators, or to the pan-muscarinic activator oxotremorine. Activation of the Gq-coupled M1 receptor is typically expected to increase pCREB levels through calciumdependent activation of PKC signaling pathways29. By contrast, activation of the Gi/o-coupled M4 receptors is expected to decrease pCREB levels by reducing cAMP production30. Surprisingly, we found that xanomeline produced differential pCREB responses in all three brain regions under investigation (Figure 2A). In the striatum, we found a significant dose-dependent decrease in pCREB levels with xanomeline administration, whereas in the hippocampus, we observed the opposite effect on pCREB. Xanomeline had no significant effect on pCREB in the prefrontal cortex at any of the doses tested (0.3 to 32 mg/kg). Both striatal and hippocampal effects on pCREB were blocked by co-administration of 12 mg/kg of the muscarinic antagonist tropicamide (Figure 2B), confirming that the region-specific effects of xanomeline on pCREB levels are likely mediated by activation of muscarinic receptors. When we examined the effects of more selective activation of M4 and M1 receptors, we found that the M4 PAMs PT-3763 (administered at 0.3 to 32 mg/kg) and PT-8658 (administered at 0.1 to 10 mg/kg) induced a significant decrease in striatal pCREB without affecting hippocampal or prefrontal cortical pCREB levels (Figure 2C). By contrast, the M1 PAM MK-7622 (administered at 1.0 to 32 mg/kg) significantly increased pCREB in all three regions (Figure 2D). When compared to more selective compounds, xanomeline modulation of pCREB appears consistent with activation of M4 receptors in the striatum, and simultaneous activation of M1 receptors in the hippocampus, despite exhibiting approximately equal potency at the two receptor subtypes in vitro. Though both M1 and M4 receptors are highly expressed in striatum and hippocampus memory systems9, major differences in the locations in which these subtypes are expressed within the two networks, as well



as differences in network connectivity, may contribute to the differential activation of muscarinic receptor subtypes in the two regions. To determine whether anatomical differences in striatal and hippocampal circuitry were likely to bias the pCREB readouts toward M4-like and M1-like effects, respectively, when multiple mAChRs were simultaneously activated, we additionally tested the effects of the non-selective muscarinic agonist oxotremorine (Table 1). We found that, unlike xanomeline, oxotremorine (administered at 0.03 to 3.2 mg/kg) significantly increased pCREB in striatum, hippocampus, and prefrontal cortex (Figure 2E). This result is similar to that observed for selective M1 activators and suggests that the profile we observed for xanomeline differs from that of other equipotent M1/M4 agonists. Unlike oxotremorine, systemic xanomeline administration resulted in differential activation of muscarinic receptors in the striatum and the hippocampus, producing decreases in pCREB in the striatum that are consistent with M4 mAChR activation, while at the same time producing increases in pCREB in the hippocampus that are consistent with activation of the M1 mAChR. We next examined the in vivo potency of xanomeline to mediate M4- versus M1-like functional effects by modeling the in vivo pCREB changes in striatum versus hippocampus in concert with unbound brain exposure of xanomeline (Table 3). We found that significant striatal pCREB attenuation occurred at xanomeline brain exposure IC50 of 14.7 nM, whereas, significant hippocampal pCREB increases occurred at EC50 of 158 nM brain exposure (Figure 2F). We note that the in vivo EC50 values obtained using this approach are similar to those obtained in vitro for the rM4 mAChR. However, EC50 values for M1 mAChR potency are approximately 10-fold higher in vivo than those obtained in vitro. Low in vitro potencies may be an artifact of overexpression systems where potency of an agonist can be influenced by receptor expression level31. Our data thus highlight the importance of collecting functional data in vivo, to verify in vitro observations32, and suggest that xanomeline may be a more potent activator of the M4 receptors than the M1 receptors.

