Cholinomimetics part 2

Question 1

What are the two classes of cholinomimetic drugs

Answer 1

Directly acting

Indirectly acting

Question 2

What are the two types of directly acting cholinomimetic drugs

Answer 2

Typical agonists at muscarinic receptors

1) choline esters (bethanechol
(2) alkaloids (pilocarpine)

Question 3

What are muscarinic receptor agonists usually referred to as

Answer 3

Muscarinic agonists, as a group, are often referred to as parasympathomimetic, because the main effects that they produce in the whole animal resemble those of parasympathetic stimulation. The structures of ACh and related choline esters are given in Table 14.3. They are agonists at both mAChRs and nAChRs, but act more potently on mAChRs (see Fig. 14.1). Bethanechol, pilocarpine and cevimeline are the main ones used clinically.

Question 4

What are the key features of the ACh molecule

Answer 4

The key features of the ACh molecule that are important for its activity are the quaternary ammonium group, which bears a positive charge, and the ester group, which bears a partial negative charge and is susceptible to rapid hydrolysis by cholinesterase. Variants of the choline ester structure (see Table 14.3) have the effect of reducing the susceptibility of the compound to hydrolysis by cholinesterase, and altering the relative activity on mAChRs and nAChRs.

Question 5

Summarise the effects of bethanechol and pilocarpine

Answer 5

Bethanechol, which is a hybrid of these two molecules, is stable to hydrolysis and selective for mAChRs, and is occasionally used clinically (see clinical box, p. 184). Pilocarpine is a partial agonist and shows some selectivity in stimulating secretion from sweat, salivary, lacrimal and bronchial glands, and contracting iris smooth muscle (see later), with weak effects on gastrointestinal smooth muscle and the heart.

Question 6

Why do the directly acting cholinomimetics not have selectivity for nicotinic receptors

Answer 6

Despite having a very similar structure to ACh- which allows them to be recognised by the muscarinic receptors
We know that small changes in structure can effect their activity- and it transpires that these small deviations of structure from ACh means that they cannot be recognised by nicotinic receptors

Question 7

Where is pilocarpine derived from

Answer 7

Derived from the leaves of a South American shrub Pilocarpus

Question 8

Summarise the pharmacological properties of pilocarpine

Answer 8

Non-selective muscarinic agonist; good lipid solubility; t1/2 ≈ 3-4h
Non-selective muscarinc agonist means that it can act on all muscarinic receptor sub-types

Question 9

What is pilocarpine particularly useful to treat and list some of its side effects

Answer 9

Particularly useful in ophthalmology as a local treatment for glaucoma

Side effects: Blurred vision, sweating, gastro-intestinal disturbance and pain, hypotension, respiratory distress
Side effects are due to simulation of muscarinic receptors

Question 10

How can pilocarpine be given locally to treat glaucoma

Answer 10

Eye drops

Question 11

What type of cholinomimetic is bethanehcol

Answer 11

Minor modification of acetylcholine, produces an M3 AChR selective agonist- most drugs aren’t great at selecting between different sub-types

Question 12

Summarise the pharmacological properties of bethanechol

Answer 12

Resistant to degradation, orally active and with limited access to the brain (t1/2 ≈ 3-4h)
Not metabolised by CAT
As it has limited access to the brain- we see less CNS related side effects

Question 13

What are the main clinical uses of bethanechol and list some of its side effects

Answer 13

Mainly used to assist bladder emptying and to enhance gastric motility- often given post-op to kick start gastric motility again after the anaesthesia

Side effects: sweating, impaired vision, bradycardia, hypotension, respiratory difficulty

Question 14

Why are the side effects of bethanechol more pronounced than that of pilocarpine

Answer 14

Given systemically (oral) as compared to the topical administration of pilocarpine
Therefore it has more access to the muscarinic receptors expressed in the body to elicit side effects.

