FUN2: 10:00-11:00Scribe: Ryan O Neill

FUN2: 10:00-11:00Scribe: Ryan O’Neill

Wednesday, December 3, 2008Proof: Brannon Heape

Dr. BesseAutonomicsPage1 of 4

Acetylcholine hydrolysis at the active center of acetylcholinesterase [S7]
We will discuss two direct acting cholinergic or muscarinic agonists and their pharmacology.
These two compounds are Bethanechol (an analog of acetylcholine) and Pilocarpine.
Bethanechol (textbook) has structural modifications. Look at the structure of acetylcholine on the slide: The addition of a hydroxyl group makes the compound resistant to hydrolysis by acetylcholinesterase.
Secondly, the addition of an amino group confers selectivity of Bethanechol for muscarinic receptors, opposed to nicotinic receptors.
“Selective” is not “specific.” For example, if you boost the dose of Bethanechol above the therapeutic range, you will also begin to stimulate the nicotinic receptors, some at the skeletal/neuromuscular junctions and sometimes at the ganglia.
Bethanechol is resistant to hydrolysis by acetylcholinesterase and it has some selectivity for the muscarinic receptor.
These characteristics of Bethanechol also apply to Pilocarpine, which is a natural alkaloid. This explains what these compounds are used for.
Both of them are still commonly used to treat the GI tract and/or the urinary tract atony (lack of tone) after surgical procedures. They can facilitate return of function to the GI tract to the normal level and promote voiding (urinating).
This is the major therapeutical use.
Pilocarpine, on the other hand, has one other use; it was the first drug that had any effectiveness in the management of glaucoma.
Remember, if you increase the activity of the parasympathetic division to the eye, you will increase aqueous outflow (11/2 11:00-12:00 lecture) especially when intraocular pressure is elevated in glaucoma.
Can be administered topically, not systemically.
In the case of the previous condition described, you would give Bethanechol or Pilocarpine orally.
Another point (not testable material): there is a new drug used in the management Sjogren's syndrome, an autoimmune disease which brings about relative xerstomia (dry mouth) in the mouth.
This drug is not in your handout, but is on page 103 in the textbook. It is called Cevimiline, which is a muscarinic agonist that is used with some success in the management of xerstomia in these particular patients with Sjogren's syndrome.
What happens if too much Pilocarpine is topically applied in glaucoma? You get blurred vision because of cyclospasm (too much muscle contraction) and it can be very irritating, especially in higher amounts.
End: That is it as far as therapeutics is concerned with the direct acting cholinergic or muscarinic agonists.

Acetylcholinesterase agents [S8]
Acetylcholinesterase agents - compounds that act indirectly to produce parasympathetic-like effects, but by an indirect mechanism – inhibition of cholinesterase, the effect of which is to increase acetylcholine at all parasympathetic neuro-effector junctions.
We will begin with a discussion of a mechanistic way in which the enzyme inactivates acetylcholine.
Acetylcholine binds at these two respective sites at the enzyme. An acetylated intermediate is formed. There is further hydrolysis and free enzyme is reactivated with the release of acetate and choline.
This process takes no more the 115 milliseconds; it’s very rapid. This is the key way to distinguish this process from what happens when you inhibit the enzyme.
Three of the major anticholinesterase agents in therapy are listed on the slide: Physostigmine, Neostigmine, and Edrophonium; another is not listed but is important in therapy – Pyridostigmine (on drug list). With Neostigmine, Edrophonium and Pyridostigmine you will have a plus charge on the nitrogen atom and it is a quaternary ammonium compound.
What is significant is that they have very low lipid solubility and will not penetrate the blood brain barrier.
Physostigmine has a tertiary ammonium grouping and it does have more than adequate lipid solubility to penetrate the blood brain barrier.
This is the only difference in these anticholinesterases.
Neostigmine is the example on the slide.
It binds in the same way to the enzyme as does acetylcholine and a carbamylated intermediate is formed and it will subsequentially hydrolyze to reactivate the enzyme again after 2-4 hours, rather than 115 milliseconds as in acetylcholine.
The enzyme will be inhibited for substantially increased amount of time compared to when acetylcholine binds to it and this accounts for its ultimate therapeutic effect.
Do not memorize the structure of the three compounds listed on the slide, but know that they have the quaternary ammonium compound.
These four compounds are said to be “reversible” indirect-acting cholinomimetics.

