Pharmacokinetics - Drug absorption and Transport of drugs across biological membranes

CHOLINERGIC NEUROHUMORAL TRANSMISSION

Biosynthesis and Transmission of Acetylcholine

 •           acetylcholine is biosynthesized  by the acetylation of choline catalyzed by choline acetyltransferase and acetylcoenzyme A as an acetyl donor

•           choline accumulates in axoplasm by uptake process from extraneuronal sites

•           ACh accumulates in storage vesicles

•           action potential ------------influx of Ca2+ ----------------release of ACh from vesicles

•           ACh in synaptic cleft is rapidly metabolized by acetylcholinesterase

Synaptic Transmission at Cholinergic Synapse

1. Synthesis of neurotransmitter - Acetylcholine (ACh)

a. Choline transported into presynaptic terminal. Hemicholinium inhibits transport.

b. ACh synthesis from choline and acetyl-CoA is catalyzed by choline acetyltransferase (CAT).

       

Transport and storage of ACh into vesicles in nerve terminal.

Vesamicol blocks transporter

ATP and peptides in vesicles along with quanta of Ach molecules.

Action potential ® Ca⁺⁺ influx ® fusion of vesicles with the surface membrane and release  of Ach and other vesicular contents by exocytosis.

· Clostridium toxins (Botulinum toxin , tetanus toxin) block this step

· Toxin from black widow spider (α-latrotoxin) ® massive vesicular release.

ACh diffuses across synaptic cleft and binds ACh receptor (“cholinoceptor”).

Nicotinic receptors are ligand-gated ion channels which cause opening of Na⁺ and K⁺ when activated ® depolarization.

Muscarinic receptors are coupled to G-proteins and response to activation ® ­IP₃ and DAG or ¯adenyl cyclase activity and effects may be excitatory or inhibitory.

ACh may occupy presynaptic “autoreceptor” sites.

Destruction of ACh by enzymatic hydrolysis of ester linkage.

The cholinesterase enzyme acetylcholinesterase (AChE) is associated primarily with neural structures (e.g., membrane of neurons) but is also in red blood cells.

Another type of cholinesterase ("non-specific" or "pseudo-cholinesterase") is found in plasma and in certain tissues (e.g., liver, glial or satellite cells).

Summary of Types of Drug Action at the Cholinergic Junction

Inhibition of enzymatic destruction of ACh producing ACh accumulation (cholinomimetic or parasympathomimetic action); e.g., cholinesterase inhibitor agents (physostigmine).

Alteration of release of ACh as a neurotransmitter.

Prevention of release of neurotransmitter (Botulism toxin).

Promotion of ACh release (metoclopramide [Reglan^®]?)

Theoretically, could alter synthesis or activity of choline acetyltransferase, e.g., hemicholinium blocks uptake of choline and slows synthesis.

Mimicry of transmitter at postsynaptic site (parasympathomimetic or cholinomimetic action); e.g., methacholine and other choline esters).

Sensitization of effector to effects of ACh (e.g., metoclopramide (Reglan^®) sensitizes intestinal smooth muscle to ACh without acting on its receptors).

Muscarinic and Nicotinic Cholinergic Receptors

Different types of receptors for ACh.

Receptors for ACh on effector organs innervated by postganglionic parasympathetic neurons are called MUSCARINIC RECEPTORS.

The alkaloid muscarine (from the poisonous mushroom Amanita muscaria and certain other mushrooms) mimics all effects of ACh or stimulation of postganglionic parasympathetic neurons.

Muscarine also mimics the effects of ACh at certain postganglionic sympathetic neurons that release ACh (sweat glands, some blood vessels in skeletal muscle.)

Most arterioles don't receive cholinergic fibers (either parasympathetic or sympathetic) but they are very sensitive to muscarine and muscarine-like drugs.

Muscarine produces little, if any, effect on other cholinergic receptors, namely, those of the autonomic ganglia and the neuromuscular junction.

Muscarine receptors (effector organs innervated by postganglionic neurons of PNS and a few effectors receiving cholinergic SNS innervation and the cholinergic receptors in some blood vessels) are further characterized by selective blockade by drugs like atropine.

Receptors in autonomic ganglia (both SNS and PNS), the adrenal medulla, and the neuromuscular junction are called NICOTINIC RECEPTORS.

The alkaloid nicotine mimics the effects of ACh at these sites.

Responses to ACh at these sites are referred to as nicotinic actions. 

