Review article

Muscarinic And Nicotinic

Acetylcholine Receptor
Agonists:
Current Scenario In
Alzheimer’s Disease Therapy
Stuti Vermaa, Ashwini Kumara,
Timir Tripathib & Awanish Kumara

Vol 70-Issue8
August 2018
INTRODUCTION
ALZHEIMER’S DISEASE (AD)
• Chronic neurodegenerative disease
• Major cause of dementia
• Symptoms –
– Memory loss
– Disorientation
– Language problems
– Behavioural issues
• Incidence increase with ageing
• Diagnostic procedures –
– Cognitive testing
– Medical imaging
– Blood tests
 Characterized by
• Extraneuronal amyloid plaques
containing Aβ
• Loss of synaptic transmission
• Intraneuronal neurofibrillary tangles
(NFTs)
• Degeneration of cholinergic neurons
 A presynaptic cholinergic
hypofunction - major consequence of AD
 So cholinergic replacement therapy-
beneficial in alleviating the cognitive
dysfunction.
• AChE inhibitors & NMDA antagonist –
success in restoration of Ach levels
• AChR agonists- in future effective
treatment of the AD
• AChE inhibitors- good therapeutic
effects
– but their adverse effects
– Inhibition of the cholinergic autoreceptors
by released acetylcholine
– limit the efficacy of AChE inhibitors
 Muscarinic acetylcholine
receptors or mAChRs
AChRs
Nicotinic acetylcholine
receptors or nAChRs
• mAChRs-various locations including CNS
• Form G-protein receptor complexes
in certain neurons
• Responsive to the natural compound
muscarine
• Memory, motor control and learning
• mAChR M1, M2, M3, M4 & M5.
• M1, M3 & M5 coupled with G-proteins -
Gs, Gq/G11& Gp
• M2 & M4 coupled with Gi & Gk.
• Gs stimulate cAMP  ↑ed levels of cAMP
• Gi inhibit cAMP ↓ed levels of cAMP
• Gq/G11 and Gp - G-proteins-coupled to
phospholipase C & phospholipase A2,
resp
• M1-type mAChRs - hippocampus &
cerebral cortexcentral role in
cognitive processing, memory and
impaired in the AD
• These cholinergic signal deficits -in the
AD brain can be restored via
cholinergic activation
• M1–M4 AChRs ↓ed in cortex &
hippocampus region in AD brain
• On the basis of action,mAChRs in the
CNS

M1/M3 & M2/M4

• M5 - very weak in the brain
• ↓ in M2 receptors reduction in β-
secretase enzyme
activityaugmentation of β-amyloid
peptides in brainchange in
• M1/M3 receptors – cognition effect
• Stimulation of M1/M3 receptors 
γ-secretase activity ↑  less β-
amyloid peptide formation
• nAChRs(Nicotinic receptors) - ligand-
gated ion channels - 5 protein
subunits
• Sensitive to the compound nicotine
• nAChRs Neuronal type & muscle-
type
• 3 AChE inhibitors &
single NMDA
antagonist
currently used for
the treatment of
AD

• mAChRs and
nAChRs used as a
potent drug targets
in the AD
treatment with
mAChR & nAChR
agonist
Nicotinic
acetylcholine
receptors:
potent drug targets
in AD
• mAChRs - a potent target in the AD
• The amyloid plaque formation in
the AD  ↓ the ability of these
receptors to transmit the cholinergic
signals  results in disabled
cholinergic activity.
• Muscarinic agonists developed to treat
the AD
• They display neurotrophic effect
• Decrease b-amyloid plaque deposition
• Improve oxidative stress-induced
damage
• Hyperphosphorylation of tau protein is
decreased
• Cholinergic functions enhanced

 M1 muscarinic receptor impaired in

neocortex region of the patients with AD
• Severity of cognitive symptoms in the
AD  related to the degree of M1/G-
protein uncoupling
• Activation of muscarinic receptors shift
the APP processing toward
nonamyloidogenic pathway
• Experiments conducted on M1 mAChR
knockout mice ↑ed amyloid
processing of APP
• Suggest the role of mAChRs in the
processing of APP.
• Activation of M1 mAChRs reduces tau
hyperphosphorylation via
 Glycogen synthase kinase-3b (GSK-3b)
inhibition
 Increased ERK activation
 & potentiation of NMDA receptor

