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TBPB or Not to Be—The Latest on Muscarinic Receptor Agonists

Adapted from a story that originally appeared on the Alzheimer Research Forum.

4 November 2008. Key roles in modulating cognition and behavior made the M1 muscarinic acetylcholine receptor (mAChR) a prime target for drug developers dating back nearly two decades. However, untold millions of big pharma dollars have yet to produce an M1 drug that works well and avoids adverse events from non-specific activation of other mAChR subtypes. To trim the side effect profile, renewed efforts have taken a different tack. Instead of focusing on the acetylcholine binding site, which is highly conserved across all mACh receptors, researchers have found ways to activate individual mAChR subtypes by targeting unique allosteric sites away from the substrate-binding action. Reporting today in the Journal of Neuroscience, researchers led by P. Jeffrey Conn of Vanderbilt University in Nashville, Tennessee, have applied this strategy to develop a highly selective M1 activator. In addition to its lack of agonist activity against any of the other mAChR subtypes, the compound had anti-amyloidogenic effects in a rat neuronal cell line and alleviated symptoms in rat models of psychosis. These data raise the possibility that selective M1 activators could someday reach the clinic as a treatment for schizophrenia and AD symptoms.

Among those compounds that met their demise in failed mAChR drug development programs, the one that made it furthest was an Eli Lilly drug called xanomeline. In a Phase 3 trial of 343 people with mild to moderate AD, this M1/M4-preferring mAChR agonist improved performance on two widely used cognitive scales (ADAS-Cog and CIBIC-plus) and reduced a number of behavioral disturbances, including hallucinations, delusions, and vocal outbursts (Bodick et al., 1997). On the downside, the high doses that brought these clinical benefits came with side effects (predominantly gastrointestinal) that caused more than half the participants to stop using the drug. Overall, though, Conn and others saw in the Phase 3 data a ray of hope that mAChR agonists might eventually be useful for relieving both cognitive and behavioral symptoms of AD and other disorders. “That paper is really what grabbed our attention and made us focus very heavily on this even though most other companies, including Lilly, had decided to drop it,” Conn told ARF.

The allosteric strategy worked well when Conn and colleagues were fishing out modulators of group II metabotropic glutamate receptors (Hemstapat et al., 2007), so he figured he should give it a try for muscarinic acetylcholine receptors, too. Since the dead Lilly drug (xanomeline) had agonist activity at both M1 and M4 receptors, Conn focused on these two subtypes in his efforts to develop more selective compounds. For M4, allosteric targeting has succeeded in his hands (see Brady et al., 2008) and for Eli Lilly, which recently reported a similar M4 potentiator (Chan et al., 2008). The current paper extends this line of success to M1 receptors.

The newly characterized M1 agonist is TBPB (or, more long-windedly, 1-(1’-2-methylbenzyl)-1,4’-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one). Discovered five years ago while Conn was at Merck & Company Inc. in West Point, Pennsylvania, the compound was first presented at the 2006 annual meeting of the American College of Neuropsychopharmacology (Kinney, 2006) and characterized further after Conn moved to Vanderbilt.

First author Carrie Jones and colleagues established TBPB’s pharmacological properties by measuring agonist-induced intracellular calcium increases in cell lines expressing wild-type rat M1 or various rat or human M1 mutants. The researchers reasoned that TBPB functions as an allosteric agonist based on several lines of evidence: 1) it was able to activate an M1 mutant that is insensitive to acetylcholine or orthosteric agonists, and 2) its effects were blocked in a non-competitive manner by a competitive orthosteric antagonist (atropine). To address selectivity, they tested TBPB in cell lines expressing each of the five mAChR subtypes. For comparison, they threw in AF267B, reportedly an M1 agonist that recently revitalized the field with its ability to reduce Aβ and tau pathologies in the triple transgenic (3xTg) AD mouse model. In Conn’s new study, AF267B was not selective for M1—it also activated M3 and M5 receptors. On the other hand, TBPB was highly selective for M1.