Isoflurophate reaction at AChE esteratic site, aging, spontaneous reactivation and oxime reactivation. [S9]
There is another group – the irreversible type.
These cholinesterase inhibitors all have phosphorus as part of their structure.
Isoflurophate is a very simple one and has an easy mechanism of action, but you don’t need to remember it.
Tabun and Sarin were once synthesized for nerve gases.
They are “irreversible” inhibitors of acetylcholinesterase.
The compound binds to the enzyme in this example only to the esteratic site, which ultimately forms this phosphorus-oxygen bond, which is extremely strong.
Initial binding is depicted on the slide where there is reorganization into another structure.
In the absence of any therapy, hydrolysis of this intermediate will not occur for days, weeks or months. During that process that occurs fairly early, the term “aging” is applied to what happens to the enzyme; it permanently becomes as depicted on the slide.
That enzyme will never function again.
There is however a compound that is part of a combination product that soldiers had when they were training should they be exposed to nerve gas.
One of the two compounds was Pralidoxime (2-PAM); this compound has a very strong affinity for a particular bond (P-O) and will break it and when it does subsequent hydrolysis reactivates the enzyme.
2-PAM in an anecdote for extreme cholinesterase inhibition. 2-PAM is part of drugs that are in the armamentarium in Children’s Hospital because so many insecticides contain irreversible acting inhibitors of cholinesterase. When a child goes into Children’s Hospital or a full grown male goes into University Hospital because of symptoms as a result of something like crop-dusting efforts, he will be given 2-PAM as soon as possible.
What is the other ingredient? 2-PAM will take care of the nicotinic effect caused by a compound such as this, which can bring about respiratory arrest and death, but the muscarinic effects of extensive cholinesterase inhibition are not. The other part of that mixture is Atropine, an antimuscarinic drug given with 2-PAM in order to minimize as much as possible the excess muscarinic effects as a result of cholinesterase inhibition. We will see how this works when we discuss that class of drugs.

