3. Pharmacology
          3.8. Acetylcholine-related drugs
 3.8.1. Anticholinesterase


[SH4:p251-p262; CEACCP 2004 Vol 4(5) "Anticholinesterase and anticholinergic drugs"]


Anticholinesterase include:

  • Edrophonium
  • Neostigmine
  • Pyridostigmine
  • Physostigmine


  • Physostigmine is a natural alkaloid derived from the Calabar bean


Acetylcholinesterase (AChE)

  • Acetylcholinesterase consists of an anionic and an esteratic site
  • Anionic site binds to the quaternary nitrogen in ACh
    --> Orientate the ester link of ACh to the esteratic site
  • Esteratic site hydrolyse ACh
  • Located at:
    * High concentration at NMJ
    * Lower concentration throughout skeletal muscle fibres
    * Encoded by a single gene on chromosome 7q22
  • Acetylcholinesterase (AChE) is one of the most efficient enzymes known
    * Each hydrolyse 300,000 molecules of ACh per minute [SH4:p251]
    * Or 4,000 molecule per second [SH4:p253]
    * 50% of the released ACh during the time of diffusion across the synaptic cleft
    --> Near diffusion-limited
  •  May also have other functions
    * Nerve growth-promotion
    * Modulation of nAChRs

Structure-activity relationship

  • Linking two quaternary ammonium nuclei by a chain of proper length
    --> Increase in anticholinesterase potency and duration
  • Neostigmine, pyridostigmine, and edrophonium are quaternary amine
    --> Does not cross BBB
    --> Effect is mostly peripheral stimulation of nAChRs and mAChRs
  • Physostigmine is tertiary amine
    --> Can cross BBB
  • Organosphates are very lipid soluble
    --> Can cross BBB


Mechanism of action

  • Enzyme inhibition
  • Presynaptic effects
  • Direct effects on neuromuscular junction (NMJ)

Overall effect is effectively stimulation of the cholinergic system

Enzyme inhibition

  • Inhibition of acetylcholinesterase --> Increased ACh in:
    * Preganglionic sympathetic nerve endings
    * Parasympathetic nerve endings
    * NMJ
  • Neostigmine and pyridostigmine are hydrolysed by AChE
    --> AChE is carbamylated
    --> Decreased ability to hydrolyse ACh
  • Edrophonium is NOT hydrolysed by AChE
    * It forms reversible electrostatic attachment to AChE


  • The differences in mechanisms have little clinical implication
Different mechanisms of AChE inhibition
  • Reversible inhibition
  • Formation of carbamyl esters
  • Irreversible inactivation by organophosphate
Reversible inhibition

e.g. Edrophonium

  • Lacks carbamyl group
  • Reversible inhibition of AChE by electrostatic attachment to the anionic site on the AChE enzyme
    --> Edrophonium-AChE complex prevents ACh binding to AChE
  • Due to reversibility of the inhibition
    --> ACh competes with edrophonium
    --> Increase in ACh concentration increases access to AChE
    --> Thus duration is short
  • Predominantly presynaptic action
    * c.f. Slower-onset anticholinesterases (e.g. neostigmine and pyridostigmine) which are predominantly postsynaptic
  • Muscarinic effects are mild
    * c.f. Longer-acting anticholinesterases
  • Clinical use:
    * Antagonise effects of non-depolarising NMBDs
    * Diagnosis and assessment of therapy adequacy in myasthenia gravis and cholinergic crisis
    * Evaluation the presence of dual blockade produced by suxamethonium
Formation of carbamyl esters

e.g. Neostigmine, pyridostigmine, physostigmine

  • Reversible inhibition of AChE by formation of a carbamyl ester complex at the esteratic site on the AChE enzyme
  • The carbamylated AChE cannot hydrolyse ACh until the carbamate-enzyme bond dissociates
  • Carbamylated AChE has a half-time of 15-30 minutes
Irreversible inactivation
  • Organophosphate combines with AChE at the esteratic site
    --> Stable inactive complex (i.e. does not hydrolyse)
  • Echothiophate interacts with BOTH the esteratic site and anionic subsites
    --> Extreme potency
    * Only one used clinically
  • Other organophosphate (e.g. parathion, malathion) are used as insecticides