However, despite its excellent selectivity for the M1 mAChR, the possibility that TBPB may also bind allosteric sites shared by other G protein-coupled receptors (GPCR) cannot be dismissed, wrote Abraham Fisher of the Israel Institute for Biological Research in Ness-Ziona, in an e-mail to ARF. AF267B and related M1 agonists were originally identified in Fisher’s lab. (See full comment below and recent reviews Fisher, 2008 and Fisher, 2008).

In a rat neuronal cell line (PC12) overexpressing human amyloid precursor protein (APP) and M1, TBPB shifted APP processing toward the non-amyloidogenic pathway. With help from coauthor Allan Levey of Emory University in Atlanta, Georgia, this showed up as a 58 percent increase in production of the α-secretase cleavage product and a 61 percent drop in Aβ40 in TBPB-treated versus vehicle-treated cells.

TBPB also appeared to have antipsychotic-like effects—demonstrated in rats at doses that did not elicit peripheral adverse effects commonly seen with other mAChR agonists. Moving into the cognitive realm, Conn said his group is now testing TBPB and newer selective M1 agonists in AD mouse models. He stressed that though AD is in large part a disease of cognition, the behavioral benefits of TBPB and related compounds should not be downplayed. In caring for his father, who recently died of AD, Conn said he and other family members felt that “the psychosis was in many ways a bigger challenge than the cognition.”

Levey calls TBPB “a novel compound that represents a new generation of highly specific drugs.” In an e-mail to ARF, he writes, “The study is important for the AD field because this drug will allow the role of the M1 muscarinic receptor in AD to be more clearly defined, including its potential for AD therapeutics.” (See full comment below.)

Acadia Pharmaceuticals Inc. has recently published animal data on its own selective M1 drug (Vanover et al., 2008), as has Merck, which presented preclinical work on another such compound (benyzl quinolone carboxylic acid, or BQCA) at this year’s Keystone conference on AD in Keystone, Colorado. Among TBPB and these two compounds, all target allosteric sites, but only TBPB and the Acadia drug are agonists—that is, they activate M1 in the absence of acetylcholine. BQCA is an allosteric potentiator, which means it does not directly activate M1 but potentiates the effect of acetylcholine. Because cholinergic neurons die in early AD, one concern with potentiators is that they might not be as effective as agonists in the context of reduced endogenous acetylcholine activity, Conn said.

Fisher’s compound, AF267B, is being tested by TorreyPines Therapeutics in a Phase 2 study (under the name NGX267) of dry mouth associated with Sjorgren’s syndrome to get data on the drug’s safety, tolerability, and M1 activity. The small San Diego biotech had been developing the compound as a possible AD treatment. However, the company has been running low on cash and last month reorganized its efforts to focus on developing a migraine drug. Acting CEO Evelyn Graham wrote in an e-mail to ARF that TorreyPines is “seeking a development partner for NGX267” and has “no current plans to initiate AD studies.” The end of TorreyPines’s AD genetics collaboration with Eisai Co. preceded its shutdown of discovery research (see press release). Graham also noted that “the shutdown of our discovery operations was a separate strategic decision from how we are addressing our development plan for NGX267.”—Esther Landhuis.

Jones CK, Brady AE, Davis AA, Xiang Z, Bubser M, Tantawy MN, Kane AS, Bridges TM, Kennedy JP, Bradley SR, Peterson TE, Ansari MS, Baldwin RM, Kessler RM, Deutch AY, Lah JJ, Levey AI, Lindsley CW, Conn PJ. Novel Selective Allosteric Activator of the M1 Muscarinic Acetylcholine Receptor Regulates Amyloid Processing and Produces Antipsychotic-Like Activity in Rats. J Neurosci. 2008 Oct 8;28(41):10422–10433.

Comments on News and Primary Papers
Comment by:  Brian DeanElizabeth Scarr
Submitted 19 November 2008
Posted 19 November 2008

