If this data holds true, it is very good news in the field. Abe Fisher has been working for years to develop highly specific muscarinic (M1) agonists. He is more advanced on that than anyone else, as the major pharmaceutical companies have abandoned that front for AD therapeutics, mostly due to low efficacy and undesirable side effects. There is a rationale for a "good" M1 agonist in AD. First, there is the clear effect of the M1 receptor-driven switch towards a non-amyloidogenic APP metabolism, i.e., stimulation of ADAM secretases. Second, there is the intrinsic cognitive effect of muscarinic agonists. Third, and less well proven, there is the possibility that the muscarinic stimulation favors endogenous production of neurotrophic factors.

View all comments by A. Claudio Cuello

This data confirms our work, done in collaboration with Abraham Fisher, showing that AF267B and two other of his M1 agonists (AF102B, AF150S) all lower CSF and cortical Aβ concentrations in normal rabbits [1]. This confirms many years of in vitro work going back to 1992, when Roger Nitsch showed that M1 receptor activation shifts APP processing into the non-amyloidogenic pathway [2]. We have also demonstrated the opposite effect, in vivo, that decreasing cortical M1 receptor activation by lesioning the nucleus basalis magnocellularis (nbm) results in increased amyloidogenic processing of APP and Aβ deposition [3] and that treatment with AF267B prevents this deposition [4]. The aggregate data suggest a fusion of the cholinergic and amyloid hypotheses: cortical cholinergic deafferentation occurs during preclinical AD [5-7] and leads to Aβ deposition and AD through decreased M1 receptor activation. If this is true, then cholinergic therapy should be preventative, if given early enough. Treatment begun after dementia has been diagnosed is too late, as Aβ deposition has already...  Read more

This data confirms our work, done in collaboration with Abraham Fisher, showing that AF267B and two other of his M1 agonists (AF102B, AF150S) all lower CSF and cortical Aβ concentrations in normal rabbits [1]. This confirms many years of in vitro work going back to 1992, when Roger Nitsch showed that M1 receptor activation shifts APP processing into the non-amyloidogenic pathway [2]. We have also demonstrated the opposite effect, in vivo, that decreasing cortical M1 receptor activation by lesioning the nucleus basalis magnocellularis (nbm) results in increased amyloidogenic processing of APP and Aβ deposition [3] and that treatment with AF267B prevents this deposition [4]. The aggregate data suggest a fusion of the cholinergic and amyloid hypotheses: cortical cholinergic deafferentation occurs during preclinical AD [5-7] and leads to Aβ deposition and AD through decreased M1 receptor activation. If this is true, then cholinergic therapy should be preventative, if given early enough. Treatment begun after dementia has been diagnosed is too late, as Aβ deposition has already reached a plateau by this stage and tangle formation has usually proceeded to at least Braak stage IV. The recent ACDS trial results showing that Aricept slows conversion of MCI to AD supports a preventative role for cholinergic therapy in AD. Both muscarinic agonists and acetylcholinesterase inhibitors should now proceed to primary prevention trials.

References:
1. Beach, T.G., Walker, D.G., Potter, P.E., Sue, L.I., and Fisher, A., Reduction of cerebrospinal fluid amyloid β after systemic administration of M1 muscarinic agonists. Brain Res. 2001 Jun 29;905(1-2):220-3. Abstract

2. Beach, T.G., Muscarinic agonists as preventative therapy for Alzheimer's disease. Curr Opin Investig Drugs. 2002 Nov;3(11):1633-6. Review. Abstract

3. Beach, T.G., Potter, P.E., Kuo, Y.M., Emmerling, M.R., Durham, R.A., Webster, S.D., Walker, D.G., Sue, L.I., Scott, S., Layne, K.J., and Roher, A.E., Cholinergic deafferentation of the rabbit cortex: a new animal model of Aβ deposition. Neurosci Lett. 2000 Mar 31;283(1):9-12. Abstract

4. Beach, T.G., Walker, D., Sue, L., Scott, S., Layne, K., Newell, A., Potter, P., Durham, R.A., Emmerling, M., Webster, S.D., Honer, W., Fisher, A., and Roher, A. Immunotoxin lesion of the cholinergic nucleus basalis causes Ab deposition: towards a physiologic animal model of Alzheimer's disease. Curr.Med.Chem. 3, 57-75. 2003.