  • Malathion is a selective insecticide
    * Enzyme necessary for its metabolism is absent in insects
  • Malathion is hydrolysed by phosphorylphosphatases --> Excreted in urine


  • Nerve gases (tabum, saran, soman) are extremely lipid-soluble
    --> May be absorbed through intact skin

Presynaptic effects

  • An anticholinesterase may produce spontaneous contractions (fasciculations) of skeletal muscles
    * Only in the absence of non-depolarising NMBDs
  • Fasciculations may be due to direct stimulation of presynaptic nAChRs
    --> Increased availability of ACh

Direct effects on NMJ

  • Anticholinesterase may produce some form of NMJ blockade
    * But only at doses far greater than administered clinically
  • Possible mechanism
    = Excess of ACh at NMJ --> Desensitisation block

Dose-response curve

  • Dose-response curve are parallel between neostigmine and pyridostigmine
    --> Similar mechanism
  • Dose-response curve for edrophonium is NOT parallel with that of neostigmine or pyridostigmine
    --> Different mechanism of action for edrophonium
    * Dose-response curve for edrophonium is flatter
  • When the NMJ blockade is still intense at the time of the reversal being initiated
    --> Dose response curve is shifted to the right
    * The shift is more important for edrophonium and pyridostigmine (than for neostigmine)
    * Neostigmine is preferable when antagonising >90% twitch depression [SH4:p258]


  • Dose-response curve for neostigmine is to the left of pyridostigmine
    --> At a given response, less neostigmine is required
    * i.e. Neostigmine is more potent
  • But when combined, the effects of edrophonium and other anticholinesterases are only additive
    --> Suggesting a similar mechanism [SH4:p258]
  • Potency ratio between anticholinesterase drugs depends on:
    * The non-depolarising NMBD being antagonised (and its inherent speed of spontaneous recovery)
    * Depth of NMJ blockade when reversal is initiated
    * The selected end-point

Ceiling effect

  • Once acetylcholinesterase is maximally inhibited
    --> Additional anticholinesterase will not further antagonise nondepolarising NMJ blockade
  • Persistent NMJ blockade despite large doses neostigmine (70mcg/kg)
    --> Indication for further mechanical ventilation

Effects by systems

  • The effects are due to accumulation of ACh at muscarininc and nicotinic cholinergic receptors
  • Muscarinic cholinergic effects are evoked by lower concentration of ACh than are required for nicotinic effects at autonomic ganglia and NMJ
  • Co-administration of an anticholinergic drug to prevent adverse muscarininc cholinergic effects
    * Anticholinergic drugs selectively block muscarinic cholinergic receptors


  • Anticholinesterases
    --> Increase the amount of ACh available at NMJ
    --> NMJ are more likely to bind with ACh than with non-depolarising NMBDs
    --> Reversal of NMJ blockade
  • In overdose, anticholinesterases
    --> Excessive amount of ACh at NMJ
    --> Depolarisation NMJ blockade


  • Bradycardia
    * Most likely due to slowing of the conduction at AV node (and slowed rate at SA node)
    * No change in ventricular conduction, and contractility [SH4:p262]
    * Denervated heart (e.g. in heart transplant) are exquisitely sensitive to the bradycardic effect of neostigmine
  • Possible decrease in BP
    * Due to decrease in systemic vascular resistance


  • Smooth muscle fibres of bronchioles and ureters are contracted
    --> Can potentially produce bronchoconstriction
  • Increased tracheobronchial secretions



