Nira Bar-Ner

M1 muscarinic agonists can modulate some of the hallmarks in Alzheimer's disease: implications in future therapy.

M1 muscarinic receptors (M1 mAChRs) play a role in an apparent linkage of three major hallmarks of Alzheimer’s disease (AD): β-amyloid (Aβ) peptide; tau hyperphosphorylation and paired helical filaments (PHFs); and loss of cholinergic function conducive to cognitive impairments. We evaluated the M1 muscarinic agonists AF102B (Cevimeline, EVOXAC™: prescribed for Sjøgren’s syndrome), AF150(S), and AF267B on some of these hallmarks of AD. Activation of M1 mAChRs with these agonists leads, inter alia, to enhanced secretion of amyloid precursor protein (α-APP), (via α-secretase activation), to decreased Aβ (via γ-secretase inhibition), and to inhibition of Aβ- and/or oxidative stress-induced cell death. In several animal models mimicking different aspects of AD, these drugs restored cognitive impairments, and in select cases induced a decrease in brain Aβ elevation, with a high safety margin, following po administration. Notably, in mice with small hippocampi, unlike rivastigmine and nicotine, AF150(S) and AF267B restored cognitive impairments also on escape latency in a Morris water maze paradigm, in reversal learning. Studies from other labs showed that AF102B and talsaclidine (another M1 agonist) decreased cerbrospinal fluid (CSF) Aβ in AD patients following chronic treatment, being the first reported drugs with such a profile. The clinical significance of these studies remains to be elucidated, yet based on in vivo (rabbits) and in vitro studies (cell cultures), our M1 agonists can decrease brain Aβ, owing to a novel and dual complementary effect (e.g., inhibition of γ-secretase and activation of α-secretase). Remarkably, although M1 agonists can decrease CSF Aβ in AD patients, an increased AD-type pathology in Parkinson’s disease was recently been associated with chronic antimuscarinic treatment. In another aspect, these agonists decreased tau hyperphosphorylation in vitro and in vivo. Notably, nicotinic agonists or cholinesterase inhibitors increased tau hyperphosphorylation. In summary, the M1 agonists tested are effective on cognition and behavior and show unique disease-modifying properties owing to beneficial effects on major hallmarks of AD. This may place such drugs in the first line of modern AD therapies (e.g., β- or γ-secretase inhibitors, vaccines against Aβ, statins, and inhibitors of tau hyperphosphorylation).

Impact of muscarinic agonists for successful therapy of Alzheimer’s disease

The M1 muscarinic agonists AF102B, AF150(S) & AF267B--i) restored cognitive impairments in several animal models for AD with an excellent safety margin; ii) elevated alpha-APPs levels; iii) attenuated vicious cycles induced by A beta, and inhibited A beta- and oxidative stress-induced apoptosis; and iv) decreased tau hyperphosphorylation. AF150(S) and AF267B were more effectve than rivastigmine and nicotine in restoring memory impairments in mice with small hippocampi. In apolipoprotein E-knockout mice, AF150(S) restored cognitive impairments and cholinergic hypofunction and decreased tau hyperphosphorylation. In aged microcebes, AF150(S) restored cognitive and behavioral impairments and decreased tau hyperphosphorylation, paired helical filaments and astrogliosis. In rabbits, AF267B & AF150(S) decreased CSF A beta(1-42 & 1-40), while AF102B reduced A beta(1-40). Finally AF102B decreased CSF A beta(total) in AD patients. Taken together, M1 agonists may represent a unique therapy in AD due to their beneficial effects on three major hallmarks of AD--cholinergic hypofunction, A beta and tau protein hyperphosphorylation.

Novel m1 muscarinic agonists in treatment and delaying the progression of Alzheimer's disease: An unifying hypothesis

M1 selective agonists from the AF series (e.g. AF102B, AF150(S)), via m1 muscarinic receptors, activate distinct signal transductions, enhance amyloid precursors proteins secretion from transfected cells and primary cell cultures, show neurotrophic effects and are beneficial in a variety of animal models for Alzheimers disease. Such m1 agonists may be effective in the treatment and therapy of Alzheimers disease.

AF710B, a Novel M1/σ1 Agonist with Therapeutic Efficacy in Animal Models of Alzheimer's Disease

We previously developed orthosteric M1 muscarinic agonists (e.g. AF102B, AF267B and AF292), which act as cognitive enhancers and potential disease modifiers. We now report on a novel compound, AF710B, a highly potent and selective allosteric M1 muscarinic and σ1 receptor agonist. AF710B exhibits an allosteric agonistic profile on the M1 muscarinic receptor; very low concentrations of AF710B significantly potentiated the binding and efficacy of carbachol on M1 receptors and their downstream effects (p-ERK1/2, p-CREB). AF710B (1-30 µg/kg, p.o.) was a potent and safe cognitive enhancer in rats treated with the M1 antagonist trihexyphenidyl (passive avoidance impairment). These effects of AF710B involve σ1 receptor activation. In agreement with its antiamnesic properties, AF710B (at 30 nM), via activation of M1 and a possible involvement of σ1 receptors, rescued mushroom synapse loss in PS1-KI and APP-KI neuronal cultures, while AF267B (1 µM) was less potent in PS1-KI and ineffective in APP-KI models, respectively. In female 3xTg-AD mice, AF710B (10 µg/kg, i.p./daily/2 months) (i) mitigated cognitive impairments in the Morris water maze; (ii) decreased BACE1, GSK3β activity, p25/CDK5, neuroinflammation, soluble and insoluble Aβ40, Aβ42, plaques and tau pathologies. AF710B differs from conventional σ1 and M1 muscarinic (orthosteric, allosteric or bitopic) agonists. These results highlight AF710B as a potential treatment for Alzheimers disease (e.g. improving cognitive deficits, synaptic loss, amyloid and tau pathologies, and neuroinflammation) with a superior profile over a plethora of other therapeutic strategies.