Figure 2: In vivo brain pCREB changes associated with systemic administration of M1 and M4 mAChR activators in mice. A) Xanomeline (0.3-32 mg/kg; N = 11 for each dose) produced a dose-dependent


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attenuation of striatal pCREB, a dose-dependent enhancement of hippocampal pCREB, and no effect on pCREB in prefrontal cortex. B) Tropicamide (12 mg/kg; N = 12 for each group), a muscarinic antagonist, on its own did not have an effect on pCREB, but it reversed the xanomeline-mediated striatal and hippocampal pCREB effects. C) M4 PAMs, PT-3763 (0.3-32 mg/kg; N = 13 for each dose) and PT-8658 (0.1-10 mg/kg; N = 4 for each dose), dose-dependently attenuated striatal pCREB with no effect in the two other brain regions. D) The M1 PAM MK-7622 (1-32 mg/kg; N = 5 for each dose) and E) the nonselective mAChR agonist oxotremorine (0.03-3.2 mg/kg; N = 4 for each dose) dose-dependently enhanced pCREB in all three brain regions. F) Pharmacokinetic and pharmacodynamic modeling of xanomeline’s striatal and hippocampal pCREB efficacy revealed that the M4-like reduction in striatal pCREB occurred at significantly lower free brain exposures than were required to produce the M1-like increase in hippocampal pCREB. Veh = vehicle; Xan = xanomeline; Trop = tropicamide; * p < 0.05, ** p < 0.01, *** p < 0.001; error bars represent standard error of the mean (SEM).




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Table 3: Unbound brain exposure of xanomeline in mouse (N = 3 for each dose) and rat (N = 3 for each dose) after systemic administration. Dose (mg/kg)

0.32

1

3.2

10

32

Mouse free brain exposure of xanomeline (nM) @ 30 min

4.5

9.7 26.1 100.6 392.1

Rat free brain exposure of xanomeline (nM) @ 60 min

1.4

3.9

7.1

24.7

--

Xanomeline increases IP1 accumulation in striatum, hippocampus and prefrontal cortex of mice Activation of the Gq-coupled M1 receptor activates phospholipase C, which activates two intracellular second messenger signaling cascades through formation of inositol 1,4,5-triphosphate (IP3) and diaglycerol. IP3 degrades rapidly in vivo, but its degradation product, inositol monophosphate (IP1), can be stabilized with lithium chloride (LiCl) and measured using commercially available kits. We have previously demonstrated that several highly selective M1 PAMs, including MK-7622, Merck compound 1, PF-96827443, produce detectable and consistent increases in IP1 levels in the striatum, hippocampus and prefrontal cortex following systemic administration in mice 10,16. We therefore tested whether xanomeline increased IP1 levels in the brain following systemic administration in a manner consistent with activation of M1 receptors in the central nervous system. We found that in all three regions examined, xanomeline (administered at 0.3 to 32 mg/kg) produced a significant dose-dependent increase in IP1 accumulation (Figure 3A), consistent with previous reports for this compound

33–35.

In the striatum and hippocampus, we verified that the increases in IP1 were largely

due to the activation of muscarinic receptors, as they were blocked by 12 mg/kg of the muscarinic antagonist tropicamide (Figure 3B). As the selective M4 PAM PT-3763 (administered at 1.0 to 32 mg/kg) did not significantly increase IP1 in any of the regions examined (Figure 3C), our results are consistent with a xanomeline-mediated activation of M1 receptors in striatum, hippocampus, and prefrontal cortex that led to increases in IP1. IP1 changes, thought to be mediated through activation of the M1 receptors, were observed at xanomeline doses corresponding to brain exposures of at least 100 nM consistent with the



possibility that xanomeline may be less potent at M1 receptors in vivo than would be expected based on its in vitro potency.

Figure 3: In vivo brain IP1 changes associated with systemic administration of M1 and M4 mAChR activators in mice. A) Xanomeline (0.3-32 mg/kg; N = 9 for each dose) produced a dose-dependent IP1 increase in striatum, hippocampus and prefrontal cortex. B) Tropicamide (12 mg/kg; N = 6 for each group) was inactive on its own, but blocked xanomeline-mediated striatal and hippocampal IP1 increases. C) The M4 PAM PT-3763 (1-32 mg/kg; N = 9 for each dose) did not significantly impact IP1 levels in any of the three brain regions. Veh = vehicle; Xan = xanomeline; Trop = tropicamide; * p < 0.05; ** p < 0.01; *** p < 0.001; error bars represent SEM.