  • Increased gastric secretion (by parietal cells)
  • Increased motility (especially in large bowel)
    * Due to accumulated ACh acting on ganglion cells of Auerbach's plexus and on smooth muscle fibres
  • Increased incidence of post-operative nausea and vomiting (PONV)
    * Especially neostigmine [CEACCP 2004 Vol 4(5):p166]
  • Sometimes used to treat achalasia
  • At high doses
    --> Vomiting, diarrhoea, and incontinence


  • Increase production of secretions innervated by postganglionic cholinergic fibres
    * e.g. bronchial, lacrimal, sweat, salivery, gastric, intestinal, and acini pancreatic gland


  • Sweat glands are cholinergic, even though they are innervated by sympathetic nervous system



When topically applied to the eye, anticholinesterase can cause:

  • Constriction of iris sphincter
    --> Miosis
  • Constriction of ciliary muscle
    --> Inability to focus for near vision
  • Intraocular pressure (IOP) declines because outflow of aqueous humour is facilitated


  • Interference with accomodation is usually shorter in duration than miosis
  • Usually topically applied


Myasthenia gravis
  • In patients with myasthenia gravis, who received non-depolarising NMBDs
    * Giving anticholinesterase drugs (to reverse the NMJ blockade) is associated with a risk of cholinergic crisis
    * [SH4:p257]
    * ??? why
Plasma cholinesterase activity
  • Neostigmine and pyridostigmine produce prolonged and marked inhibition of plasma cholinesterase
    * Edrophonium does not inhibit plasma cholinesterase


  • Normal renal and hepatic function
    --> No significant different between anticholinesterase drugs
  • Similar pharmacokinetics
    --> Differences in potency are due to pharmacokinetic reasons


Lipid solubility

  • Anticholinesterase containing quaternary ammonium group are poorly lipid soluble
    * i.e. edrophonium, neostigmine, pyridostigmine
    --> Poor GIT absorption and unpredictable CNS effect
  • Lipid soluble anticholinesterase
    * i.e. tertiary amine (physostigmine) and organophosphates
    --> Good GIT absorption and predictable CNS effects


Volume of distribution (Vd)

  • Quaternary ammonium anticholinesterases have a large Vd (0.7-1.4 L/kg)
    * Compared to nondepolarising NMBDs and  considering their low lipid solubility
    * May be due to extensive tissue storage in liver and kidney


  • In the absence of renal function, hepatic metabolism account for
    * 50% of the clearance of neostigmine
    * 30% of the clearance of edrophonium
    * 25% of the clearance of pyridostigmine
  • Physostigmine --> Hydrolysed at ester bond
    * By plasma esterase [CEACCP 2004 Vol 4(5):p166]


  • Metabolised by plasma esterase to a quaternary alcohol [CEACCP 2004 Vol 4(5):p166]
  • Rest (which is 50% [SH4] to 60% [CEACCP]) is excreted unchanged in urine


  • None of the metabolites contribute significantly to activity
  • Principle metabolite for neostigmine = 3-hydroxyphenyltrimethylammonium
    --> About 1/10 the activity of the parent compound
  • Principle metabolite for pyridostigmine = 3-hydroxy-N-methylpyridinium
    --> Inactive
  • Principle metabolite for edrophonium = edrophonium glucuronide (conjugation)
    --> Inactive


Renal clearance

  • Anticholinesterase drugs are actively secreted into the renal tubular lumen
    --> Thus clearance is higher than GFR
  • Neostigmine
    --> Renal clearance accounts for 50% of elimination
  • Edrophonium and pyridostigmine
    --> Renal clearance accounts for 75% of elimination
  • In renal failure, elimination of anticholinesterase is more prolonged than nondepolarising NMBDs are
    --> Recurarisation unlikely
  • Physostigmine does NOT depend on renal function for elimination
    * Unlike other anticholinesterases [CEACCP 2004 Vol 4(5):p166]

Elimination half-lifes

[CEACCP 2004 Vol 4(5):p166]

  • Edrophonium = 110 min
  • Neostigmine = 77 min
  • Pyridostigmine = 113 min


  • Edrophonium = 110 min
  • Neostigmine = 77 min
  • Pyridostigmine = 112 min


  • ??? Not sure why edrophonium has longer half-life, yet duration of action is shorter than, or about the same as neostigmine. I wonder if it has anything to do with the competitive nature of the antagonism by edrophonium .