*Slide 10 was covered yesterday.*

Effects of Anticholinesterase Agents [S11]
Here is a list of the effects of inhibition of cholinesterase. In a way it mirrors the effect of acetylcholine.
Skin – profuse sweating
Digestive – increased secretions, increased tone and motility in the gut and this can lead to cramping, vomiting, diarrhea and defecation, enhanced acetylcholine activity
Urinary – increased frequency of urination and incontinence
Respiratory – increased bronchial secretions, bronchoconstriction and weakness or paralysis of respiratory muscles as well as other skeletal muscles
Skeletal muscle – muscle fasciculations is an initial excitation where you have uncoordinated skeletal muscle contractions all over the body because you have so much acetylcholine continuously stimulating the nicotinic receptors at the neuromuscular junctions that will eventually fade into muscle weakness and ultimately paralysis or a depolarizing blockade.
Think of fasciculations as Jell-o on the hood of a sports car with a wiggling-type movement.
Fasciculations  weakness  paralysis
Cardiovascular – bradycardia, decreased cardiac output, hypotension; now we are getting sufficient activity of acetylcholine at the vascular endothelium to produce this effect; also effects of ganglionic actions and activation of adrenal medulla.
Central nervous system – tremor, anxiety, restlessness which can progress culminating into circulatory/respiratory depression and possibly death (although death would be more likely to occur from a respiratory effect opposed to a cardiovascular effect).
The effect of these anticholinesterase agents mirrors the effects of acetylcholine itself.
There is one very important therapeutic use of anticholinesterase agents and another use, which is common and important as well. The important one is in the treatment of Myasthenia gravis – an autoimmune disease which results over a protracted period of time in the loss of neuromuscular endplates. If you do not have neuromuscular endplates, how are you going to get skeletal muscle activity?
Certain symptoms include muscle weakness, lethargy, diplopia (double vision), drooping eyelids, etc.
The hallmark therapy of Myasthenia Gravis is the use of Neostigmine or Pyridostigmine to enhance the effects of the acetylcholine that is still being released during the course of the disease.
Neostigmine has a slightly shorter duration of effect compared to Pyridostigmine. Four doses a day with Neostigmine and two or three with Pyridostigmine.
The use of Edrophonium (alcohol-like compound): the duration of effect is on the order of 20 minutes verses several hours with Neostigmine or Physostigmine. It has a relatively important use in the diagnosis of Myasthenia gravis. If one is suspected of having Myasthenia gravis (see symptoms) an intravenous or subcutaneous injection of Edrophonium will restore “normal-looking” skeletal function in about 5 minutes. The effect will last another 5 or 10 minutes and will then return to the pre-edrophonium state. This is a very good test to diagnose Myasthenia in a patient.
One other point: Cholinergic crisis vs. Myasthenia crisis
The use of an anticholinesterase inhibitor in a patient with Myasthenia can be subject to times at which there is too much given of the agent verses times where not enough of the agent is being given.
If too much is being given, the patient will display signs of cholinergic crisis (too much acetylcholine).
If not enough is being given, the term “Myasthenia crisis” is used and the patient will suddenly display signs suggesting the drug is not working.
Because of its short duration of effect, Edrophonium can be used to distinguish between cholinergic crisis vs. Myasthenia crisis.
If it is a cholinergic crisis, Edrophonium would either have no effect or it would intensify it.
On the other hand, if it was a Myasthenia crisis, the Edrophonium would immediately improve the overall condition of the patient.
Those are the only two specialized uses for edrophonium.
Lastly, with the anti-cholinesterases, there are some new compounds that give modest therapeutic effect in the treatment of Alzheimer’s, which is characterized by patients who have a loss of cholinergic function in the nervous system.
There are certain cholinesterase inhibitors that can be used to promote this modest effect including compounds Rivastigmine and Donepezil (on drug list).
The duration of these two effects are longer and require less frequent dosing than the original one first used for this process - Tacarine, which had an incidence of disturbed hepatic function.
These two drugs, Rivastigmine and Donepezil, do not share the liver toxicity shown in Tacarine.
Point of emphasis: The efficacy of these drugs of treating Alzheimer’s is modest at best.