Xanomeline increases spontaneous firing rates in the CA1 region in rat hippocampal slice We examined the M1- and M4-mediated functional effects of xanomeline in the hippocampus in greater detail using electrophysiological recordings from adult rat hippocampal slices. Previous work has shown that activation of M1 receptors using selective agonists and PAMs including PF-96827443 results in an increase in excitability and firing rates of CA1 pyramidal cell neurons in the hippocampus10,16,25


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whereas activation of M4 receptors produces no significant change in firing rates25. We found that xanomeline increases spontaneous CA1 pyramidal cell firing rates in this assay in a concentrationdependent manner with a minimum concentration of 100 nM (Figure 4A). The M4 PAM PT-8658 failed to produce a significant effect on firing rates at concentrations up to 1 M (Figure 4B). Combined with the results of previous studies, our data are consistent with an M1-mediated increase in hippocampal cell excitability by xanomeline.

Figure 4: Spontaneous firing rates in rat hippocampal slice in response to muscarinic activation. A) Xanomeline (3-1000 nM; n = 5 slices from 2 rats) increased CA1 firing rates in a concentration-dependent manner, while B) the M4 PAM PF-8658 (1-1000 nM; n = 3 slices from 1 rat) did not elicit a significant change in firing rates at any concentration tested. *** p < 0.001; error bars represent SEM.

Xanomeline weakly suppresses Schaffer collateral synaptic transmission in rat hippocampal slice In our in vivo biochemical assays, xanomeline produced effects that were consistent with preferential M1 activation in hippocampal tissue, though M4-mediated effects were prominent in striatum. To look more closely at whether xanomeline significantly activated M4 receptors in the hippocampus, we again used electrophysiological recordings from adult rat hippocampal slices. For these experiments, we



recorded synaptically evoked extracellular field potentials (fSPs) following bipolar stimulation of the SC axons in the stratum radiatum of CA1. Muscarinic activation of hippocampal networks has been shown to strongly suppress synaptic transmission in the Schaffer collateral (SC) pathway, and we previously showed that activation of M4 mAChRs is likely to mediate this effect13,25. In this assay, we found that xanomeline partially suppressed SC-evoked fSPs to 76.4  2.9% of their initial amplitude at high concentrations (EC50 = 708.6  219.0 nM). This suppression was blocked by two reported M1-preferring muscarinic antagonists, pirenzepine (5 M) and VU0255035 (3 M) (Figure 5A). Both antagonists were applied here at high concentrations such that antagonism of non-M1 mAChRs is likely36,37. Nonetheless, the weak suppression of SC synapses by xanomeline, along with its high EC50 value and reversal by both pirenzepine and VU0255035, suggest that xanomeline fails to exhibit strong M4-mediated functional effects in this assay. We compared the xanomeline-mediated suppression of SC synapses to that of several moreselective M1 and M4 activators, replicating and extending our previous work. Consistent with our previously published studies, we found that the M4-selective PAM PT-8658 produced a strong suppression of SC-evoked fSPs (to 29.0  4.7%, EC50 = 13.2  2.75 nM; Figure 5B), confirming that activation of M4 receptors is a potent mechanism modulating synaptic transmission in the SC pathway. Surprisingly, we also found that the M1-selective PAM MK-7622 significantly reduced SC-evoked fSP amplitude (to 51.5  8.2%, EC50 = 593.5  3.3 nM; Figure 5C), as did a second previously published M1 PAM, PF-06827443 (to 33.9  15.3%, EC50 = 607.6  676.3 nM; Figure 5D). As M1 receptors are known to be expressed postsynaptically on CA1 pyramidal cells38, we tested whether retrograde cannabinoid signaling was involved in M1-mediated suppression of SC synaptic transmission. Bath application of AM251 (3 M) had no effect on the concentration-dependent suppression of SC-evoked fSP amplitudes by PF-06827443 (Figure 5D). Combined, our results provide evidence suggesting that M1 activation can reduce SC-evoked fSPs, though this effect is not observed with all M1 agonists and PAMs25, and the mechanisms underlying this effect remain unclear.


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Unlike other M4 agonists, xanomeline exhibits a weak effect on SC-evoked synaptic transmission. Xanomeline-mediated suppression of SC synapses was observed at concentrations at which, in other ex vivo and in vivo assays, M1 effects were typically seen in the hippocampus. Combined with our findings that M1 activation can yield suppression of fSPs in this assay, we cannot rule out the possibility that xanomeline may be acting in the hippocampus preferentially through M1 receptors.