The data implicating acetylcholine in the treatment and neurobiology of schizophrenia continues to grow and has generated the hypothesis that activating specific cholinergic receptors will 1) lead to improvements in the cognitive deficits associated with the disorder and 2) prove to be an alternative mechanism for reducing psychotic symptoms. However, translating these concepts into clinically effective moieties has proven difficult. One problem in designing agonists for the muscarinic receptors is the highly conserved acetylcholine (orthosteric) binding site (Langmead et al., 2008). This has made it difficult to produce centrally active drugs that do not also activate peripheral M2 and M3 receptors, causing cholinergic side effects such as bradycardia, gastrointestinal problems and salivation. Thus, the discovery of allosteric binding sites on both muscarinic (Spalding et al., 2002; Langmead et al., 2006) and nicotinic (Bertrand and Gopalakrishnan, 2007), for review including principles of allosteric modulation) receptors was significant as these sites seem to have a lower degree of homology within receptor families and may offer alternative targets for drug development. The recent characterization of the allosteric M1 agonist, TBPB (1-(1’-2-methylbenzyl)-1,4’-bipiperidin-4-yl) (Jones et al., 2008), is the latest addition to a growing pharmacopeia of novel compounds that may prove to be effective in treating the symptoms of schizophrenia.

This news story contains commentary on other potential allosteric agonists that have been reported to target muscarinic M4 receptors (LY2033298, VU0152099, and VU0152100) and their effectiveness in animal models predictive of antipsychotic action. It also mentions AC-260584, a M1 receptor allosteric agonist, which has been shown to reduce amphetamine and MK-801-induced hyperactivity as well as improving spatial memory performance (Vanover et al., 2008). Another selective M1 allosteric agonist (77-LH-28-1) has been shown to mobilize calcium and enhance NMDA-mediated excitation in rat hippocampus (Langmead et al., 2008), as well as demonstrating procognitive effects in preclinical trials (Watson et al., 2008). Together, these data sustain the promise that muscarinic allosteric agonists for the M4 receptor may be beneficial for psychotic symptoms whereas those that target the M1 receptor may improve both cognitive processing and psychotic symptoms.

With regard to the nicotinic receptors it has been reported that galantamine, a combined acetylcholinesterase inhibitor and allosteric potentiator of nicotinic α7 and α4β2 receptors, may perform better than acetylcholinesterase inhibitors (Deutsch et al., 2008). Although only modestly brain-penetrant, the α7 nicotinic allosteric potentiator NS1738 (1-(5-chloro-2-hydroxy-phenyl)-3-(2-chloro-5-trifluoromethylphenyl)-urea), reduces the scopolamine-induced deficits seen in acquisition of water-maze learning tasks and improved social recognition in rats (Timmermann et al., 2007). Other α7 nicotinic allosteric potentiators have been reported to improve performance in a variety of learning and/or memory tasks and to normalize sensory gating deficits (Ng et al., 2007; Hurst et al., 2007; Kohlhaas et al., 2006). Interestingly, 17β-estradiol appears to act as a positive allosteric modulator at human but not rat α4β2 nicotinic receptors (Paradiso et al., 2001) but to date few clinical studies have used 17β-estradiol as an adjunct to antipsychotics; those studies that have used this approach have not yielded compelling results (see, e.g., (Bergemann et al., 2008a; Bergemann et al., 2007; Bergemann et al., 2008b). Again, current data lend credence to the theory that activating nicotinic receptors may improve cognitive dysfunction and improve sensory gating, as indeed nicotine does for the P50 sensory gating deficit in some patients with schizophrenia. It should be noted however, that improved P50 gating did not correspond to any improvement in the clinical ratings of patients (Adler et al., 2005) and thus may not constitute a clinical improvement.

As well as showing improved selectivity for the receptor of interest, allosteric modulation offers a number of other benefits. It allows the system to continue integrating the temporal and spatial signaling patterns of the native transmitter, both of which are essential for normal neurotransmission. Furthermore, current data suggests that allosteric agonists do not cause appreciable receptor internalization (Watson et al., 2008), which should reduce the rate of desensitization compared to orthosteric agonists. Despite the promising preclinical results obtained with the allosteric agents, they are still a long way from clinical use and a number of problems may arise. Firstly, as pointed out in the article, there is the possibility that these agents interact with allosteric sites on other receptors. Secondly, it has been shown that allosteric agonists can cause differential activation of second messenger systems (Thomas et al., 2008), which may limit their therapeutic usefulness. Finally, it is highly likely that schizophrenia is a syndrome, consisting of a number of diseases. Thus, it is improbable that any single treatment is going to be effective across the entire syndrome; rather, as is the case for existing antipsychotic medications, cholinergic potentiators may prove to be efficacious in a subgroup of people with the disorder. A group that might benefit from treatment with drugs which selectively activate components of the cholinergic system is muscarinic-receptor-deficit schizophrenia, a subgroup within schizophrenia that has lost, on average, 75 percent of their cortical M1 receptors (Scarr et al., 2008). Despite these potential issues, the allosteric agonists/modulators are the result of novel approaches to the development of drugs to treat schizophrenia. It is hoped they also mark the start of a very stimulating era in schizophrenia research.