Effects of Muscarinic Blocking Drugs [S12]
Antimuscarinic receptor prototype agent is Atropine.
All of these antimuscarinic compounds are specific for competitive reversible blockade of muscarinic receptors. They do not have any affinity for the nicotinic receptors.
When you consider all of the subtypes of muscarinic receptors (1-5), Atropine and most of the antimuscarinic receptors used in therapy today are non-selective; all block any of the subtypes of the muscarinic subtypes equally.
Reversible, competitive inhibitors of muscarinic receptors. No affinity for nicotinic receptors.
Using Atropine as the prototype, you see all of these effects, which you should begin to be able to predict if you know what acetylcholine does at muscarinic receptors.
Atropine was initially used for cosmetic reasons in the time of the Roman Empire. “Belladonna” is a plant containing Atropine and was applied to beautiful ladies and their hallmark, characteristic appearance were wide pupils.
Skin – inhibition of sweating, which may result in the dangerous condition of hyperpyrexia, especially in children; the patient is said to be “dry as a bone” (effect of too much Atropine)
Visual
Cycloplegia (as opposed to cyclospasm)
Now the ciliary muscle and the circular smooth muscle of the iris cannot contract. The combination of inhibition of those two muscles reduces the condition of cycloplegia.
What happens to the size of the pupil exposed to Atropine? It will get bigger.
Important point: When you consider tissues or organs that have dual autonomic innervation (both parasympathetic as well as sympathetic), if you inhibit the effect of one, you enhance the effect of the other.
In the eye, if you block parasympathetic stimulation with Atropine, sympathetic stimulation is unaffected. The background activity of the sympathetic nerve will produce contraction of the radial smooth muscles and mydriasis.
Although the action of Atropine is on the muscarinic receptors, the effect in the eye is observed as mydriasis. The patient is said to be “blind as a bat,” as were the belladonna women in the Roman Empire.
There is also an increase in aqueous outflow resistance; the ciliary muscle relaxes.
This can be very dangerous in people who are said to have “narrow-angle” glaucoma.
Narrow angle - angle between the iris and the area of the trabeculum meshwork, compared to “normal-angle”
Digestive system – basically shut it down
Urinary - relaxation of the detrusor muscle & effectively contraction of the sphincters, leads to urinary retention
Respiratory – bronchial dilation and decreased secretions
Atropine unsuccessfully treated asthma in the past.
Cardiovascular - bradycardia at lower doses and tachycardia at higher doses
Why tachycardia at higher doses of Atropine? Will see at the blockade of muscarinic receptors because the sympathetic input to the heart is less unopposed.
Why bradycardia at low doses of Atropine? The Atropine is blocking the cholinergic autoreceptors at the SA node. Control on the release of the transmitter acetylcholine is abolished. This is the early possible effect of antimuscarinic compound on the heart.
Bradycardia - slight intensification of acetylcholine because more is being released.
Central nervous system – opposite effect of what acetylcholine does; instead of tremor/excitement, you see decrease concentration of memory, drowsiness and sedation (the sequence of effects that follow will not be testable); eventually get to a coma and death due to excess Atropine.
CNS effects tend to produce patients who are as “mad as a hatter”
Four characteristics of Atropine:
Blind as a bat.
Dry as a bone.
Mad as a hatter.
Red as a beat – if you can’t sweat, you are going to get red; there will be peripheral vasodilation in the skin area, hence the redness

Antimuscarinic drugs [S13] and additional notes on [S12]
Examples of some antimuscarinic drugs that are important in the therapy involving an antimuscarinic blockade with antimuscarinic compounds.
CNS – the sedative effect of what antimuscarinic compound is usefulto control motion sickness?
Polamine (truth serum) – has great lipid solubility therefore penetrates the blood brain barrier very well and has an effect on the vestibular apparatus in the brain
Visual – which compound on the list would you prefer to use to cause mydriasis to allow the examination of the retina? Tropicamide. Note its duration of effect.
Tropicamide and a sympathomymetic (Phenylphrine) combination cause pupil dilation. This returns near vision and mydriasis begins to wear off.
Digestive and Urinary systems - two drugs are now available to treat hyperactivity of the GI or urinary tract. Oxybutynin & Totaridine (bold face drugs) that are fairly new compounds.
What’s their advantage?
They have some M3 selectivity. They will preferentially block the M3 in the gut and urinary system. However, there are several possible side effects.
Respiratory - two antimuscarinic drugs: Tiotropium and Ipratropium
Why are they better than Atropine? Atropine gets into the brain. These two can be administered as aerosols and get to where they will have activity in concentrations that do not get into the systemic circulation to any significant extent in therapeutic doses.
Ipratropium and Tiotropium are the agents of choice to manage COPD, especially Ipratropium.
These compounds are useful adjuncts (not first-line therapy) for asthma when there is a significant cholinergic input to the cause of the asthma
Tiotropium is slightly better because it has a slightly longer duration of effect.
M3-selective muscarinic compounds - Darifenacin and Solifenacin (not in drug list, but in textbook) have an advandtage of Oxybutynin and Totaridine in that they have longer durations of action.
Solifenacin is in the drug Vesicare

[end 52:02 min]