Cholinergic Agents

Cholinergic Agents

Alkaloids

Nicotine

Lobeline

Arecoline

Muscarine

Pilocarpine

Synthetic Agents

Dimethylphenylpiperazinium-(DMPP)

Oxotremorine

Methacholine

Bethanechol

Carbachol

Cevimeline

Nicotine

• Nicotine mimics the actions of acetylcholine at nicotinic sites– Cell body of the postsynaptic neurons

• sympathetic and parasympathetic divisions– Chromaffin cells of the adrenal medulla– End plate of skeletal muscle fiber

• Affinity for NN sites versus NM sites• Used as an insecticide

Muscarine

• Muscarine mimics the actions of acetylcholine at smooth muscles, cardiac muscles, and glands

• Poisoning by muscarine produces intense effects qualitative to those produced by cholinergic stimulation of smooth muscles, cardiac muscle, and glands

• Muscarine is found in various mushrooms– Amanita muscaria: content of muscarine is very

low– Inocybe sp: content of muscarine is high– Clitocybe sp: content of muscarine is high

Pilocarpine

• Has muscarinic actions• Used for xerostomia• Used for glaucoma

Structure of Acetylcholine and its Derivatives

Acetylcholine Methacholine

Bethanechol Carbachol

Therapeutic Uses of Cholinergic Agonists

• Dentistry– Pilocarpine– Cevimeline

• Ophthalmology– Pilocarpine– Carbachol

• Gastrointestinal tract – Bethanechol

• Urinary bladder– Bethanechol

Contraindications to the Use of Choline Esters

• Hyperthyroidism• Asthma• Coronary insufficiency• Peptic ulcer• Organic obstruction in bladder or gastrointestinal tract

Toxicity of Choline Esters

• Flushing• SWEATING (diaphoresis)• Abdominal cramps• Spasm of the urinary bladder• Spasm of accomodation• Miosis• Headache• Salivation• Bronchospasm• Lacrimation• Hypotension• Bradycardia

Agents That Inhibit Acetylcholinesterase

Acetylcholinesterase

(True Cholinesterase)

Acetylcholinesterase (1)

• Sites of location– Cholinergic neurons– Cholinergic synapses– Neuromuscular junction– Red blood cells

• Substrates– Acetylcholine is the best substrate– Methacholine is a substrate– Hydrolyzes ACh at greater velocity than choline esters with

acyl groups larger than acetate or proprionate

Acetylcholinesterase (2)

• Esters that are not substrates– Bethanechol– Carbachol– Succinylcholine

• Its inhibition produces synergistic interaction with methacholine and additive actions with bethanechol and carbachol

• Drugs that block its hydrolysis of esters are called cholinesterase inhibitors

Drug Interactions of Choline Esters and Inhibitors of Acetylcholinesterase - Synergism versus Additivity

• Methacholine

• Carbachol

• Bethanechol

Butyrylcholinesterase

(Plasma esterase, pseudocholinesterase, serum esterase, BuChE, PseudoChE)

Butyrylcholinesterase (1)

• Sites of location

– Plasma, liver, glial cells, other tissues

• Substrates

– Butyrylcholine is the best

– Acetylcholine

– Succinylcholine

– Procaine

Butyrylcholinesterase (2)

• Esters that are not substrates– Methacholine, bethanechol, and carbachol

• Is inhibited by carbamyl and organophosphate inhibitors of acetylcholinesterase

Active Site of Acetylcholinesterase

Interaction of AChE and Acetylcholine

Acetylation of AChE and Release of Choline

Hydroxyl Group of Water Attacks the Carbonyl Group of Acetylated-AChE to Liberate AChE

Carbamyl Inhibitors of AChE

• Their action promoting accumulation of ACh at muscarinic or nicotinic receptors is the basis of their pharmacological, therapeutic, and toxic actions

• Are derivatives of carbamic acid

• Bind covalently to the esteratic site of AChE, resulting in carbamylation of the enzyme

Carbamyl Inhibitors of AChE (1)

Carbamic acid Carbamic acid ester

• Quaternary compounds bind to the ionic binding site of AChE

• Their induce accumulation of AChE at nicotinic and muscarinic sites, producing pharmacological responses qualitative to cholinergic stimulation

• Inhibition of AChE is reversible, in the order of hours

• Are metabolized in the plasma by plasma esterases

Carbamyl Inhibitors of AChE (2)

• High doses produce skeletal muscle weakness due to depolarizing blockade at the end plate of the neuromuscular junction

• High doses produce a profound fall in cardiac output and blood pressure

• Their inhibition of AChE is not reversed by pralidoxime

Carbamyl Inhibitors of AChE (3)