Figure 5: Schaffer collateral-evoked field potential responses in hippocampal rat slice are suppressed by M4 and M1 activation. A) Xanomeline (0.3-10,000 nM; n = 9 slices from 2 rats) produced a concentrationdependent suppression of fSPs. This suppression was blocked by application of mAChR antagonists pirenzepine (5 M; n = 3 slices from 1 rat) and VU0255035 (3 M; n = 7 slices from 2 rats). B) The M4 PAM PT-8658 (0.3-1,000 nM; n = 4 slices from 1 rat) strongly, and concentration-dependently attenuated fSPs. C-D) The selective M1 PAMs MK-7622 (1-10,000 nM; n = 7 slices from 2 rats) (C) and PF-96827443 (1-10,000 nM; n = 9 slices from 3 rats) (D). Addition of VU0255035 at a concentration at which preferential M1 antagonism is observed (1 M) significantly blocked activity of both MK-7622 (n = 6 slices from 2 rats) and PF-96827443 (n = 4 slices from 1 rat). AM251 (3 M; n = 3 slices from 1 rat), a cannabinoid receptor 1 antagonist, failed to block PF-96827443 mediated fSP suppression (n = 3 slices from 1 rat). *** p < 0.001; error bars represent SEM.



Xanomeline increases in vivo nPO-evoked hippocampal theta in rats Atropine-sensitive type II theta (3-12 Hz) oscillations can be generated in the hippocampus by stimulation of the nucleus pontis oralis (nPO) in rodents under urethane anesthesia 39,40. Evoked theta-band synchronization depends on muscarinic activation throughout the ascending pathway, including in the posterior hypothalamus, medial septum, and the hippocampus41. The amplitude of nPO-evoked theta oscillations has been shown to be enhanced by infusion of muscarinic agonists along this synchronization pathway, and reduced or eliminated by infusion of muscarinic antagonists42,43. We asked whether systemic xanomeline administration would similarly enhance nPO-evoked type II theta oscillations, and whether this enhancement was consistent with preferential activation of either M1 or M4 mAChRs. Consistent with its preferential activation of muscarinic receptors, we found that in rats, systemic xanomeline administration (1 mg/kg and 10 mg/kg) dose-dependently increased the amplitude of nPO-evoked theta oscillations, and this effect was blocked by prior administration of 10 mg/kg tropicamide (Figure 6A). We further found


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that selective M4 activation with PT-3763 (32 mg/kg) also enhanced nPO-evoked theta, whereas selective M1 activation with Merck compound 1 (10 mg/kg) had no significant effect in this assay (Figure 6B). Combined, our results suggest that systemic M4 activation has a greater enhancement effect than M1 activation on the generation of type II theta oscillations in vivo. Moreover, xanomeline is likely to be acting at M4 receptors throughout the theta synchronizing pathway, including in the hippocampus, to enhance nPO-evoked theta amplitude. The effect of xanomeline in this assay was observed at a free brain exposure of 3.9 nM (Table 3), supporting the idea that the xanomeline may activate M4 receptors in vivo at lower concentrations than are required to activate M1 receptors.

Figure 6: The amplitude of nPO-evoked theta in urethane anesthetized rats is modulated by M4 activators and xanomeline, but not M1 activators. A) Systemic administration of xanomeline at 1 mg/kg (n = 5) and 10 mg/kg (n = 6) increased nPO-evoked hippocampal theta power compared to vehicle administration (n = 5), and this effect was blocked by co-administration of the muscarinic antagonist tropicamide (10 mg/kg; n = 3). B) The M4 PAM PT-3763 (32 mg/kg; n = 5) increased evoked theta power, whereas the M1 PAM Merck compound 1 (10 mg/kg; n = 3) had no significant effect on theta power. ** p < 0.01, *** p < 0.001; error bars represent SEM. BL = baseline



Xanomeline exhibits a unique muscarinic profile with implications for drug development and clinical efficacy These studies extend our knowledge of the mechanisms of action of xanomeline, a compound that showed some promise toward improving cognitive and psychotic symptoms in AD and SZ patients19,20. Our in vitro characterizations confirm that xanomeline is a potent M1 and M4 mAChR agonist with the potential to bind and functionally impact non-muscarinic receptor targets, particularly ∂-opioid receptors 17,18.