5. Beach, T.G., Honer, W.G., and Hughes, L.H., Cholinergic fibre loss associated with diffuse plaques in the non- demented elderly: the preclinical stage of Alzheimer's disease? Acta Neuropathol (Berl). 1997 Feb;93(2):146-53. Abstract

6. Beach, T.G., Kuo, Y.M., Spiegel, K., Emmerling, M.R., Sue, L.I., Kokjohn, K., and Roher, A.E., The cholinergic deficit coincides with Aβ deposition at the earliest histopathologic stages of Alzheimer disease. J Neuropathol Exp Neurol. 2000 Apr;59(4):308-13. Abstract

7. Katzman, R., Terry, R., DeTeresa, R., Brown, T., Davies, P., Fuld, P., Renbing, X., and Peck, A., Clinical, pathological, and neurochemical changes in dementia: a subgroup with preserved mental status and numerous neocortical plaques. Ann Neurol. 1988 Feb;23(2):138-44. Abstract

View all comments by Thomas Beach

The triple transgenic animals are a fascinating addition to the tools available. Especially exciting is this new data indicating that these genes have to work together in order to convert mice to an AD model. Obviously, these mice remain an animal model of AD, but are likely a big leap forward from the standard amyloid model mice we used to work with. There might be other animal models that do the same without the need for mutations in three different genes, but to be able to study this in mice will speed up the necessary research enormously.

Intracellular Aβ accumulation sheds light on what might be expected. Intracellular Aβ accumulations had been found in several studies previously, including human brain. However, in transgenic mice this does not appear to be a consistent feature. For a “perfect” model of AD, that’s just strange. If it exists in human AD brains, it should be present in all transgenic mouse models. The conclusion that comes to mind is that the triple transgenic mice enhance important aspects of the pathology that would otherwise be easily missed. Do we...  Read more

The triple transgenic animals are a fascinating addition to the tools available. Especially exciting is this new data indicating that these genes have to work together in order to convert mice to an AD model. Obviously, these mice remain an animal model of AD, but are likely a big leap forward from the standard amyloid model mice we used to work with. There might be other animal models that do the same without the need for mutations in three different genes, but to be able to study this in mice will speed up the necessary research enormously.

Intracellular Aβ accumulation sheds light on what might be expected. Intracellular Aβ accumulations had been found in several studies previously, including human brain. However, in transgenic mice this does not appear to be a consistent feature. For a “perfect” model of AD, that’s just strange. If it exists in human AD brains, it should be present in all transgenic mouse models. The conclusion that comes to mind is that the triple transgenic mice enhance important aspects of the pathology that would otherwise be easily missed. Do we then have to conclude that tau plays a role in intracellular Aβ toxicity? This may very much be the case as intracellular Aβ has all the properties to interfere with intracellular trafficking, including the role of the Aβ domain in APP as essential mediator of axonal APP transport.

The other intriguing item is Abraham Fisher's M1 agonist that interferes in a complex manner with APP processing and Aβ production. Again, previous transgenic approaches might simply not have been able to model AD closely enough to reveal the strong effect AF267B has, as these new data indicate. The more we try to shape and optimize approaches to find a cure, the more complex the models will have to be to gain the necessary insights. If that needs to be done with three mutated genes, it's fine with me.

View all comments by Tobias Hartmann

I think this is among the most important observations shown at the meeting. I had seen Frank LaFerla’s behavior data and antibody reversal earlier in September at a meeting on cognition. It supports and is consistent with the results from most of the mice that cognitive function and Aβ correlate. We have not been able to detect the intracellular Aβ in our Tg2576-based mice, but that may be a technical difference. In any event, it seems very likely that Aβ can cause the memory deficits in the APP-transgenic animals.

This is the first time for the M1 data in vivo to my knowledge. There is a long history of muscarinic cholinergic regulation of APP processing (see Nitsch et al., 1992 or Buxbaum et al., 1992), in addition to the mechanism suggested by Abe. The one control, however, that I know Frank will run if he hasn't already is to evaluate the levels of the transgene mRNAs. Because the transgenes are driven by an autologous promoter (Thy-1,...  Read more

I think this is among the most important observations shown at the meeting. I had seen Frank LaFerla’s behavior data and antibody reversal earlier in September at a meeting on cognition. It supports and is consistent with the results from most of the mice that cognitive function and Aβ correlate. We have not been able to detect the intracellular Aβ in our Tg2576-based mice, but that may be a technical difference. In any event, it seems very likely that Aβ can cause the memory deficits in the APP-transgenic animals.