Pharmacological Actions of ACh

Administration

Positively charged, highly polar and therefore not lipid soluble.

Quickly hydrolyzed by cholinesterases.

Cardiovascular System

Injectable doses of ACh decrease blood pressure.

Causes vasodilation of major vascular beds even when blood vessels have no cholinergic innervation.

Vasodilation of cerebral, coronary, cutaneous, and splanchnic circulation - none of which are under cholinergic control.

Action of ACh on heart produces a decrease in cardiac rate (negative chronotropic effect) and a decrease in force of cardiac contraction (negative inotropic effect).

Action of ACh to produce cardiac effects (bradycardia, block of A-V nodal conduction, etc.) can be produced either by large intravenous doses of ACh or by vagus nerve impulses (cholinergic parasympathetic fibers distributed to S-A node, A-V node and atrial muscle).

Decreases rate of discharge of S-A node (pacemaker) negative chronotropic effect.

Prolongs A-V conduction time; decrease in conduction velocity (negative dromotropic effect).

Decreases strength of contraction of atrial muscle (negative inotropic effect).

Compensatory cardiovascular reflexes (e.g. baroreceptor reflex) can  obscure certain effects.

Smooth Muscle (other than blood vessels)

G.I. tract - Increase in tone and motility; relaxation of sphincter.

Bladder - Increase in tone and motility; relaxation of sphincter.

Bronchial muscle contracted; bronchoconstriction.

Contraction of gall bladder and ducts.

Contraction of constrictor (sphincter) muscle of iris to produce miosis (pupillary constriction).  Also contraction of ciliary muscle to produce contraction for near vision, i.e., accommodation.

Exocrine Glands - Secretion increased in all glands receiving PNS innervation, including salivary, lacrimal, bronchial, gastric, intestinal, and sweat glands; also sweat glands receiving SNS innervation.

Nicotinic Effects

Autonomic Ganglia - large doses of ACh can activate nicotinic receptors of PNS, SNS, and adrenal medulla.

Depolarization of neuromuscular junction produces skeletal muscle contraction.

Direct-Acting Parasympathomimetics

            Although ACh can be considered the prototype drug of this class, its actions are too brief to be clinically useful. 

Parasympathomimetic drugs can be divided into two major groups: Direct-Acting agents (directly activate cholinergic receptor) and Indirect-Acting cholinesterase inhibitors. 

The Direct-Acting Parasympathomimetics include choline esters (methacholine, bethanechol, and carbachol) which are structural analogs of ACh, and the naturally occurring alkaloids pilocarpine and muscarine.

Cholinergic Agonists

 

Direct-acting (receptor agonists)  

                                                                                    Indirect-acting (cholinesterase inhibitors)

            Muscarinic             Nicotinic

      [primary effect]     [primary effect]      

           

                              Ganglionic              Neuromuscular

Choline esters              Alkaloids                     Reversible                   Irreversible

[acetylcholine]             pilocarpine                   physostigmine              [organophosphates]

methacholine               muscarine                    neostigmine                 echothiophate

carbachol                                                         edrophonium

bethanechol                                                     pyridostigmine

cevimeline                                                       ambenonium

                                                                        tacrine, donepezil

Cholinergic Receptor Agonists

(Parasympathomimetic Drugs)

Introduction:

•           Parasympathetic nerves use ACh as a neurotransmitter

•           Cholinomimetic drugs mimic the action of ACh at its receptors

•           Knowledge of distribution of receptor subtypes (muscarinic or nicotinic) helps in predicting drug response

Cholinergic site	Receptor subtype
Neuroeffector junctions	Muscarinic
Ganglionic synapses	Nicotinic

Classification of Cholinomimetics

1. Direct-acting (receptor agonists)

•           muscarinic receptors

•           nicotinic receptors

2. Indirect-acting (cholinesterase inhibitors)

•           reversible

•           irreversible

1. Direct-acting cholinergic receptor agonists

a) Muscarinic receptor agonists

            -drugs that mimic ACh at neuroeffector junctions of PNS

Mechanisms:

Cholinergic receptors are coupled to G proteins (intramembrane transducers that regulate second messengers

•           agonist®­ cGMP®activation of IP3, DAG cascade

•           DAG opens smooth muscle Ca2+ channels

•           IP3®release of Ca2+ from sarcoplasmic reticulum

•           agonist selectivity is determined by muscarinic receptor subtype and G protein in cell.

