1. Pharmacology of Cholinesterase Inhibitors and Nicotinic Antagonists
Dr. Thomas Abraham
PHAR 417: Fall 2004

2. Cholinesterase Inhibitors
Are generally carbamate derivatives or organophosphates

3. Cholinesterase Inhibitors
Ø      Potentiate the effects of acetylcholine released from cholinergic nerves by inhibiting acetylcholinesterase.
Ø      Acetylcholinesterase highly active enzyme that rapidly metabolizes acetylcholine to inactive products; usually found in high density in the synaptic cleft and nerve endings.   
Ø      Reversible cholinesterase inhibitors: edrophonium, neostigmine, physostigmine
-        physostigmine, neostigmine lead to carbamoylation of cholinesterase which prevents binding and metabolism of acetylcholine.
   
-         edrophonium competes with acetylcholine for binding to cholinesterase to prevent binding and metabolism of ACh.

4. Cholinesterase Inhibitors
Metabolism of Acetylcholine by Cholinesterase
Extremely rapid metabolism
Example of base hydrolysis of an ester.
Acetylcholine binds to enzyme active site via interactions with specific amino acid residues.
The acetate group is hydrolyzed by the addition of water to regenerate the active enzyme.

5. Cholinesterase Inhibitors
Inhibition of Cholinesterase by Carbamates
Carbamate derivatives bind to same active site of the cholinesterase as ACh.
Cleavage of the carbamate yields an amino alcohol and carbamoylated enzymes complex.
Rate of hydrolysis of the carbamoylated residue to generate the active enzyme is slower than that of the acetylated enzyme complex.
Thus the active site of the enzyme remains blocked for longer periods of time preventing endogenous ACh metabolism.

6. Cholinesterase Inhibitors
 Ø      Irreversible cholinesterase inhibitors: diisopropylfluorophospate (DFP), malathione, echothiophate, soman (GB), tabun.
-         lead to phosphorylation of serine residue in the cholinesterase active site; stable complex does not dissociate and endogenous ACh is unable to bind and be metabolized.   
-         aging of the phospho-complex leads to further stable bonds between drug and enzyme that permanently inactivates the enzymatic activity of the cholinesterase.
-         prior to aging of phospho-enzyme complex strong nucleophiles such as pralidoxime (2-PAM) can regenerate the enzyme active site by removing the phosphate group.
Ø      Lipophilicity of organophosphates allows rapid absorption from GI tract, lungs, skin and conjunctiva and non-polar phosphates distribute well to all parts of the body and partition into fat.

7. Cholinesterase Inhibitors
Inhibition of Acetylcholinesterase by Organophosphates
The organophosphates bind with high affinity to the serine residue resulting a phosphorylated enzyme complex.
This complex can undergo aging where one of the O—C bonds is broken and the chemical becomes even more resistant to hydrolysis.
Pralidoxime (2-PAM) is able to regenerate the active enzyme complex only when aging has not occurred.

8. Cholinesterase Inhibitors
Different rates of hydrolysis of the ester bond determine extent of Cholinesterase inhibition:
Diisofluorophosphate:
Very slow hydrolysis;
High, irreversible inhibition
Acetylcholine:
fast hydrolysis;
Low inhibition
Neostigmine:
Slow hydrolysis;
High, reversible inhibition

9. Cholinesterase Inhibitors
Ø      Pharmacological effects of cholinesterase inhibitors:
1.      Cardiovascular system – decreased heart rate, force (decreased cardiac output), small or no change in blood pressure.
2.      GI tract – increased motility, increased digestive secretions, flatulence, cramps, defecation.
  3.      Eyes – miosis, increased accommodation for near vision,increased then decreased intraocular pressure.
  4.      Respiratory tract – increased bronchial tone, increased mucosal secretions.
  5.      Central nervous system – increased alertness, convulsions, seizures, coma.
6.      Neuromuscular junction – increased muscle strength, faciculations, ataxia, tremors.

10. Cholinesterase Inhibitors
Ø      Therapeutic uses of cholinesterase inhibitors:
1.      Glaucoma – results from increased intraocular pressure due to poor drainage of aqueous humor through anterior eye and out through canal of Schlemm. Physostigmine, echothiophate, DFP enhance cholinergic responses to the iris to increase aqueous flow and decrease intraocular pressure.
2.      Improve GI and urinary motility – neostigmine (Prostigmine®) administered p.o. or s.c.   
3.      Improve neuromuscular transmission in myasthenia gravis – autoimmune disorder resulting in decreased nicotinic receptors at the motor end-plate. Edrophonium (Tensilon®) used as diagnostic test of myesthenic condition and for dose assessment. Neostigmine, pyridostigmine (Mestinon®) most often used for therapy.