This is the first time for the M1 data in vivo to my knowledge. There is a long history of muscarinic cholinergic regulation of APP processing (see Nitsch et al., 1992 or Buxbaum et al., 1992), in addition to the mechanism suggested by Abe. The one control, however, that I know Frank will run if he hasn't already is to evaluate the levels of the transgene mRNAs. Because the transgenes are driven by an autologous promoter (Thy-1, in this case), changes in gene expression might be trivial due to regulation of Thy-1 expression (which is presumably irrelevant for AD).

I emphasize these are exciting data and I can’t wait to see how they translate into the clinic.

View all comments by Dave Morgan

The ability of the M1 agonist AF267B to decrease amyloid plaque load, decrease tau phosphorylation, and enhance memory function in the "triple transgenic" mice is indeed encouraging. Selective muscarinic agonists are among the few therapeutic approaches that could help alleviate the symptoms (memory deficits, cognitive dysfunction) of Alzheimer disease and have an impact on the underlying disease process. Over the years, Dr. Fisher has been a strong proponent of using selective muscarinic agonists to treat Alzheimer disease. Although several muscarinic agonists have failed in clinical studies, most of the compounds tested lacked selectivity for M1 receptors or appreciable activity at M1 receptors in the CNS.

We also presented data at the 2004 Society for Neuroscience meeting (1) on the potential neuroprotective effects of a selective M1 agonist CDD-0102. In the studies presented in San Diego, CDD-0102 promoted activation of α-secretase, (as measured by elevated levels of soluble APP-α) and decreased levels of Aβ in HEK 293T cells expressing human M1 receptors, wild-type...  Read more

The ability of the M1 agonist AF267B to decrease amyloid plaque load, decrease tau phosphorylation, and enhance memory function in the "triple transgenic" mice is indeed encouraging. Selective muscarinic agonists are among the few therapeutic approaches that could help alleviate the symptoms (memory deficits, cognitive dysfunction) of Alzheimer disease and have an impact on the underlying disease process. Over the years, Dr. Fisher has been a strong proponent of using selective muscarinic agonists to treat Alzheimer disease. Although several muscarinic agonists have failed in clinical studies, most of the compounds tested lacked selectivity for M1 receptors or appreciable activity at M1 receptors in the CNS.

We also presented data at the 2004 Society for Neuroscience meeting (1) on the potential neuroprotective effects of a selective M1 agonist CDD-0102. In the studies presented in San Diego, CDD-0102 promoted activation of α-secretase, (as measured by elevated levels of soluble APP-α) and decreased levels of Aβ in HEK 293T cells expressing human M1 receptors, wild-type APP695 and mutant PS1. In PC12 cells treated with NGF to promote differentiation into a neuronal phenotype, CDD-0102 protected cells from apoptosis and elevated caspase-3 cleavage induced by staurosporine.

CDD-0102 is functionally selective for M1 receptors with minimal activity at other muscarinic receptor subtypes (2,3). It also enhances memory function in rats treated with 192IgG saporin to deplete cortical and hippocampal acetylcholine (4). Further studies are necessary to determine whether CDD-0102 produces similar effects to those observed in transgenic mice for AF267B.

References:
1. Messer WS, Jr, Tang B, Hoss WP, and Ghosh D. Neuroprotective effects of the selective M1 agonist CDD-0102: Stimulation of α-secretase, inhibition of Aβ and prevention of apoptosis. Society for Neuroscience, 29, no. 673.19, 2004.

2. Messer WS Jr, Abuh YF, Liu Y, Periyasamy S, Ngur DO, Edgar MA, El-Assadi AA, Sbeih S, Dunbar PG, Roknich S, Rho T, Fang Z, Ojo B, Zhang H, Huzl JJ 3rd, Nagy PI. Synthesis and biological characterization of 1,4,5,6-tetrahydropyrimidine and 2-amino-3,4,5,6-tetrahydropyridine derivatives as selective m1 agonists. J Med Chem. 1997 Apr 11;40(8):1230-46. 9111297

3. Messer WS, Jr, Abuh YF, Ryan K, Shepherd MA, Schroeder M, Abunada S, and El-Assadi AA. Tetrahydropyrimidine derivatives display functional selectivity for M1 muscarinic receptors in brain. Drug Dev Res 1997;40:171-174.