Question 15

What is cevimeline

Answer 15

Cevimeline – newer M3-selective cholinomimetic- greater degree of selectivity than bethanechol

Question 16

Which diseases can bethanechol be used to treat

Answer 16

Treatment of bladder and gastrointestinal hypotoniaa

Question 17

What is the ultimate effect of indirectly acting cholinomimetic drugs

Answer 17

To raise the endogenous concentration of ACh (less is broken down by CAT)

Question 18

Summarise the actions of the indirectly acting cholinomimetic drugs

Answer 18

Increase effect of normal parasympathetic nerve stimulation
Reversible anticholinesterases: physostigmine, neostigmine, donepezil (‘Aricept’)
Irreversible anticholinesterases: ecothiopate, dyflos, sarin

Question 19

How can drugs that enhance cholinergic transmission act

Answer 19

Drugs that enhance cholinergic transmission act either by inhibiting cholinesterase (the main group) or by increasing ACh release.

Question 20

What is the action of cholinesterase enzymes

Answer 20

Metabolise acetylcholine to choline and acetate

Question 21

What are the two types of cholinesterase enzymes

Answer 21

Two types which differ in distribution, substrate specificity and function:

Acetylcholinesterase (true or specific cholinesterase)

Butyrylcholinesterase (pseudocholinesterase)

Question 22

Describe the structure of the cholinesterases

Answer 22

Both consist of globular catalytic subunits, which constitute the soluble forms found in plasma (BuChE) and cerebrospinal fluid (AChE). Elsewhere, the catalytic units are linked to accessory proteins, which tether them like a bunch of balloons to the basement membrane (at the neuromuscular junction) or to the neuronal membrane at neuronal synapses (and also, oddly, the erythrocyte membrane, where the function of the enzyme is unknown).

Question 23

Summarise the characteristics pop acetylcholinesterase

Answer 23

Found in all cholinergic synapses (peripheral and central)
Very rapid action (hydrolysis; >10 000 reactions per second)
Highly selective for acetylcholine

Question 24

Describe the actions of acetylcholinesterase

Answer 24

The bound AChE at cholinergic synapses serves to hydrolyse the released transmitter and terminate its action rapidly. Soluble AChE is also present in cholinergic nerve terminals, where it has a role in regulating the free ACh concentration, and from which it may be secreted; the function of the secreted enzyme is so far unclear. AChE is quite specific for ACh and closely related esters such as methacholine. Certain neuropeptides, such as substance P (Ch. 19) are inactivated by AChE, but it is not known whether this is of physiological significance.

Question 25

Describe the correspondence between the distribution of cholinergic synapses and that of AChE

Answer 25

Overall, there is poor correspondence between the distribution of cholinergic synapses and that of AChE, both in the brain and in the periphery, and AChE most probably has synaptic functions additional to disposal of ACh, although the details remain unclear

Question 26

What residue is present at the active site of acetylcholinesterase

Answer 26

Serine residue- which has an OH group- which hydrolyses ACh- inactivating it- splitting it into acetate and choline- happens very quickly

Question 27

Summarise the key characteristics of butyrylcholinesterase

Answer 27

Found in plasma and most tissues but not cholinergic synapses

Broad substrate specificity - hydrolyses other esters e.g. suxamethonium

Is principal reason for low plasma acetylcholine

Shows genetic variation- drugs such as suxamethonoium broken down by different rates by different people- different sensitivities

Question 28

Describe the activity of butyrylcholinesterase

Answer 28

BuChE has a widespread distribution, being found in tissues such as liver, skin, brain and gastrointestinal smooth muscle, as well as in soluble form in the plasma. It is not particularly associated with cholinergic synapses, and its physiological function is unclear. It has a broader substrate specificity than AChE. It hydrolyses the synthetic substrate butyrylcholine more rapidly than ACh, as well as other esters, such as procaine, suxamethonium and propanidid (a short-acting anaesthetic agent; see Ch. 42). The plasma enzyme is important in relation to the inactivation of the drugs listed previously.