In a variety of in vivo and ex vivo assays, however, we found that xanomeline’s effects were replicated

by selective M1 or M4 PAMs, and reversed or blocked with muscarinic antagonists, reinforcing previous studies that have shown significant M1- and M4-mediated effects of xanomeline34,44–46. Novel to our study, we observed both M1- and M4-like effects of xanomeline in all three brain regions examined in our in vivo or ex vivo assays, but these effects varied significantly across prefrontal cortex, striatum, and hippocampal networks. Moreover, in our in vivo assays, regardless of brain region examined, we observed M4-mediated effects at significantly lower free brain concentrations of xanomeline than we observed M1-mediated effects. Combined, these studies demonstrate that xanomeline exhibits unique and region-specific activation of M4 and M1 receptors that likely contributes to its distinctive clinical profile. We opted for measuring CREB phosphorylation and IP1 accumulation as two biochemical endpoints known to be impacted by activation of the mAChRs10,30 and pliable to in vivo quantification. Consistent with M4 activation, xanomeline decreased striatal pCREB and did not impact prefrontal cortex pCREB levels. In the same mice, xanomeline simultaneously increased hippocampal pCREB levels, an effect most consistent with activation of M1 mAChRs. IP1 accumulation, a more direct test of Gq-coupled receptor activation10, increased across all three brain regions in a manner consistent with functional M1 activation of prefrontal, striatal and hippocampal circuits. We further found that nPO-stimulated theta oscillations in the hippocampus, which are known to be modulated by cholinergic activity47, were enhanced by xanomeline and M4-selective PAMs, but not M1-selective PAMs. As we also observed a potent in vitro xanomeline-mediated agonism of the ∂-opioid receptor, a Gi-coupled GPCR widely expressed in striatal


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and cortical tissue, all in vivo effects were examined with—and found to be blocked by—co-administration of tropicamide, suggesting xanomeline was indeed acting through M1 and M4 mAChRs. Importantly, across all our in vivo assays, we observed M4-mediated effects—including the decrease in striatal pCREB and the increase in hippocampal stimulated theta—at free brain exposures of xanomeline that aligned well with EC50 values obtained from our in vitro M4 mAChR functional assay. By contrast, M1-mediated effects—including hippocampal pCREB increases and IP1 accumulation across all regions—occurred at exposures that were consistently 10-fold higher than those predicted by our in vitro M1 functional assay. As previously noted, low in vitro potencies may be an artifact of the overexpression systems in which functional effect is tested31, highlighting the importance of confirming the functional efficacy of any compound in vivo. Our current findings suggest that xanomeline may be a more potent activator of M4 receptors in vivo, while higher doses may be necessary to obtain M1-mediated effects. We were surprised not to observe more consistent M4-mediated effects of xanomeline in hippocampal tissue. In this region, we did not observe the decrease in pCREB typically associated with in vivo activation of Gi-coupled M4 receptors, and ex vivo, we observed only a weak suppression of Schaffer collateral synapses, an effect previously attributed to presynaptic activation of M4 mAChRs13,25. Consistent with more prominent M1 mAChR activity by xanomeline in hippocampal circuits, we did observe an increase in IP1 accumulation in vivo, as well as an increase in spontaneous CA1 firing rates ex vivo. Interestingly, all in vivo and ex vivo functional effects in hippocampal tissue—including pCREB increases, IP1 accumulation, spike rate increases and Schaffer collateral suppression—were observed at xanomeline exposures and concentrations above 100 nM, well above concentrations at which clear M4-mediated effects were observed in vivo, and consistent with concentrations required to obtain M1-mediated effects. In several of our assays, selective M1 activation yielded somewhat unexpected results. In vivo, we saw that selective M1 activators increased pCREB levels in the hippocampus, as well as in the prefrontal cortex and striatum, though phosphorylation of CREB is canonically attributed to Gs-coupled, rather than Gq-coupled, signaling pathways. Similarly, ex vivo, we found that though M4 activation is a potent mechanism by which Schaffer collateral synaptic transmission can be suppressed13, M1-selective PAMs