4. Messer, W.S., Jr., K.A. Bachmann, C. Dockery, A.A. El-Assadi, E. Hassoun, N. Haupt, B. Tang and X. Li. Development of CDD-0102 as a selective M1 agonist for the treatment of Alzheimer’s disease. Drug Dev Res. 2002;57(4):200-213.

View all comments by William Messer

I concur with the other comments: The in-vivo M1 agonist treatment effect on Aß is very encouraging. It has long been clear that different muscarinic receptor subtypes have opposing actions on amyloidogenesis and other physiological processes, and that selective M1 agonists may provide a major step forward from current nonselective cholinergic therapies. M1 is the predominant muscarinic receptor involved in cognition, neuronal excitability, synaptic plasticity and likely regulation of amyloidogenesis.

However, the hypothesis has never been adequately tested since highly selective and potent M1 agonists have been so difficult to develop. Hopefully, Dr. Fisher's persistence in developing M1 agonists will pay off and add a significantly improved therapeutic approach that targets cognition, behavior, and amyloidogenesis. The unanticipated benefits of cholinergic therapies on behavorial problems in AD, including psychosis, have also renewed the interests of big pharma in developing M1 agonists given their potential for schizophrenia (and pain). Hence, we may finally see...  Read more

I concur with the other comments: The in-vivo M1 agonist treatment effect on Aß is very encouraging. It has long been clear that different muscarinic receptor subtypes have opposing actions on amyloidogenesis and other physiological processes, and that selective M1 agonists may provide a major step forward from current nonselective cholinergic therapies. M1 is the predominant muscarinic receptor involved in cognition, neuronal excitability, synaptic plasticity and likely regulation of amyloidogenesis.

However, the hypothesis has never been adequately tested since highly selective and potent M1 agonists have been so difficult to develop. Hopefully, Dr. Fisher's persistence in developing M1 agonists will pay off and add a significantly improved therapeutic approach that targets cognition, behavior, and amyloidogenesis. The unanticipated benefits of cholinergic therapies on behavorial problems in AD, including psychosis, have also renewed the interests of big pharma in developing M1 agonists given their potential for schizophrenia (and pain). Hence, we may finally see some progress in the pharmacology.

View all comments by Allan Levey

Given the recent interest in the M1 agonist formerly designated AF267B, we thought it would be useful to provide information concerning the basic pharmacologic properties and future development plans for this compound at Neurogenetics, Inc. (licensee) in La Jolla, CA.

Currently designated as NGX267B, this compound is an orally active, rigid analog of acetylcholine. Its pharmacological properties partially mimic the actions of acetylcholine through a stimulation of neurons that are generally spared in the neurodegenerative processes characterizing Alzheimer disease (AD). Consistent with this hypothesis, animal models with predictive utility for the symptomatic treatment of AD have demonstrated efficacy at dosages of NGX267B below those associated with nonspecific effects. NGX267B has also demonstrated disease modification properties involving reduction of β-amyloid and tau deposition in the LaFerla triple transgenic mice, thereby offering insights into mechanisms with long-term clinical implications.

One mechanism of action for NGX267B involves direct stimulation of...  Read more

Given the recent interest in the M1 agonist formerly designated AF267B, we thought it would be useful to provide information concerning the basic pharmacologic properties and future development plans for this compound at Neurogenetics, Inc. (licensee) in La Jolla, CA.

Currently designated as NGX267B, this compound is an orally active, rigid analog of acetylcholine. Its pharmacological properties partially mimic the actions of acetylcholine through a stimulation of neurons that are generally spared in the neurodegenerative processes characterizing Alzheimer disease (AD). Consistent with this hypothesis, animal models with predictive utility for the symptomatic treatment of AD have demonstrated efficacy at dosages of NGX267B below those associated with nonspecific effects. NGX267B has also demonstrated disease modification properties involving reduction of β-amyloid and tau deposition in the LaFerla triple transgenic mice, thereby offering insights into mechanisms with long-term clinical implications.

One mechanism of action for NGX267B involves direct stimulation of specific muscarinic (M1) receptors that exist on intact cholinergic neurons within the CNS; therefore, this proposed therapy could complement currently available AD treatments (e.g., acetylcholine esterase inhibitors). It is specific to memory and cognitive behaviors, and largely devoid of adverse effects such as dizziness, salivation, nausea, vomiting, or diarrhea at clinically relevant doses. We consider both monotherapy and concomitant use with existing products plausible at this juncture.