• Neurotransmitter ACh binds to

muscarine receptors (mAChR)
initiate a cascade of reactions
involved in memory and cognition
• nAChR
• α7-nAChR – hippocampus - most
affected area in case of the AD
• Present in presynaptic regions –
modulates neurotransmitter release.
• a7nAChR - involved in cognitive
functions
• Inhibits the formation of β-amyloid
• Promotes a-secretase cleavage
of APP
• Interaction b/w a7-nAChR & β-amyloid
fragments
– neurotoxic effects through blocking of a7-
nAChR
– depletion & inactivation of nAChRs
• Αβ peptides impair cognitive potential of
hippocampus of rats infused with β-
amyloid through nAChR-dependent
mechanism
• Bednar et al.- in vivo - ↑ in nAChR
antagonist binding  ↑ed impairment
in learning & memory & accelerated
amyloid-β pathology
• Lombardo & Maskos - Rat hippocampal
neurons
– Aβ inhibits a7 nAChRs in higher
concentration
– But Aβ activated nAChR in low
concentrations.

 So nAChR agonist can be used

against AD
Muscarinic and Nicotinic
acetylcholine receptors
agonist &
their therapeutic potential
• Initial M1 mAChR allosteric agonists
- AC-42 & 77-lH-28-1
• AC-42 activated M1 at the region
unconserved in other mAChR types
o failed in vivo because of its inactivity in
native brain slices
• Brucine - initial PAM for M1 mAChR
• Xanomeline - as M1 mAChR agonist
o Improved cognitive functions in AD
patients
o But less selectivity, Gastrointestinal
adverse effects ,Poor physiochemical
characteristicsfailed in the clinical trial
• More selective novel agonists - TBPB, 77-
LH-28-1, AC-260584
• TBPB activated M1 mAChR  induced
potentiating of neurons carrying NMDA
receptor in an M1-dependent pathway
• 77-LH-28-1 selectively activates M1 but
also partially activates M2,M3 & M4 in
high conc
o It can bind to orthosteric site & to the
allosteric site & modulate affinity at the
orthosteric site  bitopic
ligand for M1 receptor
o It penetrated brain after s.c administration 
enhanced NMDA-mediated neuronal
excitation
• 4-phenylpyridine-2-one derivatives -
highly potent PAMs for M1 receptor.
• Muscarinic agonists
– Improved cognitive functions in the patients
– But could not complete the clinical trials as
they were nonspecific & activated other
receptor subtypes
• Selective M1 agonists producing
cellular excitation
 beneficial in the AD
• Cholinergic stimulation of M1 mAChR
agonists
– Prevents the formation & deposition of
amyloid plaques &
– Decreases tau hyperphosphorylation
• Activating cholinergic signaling 
beneficial in cognitive treatment,
delaying the onset of the disease
• Most of the potent muscarinic agonists
tested
– Lacked specificity for specific mAChRs 
reduction in their efficiency and efficacy
– Displayed various central and peripheral
adverse effects - hypothermia, depression &
cardiovascular events.
• M1 selective agonists - AF102B,
AF267B, AF150 & AF292 tried on
patients with AD
• The lead compound AF267B exhibited
excellent PK properties & penetrated
BBB even with oral administration
• AF102B, AF150 & AF267B
– significant neurotrophic effects
– elevated nonamyloidogenic APP pathway
– decreased beta-amyloid deposition
Effect of mAChR agonist AF102B on the expression of mAChR.
Activation of mAChR by neurotransmitter ACh -a cascade of reactions.
Activated PKC activates ERK1/2, followed by a-secretase (A) and c-
secretase (G) release which leads to the formation of the
nonamyloidogenic fragment.
PKC also blocks b-secretase (B) which leads to the formation of pathogenic
• 77-LH-28-1, a Glaxo SmithKline M1
allosteric candidate
– Good pharmacological profile
– effective CNS penetration
• M1 selective agonist lead candidates
VU0364572 & VU0357017
– tested on cell lines & animal models
– Exhibited effective properties on various
parameters
• Study by Lebois et al
– half-life of VU0364572 - 45 min
– VU0364572 preserved hippocampal memory
& reduced burden of pathogenic b-amyloids
in the experimental 5XFAD AD mouse models
• Agonists for M1 mAChR being tested (or failed)
–
– arecoline, CI-1017, xanomeline, tazomeline,
talsaclidine, milameline
– little selectivity for the M1 subtype
• Presence in the brain
– M2 – throughout the brain
– M3 and M4-in cortex and hippocampus
– M5 - substantia nigra pars compacta,
hypothalamus & ventral tegmental region.