could also reduce Schaffer collateral-evoked field potential amplitudes. Activation of Gq-coupled receptors has recently been shown to activate protein kinase A signaling in hippocampal circuits, and to be critical for M1-mediated LTD to occur48, providing one potential explanation for both the M1-mediated increases in pCREB and the M1-mediated suppression of Schaffer collateral signaling that we observed here. Future studies are needed, however, to further clarify the potential for non-canonical M1 mAChR signaling and functional effects. Xanomeline has long been known to convey multipharmacology, yet its clinical efficacy has been attributed to its activation of the M1 and the M4 mAChRs17,49. This conclusion was in part based on preclinical antipsychotic and procognitive efficacy of both M1 and M4 selective activators14,15,50. Our results support a muscarinic mechanism of action for xanomeline, as every ex vivo and in vivo effect was blocked by co-administration of a muscarinic antagonist. Our results also reinforce the likelihood of these effects being specifically mediated through the M1 and M4 receptor subtypes as the major biochemical and electrophysiological effects that we observed were replicated by highly selective M1 or M4 activators. Moreover, at lower concentrations, we found that xanomeline produced functional effects in vivo that were more consistent with M4 activation, whereas, in general, higher concentrations were required to elicit M1like effects. Importantly, our findings reveal significant variation across brain regions in the preferential activation of M1 and M4 receptors by xanomeline. Taken together, our results suggest that the clinical and pre-clinical efficacy of xanomeline may be mediated by its unique and region-specific profile of mAChR activation in vivo. 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.

In vitro muscarinic functional activity. We verified the selectivity of the muscarinic compounds used in this study using methods reported previously 10,15,16,25. Briefly, compounds were tested in each of six different


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cell lines, each stably expressing a different human muscarinic receptor subtype (hM1-hM5) or the rat M4 (rM4) receptor subtypes. Muscarinic receptor expression levels were determined using [H3]-Nmethylscopolamine binding51 resulting in the following saturable binding capacity (Bmax) values: hM1, 384 fmol/mg of protein; hM2, 138.90 fmol/mg of protein; hM3, 976.88 fmol/mg of protein; hM4, 167.59 fmol/mg of protein; rM4 and hM5, not tested. M2 and M4 mAChR activity was evaluated using GloSensor™ cAMP detection technology (Promega, Madison, WI) in HEK293 stable cell lines expressing the hM2, hM4, or rM4 generated from a host cell line stably expressing the GloSensor™. To elevate otherwise low cellular cAMP levels, cells were treated with an EC80 concentration of isoproterenol (50 nM). FLIPR™ (Molecular Devices, Sunnyvale, CA) was used to quantify Ca2+ release induced by the muscarinic compounds in CHO K1 cell lines stably expressing hM1, hM3, or hM5 receptors. In all platebased experiments, the effect of each compound was expressed as a percentage of the response generated by a saturating concentration of acetylcholine (10 µM) included on each plate. PAMs were tested in the presence of an EC20 concentration of acetylcholine, determined separately for each cell line (hM1: 5 nM; hM2: 3 nM; hM3: 0.8 nM; hM4: 15 nM; hM5: 2 nM; rM4: 0.8 nM). Additional GloSensor, FLIPR, or MicroBeta radiometric (PerkinElmer, Waltham MA) assays were used to quantify any off-target activity at several nonmuscarinic receptors, amine transporters, ion channels, and phosphodiesterases.

Mouse in-vivo pCREB assay. 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 hours prior to initiation of the experiment. Mice were subcutaneously administered with indicated dose of test compound at 1/100th volume to weight ratio. Test compounds were dissolved in 5% DMSO, 15% solutol and 80% sterile water. Tropicamide was the exception as it was dissolved in saline and administered intraperitoneally. Thirty minutes later, animals were euthanized by brain-focused microwave application (customized instrument from Litton Model 70/50). Neural tissues were immediately isolated and frozen on dry ice, and samples were then stored at -80oC until subsequent analysis.



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. pCREB was measured using a pCREB ELISA kit (KHO0241, Invitrogen). Brain tissue homogenates (3 µL) in 97 L of standard diluent buffer were spotted onto a 96-well plate provided with the kit and incubated overnight at 4oC. The next day the wells were washed with the wash buffer and incubated for an hour, at room temperature, in 100 L of CREB (pS133) detection antibody, followed by a thirty-minute incubation with anti-rabbit IgG HRP (1:100 dilution) and development with 100 µL of stabilized chromagen. The reaction was stopped with 100 L of stop solution allowing for absorbance reading at 450 nm. Data was graphed using GraphPad software (GraphPad Software Inc, California, USA), and analyzed using one-way ANOVA with Bonferroni correction for multiple comparisons. Significant differences from vehicle treatment are reported for corrected p < 0.05.