Langmead CJ, Watson J, Reavill C. Muscarinic acetylcholine receptors as CNS drug targets. Pharmacol Ther. 2008 Feb 1;117(2):232-43. Abstract

Spalding TA, Trotter C, Skjaerbaek N, Messier TL, Currier EA, Burstein ES, Li D, Hacksell U, Brann MR. Discovery of an ectopic activation site on the M(1) muscarinic receptor. Mol Pharmacol. 2002 Jun 1;61(6):1297-302. Abstract

Langmead CJ, Fry VA, Forbes IT, Branch CL, Christopoulos A, Wood MD, Herdon HJ. Probing the molecular mechanism of interaction between 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42) and the muscarinic M(1) receptor: direct pharmacological evidence that AC-42 is an allosteric agonist. Mol Pharmacol. 2006 Jan 1;69(1):236-46. Abstract

Bertrand D, Gopalakrishnan M. Allosteric modulation of nicotinic acetylcholine receptors. Biochem Pharmacol. 2007 Oct 15;74(8):1155-63. Abstract

Jones CK, Brady AE, Davis AA, Xiang Z, Bubser M, Tantawy MN, Kane AS, Bridges TM, Kennedy JP, Bradley SR, Peterson TE, Ansari MS, Baldwin RM, Kessler RM, Deutch AY, Lah JJ, Levey AI, Lindsley CW, Conn PJ. Novel selective allosteric activator of the M1 muscarinic acetylcholine receptor regulates amyloid processing and produces antipsychotic-like activity in rats. J Neurosci. 2008 Oct 8;28(41):10422-33. Abstract

Vanover KE, Veinbergs I, Davis RE. Antipsychotic-like behavioral effects and cognitive enhancement by a potent and selective muscarinic M-sub-1 receptor agonist, AC-260584. Behav Neurosci. 2008 Jun 1;122(3):570-5. Abstract

Langmead CJ, Austin NE, Branch CL, Brown JT, Buchanan KA, Davies CH, Forbes IT, Fry VA, Hagan JJ, Herdon HJ, Jones GA, Jeggo R, Kew JN, Mazzali A, Melarange R, Patel N, Pardoe J, Randall AD, Roberts C, Roopun A, Starr KR, Teriakidis A, Wood MD, Whittington M, Wu Z, Watson J. Characterization of a CNS penetrant, selective M1 muscarinic receptor agonist, 77-LH-28-1. Br J Pharmacol. 2008 Jul 1;154(5):1104-15. Abstract

Watson J, Langmead C, Davies C, and Hagan J. Challenges in developing cholinergic agents for the treatment of schizophrenia. Int.J Neuropsychopharmacol.11(Supp 1), 31 (2008).

Deutsch SI, Schwartz BL, Schooler NR, Rosse RB, Mastropaolo J, Gaskins B. First administration of cytidine diphosphocholine and galantamine in schizophrenia: a sustained alpha7 nicotinic agonist strategy. Clin Neuropharmacol. 2008 Jan-Feb ;31(1):34-9. Abstract

Timmermann DB, Grønlien JH, Kohlhaas KL, Nielsen EØ, Dam E, Jørgensen TD, Ahring PK, Peters D, Holst D, Chrsitensen JK, Malysz J, Briggs CA, Gopalakrishnan M, Olsen GM. An allosteric modulator of the alpha7 nicotinic acetylcholine receptor possessing cognition-enhancing properties in vivo. J Pharmacol Exp Ther. 2007 Oct 1;323(1):294-307. Abstract