Question 29

Describe the genetic variation in the activity of butyrylcholinesterase

Answer 29

Genetic variants of BuChE causing significantly reduced enzymic activity occur rarely (see Ch. 12), and these partly account for the variability in the duration of action of these drugs. The short duration of action of ACh given intravenously (see Fig. 14.1) results from its rapid hydrolysis in the plasma

Question 30

Why is ACh strictly a neurotransmitter and not a hormone

Answer 30

Normally, AChE and BuChE between them keep the plasma ACh at an undetectably low level, so ACh is strictly a neurotransmitter and not a hormone.

Question 31

Describe the two regions of the active sites of the anticholinesterases

Answer 31

Both AChE and BuChE belong to the class of serine hydrolases, which includes many proteases such as trypsin. The active site of AChE comprises two distinct regions (Fig. 14.8): an anionic site (glutamate residue), which binds the basic (choline) moiety of ACh; and an esteratic (catalytic) site (histidine + serine). As with other serine hydrolases, the acidic (acetyl) group of the substrate is transferred to the serine hydroxyl group, leaving (transiently) an acetylated enzyme molecule and a molecule of free choline.

Question 32

Describe the hydrolysis of the serine acetyl group

Answer 32

Spontaneous hydrolysis of the serine acetyl group occurs rapidly, and the overall turnover number of AChE is extremely high (over 10,000 molecules of ACh hydrolysed per second by a single active site).

Question 33

Describe the effect of cholinesterase inhibitors when given at a low dose

Answer 33

Enhanced muscarinic activity- raises endogenous conc of ACh

Question 34

Describe the effect of cholinesterase inhibitors when given at a moderate dose

Answer 34

Further enhancement of muscarinic activity
Increased transmission at ALL autonomic ganglia (nAChRs)
Need more ACh to stimulate nicotinic receptors

Question 35

Describe the effect of cholinesterase inhibitors when given at a high dose

Answer 35

Depolarising block at autonomic ganglia & NMJ
Toxic
Receptors switch off and become inactivated- respiratory depression - diaphragm

Question 36

What are the medium-duration anticholinesterases

Answer 36

These include neostigmine and pyridostigmine, which are quaternary ammonium compounds of clinical importance, and physostigmine (eserine), a tertiary amine, which occurs naturally in the Calabar bean.7

Question 37

Summarise action of the reversible anticholinesterases

Answer 37

Physostigmine, neostigmine

Compete with acetylcholine for active site on the cholinesterase enzyme

Donate a carbamyl group to the enzyme, blocking the active site and preventing acetylcholine from binding

	Carbamyl group removed by 	slow hydrolysis (mins rather 	than msecs)

	Increase duration of acetylcholine 	activity in the synapse

Question 38

What is a key property of the reversible cholinesterases

Answer 38

These drugs are all carbamyl, as opposed to acetyl, esters and all possess basic groups that bind to the anionic site. Transfer of the carbamyl group to the serine hydroxyl group of the esteratic site occurs as with ACh, but the carbamylated enzyme is very much slower to hydrolyse (see Fig. 14.8), taking minutes rather than microseconds.
The anticholinesterase drug is therefore hydrolysed, but at a negligible rate compared with ACh, and the slow recovery of the carbamylated enzyme means that the action of these drugs is quite long-lasting.

Question 39

Summarise physostigmine

Answer 39

Naturally occurring tertiary amine from Calabar beans

Primarily acts at the postganglionic parasympathetic synapse (t1/2 ≈ 30 mins)

Used in the treatment of glaucoma, aiding intraocular fluid drainage

Also used to treat atropine poisoning, particularly in children
Lipid soluble

Question 40

What is atropine

Answer 40

Muscarinic receptor antagonist- competitive antagonist- thus surmountable with physositgmine.
The atropine is then metabolised quickly

Question 41

Summarise the irreversible anti cholinesterase enzymes

Answer 41

Organophosphate compounds:
ecothiopate, dyflos, parathion and sarin

Rapidly react with the enzyme
active site, leaving a large blocking
group

This is stable and resistant to hydrolysis - recovery may require the production of new enzymes (days/weeks)

	Only ecothiopate in clinical use, 	but the others are commonly used 	as insecticides (and as nerve gas!)