Mouse in-vivo IP1 Assay. Mice (Charles River Laboratories) were acclimated to the facility and procedure room as described above for the in-vivo pCREB assay. Mice received an initial subcutaneous injection of 100 mg/kg LiCl (Sigma Aldrich #203637-10G dissolved in water) administered at 1/100th volume to weight ratio. One hour later, test compounds or vehicle were administered subcutaneously. Test compounds were dissolved in 5% DMSO, 15% solutol and 80% sterile water. Two hours later, animals were euthanized by brain-focused microwave application (customized instrument from Litton Model 70/50). Tissues were isolated and stored as described above. Frozen tissues were weighed and homogenized as for the pCREB assay. IP1 was measured using a CisBio IP-One TB kit (62IPAPEC). Brain tissue homogenates (3 µL) were spotted in duplicate into a 384well solid white low volume plate (Greiner Bio-One #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 hour, before reading the plates on an Envision 2101 Plate Reader (PerkinElmer, Waltham, Massachusetts) at 620 and 665 nm. Results were calculated as a ratio of 665nm/620nm and IP1


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concentrations were interpolated 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 Bonferroni correction for multiple comparisons. Significant differences from vehicle treatment are reported for corrected p < 0.05.

Brain tissue exposure quantification. For brain tissue exposure quantification all standards, quality controls, and tissue samples were prepared by protein precipitation. A working solution of protonated compound was prepared separately in 1:1 DMSO:acetonitrile. Dilution of the working solution in blank brain homogenate yielded serial solutions of standards. Brain samples were further diluted and homogenized using a 1:4 dilution in 60:40 IPA:H20 and shaken for 2 minutes in a bead beater using a combination of steel and zirconia beads. Samples requiring additional dilution were titrated into blank homogenate. Samples were extracted using a mixed matrix approach where equal aliquots of samples and standards were subjected to protein precipitation with 4 volumes of acetonitrile with internal standards. After centrifugation at 1811 x g for 10 minutes, supernatants of the mixture were mixed with solvents for LC-MS/MS analysis. Quantitative analyses were carried out by reversed phase chromatography coupled with an AB/Sciex mass spectrometer using MRM as the detection mode. Unbound brain exposures , Cb,u, were determined by multiplying brain exposure with the relevant test compound’s unbound fraction in brain. The unbound fractions were determined using equilibrium dialysis 52.

Slice electrophysiology. Adult (8–12 weeks) male Sprague Dawley rats were deeply anesthetized with isoflurane and either rapidly decapitated, or perfused transcardially with ice-cold high-sucrose artificial cerebrospinal (ACSF) cutting solution containing 206 mM sucrose, 26 mM NaHCO3, 3 mM KCl, 1.25 mM NaH2PO4, 7 mM MgCl2, 0.5 mM CaCl2, 10 mM glucose, 1 mM sodium pyruvate, and 0.89 mM sodium lascorbate, bubbled with 95% O2/5% CO2. Brains were removed into ice-cold cutting ACSF and coronal hippocampal slices were made (300 μM) using a vibrating microtome. Slices were incubated at 35°C in recording ACSF (124 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM glucose, 1.3



mM MgCl2, 2 mM CaCl2, 1 mM sodium pyruvate, and 0.89 mM sodium l-ascorbate) bubbled with 95% O2/5% CO2 for 1–4 hr prior to recording. Slices were placed onto MED-P515A 64-channel multielectrode arrays (Alpha MED Scientific Inc., Osaka JP) with the CA1 pyramidal cell layer positioned directly over the array contacts. Recordings were made at 30°C in recirculating recording ACSF bubbled with 95% O2/5% CO2. Spiking activity or evoked potentials were captured using MED64 hardware and Mobius software (MED64 system, Alpha MED Scientific Inc., Osaka JP). Synaptic field potentials were evoked by stimulation of the SC pathway at a contact located in stratum radiatum. Stimulus intensity was determined prior to the start of the experiment as the intensity that produced evoked field response amplitude 60–80% of the maximum response (10–130 μA, 0.2 ms). For the in vitro electrophysiology studies, concentrated stock solutions of test compounds (10 mM) were made by dissolving compounds in either H2O (pirenzepine) or DMSO (all other compounds). During recording experiments, small volumes of stock solutions were diluted in the circulating recording ASCF to the desired concentrations.