M1 Muscarinic Agonists as a Therapeutic Strategy in Alzheimer’s Disease

M1 muscarinic receptors (M 1 mAChR) have an important role in cognitive processing relevant to Alzheimer’s disease brains (AD)1–5. M1 mAChR are relatively unchanged in AD1–5 and therefore may serve as a target for an anti-dementia drug treatment. However, some of the tested muscarinic agonists were not highly M1 selective, had major clinical limitations and showed disappointing clinical results in AD 4,6,7. Thus the proof of clinical concept could not be shown.

M1 MUSCARINIC AGONISTS AND A MULTIPOTENT ACTIVATOR OF SIGMA1/M1 MUSCARINIC RECEPTORS: FUTURE THERAPEUTICS OF ALZHEIMER'S DISEASE

These compounds are cognitive enhancers and disease modifiers in vitro and in vivo. Notably, i) AF102B and AF267B decreased CSF Abeta levels in preclinical studies (Beach et al, 2001); ii) AF267B was effective against cognitive deficits, Abeta42 and tau pathologies in 3xTg-AD mice (Caccamo et al, 2006); iii) AF102B and AF267B decreased brain alpha-synuclein in transgenic mice overexpressing human alpha-synuclein (Fisher et al, ADPD2011); and iv) AF102B decreased CSF Abeta in AD patients (Nitsch et al, 2000). AF710B (nM range, in vitro) decreased Abeta, Tau-hyperphosphorylation, GSK3beta activation, and prevented apoptosis and mitochondrial dysfunction via increased Bcl2/Bax. AF710B was a highly potent cognitive enhancer (rats: 1-30 and 10100 mcg/kg, po in trihexyphenidyland MK801-induced passive avoidance impairments, respectively). AF710B had also an unprecedented safety margin (> 50,000; po). Furthermore, in female 3xTg-AD mice AF710B (10 mcg/kg, ip/daily for 2 months) – i) mitigated cognitive impairments in Morris water maze; ii) decreased BACE1, GSK3beta activity, p25CDK5, neuroinflammation, soluble and insoluble Abeta40, Abeta42, plaques and tau pathologies. AF710B differs from any sigma1, M1 muscarinic (allosteric, orthosteric or bi-topic) and sigma1/M1 agonists, respectively. AF710B induces a synchronized Sig1R activation and M1 muscarinic allosteric/bi-topic modulation via super-sensitization of M1mAChR, through a hypothetical heteromerization with Sig1R.

21 Concomitant activation of G-protein coupled receptors (GPCR) and sigma1 receptor (Sig1R): a novel pharmacological concept in treatment of Alzheimer's disease (AD)

The M1 muscarinic agonists AF102B (Evoxac: prescribed in Sjogren’s syndrome) and AF267B {successful in Phases I and IIa (Sjogren’s syndrome)} are effective cognitive enhancers and disease modifiers with a wide safety margin. Notably, i) AF102B decreased CSF Abeta in Alzheimer’s disease (AD) patients (Nitsch et al, Ann Neurol 2000); ii) AF267B rescued cognitive deficits and decreased Abeta42 and tau pathologies in 3xTg-AD mice (Caccamo et al, Neuron, 2006); and iii) in transgenic mice overexpressing human alpha-synuclein, AF102B and AF267B decreased brain alpha-synuclein aggregates (A. Fisher, E. Mazliah, B. Hutter-Paier, D. Havas, M. Windisch, ADPD 2011). Thus the M1 muscarinic GPCR appears to be a pivotal target for treatment of AD, Parkinson’s disease (PD) and Lewy body dementia (LBD). We have also hypothesized that concomitant activation of such a GPCR and the molecular chaperone Sig1R may have broader therapeutic implications in AD and related diseases. To achieve such combined activity we designed a low MW (357.5) compound, AF710B. This compound shows a novel mechanism of enhancing neuroprotection and cognition via Sig1R activation and M1 muscarinic modulation, but not resembling sigma1, M1 muscarinic (allosteric or orthosteric) and dual sigma1/M1 agonists, respectively. The effects of AF710B at low concentrations in vitro against neurodegeneration, oxidative stress, Abeta, Tau-phosphorylation and GSK-3beta activation translate into down-regulation of the apoptotic protein Bax and mitochondrial dysfunction, up-regulation of antiapoptotic Bcl2 and possible modulations of downstream kinases (e.g. PKC, PKB, GSK-3beta). AF710B has an exceptional pharmacology being an excellent cognitive enhancer in rats (at 1-30 and 10-100 mcg/kg, po in trihexyphenidyland in MK801-induced passive avoidance impairments, respectively). Furthermore, AF710B is devoid of side effects, having an unprecedented safety margin 50000 (po). In summary, while M1 agonists can alter pathologies in AD, PD and LBD, the unique profile of AF710B indicates widespread therapeutic advantage, inter alia, also on mitochondrial dysfunctions in these and other protein-aggregation related diseases. 22 NPT002: A NOVEL APPROACH FOR TARGETING -AMYLOID AND TAU AGGREGATES IN ALZHEIMER’S DISEASE

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contextual memory was also measured. Conclusions: We have identified a number of development candidates aimed at targeting either improved symptomatic therapy or disease slowing. This ‘multiple shots on goal’ approach using multiple platforms and multiple targets will help to increase the likelihood of delivering novel therapeutics to patients. Ultimately, the success of our programs, will be judged by our ability to deliver novel therapies to patients with significant positive impact on their lives, and a slowing, halting or perhaps even reversing of this devastating disease process.