Action profile

  • Edrophonium, neostigmine, and pyridostigmine
    * Reach peak and decrease rapidly within 5-10 minute
    * Vd = 0.7 - 1.4 L/kg
    * Elimination half-time = 60 - 120 minutes
    * Clearance = 8 - 16 mL/kg/min (much greater than GFR)

Onset of action


  • Onset of action for edrophonium = 1 - 2 minutes
  • Onset of action for neostigmine = 7 - 11 minutes
    * Initial onset is about 1 min, but peak action is in around 10 minute [CEACCP]
  • Onset of action for pyridostigmine = 16 minutes


  • Faster onset of action for edrophonium may be due to its predominately presynaptic action
    * c.f. neostigmine and pyridostigmine exert predominantly postsynaptic action
    * i.e. action on ACh release instead of acetylcholinesterase inhibition
  • Slower onset for neostigmine and pyridostigmine are NOT related to the need to form active metabolites

Duration of action

  • Duration of action of anticholinesterase are related to its plasma clearance
  • e.g. Half-time of carbamylated enzymes are 15-30 minutes
    --> Shorter than the elimination half-time of anticholinesterase (60-120 min)
  • Edrophonium, neostigmine, and pyridostigmine have similar duration of action [SH4:p258]
    * [SH4:p255, fig 9-9] definitely showed difference in duration of action (Pyridostigmine > Neostigmine > Edrophonium)
    * [SH4:p254, tab 9-1] also showed duration Pyridostigmine > Edrophonium > Neostigmine
  • [SH4:p254]
    * Edrophonium duration of action = 60 min
    * Neostigmine duration of action = 54 min
    * Pyridostigmine duration of action = 76 min
  • [CEACCP 2004 Vol 4(5) "Anticholinesterase and anticholinergic drugs"]
    * Edrophonium duration of action = 10 minutes
    * Neostigmine duration of action = 20-30 minutes
    * Pyridostigmine duration of action = 360 minutes


  • In the past edrophonium was considered a short-acting drug
    --> Apparently controlled studies showed not much difference in duration with neostigmine
    * [SH4:p255]



  1. Reversal of NMJ blockade produced by nondepolarising NMBDs
  2. Treatment of certain drug-induced CNS effects
  3. Treatment of myasthenia gravis
  4. Treatment of glaucoma
  5. Treatment of mild-to-moderate Alzheimer's disease
  6. Other uses
    * Treatment of paralytic ileus and atony of urinary bladder
    * Analgesia (used intrathecally or epidurally)
    * Diagnosis of cardiac dysrhymthia (paroxysmal supraventricular tachycardia)
    * Treatment of post-operative shivering

Reversal of nondepolarising NMJ blocade

  • Edrophonium, neostigmine, or pyridostigmine are used to increase availability of acetylcholine at NMJ
  • Physostigmine is not used for this purpose due to the excessive dosage required
  • Neostigmine is preferable when antagonising >90% twitch depression [SH4:p258]
  • Effect is dose-related
    * With a ceiling effect


  • Due to the predominantly presynaptic action of edrophonium
    --> Train-of-four ratio is higher after edrophonium than after neostigmine or pyridostigmine
Co-administration of anticholinergic drugs
  • Usually given with anticholinergic drugs (atropine or glycopyrrolate)
    --> Attenuation of the unwanted muscarinic effects
  • Usually the anticholinergic drugs chosen need to have faster onset to minimise risk of bradycardia