Ng HJ, Whittemore ER, Tran MB, Hogenkamp DJ, Broide RS, Johnstone TB, Zheng L, Stevens KE, Gee KW. Nootropic alpha7 nicotinic receptor allosteric modulator derived from GABAA receptor modulators. Proc Natl Acad Sci U S A. 2007 May 8;104(19):8059-64. Abstract

Hurst RS, Hajós M, Raggenbass M, Wall TM, Higdon NR, Lawson JA, Rutherford-Root KL, Berkenpas MB, Hoffmann WE, Piotrowski DW, Groppi VE, Allaman G, Ogier R, Bertrand S, Bertrand D, Arneric SP. A novel positive allosteric modulator of the alpha7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization. J Neurosci. 2005 Apr 27;25(17):4396-405. Abstract

Kohlhaas KL, Bitner RS, Robb H, Radek R, Markosyan S, and Gopalakrishnan M. Improved preattention and short-term memory via positive allosteric modulation of the a7 neuronal nicotinic receptor. Int.J Neuropsychopharmacol.Supp 1, S202-P02.141 (2006).

Paradiso K, Zhang J, Steinbach JH. The C terminus of the human nicotinic alpha4beta2 receptor forms a binding site required for potentiation by an estrogenic steroid. J Neurosci. 2001 Sep 1;21(17):6561-8. Abstract

Adler LE, Cawthra EM, Donovan KA, Harris JG, Nagamoto HT, Olincy A, Waldo MC. Improved p50 auditory gating with ondansetron in medicated schizophrenia patients. Am J Psychiatry. 2005 Feb 1;162(2):386-8. Abstract

Thomas RL, Mistry R, Langmead CJ, Wood MD, Challiss RA. G protein coupling and signaling pathway activation by m1 muscarinic acetylcholine receptor orthosteric and allosteric agonists. J Pharmacol Exp Ther. 2008 Nov 1;327(2):365-74. Abstract

Scarr E, Cowie TF, Kanellakis S, Sundram S, Pantelis C, Dean B. Decreased cortical muscarinic receptors define a subgroup of subjects with schizophrenia. Mol Psychiatry. 2008 Mar 4; Abstract

Bergemann N, Abu-Tair F, Strowitzki T. Estrogen in the treatment of late-onset schizophrenia. J Clin Psychopharmacol. 2007 Dec 1;27(6):718-20. Abstract

Bergemann N, Parzer P, Jaggy S, Auler B, Mundt C, Maier-Braunleder S. Estrogen and comprehension of metaphoric speech in women suffering from schizophrenia: results of a double-blind, placebo-controlled trial. Schizophr Bull. 2008a Nov 1;34(6):1172-81. Abstract

Bergemann N, Parzer P, Kaiser D, Maier-Braunleder S, Mundt C, Klier C. Testosterone and gonadotropins but not estrogen associated with spatial ability in women suffering from schizophrenia: a double-blind, placebo-controlled study. Psychoneuroendocrinology. 2008b May 1;33(4):507-16. Abstract

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Related News: Muscarinic Receptor Agonists: Not Created Equal

Comment by:  Bryan Roth, SRF Advisor
Submitted 15 July 2012
Posted 16 July 2012
  I recommend the Primary Papers

This is an interesting and important paper which serves as a cautionary reminder regarding the potential activity of allosteric modulators to have "functionally selective" effects on GPCR signaling (Urban et al., 2007; Allen and Roth, 2011). Thus, it has been known for decades (see Urban et al., 2007, for review) that GPCR "agonists" and "antagonists" can activate distinct signaling pathways and that a drug can appear to be an "agonist" for one pathway and have "antagonist" activity at another. As we have recently suggested, such activity could have potentially therapeutic implications for schizophrenia and related disorders (Allen et al., 2011).

Although it has been recognized for some time that allosteric modulators of GPCR activity may also have functionally selective actions (Sheffler and Conn, 2008) in vitro, it was unknown if these effects of signaling bias were therapeutically relevant.

What is important here is that Digby et al. rigorously demonstrate signaling bias in vitro and in situ, and then follow these findings up by showing that the signaling "fingerprint" is relevant to the apparent electrophysiological effects of the drugs in vivo.

Although the drugs lacked apparent antipsychotic drug-like actions, they clearly had different effects on synaptic physiology.

