Key roles in modulating cognition and behavior made the M1 muscarinic acetylcholine receptor (mAChR) a prime target for Alzheimer disease 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 ARF related news story and 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 (see ARF related news story). 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 (see ARF related news story). 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

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  1. The difficulty in developing drugs for specific subtypes of some neurotransmitter receptors is that the transmitter binding site is normally highly conserved. One way around this is to design molecules that modulate a putative allosteric site(s) in the receptor. This is the take-home message of the paper of Jones et al., 2008, that describes some interesting effects in vitro and in vivo of TBPB, a novel allosteric activator of the M1 muscarinic receptor (mAChR). The authors show a high selectivity of TBPB for the rat M1 mAChR as compared to the human M2-M5 mAChR subtypes using one readout in vitro, namely agonist-evoked increases of intracellular calcium ions. Furthermore, TBPB, like other selective and non-selective M1 agonists, elevates α-APPs in cell-based assays that contain mainly the M1 mAChR (in this study, PC12 cells co-transfected with human M1 mAChR and APP Swedish mutation). In another interesting aspect of this paper, the authors describe the effects of TBPB in animal models that may have some predictive value for the treatment of symptoms associated with schizophrenia. While clearly this paper is an important step forward in our understanding of M1 mAChR activation, its allosteric site(s), and the important role of this receptor in affective disorders such as schizophrenia, several issues need to be addressed regarding this paper and development of M1 agonists for treatment of CNS diseases such as Alzheimer’s (AD) or schizophrenia, in general:

    1. We have succeeded over the years in developing functionally selective M1 agonists of the AF series (e.g., AF102B, AF150(S), AF267B, and more) with high specificity and ability to cross the blood-brain barrier (see attached papers). AF267B, for example, is orally available, penetrates the blood-brain barrier, and has an excellent pharmacokinetic profile both in preclinical and clinical studies, a high bioavailability, and a wide safety margin. AF267B (originally from our lab, >99.9 percent chemical and enantiomeric purity) was shown to reduce both Aβ and tau pathologies in the hippocampus and cortex, and to reverse cognitive deficits in 3xTg-AD mice (Caccamo et al., 2006). These effects of AF267B were observed without side effects at 1 and 3 mg/kg (ip, daily administration for two months), respectively—doses 45 or 15 times lower than those in which overt effects such as salivation were seen. This showed clearly that the compound has a wide safety margin and that the beneficial effects of AF267B can be attributed to M1 mAChR activation. Furthermore, in the same study, reduced M1 mAChR activation (by dicyclomine, a relatively selective M1 antagonist) increased Aβ production, tau hyperphosphorylation, and cognitive deficits in the same model.

    2. Different receptor reserves of mAChR subtypes in cell cultures and different experimental design can provide results that may differ from one lab to another. Given this caveat and the difficulty in predicting whether a putative M1 agonist, be it orthosteric or allosteric, will be solely selective for the M1 mAChR both in vitro and in vivo, one has to use several tests in order to obtain a valid estimate, and of course the final test will be in clinical studies. In this context, AF267B (originally from our lab, >99.9 percent chemical and enantiomeric purities) emerged in our studies as a highly selective functional M1 agonist as evidenced, inter alia, in cell cultures stably transfected with the human M1-M5 mAChR subtypes by mobilization of intracellular calcium ions (EC50 = 1.7 μM [almost full agonist vs. carbachol] with marginal effects [20-10 percent] on M3 and M5 mAChR and no effects on M2 and M4 mAChR subtypes when tested at 100 μM). AF267B emerged also as a selective M1 partial agonist vs. its effect on M3 and M5 mAChR as assayed by an increase in phosphoinositides turnover in the same cell cultures. Additionally, in cell cultures such as PC12 cells stably transfected with rat M1 mAChR, AF267B was a partial agonist (readout: phosphoinositides elevation or arachidonic acid release, but inactive on cAMP release mediated by the M1 mAChR). Moreover, AF267B was inactive as an agonist on cell cultures that lack a significant population of the M1 mAChR, but still have intrinsic M3 mAChR (e.g., PC12 or SH-SY5Y cells). For in vivo selectivity of AF267B toward M1 mAChR-mediated functions, see point 1 above.