  • Edrophonium (fastest in onset) is usually given with atropine (faster onset than glycopyrrolate)
  • Neostigmine + atropine
    --> Early tachycardia and late bradycardia is more likely
    * Due to faster onset time and shorter duration of action of atropine
  • Neostigmine + glycopyrrolate
    --> HR more stable, and late bradycardia is less likely
    * Glycopyrrolate action profile more closely matches neostigmine
Factors influencing reversal of NMJ blockade
  • Intensity of NMJ blockade at the time of reversal
  • The nondepolarising NMBD used
  • End-point selected
  • Other factors [SH4:p259]
    * Certain antibiotics
    * Hypothermia
    * Respiratory acidosis (pCO2 > 50 mmHg)
    * Hypokalaemia and metabolic acidosis


  • Edrophonium is less effective than neostigmine in reversing deep NMJ blockade
  • Edrophonium is more effective against atracurium
  • Neostigmine is more effective against vecuronium

Treatment of certain drug-induced CNS effects

Central cholinergic syndrome
  • Physostigmine is used to treat central cholinergic syndrome due to atropine or scopolamine
    * Physostigmine 15-60 mcg/kg IV
  • Duration of action for physostigmine is shorter than anticholinergic drugs
    --> Repeated dosing may be necessary
  • Physostigmine may reduce postoperative somnolence after anaesthesia with a volatile anaesthetic agent
  • Physostigmine may reduce the somnolent effect of opioids

Treatment of myasthenia gravis

  • Neostigmine, pyridostigmine, and ambenonium
    --> Standard anticholinesterase drugs used in symptomatic treatment of myasthenia gravis
    * Presumably improve muscle response by increasing ACh availability
  • Due to quaternary ammonium structure in neostigmine and pyridostigmine
    --> Poor oral absorption
    --> Oral dose of neostigmine is 30 times the IV dose
  • Pyridostigmine is longer duration of action
    --> Used in treatment of myasthenia gravis
Testing the adequacy of anticholinergic drug therapy
  • Edrophonium is used to test the adequacy anticholinergic drug therapy
    --> Edrophonium 1mg IV every 1-2 minutes
  • If symptom improves
    --> Anticholinergic therapy has been inadequate
  • If increased muscle weakness (due to cholinergic crisis)
    --> Therapy adquate
  • When used to diagnose myasthenia gravis
    --> Edrophonium 2mg followed by 8 mg IV 30 seconds later [CEACCP 2004 Vol 4(5):p166]

Treatment of glaucoma

  • Anticholinesterase drugs decreases intraocular pressure in narrow-angle and wide-angle glaucoma
    * Due to a decrease in resistance to outflow of aqueous humour
  • Longer term treatment (>6 month) with topical long-acting anticholinesterase (e.g. echothiophate, demecarium, isoflurophate)
    --> Risk of cataracts
  • Prolonged use of ecothiophate and physostigmine eye drops
    --> Risk of acquired cholinesterase deficiency
    --> Risk of prolonged NMJ blockade by NMBDs metabolised by this enzyme [CEACCP 2004 Vol 4(5):p167]
  • No risk of cataracts with shorter-acting anticholinesterases
  • In contrast, topical beta-adrenergic antagonists (e.g. timolol)
    * No miosis
    * Decrease IOP by decreases secretion of aqueous humour

Treatment of Alzheimer's Disease

  • Anticholinesterases are recommended for treatment of mild-to-moderate Alzheimer disease
  • Four centrally acting drugs are availabe:
    * Tacrine
    * Donepezil
    * Rivastigmine
    * Galantamine
  • Tacrine is rarely used due to hepatotoxic effects in nearly 40% of patients
  • Donepezil is effective once daily dosing, without hepatotoxicity

Treatment of post-operative shivering

  • Physostigmine 40mcg/kg IV
  • Similar efficacy to pethidine and clonidine

Postoperative analgesia


  • Neostigmine 50-100mcg intrathecally or 1-4mcg/kg epidurally
  • High incidence of N&V, pruritus, and prolonged block
  • No respiratory depression
  • No neurotoxicity with intrathecal administration of neostigmine with paraben preservative



