O Cholinergic Receptor Antagonists

Characterization of muscarinic receptors can now be extended beyond the pharmacological observations on organ systems (e.g., smooth muscle, heart) to determine structure-activity relationships. Dissociation constants of antagonists from radioligand-binding experiments on the various muscarinic receptors have played a major role in identifying these receptors and the selectivity of antagonists to the five muscarinic receptor subtypes. Antagonists with high affinity for one receptor and a low affinity for the other four receptor types are very few, however, and many antagonists bind to several subtypes with equal affinity. Mi receptors have been identified as those with high affinity for pirenzepine and low affinity for a compound such as AF-DX 116. Pirenzepine can distinguish between M1 and M2, M3, or M5 but has significant affinity for M4 receptors. Himbacine can distinguish between M1 and M4 receptors. Methoctramine, a polymethylenetetramine, not only discriminates between M1 and M2 receptors but also has good selectivity for M2 muscarinic receptors. M2 receptors bind to AF-DX 116 and gallamine, a neuromuscular blocking agent. M3 receptors have a high affinity for 4-diphenylacetoxy-N-methylpiperi-dine (4-DAMP) and hexahydrosiladifenidol (HHSiD) but

Tripitiramine Antagonist
Figure 17.13 • Chemical structures of partially selective muscarinic antagonists.

also exhibit affinity for M1 and M2 receptors.21 Tropicamide has been reported to be a putative M4 receptor antagonist. Figure 17.13 includes structures of some receptor subtype antagonists.


Acetylcholine Chloride. ACh chloride exerts a powerful stimulant effect on the parasympathetic nervous system. Attempts have been made to use it as a cholinergic agent, but its duration of action is too short for sustained effects, because of rapid hydrolysis by esterases and lack of specificity when administered for systemic effects. It is a cardiac depressant and an effective vasodilator. Stimulation of the vagus and the parasympathetic nervous system produces a tonic action on smooth muscle and induces a flow from the salivary and lacrimal glands. Its cardiac-depressant effect results from (a) a negative chronotropic effect that causes a decrease in heart rate and (b) a negative inotropic action on heart muscle that produces a decrease in the force of myo-cardial contractions. The vasodilatory action of ACh is primarily on the arteries and the arterioles, with distinct effect on the peripheral vascular system. Bronchial constriction is a characteristic side effect when the drug is given systemically.

One of the most effective antagonists to the action of ACh is atropine, a nonselective muscarinic antagonist. Atropine blocks the depressant effect of ACh on cardiac muscle and its production of peripheral vasodilation (i.e., muscarinic effects) but does not affect the skeletal muscle contraction (i.e., nicotinic effect) produced.

ACh chloride is a hygroscopic powder that is available in an admixture with mannitol to be dissolved in sterile water for injection shortly before use. It is a short-acting miotic when introduced into the anterior chamber of the eye and is especially useful after cataract surgery during the placement of sutures. When applied topically to the eye, it has little therapeutic value because of poor corneal penetration and rapid hydrolysis by AChE.

Methacholine Chloride, United States Pharmacopeia (USP). Methacholine chloride, acetyl-^-methylcholine chloride or (2-hydroxypropyl)trimethylammonium chloride acetate, is the acetyl ester of jS-methylcholine. Unlike ACh, methacholine has sufficient stability in the body to give sustained parasympathetic stimulation. This action is accompanied by little (1/1,000 that of ACh) or no nicotinic effect.

Methacholine can exist as (S) and (R) enantiomers. Although the chemical is used as the racemic mixture, its muscarinic activity resides principally in the (s)-isomer. The (s)/(r) ratio of muscarinic potency for these enantiomers is 240:1.

(+)-Acetyl-(s)-j8-methylcholine is hydrolyzed by AChE, whereas the (r)( —)-isomer is not. ( — )-Acetyl-(r)-j-methylcholine is a weak competitive inhibitor (Ki, 4 X 10—4 M) of AChE obtained from the electric organ of the eel (E. electricus). The hydrolysis rate of the (s)(+)-isomer is about 54% that of ACh. This rate probably compensates for any decreased association (affinity) owing to the j-methyl group with the muscarinic receptor site and may account for the fact that ACh and (+)-acetyl-j-methylcholine have equimolar muscarinic potencies in vivo. ( — )-Acetyl-(r)-jS-methylcholine weakly inhibits AChE and slightly reinforces the muscarinic activity of the (s)(+)-isomer in the racemic mixture of acetyl-jS-methylcholine.