    3. Regarding the allosteric agonist TBPB, the authors emphasize its value in the non-amyloidogenic processing of APP as mediated by M1 mAChR. This is an important finding that confirms studies with other muscarinic agonists, including the functionally selective M1 agonists from the AF series. However, effects of TBPB in cell culture or brain slices that contain several mAChR subtypes were not reported in the paper. Notably, in such preparations the M1 agonists of the AF series appeared even more potent than the non-selective agonist carbachol most probably due to their selective effects on the M1 mAChR.

    4. By binding to allosteric sites on the receptor, allosteric modulators can sometimes be more selective than orthosteric agonists for the M1 vs. M2-M5 mAChR. However, paradoxically one cannot disregard the scenario that such an allosteric agonist will bind or modulate other G protein-coupled receptors (GPCR) in spite of its excellent selectivity for the M1 mAChR. While the orthosteric site is the target that dictates the selectivity of a given neurotransmitter for a receptor including its subtypes, the exact orthosteric site is not found in other GPCRs. However, we lack enough compelling information for whether the allosteric site(s) for one receptor (e.g., the M1 mAChR) is found exclusively in this particular receptor and cannot be found in some other GPCR. Thus, while the orthosteric agonist AF267B was shown to bind specifically only to the mAChR subtypes when tested on a plethora of GPCRs and other receptors and enzymes, it is not clear from the paper whether TBPB is solely specific for the M1 mAChR. In fact, in the range of its full effects on the M1 mAChR, TBPB also binds to the D2 receptors in the μM range.

    5. Finally, the clinical value of a selective M1 agonist, be it orthosteric or allosteric, is dictated by its pharmacokinetic profile and bioavailability. In this context, AF267B has an excellent pharmacokinetic profile producing the metabolite, N-des-methyl analog, a highly selective partial M1 agonist, in vitro and in vivo. The pharmacokinetic profile of TBPB was not mentioned, and no effects of low doses were reported in in vivo studies. Notably, based on the lesson learned with xanomeline (highly selective in vitro, yet bad pharmacokinetics and selectivity in vivo) selectivity for a mAChR subtype in vitro is not a guarantee for a good selectivity in vivo or a promising bioavailability/pharmacokinetic profile. Definitively, more studies are required to substantiate the practical value of TBPB and to show whether it has a preclinical profile that can match a good M1 candidate for AD treatment.

    References:

    . M1 muscarinic agonists target major hallmarks of Alzheimer's disease--the pivotal role of brain M1 receptors. Neurodegener Dis. 2008;5(3-4):237-40. PubMed.

    . Cholinergic treatments with emphasis on m1 muscarinic agonists as potential disease-modifying agents for Alzheimer's disease. Neurotherapeutics. 2008 Jul;5(3):433-42. PubMed.

    . M1 receptors play a central role in modulating AD-like pathology in transgenic mice. Neuron. 2006 Mar 2;49(5):671-82. PubMed.

  2. The paper by Jones et al. describes TBPB, a novel compound that represents a new generation of highly specific drugs. 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. There is a long and well-known history of research on the cholinergic system in AD that led to the development of cholinesterase inhibitors as approved therapies. However, the therapeutic utility of cholinesterase inhibitors is modest, and because these drugs lead to non-specific activation of many different subtypes of muscarinic and nicotinic receptors, side effects are frequent and tolerability suboptimal. For these reasons, development of selective agonists has been a long sought goal for AD treatment, dating back to the discovery of the cholinergic deficiency in AD. Indeed, a wealth of preclinical and clinical data have supported the prediction that highly specific M1 agonists would be more efficacious for cognitive and behavioral symptoms of AD. Such drugs would also be expected to be better tolerated with fewer cholinergic side effects than cholinesterase inhibitors. However, the development of selective drugs has been an elusive goal despite decades of intensive efforts by big pharma.