Taken together with many other papers of this sort which have appeared over the past several years, the findings reinforce the principle that relying on a single cellular readout for demonstrating the "agonist," "antagonist," or "allosteric" actions of a small molecule could ultimately be problematic for therapeutics.


Allen JA, Roth BL. Strategies to discover unexpected targets for drugs active at G protein-coupled receptors. Annu Rev Pharmacol Toxicol. 2011 Feb 10;51:117-44. Abstract

Allen JA, Yost JM, Setola V, Chen X, Sassano MF, Chen M, Peterson S, Yadav PN, Huang XP, Feng B, Jensen NH, Che X, Bai X, Frye SV, Wetsel WC, Caron MG, Javitch JA, Roth BL, Jin J. Discovery of β-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy. Proc Natl Acad Sci U S A. 2011 Nov 8;108(45):18488-93. Abstract

Sheffler DJ, Conn PJ. Allosteric potentiators of metabotropic glutamate receptor subtype 1a differentially modulate independent signaling pathways in baby hamster kidney cells. Neuropharmacology. 2008 Sep;55(4):419-27. Abstract

Urban JD, Clarke WP, von Zastrow M, Nichols DE, Kobilka B, Weinstein H, Javitch JA, Roth BL, Christopoulos A, Sexton PM, Miller KJ, Spedding M, Mailman RB. Functional selectivity and classical concepts of quantitative pharmacology. J Pharmacol Exp Ther. 2007 Jan;320(1):1-13. Abstract

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Related News: Muscarinic Receptor Agonists: Not Created Equal

Comment by:  Rick Neubig
Submitted 19 July 2012
Posted 20 July 2012

This is a very useful paper, but it is worth pointing out that there is really no evidence for "ligand bias" or "functional selectivity" of these compounds. The VU analogs are partial agonists but show no bias toward one signal pathway or another. The low ERK and α-arrestin signaling are due to the lower degree of receptor reserve for those two pathways and the requirement for more efficacious agonists to signal. The correlation of the in-vitro studies with the different in-vivo readouts does provide very important guidance for pharmacologists about what properties are needed for M1 agonists to produce different physiological effects. It doesn't, however, give any real information about "biased agonists."

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Related News: Muscarinic Receptor Agonists: Not Created Equal

Comment by:  Anantha ShekharAmanda Bolbecker
Submitted 27 July 2012
Posted 27 July 2012

The discovery of M1 allosteric agonists caused a great deal of excitement because they act on the M1 receptor fairly exclusively. The new research by Digby et al. suggests that this newer class of compounds can influence very specific molecular signaling pathways initiated by M1 activation, and that these pathways mediate specific aspects of cognition. These findings could have profound effects on the development of novel therapeutic agents for cognitive impairments seen in schizophrenia and other severe neuropsychiatric disorders.

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Related News: Muscarinic Receptor Agonists: Not Created Equal

Comment by:  Bryan Roth, SRF Advisor
Submitted 1 August 2012
Posted 1 August 2012

Regarding the comment by Rick Neubig: while it could be argued that the paper does not provide "gold standard" pharmacological data for bona fide functional selectivity/stimulus bias, in fairness to the authors I provide their conclusions directly from the paper:

"Thus, the differential effects of these M1 agonists on CNS responses may reflect a combination of partial agonist activity that is impacted by differences in receptor reserve and by an inherent stimulus bias at M1 so that these compounds are not capable of fully activating some responses, even in systems in which the receptor is highly expressed…. Thus, different levels of M1 expression are likely to contribute to the differential responses to VU0357017 and VU0364572 observed in these studies. However, it was interesting to find that VU0357017 never achieved full efficacy in activation of ERK1/2 phosphorylation, even in cell lines with strong induction of M1 expression to levels that induced high receptor reserve in the calcium mobilization assay. Also, VU0357017 did not induce robust β-arrestin responses in the original cell line or in the TREx hM1 cells. Thus, the differential effects of these M1 agonists on CNS responses may reflect a combination of partial agonist activity that is impacted by differences in receptor reserve and by an inherent stimulus bias at M1 so that these compounds are not capable of fully activating some responses, even in systems in which the receptor is highly expressed."

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