In the hydrolysis of the acetyl a- and jS-methylcholines, the greatest stereochemical inhibitory effects occur when the choline is substituted in the jS-position. This also appears to be true of organophosphorous inhibitors. The (r)( —)- and (s)(+)-isomers of acetyl-a-methylcholine are hydrolyzed at 78% and 97% of the rate of ACh, respectively.

Methacholine chloride occurs as colorless or white crystals or as a white crystalline powder. It is odorless or has a slight odor and is very deliquescent. It is freely soluble in water, alcohol, or chloroform, and its aqueous solution is neutral to litmus and bitter. It is hydrolyzed rapidly in alkaline solutions. Solutions are relatively stable to heat and will keep for at least 2 or 3 weeks when refrigerated to delay growth of molds.

Carbachol. Choline chloride carbamate is nonspecific in its action on muscarinic receptor subtypes. The pharmacological activity of carbachol is similar to that of ACh. It is an ester of choline and thus possesses both muscarinic and nicotinic properties by cholinergic receptor stimulation. It can also act indirectly by promoting release of ACh and by its weak anticholinesterase activity. Carbachol forms a car-bamyl ester in the active site of AChE, which is hydrolyzed more slowly than an acetyl ester. This slower hydrolysis rate reduces the amount of free enzyme and prolongs the duration of ACh in the synapse. Carbachol also stimulates the autonomic ganglia and causes contraction of skeletal muscle but differs from a true muscarinic agent in that it does not have cardiovascular activity despite the fact that it seems to affect M2 receptors.42

Carbachol is a miotic and has been used to reduce the intraocular tension of glaucoma when a response cannot be obtained with pilocarpine or neostigmine. Penetration of the cornea is poor but can be enhanced by the use of a wetting agent in the ophthalmic solution. In addition to its topical use for glaucoma, carbachol is used during ocular surgery, when a more prolonged miosis is required than can be obtained with ACh chloride.

Carbachol differs chemically from ACh in its stability to hydrolysis. The carbamyl group of carbachol decreases the electrophilicity of the carbonyl and, thus, can form resonance structures more easily than ACh can. The result is that carbachol is less susceptible to hydrolysis and, therefore, more stable in aqueous solutions.

Bethanechol Chloride, USP. Bethanechol, jS-methyl-choline chloride carbamate, (2-hydroxypropyl)trimethy-lammonium chloride carbamate, carbamylmethylcholine chloride (Urecholine), is nonspecific in its action on muscarinic receptor subtypes but appears to be more effective at eliciting pharmacological action of M3 receptors. It has pharmacological properties similar to those of metha-choline. Both are esters of jS-methylcholine and have feeble nicotinic activity. Bethanechol is inactivated more slowly by AChE in vivo than is methacholine. It is a carbamyl ester and is expected to have stability in aqueous solutions similar to that of carbachol.

The main use of bethanechol chloride is in the relief of urinary retention and abdominal distention after surgery. The drug is used orally and by subcutaneous injection. It must never be administered by intramuscular or intravenous injection because of the danger from cholinergic overstimulation and loss of selective action. Proper administration of the drug is associated with low toxicity and no serious side effects. Bethanechol chloride should be used with caution in asthmatic patients; when used for glaucoma, it produces frontal headaches from the constriction of the sphincter muscle in the eye and from ciliary muscle spasms. Its duration of action is 1 hour.

Pilocarpine Hydrochloride, USP. Pilocarpine monohy-drochloride is the hydrochloride of an alkaloid obtained from the dried leaflets of Pilocarpus jaborandi or p. micro-phyllus, in which it occurs to the extent of about 0.5% together with other alkaloids.