Question 42

Describe the structural properties of the irreversible anti cholinesterase enzymes

Answer 42

Irreversible anticholinesterases (see Table 14.8) are pentavalent phosphorus compounds containing a labile group such as fluoride (in dyflos) or an organic group (in parathion and ecothiophate). This group is released, leaving the serine hydroxyl group of the enzyme phosphorylated (see Fig. 14.8).

Question 43

What are the main uses of the irreversible anticholinesterases

Answer 43

Most of these organophosphate compounds, of which there are many, were developed as weapons, such as sarin, and the more potent VX (10 mg of which through skin contact is said to be fatal) which acquired notoriety as an agent of state-sponsored assassination.8 Some are used as pesticides as well as for clinical use; they interact only with the esteratic site of the enzyme and have no cationic group. Ecothiophate is an exception in having a quaternary nitrogen group designed to bind also to the anionic site.

Question 44

Describe the activity of the inactivated, phosphorylated enzyme

Answer 44

The inactive phosphorylated enzyme is usually very stable. With drugs such as dyflos, no appreciable hydrolysis occurs, and recovery of enzymic activity depends on the synthesis of new enzyme molecules, a process that may take weeks. With other drugs, such as ecothiophate, hydrolysis occurs over the course of a few days, so that their action is not strictly irreversible.

Question 45

Which property does the use of the irreversible anti-cholinesterases depend on

Answer 45

Dyflos and parathion are volatile non-polar substances of very high lipid solubility, and are rapidly absorbed through mucous membranes and even through unbroken skin and insect cuticles; the use of these agents as war gases or insecticides relies on this property.

Question 46

Describe the other actions of the irreversible anticholinesterases

Answer 46

The lack of a specificity-conferring quaternary group means that most of these drugs block other serine hydrolases (e.g. trypsin, thrombin), although their pharmacological effects result mainly from cholinesterase inhibition.

Question 47

Summarise ecothiopiate

Answer 47

Potent inhibitor of acetylcholinesterase

Slow reactivation of the enzyme by hydrolysis takes several days

Used as eye drops in treatment of glaucoma, acting to increase intraocular fluid drainage with a prolonged duration of action

Systemic side effects: sweating,
blurred vision, GI pain, bradycardia,
hypotension, respiratory difficulty

Question 48

Summarise the effects of the anti cholinesterase drugs on the CNS

Answer 48

Non-polar anticholinesterases (e.g. physostigmine; nerve agents) can cross the blood brain barrier

Low doses: Excitation with possibility of convulsions

High doses: Unconsciousness, respiratory depression, death- due to cessation of respiration

Question 49

Describe the effects of the anti cholinesterase drugs on the CNS

Answer 49

Tertiary compounds, such as physostigmine, and the non-polar organophosphates penetrate the blood–brain barrier freely and affect the brain. The result is an initial excitation, which can result in convulsions, followed by depression, which can cause unconsciousness and respiratory failure. These central effects result mainly from the activation of mAChRs, and are antagonised by atropine. The use of anticholinesterases to treat senile dementia is discussed in

Question 50

Describe organophosphate poisoning

Answer 50

Accidental exposure to organophosphates used in insecticides, or deliberate use as nerve agents can cause severe toxicity ( increased muscarinic activity; CNS excitation; depolarising NM block)

Question 51

Summarise the treatment of organophosphate posinoning

Answer 51

Treatment: atropine (iv); artificial respiration; pralidoxime (iv)- only drug which can reverse the block- but needs to be given within first couple of hours on being intoxicated
NB: Phosphorylated enzyme ‘ages’ within few hours

Question 52

What does the ‘ageing’ of the phosphorylated enzyme mean

Answer 52

Its inactivation becomes irrversible after a couple of hours

Question 53

Distinguish between the actions of the pralidoxine and atropine

Answer 53

Pralidoxine works on the inactivated enzyme- so reduces cholinergic transmission at both the nicotinic and muscarinic synapses
Atropine- will block muscarinic receptors

Question 54

Summarise the effects of the cholinomimetics

Answer 54

Cholinomimetics decrease heart rate and cardiac output, increase exocrine gland activity, increase non-vascular smooth muscle contractility and cause miosis