Quantification of electrophysiology recordings. Evoked synaptic field potentials (fSPs) or spontaneous spike rates were recorded from 16 channels spanning stratum pyramidale and striatum radiatum of CA1. Data were captured using MED64 hardware and Mobius software (MED64 system, Alpha MED Scientific Inc.) and analyzed using custom Matlab scripts (MathWorks Inc., Natick MA). During fSP recording experiments, escalating concentrations of compounds were bath applied to each slice and each concentration was applied for at least 20 minutes. Dose-response curves for fSP responses were constructed by first computing the amplitude of each evoked fSP as the minimum voltage reached within 25 milliseconds following stimulus onset. The mean fSP amplitude for each channel was then computed for the last 15 trials in the time window corresponding to administration of each concentration of compound and normalized to the mean fSP amplitude during an initial baseline period in which no drugs were present (xanomeline) or in which an EC20 (20 nM) of the muscarinic agonist


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oxotremorine was present to provide orthosteric activation that could be potentiated by the allosteric modulators (all M1- and M4-selective PAMs). Mean normalized fSP amplitude for each concentration was computed for each slice by averaging across all channels on which baseline fSPs with amplitudes > 200 μV were recorded. A dose-response curve was fit using the “drc” package53 in R54. Mean data from each slice were used to find the best fit to a three-parameter logarithmic function of the form DR(x) = C + (1 − C)/(1 + exp(B × (ln(x) − ln(E))), for drug concentrations x. Linear regression was used to fit dose responses for conditions in which no logarithmic fit converged. Dose-responses were compared using 2-way ANOVA (Dose x Treatment), and significant differences between Treatments are reported for p < 0.05. Extracellular action potentials were detected offline using custom-written Matlab scripts. CA1 spiking activity was measured as the multi-unit firing rate for each electrode contacting the stratum pyramidale. Rates were computed for each channel within a slice, then averaged across channels to generate a single median activity rate for the slice. The median activity during the last 10 min of each step was taken as the activity level for that concentration. For each slice, 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 (compound alone for xanomeline, in 100 nM carbachol for the M4 PAM PF-8658). Mean firing rates and SEM across slices are reported. Repeated measures ANOVA was used to determine whether firing rates diverged from baseline for any concentration and a statistically significant increase in firing rate is noted if p < 0.05.

Stimulated-theta recordings. Electrophysiological recordings were performed in anesthetized rats using previously described procedures47. Briefly, rats were anesthetized with urethane (1.5 mg/kg) administered intraperitoneally; body temperature was maintained at 35C with a warming pad throughout the procedure. Under stereotaxic guidance, craniotomies were made over the recordings sites in hippocampal CA1 (AP: 3.5 mm, ML: -2.0 mm, DV: -3.2 mm) and prefrontal cortex (PFC) (AP: 3.0 mm, ML: 0.6 mm, DV: -5.0 mm), the stimulation site in nucleus pontis oralis (nPO) (AP: -7.6 mm, ML: 1.8 mm, DV: -6.0 mm), and an



additional frontal reference site. Bipolar concentric recording microelectrodes were inserted into CA1 and PFC, and a bipolar concentric stimulating microelectrode into the nPO. A bone screw inserted at the reference location served as ground. Local field potentials were filtered (0.3-300 Hz) and recorded continuously (1000 Hz sampling rate) during sessions lasting approximately one hour. During recording, stimulus trains (0-1.4 A, 0.3 ms pulse duration, 250 Hz train frequency) were applied to the nPO every 100 seconds for 6 seconds to evoke theta oscillations (5-8 Hz peak frequency) in CA1. Test compounds were dissolved in 5% DMSO, 15% solutol and 80% sterile water and administered subcutaneously after ~30 minutes of recording stable baseline responses. Normalized theta power was computed by averaging the total power in the 4-9 Hz frequency band for each 30-minute epoch following drug administration and normalizing to the 4-9 Hz power recorded during the baseline period.

Test compounds. Xanomeline, the M4 PAM PT-3763 (3-amino-4,6-dimethyl-N-(1-(pyridin-3-yl)azetidin3-yl)thieno[2,3-b]pyridine-2-carboxamide; 24,25,55, and the M1 PAMs MK-762215 and PF7443 16 were prepared based on published methods by Pfizer World Wide Medicinal Chemistry. Other chemicals were obtained from Tocris (oxotremorine, pirenzepine, AM-251), Fisher (DMSO) and Sigma (urethane).


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Table of contents graphic (TOC):



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