• Such varying expression profile of mAChRs in
CNS makes them unique & potent drug
targets for cognitive dysfunctions
• Positive allosteric modulator ML380
binds to M5 mAChR
• AC-260584 - potent & selective M1
mAChR agonist & partial M2 agonist
– Has procognitive effects mediated by M1
mAChR-dependent ERK1/2 phosphorylation
in CNS regions involved in memory and
learning processes.
– Supported by the experiments
performed on M1 mAChR knockout
mice - exhibited no effect of AC-260584
• In-vitro- selective M4 agonists
VU0152099 & VU0152100
– Bind to an allosteric site on the M4 receptor
– Increase its affinity for ACh & coupling to G-
proteins
– They do not directly increase mAChR
expression
• 2 decades ago- an experimental
candidate CI-1017
– Improved spatial memory
– Exhibited dose-limiting adverse effects only
at efficacy level 100-fold
– a possibility of improving cognition process
related to cholinergic activity
• The Merck candidate MK-7622, an M1
muscarinic receptor agonist
– Halted prematurely at phase 2 trials with
no reasons found
• Nicotinic receptors (nAChRs) - a7 and
a4b2 subtype expressing neurons -
damaged in patients suffering from AD
• EVP-6124 (Encenicline), a7 nicotinic
receptor agonist
– developed by Bayer Pharmaceuticals &
evaluated by FORUM pharmaceuticals
– put on hold during phase 3 trial due to
severe gastrointestinal adverse effects
• a7-nAChR agonists AZD0328 and
SSR180711
– in low doses upregulated a7-nAChR in
neocortex and hippocampus  increased
cognition in mice model 
• Preclinical studies with A-582941 in a
mice model 
– cognition completely restored to the level of
age-matched wild-type mice
• nAChR agonists are
neuroprotective in a mechanism
that is Ca2+-dependent and that
involves the glutamatergic system
• Benzyl quinolone carboxylic acid
(BQCA)- PAM for the M1 mAChRs
– Enhance the memory function
– Reverses cognitive impairment
– Acts as an allosteric activator  increases
the affinity of M1 receptor for the
physiological ligand Ach
– Animal study - BQCA promoted the
nonamyloidogenic APP processing
pathwaydisease-modifying effect
– BQCA had no effect on other subtypes of
mAChRs
– Pharmacokinetic study of BQCA (at 10
mg/kg) – rapid distribution in brain an easy
passage through BBB, with the maximum
concentration (~8000 ng/ml) at 1.5 h after
an intraperitoneal injection that was
sustained up to 3–4 h
– Highest concentration of BQCA after 8 h in
kidney  renal excretion - major
route of drug elimination
• Heptares Therapeutics- xanomeline
(HTL9936)
– again in the phase 2 clinical trial against AD
– newly modified molecule designed using
their StaR_ technology
– Xanomeline earlier withdrawn from clinical
trial due to adverse events like
gastrointestinal effects & syncope
• Initiation of phase 1 clinical trial
with an M4 selective agonist
HTL0016878 for AD
CONCLUSION
• AChE inhibitors have been the mainstays
of Alzheimer’s therapy
• Various researches conducted on
activating the cholinergic receptors in AD
brain through the development of
various agonists
• The two ACh receptors(mAChR and
nAChR) are associated with neural
regeneration and decreasing b-amyloid
concentration
• Computational drug designing- for drug
development

Structure-based Ligand-based
drug designing
drug designing
Target structure chemical
as the basis of structure of
designing drug ligand and
candidates position of
functional groups
as the basis
• Ligand-based drug designing is based on
manipulation of the existing drugs which
have been withdrawn at various
developmental stages
• It is a huge time saver & provide a great
deal of information for the novel
candidates
• The major difficulty with
developing cholinergic agonists
for AD is the adverse effects
due to cross-reactivity at sites
other than CNS.
• So effective targeting is
essential element.
• Can minimize adverse effects
with targeted therapy to CNS.
• Cholinergic activation is
very essential for
alleviating the declining
cognitive functions in AD
• Development of better
cholinergic agonists is the
need of the hour for the
effective treatment of AD.
• M1 mAChR is the most
sought after target
against AD
• Several M1 and nAChR
agonists are in different
experimental, preclinical
and clinical phases
• Still need a long way to go
before making it into the
market.