    There has been a resurgence of interest in M1 receptor-based therapeutics for AD with the findings of LaFerla's and Fisher's groups in 2006 that AF267B (Caccamo et al., 2006), a purported M1 agonist, reduced amyloid and tau pathologies in the triple transgenic AD mouse model. The current study is important as it represents a true breakthrough in the pharmacology of the muscarinic cholinergic system, with the development of a truly selective M1 agonist termed TBPB. As the authors show, TBPB has anti-amyloidogenic effects on cells, and activates central M1 receptors to produce expected behavioral, cognitive, and neurophysiological benefits. Moreover, the study demonstrates that AF267B is not as selective for M1 as previously believed. Conn's group carefully assesses the specificity of AF267B and shows that this compound also activates M3 receptors—perhaps even better than M1. While this might be a potential beneficial property for anti-amyloid effects, it makes it impossible to tease out the role of M1 vs. M3 from previous studies, and its potential as a drug could be limited by M3 side effects (which are likely a major source of the peripheral cholinergic side effects plaguing current drugs, particularly GI, and can limit dosing). Thus, TBPB and other M1 selective drugs may have improved efficacy and tolerability.

    The pharmacological breakthrough comes from targeting the receptor to a different binding site than has been conventionally targeted for cholinergic drugs. TBPB binds to an allosteric binding site, which is more highly divergent among the five closely related muscarinic receptor family members. Other drugs acting at this site have been recently described by Acadia Pharmaceuticals. With this knowledge in hand, one might expect newer generations of highly selective drugs for M1 and the other muscarinic receptor subtypes. Additional studies of these drugs will be necessary to determine if they will be advantageous for clinical applications, including neuroprotection for AD.

    References:

    . M1 receptors play a central role in modulating AD-like pathology in transgenic mice. Neuron. 2006 Mar 2;49(5):671-82. PubMed.

References

News Citations

  1. Acetylcholine Boost With a Twist—Compound Targets M4 Receptors
  2. Cholinergic Transmission and Aβ: Boosting M1 Receptors Treats Model
  3. Keystone Drug News: Agonists for M1, Serotonin Receptors Prime Cholinergic Pump

Paper Citations

  1. . Effects of xanomeline, a selective muscarinic receptor agonist, on cognitive function and behavioral symptoms in Alzheimer disease. Arch Neurol. 1997 Apr;54(4):465-73. PubMed.
  2. . A novel family of potent negative allosteric modulators of group II metabotropic glutamate receptors. J Pharmacol Exp Ther. 2007 Jul;322(1):254-64. PubMed.
  3. . Centrally active allosteric potentiators of the M4 muscarinic acetylcholine receptor reverse amphetamine-induced hyperlocomotor activity in rats. J Pharmacol Exp Ther. 2008 Dec;327(3):941-53. PubMed.
  4. . Allosteric modulation of the muscarinic M4 receptor as an approach to treating schizophrenia. Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10978-83. PubMed.
  5. . Cholinergic treatments with emphasis on m1 muscarinic agonists as potential disease-modifying agents for Alzheimer's disease. Neurotherapeutics. 2008 Jul;5(3):433-42. PubMed.
  6. . M1 muscarinic agonists target major hallmarks of Alzheimer's disease--the pivotal role of brain M1 receptors. Neurodegener Dis. 2008;5(3-4):237-40. PubMed.
  7. . Antipsychotic-like behavioral effects and cognitive enhancement by a potent and selective muscarinic M-sub-1 receptor agonist, AC-260584. Behav Neurosci. 2008 Jun;122(3):570-5. PubMed.

Other Citations

  1. 3xTg

External Citations

  1. Kinney, 2006
  2. Phase 2 study
  3. low on cash
  4. press release

Further Reading

Papers

  1. . Effects of xanomeline, a selective muscarinic receptor agonist, on cognitive function and behavioral symptoms in Alzheimer disease. Arch Neurol. 1997 Apr;54(4):465-73. PubMed.
  2. . Centrally active allosteric potentiators of the M4 muscarinic acetylcholine receptor reverse amphetamine-induced hyperlocomotor activity in rats. J Pharmacol Exp Ther. 2008 Dec;327(3):941-53. PubMed.
  3. . Antipsychotic-like behavioral effects and cognitive enhancement by a potent and selective muscarinic M-sub-1 receptor agonist, AC-260584. Behav Neurosci. 2008 Jun;122(3):570-5. PubMed.

Primary Papers

  1. . 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. PubMed.