11. Cholinesterase Inhibitors
4.      To reverse neuromuscular blockade after surgery: d-Tubocurarine, pancuronium induce paralysis that is overcome by enhanced ACh at the motor end-plate after neostigmine administration.   
5.      Atropine toxicity reversed by administration of physostigmine, especially CNS effects.
Ø      Toxicity related to cholinesterase inhibition:
1.      usually due to insecticides (malathione, parathione)
2.      nerve gases such as sarin (GB), VX irreversibly inhibit central and peripheral cholinesterase.
  3.      symptoms of intoxication include: salivation, lacrimation, urination, defecation, gas, emesis (SLUDGE). CNS excitation, neuromuscular blockade also seen.
   4.      atropine used to block the muscarinic receptors; pralidoxime used to regenerate the phosphorylated site on the cholinesterase.

12. Cholinesterase Inhibitors
Organophosphate intoxicants include insecticides (malathione, parathione) and “nerve gases” (Soman, tabun, VX)
Protection from the toxic effects of organophosphates requires covering all the potential access sites of the chemical:
Lungs
Mouth
Mucosal surfaces (eyes, nasal)
Skin
Chemical Warfare Suit
The autoinjector cartridge delivers both atropine and 2-PAM via IM injection
Autoinjector kit

13. Nicotinic Antagonists
Nicotinic receptors are divided into NN (on nerve cells in the ganglia) and NM (on skeletal muscle motor end- plates).
Ø      Divided into depolarizing and
nondepolarizing antagonists.

14. Nicotinic Antagonists
Depolarizing Antagonists
Ø      Depolarizing agents (decamethonium, succinylcholine) bind to the nicotinic receptor of the neuromuscular junction and activate the receptor to cause partial depolarization and contraction of the muscle.
-   Followed by flaccid paralysis when acetylcholine released from the motor neuron activates the receptor further.
-    the partially depolarized motor end-plate results in nicotinic receptor channels that are in the inactive state.
 - this form of paralysis cannot be overcome with increased concentrations of acetylcholine.

15. Nicotinic Antagonists
Nondepolarizing Blockers:
d-tubocurarine
pancuronium
vencuronium
Rocuronium
Depolarizing Blockers:
Succinylcholine
Decamethonium

16. Nicotinic Antagonists
Nondepolarizing Neuromuscular Blockers
Nondepolarizing neuromuscular blockers (d-tubocurarine, pacuronium, atracurium, etc.) compete with acetylcholine for binding to the nicotinic receptor at the neuromuscular junction.
-         binding of these agents prevents the binding of acetylcholine to the active site of the receptor to prevent depolarization of the motor end-plate.

17. Nicotinic Antagonists
 Nondepolarizing blockers: (cont)
-         activation of the nicotinic receptor requires the binding of two molecules of acetylcholine; binding of one molecule of the antagonist to the receptor decreases the activity of the receptor channel.
-         i.v. administration leads to paralysis of small rapid muscles initially (ocular muscles, muscles of the limbs, etc.) followed by larger muscles, intercostals muscle and finally the diaphragm.

18. Nicotinic Antagonists
Nondepolarizing blockers: (cont)
-      used to produce paralysis of skeletal muscle to facilitate surgical manipulation; also reduces the amount of general anesthesia required to produce muscle relaxation. Additional uses: setting of fractures, intubations, bronchoscopy, endoscopic investigation.
-       administration of cholinesterase inhibitor (neostigmine, physostigmine) will antagonize the paralysis and can reverse the condition.

19. Nicotinic Antagonists
Adverse effects:
1.      apnea, cardiovascular collapse
2.      Histamine release (d-TC, metocurine, succinylcholine, atracurium)
  3.      Succinylcholine may release excessive K+ from muscle – hyperkalemia.
   4.      Malignant hyperthermia – genetic predisposition, due to excessive release of Ca2+ from the sarcoplasmic reticulum of muscle: increased body heat, muscle rigidity, metabolic acidosis, tachycardia. Can be treated with Dantrolene (Dantrium®). Depolarizing blockers are more prone to cause this condition.
   5.      Neonates less susceptible to depolarizing blockers and more susceptible to nondepolarizing agents.

20. Nicotinic Antagonists
DRUG INTERACTIONS
Ø       Inhalational anesthetics produce synergistic neuromuscular blockade with these agents.
Ø       Aminoglycosides decrease ACh release from cholinergic nerves to produce additive neuromuscular blockade.
Ø       Calcium channel blockers may decrease availability of Ca2+ ion for contraction and enhance the effects of neuromuscular blockers.
Ø       Opiods, lidocaine, phenytoin, magnesium salts, chloroquine.