Pilocarpine Hydrochloride

It occurs as colorless, translucent, odorless, faintly bitter crystals that are soluble in water (1:0.3), alcohol (1:3), and chloroform (1:360). (In this chapter, a solubility expressed as 1:360 indicates that 1 g is soluble in 360 mL of the solvent at 25°C. Solubilities at other temperatures are so indicated.) It is hygroscopic and affected by light; its solutions are acid to litmus and may be sterilized by autoclaving. Alkalies saponify the lactone group to give the pharmacologically inactive hydroxy acid (pilocarpic acid). Base-catalyzed epimerization at the ethyl group position occurs to an appreciable extent and is another major pathway of degradation.44 Both routes result in loss of pharmacological activity.

Pilocarpine is a nonselective agonist on the muscarinic receptors. Despite this, it reportedly acts on M3 receptors in smooth muscle to cause contractions in the gut, trachea, and eye.45,46 In the eye, it produces pupillary constriction (miosis) and a spasm of accommodation. These effects are valuable in the treatment of glaucoma. The pupil constriction and spasm of the ciliary muscle reduce intraocular tension by establishing better drainage of ocular fluid through the canal of Schlemm, located near the corner of the iris and cornea. Pilocarpine is used as a 0.5% to 0.6% solution (i.e., of the salts) in treating glaucoma. Systemic effects include copious sweating, salivation, and gastric secretion.

Pilocarpine Nitrate, USP. Pilocarpine mononitrate occurs as shining white crystals that are not hygroscopic but are light sensitive. It is soluble in water (1:4) and alcohol (1:75) but insoluble in chloroform and ether. Aqueous solutions are slightly acid to litmus and may be sterilized in the autoclave. The alkaloid is incompatible with alkalies, iodides, silver nitrate, and reagents that precipitate alkaloids.

Cevimeline Hydrochloride. Cevimeline (Evoxac) is ds-2'-methylspiro {1-azabicyclo [2.2.2] octane-3, 5' -[1,3] oxathiolane} hydrochloride, hydrate (2:1). Cevimeline has a molecular weight of 244.79 and is a white to off white crystalline powder. It is freely soluble in alcohol, chloroform, and water. Cevimeline is a cholinergic agonist which binds to the M3 muscarinic receptor subtype, which results in an increase secretion of exocrine glands, such as salivary and sweat glands. Because of these effects, it was approved for use in the treatment of dry mouth associated with Sjögren syndrome. By stimulating the salivary muscarinic receptors cevimeline promotes secretion thereby alleviating dry-mouth in these patients. Cevimeline is metabolized by the isozymes CYP2D6 and CYP3A3 and CYP3A4. It has a half-life of 5 hours.47

Cholinesterase Inhibitors

There are two types of cholinesterases in humans, AChE and butyrylcholinesterase (BuChE). The cholinesterases differ in their location in the body and their substrate specificity. AChE is associated with the outside surface of glial cells in the synapse and catalyzes the hydrolysis of ACh to choline and acetic acid. Inhibition of AChE prolongs the duration of the neurotransmitter in the junction and produces pharmacological effects similar to those observed when ACh is administered. These inhibitors are indirect-acting cholinergic agonists. AChE inhibitors have been used in the treatment of myasthenia gravis, atony in the GI tract, and glaucoma. They have also been used as agricultural insecticides and nerve gases. More recently, they have received attention as symptomatic drug treatments in patients suffering from Alzheimer disease.48

BuChE (pseudocholinesterase) is located in human plasma. Although its biological function is not clear, it has catalytic properties similar to those of AChE. The substrate specificity is broader (Table 17.5), and it may hydrolyze dietary esters and drug molecules in the blood.

Three different chemical groupings, acetyl, carbamyl, and phosphoryl, may react with the esteratic site of AChE. Although the chemical reactions are similar, the kinetic parameters for each type of substrate differ and result in differences between toxicity and usefulness.

The initial step in the hydrolysis of ACh by AChE is a reversible enzyme-substrate complex formation. The association rate (k+1) and dissociation rate (k_1) are relatively large. The enzyme-substrate complex, EA-ACh, may also

TABLE 17.5 Hydrolysis of Various Substrates by AChE and BuChE


Enzyme Substrate Source Relative Rate3 Source Relative Rate3

TABLE 17.5 Hydrolysis of Various Substrates by AChE and BuChE


Enzyme Substrate Source Relative Rate3 Source Relative Rate3


Human or bovine RBC

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