  • Edrophonium = 0.5 mg/kg IV
    + Atropine 7 mcg/kg IV
  • Neostigmine = 0.043 mg/kg IV
    + Atropine 20 mcg/kg IV OR Glycopyrrolate 10 mcg/kg IV
  • Pyridostigmine = 0.35 mg/kg IV
    + Atropine 20 mcg/kg IV OR Glycopyrrolate 10 mcg/kg IV

[CEACCP 2004 Vol 4(5):p166]

  • Edrophonium = 1 mg/kg IV
  • Neostigmine = 0.05-0.07 mg/kg IV
  • Pyridostigmine = 0.1 mg/kg IV (for myasthenia gravis)



Overdose produces two types of symptoms: muscarinic and nicotinic

  • Muscarinic symptoms include:
    * Miosis
    * Difficulty focusing (eye)
    * Salivation
    * Bronchoconstriction
    * Bradycardia
    * Abdominal cramps
    * Loss of bladder and rectal control
  • Nicotinic symptoms include:
    * Skeletal muscle weakness (may progress to paralysis)
    * CNS actions (confusion, ataxia, seizures, coma, respiratory depression)

Treatment of anticholinesterase overdose (including organophosphate)

  • Atropine
    * Antagonise muscarinic effects, but not nicotinic effects
    * Does not reactivate AChE
  • Pralidoxime (an AChE reactivator)
    * Occasionally used to supplement atropine
  • 15 mg/kg IV over 2 minutes, repeat again after 20 minutes
  • More useful in countering the NMJ effect (nicotinic effect)
  • More useful in countering drugs that phosphorylate AChE (e.g. organophosphate)
  • Not as useful in countering drugs that carbamylate AChE (e.g. neostigmine)
  • Only effective if administered with minutes after exposure
    * Before formation of irreversible bonds
    * The inactivated phosphorylated AChE is stable, and stability is enhanced by a process called "ageing"
    * [CEACCP 2004 Vol 4(5):p167]
  • Not useful against CNS effects

Special considerations

Patient age

  • Neostigmine in infants and children
    --> Faster onset and greater effect
    * Difference is due to pharmacodynamic reasons
  • Edrophonium
    --> No difference for infant, children, and adults
    * Supports the idea that different mechanisms are involved
  • In elderly
    --> Duration of action by neostigmine and pyridostigmine are prolonged
    * Due to smaller ECF and slower plasma clearance
    * i.e. due to pharmacokinetic changes
    * Pharmacodynamics are not changed
  • In elderly
    * Duration of action is not changed
    * But a higher plasma concentration is required to produce the same effect (compared to in younger patients)

Other related drugs

Organophosphate compounds

Also see "Irreversible inactivation" under "Metabolism"

[CEACCP 2004 Vol 4(5):p167]

  • Parathion and malathion are used as insecticides
  • Malathion is the main ingredient in dermatological preparation used in pediculosis treatment

Nerve gas

  • Tabun, sarin, VX, and soman are highly potent anticholinesterase
    --> Irreversible inactivation of AChE by alkylphosphorylation

Triphasic clinical syndrome

  • Initial cholinergic phase (24-48 hours)
  • Intermediate phase (4-18 days)
  • Third phase of delayed polyneuropathy (7-14 days after exposure)


[CEACCP 2004 Vol 4(5):p166]

  • Another short-acting anticholinesterase
    * Like edrophonium
  • Crosses BBB
  • Used in management of Alzheimer's disease in USA


[CEACCP 2004 Vol 4(5):p167]

  • Medium-duration anticholinesterase
  • Reversible anticholinesterase
  • Used to treat Alzheimer's disease
    * Once daily


[CEACCP 2004 Vol 4(5):p167]

  • Medium-duration anticholinesterase
  • Non-competitive reversible anticholinesterase
  • Used to treat Alzheimer's disease
    * Twice daily

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