Types of direct-acting muscarinic receptor agonists:

a)         Esters of choline (eg. acetylcholine, pilocarpine, carbachol, bethanechol chloride)

                        -poorly absorbed,

                        -variable susceptibility to hydrolysis by cholinesterase

                        -affects duration of action

b)         Alkaloids (eg. muscarine)

                        -well absorbed, not used clinically

                        - mushroom (Amanita muscaria) poisoning

i) Acetylcholine

-highly susceptible to hydrolysis

-IV bolus lasts 5-20 seconds

-limited use in topical application in ophthalmology

ii) Pilocarpine

-acts on smooth muscles of eye to constrict the pupil (miosis)

-used to treat glaucoma

-contracts ciliary muscles by stimulating muscarinic receptors

-rapid penetration (15-30 min) and long duration (8 hrs)

-­ aqueous outflow

iii) Carbachol

-carbamyl ester of choline

-used mainly in ophthalmology for cataract surgery (causes rapid myosis)

-¯ intraocular pressure by opening drainage angle of anterior chamber of eye

\ used in glaucoma (when resistant to pilocarpine or physostigmine)

iv) Bethanechol Chloride

-choline ester

-persistent effects because resistant to cholinesterases

-selectively stimulates urinary and gastrointestinal tracts

-facilitates emptying of neurogenic bladder in patients after surgery or parturition or with spinal cord injury

b) Nicotinic receptor agonists

-nicotine is anatural alkaloid found in tobacco which mimics the effects of ACh at nicotinic receptors at:

•           autonomic ganglionic synapses (both SNS & PNS)

•           skeletal neuromuscular junctions

-nicotine is still used in some insecticides

Mechanism:

-activates nicotinic receptor (transmembrane polypeptide comprised of cation-selective ion channel subunits

Nicotinic agonists

Conformational change in receptor

Opens cation channels

Na+/K+ diffusion into cell

Depolarization of nerve cell  or neuromuscular endplate

Clinical use:   

-no therapeutic action but important for its toxicity

-available as a transdermal patch or as chewing gum

-used as an aid in cessation of smoking

Toxicity:

-both stimulant and depressant (affects both SNS & PNS ganglia)

- stimulates nicotinic receptors in CNS ® mild alerting action

-also acts centrally ® tremor & convulsions

-can ­ or ¯ HR

-­ respiratory rate

-vomiting due to activation of chemoreceptor trigger zone-larger doses

®CNS and respiratory depression by muscle endplate depolarization blockade

2. Indirect-acting cholinomimetic drugs

Characteristics:

-anticholinesterase drugs ie. inhibitors of ACh metabolism

-similar effects to direct-acting cholinomimetics

Mechanism:

-normally ACh is rapidly degraded in cholinergic synapse (T1/2=40ms)

-indirect-acting cholinomimetics block the enzymatic hydrolysis of acetylcholine®­ local concentrations of ACh

\ effect of ACh is amplified leading to muscarinic or nicotinic effects, depending on the organ

-effect can be therapeutic or life threatening

Classification

 A) Reversible Inhibitors

-all are poorly absorbed from conjunctiva, skin & lungs

except physostigmine which is well absorbed from all sites

-is used topically in eye

-more commonly used clinically than organophosphates

•           Quaternary alcohols

-bind reversibly to active site of ACh esterase and prevents access by ACh

-no covalent bond between enzyme inhibitor complex

\ short T1/2 (2-10 min)

-eg. edrophonium

•           Carbamate esters

-2 step hydrolysis like ACh

-but the covalent bond of carbamylated enzyme is more resistant to hydration (T1/2=30-60 min)

eg. neostigmine, physostigmine

 Clinical use:

Primary target organs of anticholinesterase drugs:

•           eye

•           neuromuscular junctions

•           gastrointestinal tract

•           urinary tract

•           respiratory tract

•           heart

-effects are similar to direct acting cholinergic agonists

Major uses in treatment of

a) Glaucoma

b) Myasthenia gravis

c) Stimulation of gastrointestinal and urinary tract motility

            (eg. neostigmine)-same effects as with agonists

d) Reversal of neuromuscular blockade

e) Atropine poisoning

a) Glaucoma:

-an ocular disease caused by ­ intraocular pressure due to inadequate drainage of aqueous humor at filtration angle ® damage to the retina & optic nerve

-intraocular pressure is determined by the balance between fluid input & drainage out of the globe

-aqueous humour produced by ciliary epithelium and drained at the filtration angle of the anterior chamber