The known biological and pharmaceutical properties of NGX267B should permit an exploration of efficacy and safety across a range of disease severity, co-morbidities, and concomitant medications. For example, no clinically important adverse events have been detected in animals at dosages that enhanced memory and cognitive behaviors; this suggests that the therapeutic index (a measure of the safety margin) may be attractive for the elderly or debilitated patient. The major metabolite of NGX267B has a biological profile similar to that of the parent compound. This makes it less likely that we will experience the disappointing clinical results that occurred in the evaluation of other agents as a result of poor oral absorption or extensive metabolic breakdown into products with less specificity. Finally, an anticipated long duration of action may enhance both efficacy and convenience.

The clinical development program for NGX267B is scheduled to begin approximately mid- to late 2005. Although a variety of cognitive disorders are potential therapeutic targets based upon known pathophysiology, Neurogenetics has identified the symptomatic treatment of mild to moderate Alzheimer disease as an immediate therapeutic goal. The first few clinical studies will explore safety, tolerance, pharmacokinetics, and biodisposition in both asymptomatic young and older volunteers in order to define the permissible clinical dose range for subsequent "proof-of-concept" studies in AD. The influence of dosing frequency on cognitive performance will be evaluated in light of the known neuropsychological effects of cognitive enhancing agents. Additionally, clinical measures such as neuroimaging, computerized neuropsychological test batteries, and cerebrospinal fluid biomarkers may be added as outcome measures to help fully characterize the biological properties of NGX267B before we begin a more traditional dose-ranging paradigm in mild to moderate AD.

View all comments by Michael Murphy
View all comments by Steven Wagner

I concur that the work with the triple transgenic mouse and also the possibility of M1 agonists as therapy are exciting, but I specifically want to comment on a technical issue that is being brought up regarding intraneuronal Aβ, something that the triple transgenic mouse is providing intriguing new insights into. There is no evidence, to my knowledge, that AD mutant mice exist that develop plaques but never show intraneuronal Aβ. One comment mentioned not observing intraneuronal Aβ in a Tg2576-based mouse. I understand the difficulty with convincingly detecting intracellular Aβ. I use, as an analogy, doing a Western blot of brain extract with an anti-Aβ antibody. If you do a short exposure, you can see a faint band for full-length APP but no Aβ band. If you stop there, you can convince yourself that there is no Aβ in brain. But if you expose your gel longer, the APP band will become more pronounced while APP CTFs and Aβ also eventually appear. Similarly with Aβ42 immunohistochemistry, if you do a brief reaction time, you can have a clean image only of plaques. But if you wait...  Read more

I concur that the work with the triple transgenic mouse and also the possibility of M1 agonists as therapy are exciting, but I specifically want to comment on a technical issue that is being brought up regarding intraneuronal Aβ, something that the triple transgenic mouse is providing intriguing new insights into. There is no evidence, to my knowledge, that AD mutant mice exist that develop plaques but never show intraneuronal Aβ. One comment mentioned not observing intraneuronal Aβ in a Tg2576-based mouse. I understand the difficulty with convincingly detecting intracellular Aβ. I use, as an analogy, doing a Western blot of brain extract with an anti-Aβ antibody. If you do a short exposure, you can see a faint band for full-length APP but no Aβ band. If you stop there, you can convince yourself that there is no Aβ in brain. But if you expose your gel longer, the APP band will become more pronounced while APP CTFs and Aβ also eventually appear. Similarly with Aβ42 immunohistochemistry, if you do a brief reaction time, you can have a clean image only of plaques. But if you wait longer, you can start to see intraneuronal Aβ42, especially if there are not too many plaques around. If you think this is all non-specific background, consider performing the optimal control, comparing staining in equally aged/processed APP knockout mouse sections. Or consider using pre-embedding immuno-gold electron microscopy (thereby avoiding a reaction product and endogenous peroxidase), since in our experience intraneuronal Aβ42 especially accumulates in AD transgenic mice in processes/synapses, which cannot easily be seen by light microscopy. If you have difficulty with seeing intraneuronal Aβ42, please feel free to contact me.

View all comments by Gunnar K. Gouras