• Quaternary ammonium compounds do not cross the blood-brain barrier

• For oral administration, high doses must be given

Carbamyl Inhibitors of AChE (4)

Neostigmine Carbamylates Acetylcholinesterase

Slow Hydrolysis of Carbamylated-AChE and Enzyme Liberation

Organophosphate Inhibitors of Acetylcholinesterase

• Chemical characteristics

• Promote accumulation of ACh at– NM nicotinic receptor – NN nicotinic receptor– Muscarinic receptor

Organophosphate Inhibitors of Acetylcholinesterase (1)

• Their action promoting accumulation of ACh at the muscarinic receptor of the ciliary muscle is the basis of their therapeutic effectiveness in open angle glaucoma

• Only two of these agents are used for therapeutics– Echothiophate for glaucoma– Diisopropylflurophosphate (DFP) for glaucoma (?)

Organophosphate Inhibitors of AChE (2)

• Inhibition of AChE by these agents is irreversible– New enzyme synthesis is required for recovery of

enzyme function

• They also inhibit pseudocholinesterase

• Metabolized by A-esterases (paroxonases) present in plasma and microsomes. They are metabolized by CYP450.

Organophosphate Inhibitors of AChE (3)

• Enzyme inhibition by these agents can be reversed by cholinesterase reactivators such as pralidoxime if administered before “aging” of AChE has occurred. Inhibition by agents that undergo rapid “aging” is not reversed.

• Except for echothiophate, these agents are extremely lipid soluble, and some are very volatile.

Organophosphate Inhibitors of AChE (4)

Diisopropylflurophosphate (DFP) is a Substrate for AChE

The Extremely Slow Hydrolysis of Phosphorylated-AChE

New enzyme synthesis is required for recovery of enzyme function

Various “States” of Acetylcholinesterase

Clockwise: free AChE, acetylated AChE, carbamylated AChE, phosphorylated AChE

Acetylated-AChE Is Very Rapdily Hydrolyzed

AChE + Acetylcholine AChE-acetylated + choline

AChE-acetylated + H2O AChE + acetate

Hydrolysis of AChE-acetylated is rapid, in the order of microseconds

P

Carbamylated-AChE Is Hydrolyzed Slowly

AChE + Carbamyl inhibitor AChE-carbamylated + noncarbamylated metabolite

AChE-carbamylated + H2O AChE + carbamic acid derivative

Hydrolysis of the AChE-carbamylated is slow, in the order of hours. The carbamylated enzyme is reversibly inhibited, and recovery of function is in the order of hours

Enzyme after phosphorylation by neostigmine

Phosphorlylated-AChE Is Hydrolyzed Extremely Slowly

AChE + organophosphate inhibitor AChE-phosphorylated + nonphosphorylated metabolite

AChE-phosphorylated + H2O AChE + phosphorylated derivative

Hydrolysis of the AChE-phosphorylated is extremely slow, in the order of days. The phosphorylated enzyme is considered to be irreversibly inhibited, and recovery of function is in the order of days. Pralidoxime, a reactivating agent, may be adminstered to a subject before the enzyme has “aged.”

Enzyme after phosphorylation by DFP

AGING OF ACETYLCHOLINESTERASE

Loss of An Alkyl Group From Phosphorylated AChE “Ages” the Enzyme

AChE, phosphorylated and inhibited by DFP

“Aged” AChE

“Aging” of Phosphorylated- AChE

Cholinesterase Reactivation

Reactivation of Phosphorylated Acetylcholinesterase

• Oximes are used to reactivate phosphorylated AChE• The group (=NOH) has a high affinity for the phosphorus

atom• Pralidoxime has a nucleophilic site that interacts with the

phosphorylated site on phosphorylated-AChE

Pralidoxime Reacts Chemically with Phosphorylated-AChE

The oxime group makes a nucleophilic attack upon the phosphorus atom

Oxime Phosphonate and Regenerated AChE

Limitations of Pralidoxime

• Pralidoxime does not interact with carbamylated-AChE

• Pralidoxime in high doses can inhibit AChE

• Its quaternary ammonium group does not allow it to cross the blood brain barrier

• “Aging” of phosphorylated-AChE reduces the effectiveness of pralidoxime and other oxime reactivators

Other Cholinesterase Reactivators

• Diacetylmonoxime– Crosses the blood brain barrier and in

experimental animals, regenerates some of the CNS cholinesterase

• HI-6 is used in Europe– Has two oxime centers in its structure– More potent than pralidoxime

Edrophonium

Edrophonium is a Short Acting Inhibitor that Binds to the Ionic Site but Not to the Esteratic Site of AChE