Therapy: 

Objective:

­ outflow & ¯ production of aqueous humour by local treatment with:

i) muscarinic cholinomimetics:

•           direct-acting: pilocarpine, carbachol

•           indirect-acting: physostigmine

®contracting the smooth muscle of iris sphincter (contraction of pupil)

®contraction of ciliary muscle

®iris pulled from angle of anterior chamber

®widening the filtration angle and opening the trabecular network

®­ outflow of aqueous humour

®¯ intraocular pressure

ii) a adrenoceptor agonists: epinephrine

®contraction of dilator muscle of iris

®­ aqueous outflow

-also ¯ aqueous secretion

-used mainly for treatment of closed angle glaucoma along with surgery

iii) b adrenoceptor blockers: timolol

®¯ production of aqueous humour by ciliary epithelium

b) Myasthenia gravis

-an autoimmune disease resulting in destruction of nicotinic receptors

®progressive weakness, fatigue, difficulty speaking & swallowing

-resembles neuromuscular block by curare

Treated with indirect acting cholinesterase inhibitors(eg neostigmine)

®­ strength of contraction of muscles

c) Reversal of neuromuscular blockade

-short-acting cholinesterase inhibitors (eg. neostigmine, edrophonium Cl) ®­ ACh concentration which then competes with neuromuscular blocker for nicotinic receptors

B) Irreversible Inhibitors

-eg. organophosphates (Parathion, malathion)

Mechanism:

-act by covalently phosphorylating the hydroxyl group of serine on cholinesterase

-a few organophosphate pesticides are selective in toxicity to insects

-eg. malathion is rapidly metabolized by plasma esterases in humans

\ safer

Aging occurs when an alkyl or alkoxy group is lost

®­ strength of phosphorus-enzyme bond

®stable enzyme-inhibitor complex which is difficult to split

Before aging occurs patients can be treated with strong nucleophiles eg. pralidoxime which breaks the phosphorus-enzyme complex and regenerates the enzyme

Signs of Toxicity:

a) Mild exposure

-pupillary constriction

-tightness of the chest

-watery discharge from the nose

-wheezing

b) Severe exposure

-more intensified symptoms

-visual disturbances

-muscle fasciculation

-bronchoconstriction & pulmonary edema

-pronounced muscle weakness

-shallow respiration

-vomiting & diarrhea

-CNS effects, anxiety, headache, tremor, seizures, depression

-death

Mnemonic for symptoms of muscarinic excess:

DUMBELS      D-diarrhea

            U-urination

                        M-miosis

                        B-bronchoconstriction

            E-excitation (skeletal muscle and CNS)

                        L-lacrimation

                        S-salivation and sweating

Treatment - Muscarinic blocking drugs e.g. atropine

Overview of Therapeutic Applications

of Cholinomimetic Drugs

Tissue	Effect	Use/drug
Muscarinic agonists
•           Eye •           GI tract •           Urinary bladder	•           contraction of ciliary m./sphincter m. of iris •           ­ peristalsis, sphincter relaxation •           ­ contraction of detrusor m./sphincter relaxation	•           Glaucoma                         (pilocarpine) •           Paralytic ileus                         (bethanechol Cl) •           Urinary retention                         (bethanechol Cl)
ACh esterase inhibitors
•           Skeletal m. •           Eye	•           ­ muscle activity •           similar to agonists	•           Myasthenia gravis                         (neostigmine) •           Glaucoma                         (physostigmine)

Cholinergic Receptor Antagonists

Drugs which block the actions of ACh at:

•           central and peripheral muscarinic receptors

•           nicotinic receptors at neuromuscular junction

•           nicotinic receptors in ganglia

           

Types of Cholinergic Antagonists

Antimuscarinic	Antinicotinic
•           Muscarinic blockers             eg atropine	•           Ganglion blockers             eg. trimethaphan camsylate             (limited use)
	•           Neuromuscular blockers             eg. curare

1. Muscarinic Blocking Drugs

Muscarinic antagonists are parasympatholytic drugs which block parasympathetic autonomic discharge

Mechanisms

-reversible blockade of ACh at muscarinic receptors by competitive binding

-reversal by ­ ACh or agonist ®¯ blockade

•           Nonselective blockers compete for both M1 and M2 receptors

            (eg. atropine)

Tissue sensitivity to muscarinic blockers varies:

high	salivary, sweat glands, bronchial smooth muscle
medium	smooth muscle effectors
low	gastric parietal cells

Classes of antimuscarinic drugs

1. Tertiary Amines:   

-cross membranes readily and penetrate the CNS

a) Atropine

-prototype antimuscarinic drug

-derived from Atropa belladonna (deadly nightshade) and Datura stramonium (thorn apple)

History:

-during the Italian renaissance dilated pupils were considered desirable

-plant extracts were used as cosmetic eye drops

-hence the name belladonna or "beautiful lady" in Italian

Pharmacokinetics:

  -lipid soluble and readily crosses membranes

  -well distributed into CNS

  \ used in opthalmology and parkinsonism

-rapidly absorbed orally, parenterally and from eye

-T1/2 = 2 hrs

-most excreted unchanged in urine after 12 hours

Mechanisms:

-binds to muscarinic receptors to block parasympathetic effects

Tissue Responses and Clinical Uses of Antimuscarinics

Tissue	Response	Use
Eye	-relaxes constrictor m. of pupil (mydriasis) -paralysis of ciliary muscle (cycloplegia)	-preoperative mydriasis/ anterior uveitis/refraction studies/prevents synechia
GI tract	¯ GI hypermotility and secretions	-antispasmodic, hypermotility, preoperative medication
Bronchiolar smooth muscle	¯ bronchoconstriction and secretions	-surgery, asthma
General parasympathetic	¯ muscarinic functions	-anticholinesterase poisoning
Urinary bladder	-relaxation, sphincter constrict'n	-urinary incontinence
Brain	Blockade of CNS receptors	Parkinson's Disease, motion sickness
Glandular tissue	¯ gastric secretions ¯ secretions -lung, mouth -sweat glands blocked	-peptic ulcer -anesthetic premedication
Cardiac muscle	Vagal blockade, ­ HR, ­ AV node transmission	¯ parasympathetic stimulation by cardiac glycosides
Skeletal muscle	No effect

Other uses:

Cholinergic poisoning:

-medical emergency due to exposure to cholinesterase inhibitors

(eg. organophosphate insecticides, OP)

        Treatment:

a) atropine blocks both CNS and peripheral effects of OP

b) pralidoxime regenerates active enzyme

-ie. oxime group (=NOH) has high affinity for P atom

    -hydrolyzes the phosphorylated enzyme if not "aged".

Mushroom poisoning:

-eg. overexposure to Amanita muscaria (contains muscarine)

                -symptoms are of muscarinic excess:

•           nausea

•           vomiting

•           diarrhea

•           vasodilation

•           reflex tachycardia

•           sweating

Parkinson's disease:

-cholinergic neurons in basal ganglia have D2 receptors which inhibit ACh release when stimulated by dopamine

-¯ some signs of Parkinsonism (see lecture on Parkinson’s disease)

-loss of dopamine in Parkinson's disease ®disinhibition®­ ACh release

-anticholinergic drugs, eg. benztropine, block effects of ­ ACh

-used as adjuncts when patient becomes unresponsive to L-Dopa

 b) Scopolamine

-methyl analog of atropine

-derived from henbane, also many synthetic derivatives

-used for millenia as medicines, poisons and cosmetics

-more potent in CNS than atropine

-can induce hallucinations and aberrant behaviour

\ limited use clinically - effective treatment of motion sickness

-acts on iris ® mydriasis and on ciliary muscle ® cycloplegia

2. Quaternary ammonium compounds:       

-synthetic drugs developed for more peripheral, less CNS effects

-i.e. charged polar molecules can’t cross blood-brain barrier

-used for treatment of GI/urinary conditions, asthma

eg. propantheline

Other drugs that block muscarinic receptors (eg. antidepressants, neuroleptics & antihistamines) may have atropine-like effects

Side effects of antimuscarinic drugs:

Atropine Toxicity:

-overdose can occur due to substance abuse to induce hallucinations

® block of all parasympathetic functions

®Treatment: physostigmine

Low doses	High doses
-urinary retention -dry mouth -cycloplegia (blurred vision) -mydriasis -anhidrosis (dry skin) -tachycardia -elevated temperature	-photophobia -nausea -vomiting -hypertension -excitement, hallucinations -convulsions, death

Mnemonic for atropine poisoning:

“Dry as a bone”	¯ sweating ¯ salivation ¯ lacrimation
“Mad as a hatter”	sedation amnesia delirium hallucinations
“Red as a beet”	dilation of cutaneous vessels