Pharmacology of Acetylcholinesterase Inhibition

Inhibition of Acetylcholinesterase Produces Stimulation of All Cholinergic Sites

Carbamyl Inhibitors of AChE

• Physostigmine• Neostigmine (N+)• Pyridostigmine (N+)• Ambenonium (N+)• Demecarium (N+)• Carbaryl

Pharmacology of Carbamyl Inhibitors of Acetylcholinesterase

• Eye• Exocrine glands• Cardiac muscle• Smooth muscles• Skeletal muscle• Toxicity

Therapeutic Uses of Inhibitors of Acetylcholinesterase

• Glaucoma (wide angle)• Atony of the bladder• Atony of the gastrointestinal tract

• Intoxication by antimuscarinic agents (use physostigmine)

• Intoxication by tricyclic antidepressants (TCA’s) or

phenothiazines (use physostigmine)

• Recovery of neuromuscular function after competitive blockade of NN receptor of skeletal muscle fibers

• Myasthenia gravis

Therapeutic Uses of Edrophonium

• Diagnosis of myasthenia gravis

• In conjunction with chosen therapeutic agent to determine proper dose of agent

Determining Proper Dose of AChE Inhibitor

Inhibitors of AChE Are Used for Therapy of Alzheimer’s Disease

• Tacrine

• Donepezil

• Rivastigmine

• Galantamine

Organophosphate Inhibitors of AChE

Some Organophosphate Inhibitors of Acetylcholinesterase

• Tetraethylpyrophosphate• Echothiophate (N+)• Diisopropylflurophosphate (DFP)• Sarin• Soman• Tabun• Malathion• Parathion• Diazinon• Chlorpyrifos• Many others

Organophosphate Inhibitors - 2

Diisopropylfluorophosphate (DFP)

Soman

SarinTabun

Echothiophate

Therapeutic use - local application to the eye for wide angle glaucoma

Conversion of Parathion to Paraoxon

Conversion of Malathion to Malaoxon

Malathion Is Hydrolyzed by Plasma Carboxylases in Birds and Mammals but Not Insects

Carboxyl Esterases

• Preferentially hydrolyzes aliphatic esters

• Malathion is a substrate

• Are inhibited by organophosphates

• May also be called aliesterases

Uses of Malathion

• Insecticide

• Therapeutics– Used as a lotion for Pediculus humanus capitis

associated with pediculosis– 0.5% solution in 78% isopropranolol is

pediculicidal and ovicidal– Ovide is the brand name– Primoderm was the former brand name

Malathion Metabolism

• Rapidly metabolized by birds and mammals

• Plasma carboxylases are involved

• Insects do not possess the enzyme

• Organophosphates inhibit malathion metabolism

• Malathion is toxic to fish

Aryl Esterases

• Are found in the plasma and liver

• Hydrolyzes organophosphates at the– P-F bond– P-CN bond– Phosphoester bond– Anhydride bond

EPA And Organophosphates

• Diazinon – No longer allowed to be manufactured for indoor

use in as of March 1, 2001 or for garden use as of June 3, 2001

– Found in Real Kill®, Ortho®, Spectracide®

– Limited agricultural use is allowed

• Chlorpyrifos (Dursban) has been phased out

• Parathion has been phased out for agricultural use in the United States

NERVE AGENT VXChemical name:

O-ETHYL-S-(2-DIISOPROPYLAMINOMETHYL)METHYL-PHOSHONOTHIOLATE

Trade name: PHOSPHONOTHIOIC ACID

NERVE AGENT VXNERVE AGENT VX

Chemical name:

O-ETHYL-S-(2-DIISOPROPYLAMINOMETHYL)METHYL-PHOSHONOTHIOLATE

Trade name: PHOSPHONOTHIOIC ACID

Organophosphates as Nerve Gas Agents in Chemical Warfare (1)

• Extremely volatile agents such as sarin, tabun, soman, and agent VX may be used as nerve agents in chemical warfare.

• Accumulation of ACh at cholinergic receptors produces effects reflecting stimulation of cardiac muscle, smooth muscles and glands. Such effects would be identical to those caused by muscarine poisoning.

• Bradycardia and hypotension occur. However, in some cases, tachycardia may be observed, due to intense sympathetic discharge in response severe hypoxemia.

Organophosphates as Nerve Gas Agents in Chemical Warfare (2)

• Irreversible inhibition of acetylcholinesterase by these agents produces accumulation of ACh at the end plate of skeletal muscle fibers. This in turn leads to depolarizing blockade of the NM nicotinic receptor. Skeletal muscle paralysis occurs. Movement is impossible. The diaphragm is also paralyzed. The individual eventually dies due to respiratory paralysis.

• Pralidoxime, atropine, and removal of the person from the source of exposure are all to be employed in cases of posioning.

Use of Pyridostigmine During the Gulf War