2. This chapter supports the fact that acetylcholine (ACh) plays a critical role in the mechanisms of regulation of human physiology. This, in turn, not only serves in the development of agents that mimic the effect of ACh, but also those that block its effects as therapeutic agents.

3. studies of Dale,1 who described the actions of the esters and ethers of choline on isolated organs and their relationship to muscarine, pharmacologists, physiologists, chemists, and biochemists have applied their knowledge to understand the actions of the cholinergic nerve and its neurotransmitter. This chapter includes the drugs and chemicals that act on cholinergic nerves or the tissues they innervate to either mimic or block the action of ACh.

4. Acetylcholine (ACh) Was first demonstrated in the frog heart in 1921 by Loewi as the substance released by vagus nerve stimulation. Drugs that mimic the action of ACh do so either by acting directly on the cholinergic receptors in the tissue or by inhibiting acetylcholinesterase(AChE). acetylcholinesterase(AChE)- the enzyme that inactivates ACh at the nerve terminal Chemicals that bind or compete with ACh for binding to the receptor may block cholinergic neurotransmission.

5. Cholinergic nerves - are found in the peripheral nervous system and central nervous system (CNS) of humans. Regions Rich in ACh 1.Synaptic terminals in the cerebral cortex, 2.corpus striatum, 3.hippocampus, and 4.several other regions in the CNS

6. The peripheral nervous system- consists of those nerves outside the cerebrospinal axis and includes the somatic nerves and the autonomic nervous system The somatic nerves are made up of a sensory (afferent) nerve and a motor (efferent) nerve. The motor nerves arise from the spinal cord and project uninterrupted throughout the body to all skeletal muscle. ACh mediates transmission of impulses from the motor nerve to skeletal muscle (i.e., neuromuscular junction)

7. The autonomic nervous system is composed of two divisions: sympathetic and parasympathetic. The autonomic nervous system regulates the activities of smooth muscle and glandular secretions. ACh serves as a neurotransmitter at both sympathetic and parasympathetic preganglionic nerve endings, postganglionic nerve fibers in the parasympathetic division, and some postganglionic fibers (e.g., salivary and sweat glands) in the sympathetic division of the autonomic nervous system

8. The two divisions have contrasting effects on the internal environment of the body. The sympathetic division frequently discharges as a unit, especially during conditions of rage or fright, and expends energy. The parasympathetic division is organized for discrete and localized discharge and stores and conserves energy

9. Parasympathomimetic - Drugs and chemicals that cause the parasympathetic division to react Parasympatholytic- blocking the actions of Parasympathomimetic sympathomimetic -Agents that mimic the sympathetic division sympatholytic - block the actions of sympathomimetic

10. Drugs acting on the autonomic nervous system are divided into adrenergic, for those postganglionic sympathetic fibers that release norepinephrine and epinephrine, cholinergic, for the remaining fibers in the autonomic nervous system and the motor fibers of the somatic nerves that release ACh.

11. CHOLINERGIC RECEPTORS two distinct receptor types for Ach 1.nicotinic 2.muscarinic

12. Nicotinic Receptors

13. Nicotinic receptors are coupled directly to ion channels and, when activated by Ach, mediate very rapid responses. Ion channels - responsible for the electrical excitability of nerve and muscle cells and for the sensitivity of sensory cells - open or close in an all-or-nothing fashion on time scales ranging from 0.1 to 10 milliseconds to provide aqueous pathways through the plasma membrane that ions can transverse

14. Factors affecting selectivity of ion pores 1.charge of the ion 2.size of the ion Ions in aqueous solution are hydrated. The water around the ion is characterized by the presence of two distinct water structures: a tightly bound, highly ordered layer immediately surrounding the ion and a second, less structured layer.

16. Lists of the effective radii of alkali and alkaline earth cations.

17. The nicotinic ACh receptor was the first neurotransmitter isolated and purified in an active form. It is a glycoprotein embedded into the polysynaptic membrane that can be obtained from the electric organs of the marine ray, Torpedo californica and the electric eel, Electrophorus electricus. The peptide chains of the receptor are arranged to form an opening in the center, which is the ion channel. Each chain contains a negatively charged binding site for the quaternary ammonium group of ACh. The receptor appears to exist as a dimer of the two five-subunit polypeptide chain monomers linked through a disulfide bond between chains.

18. When the neurotransmitter ACh binds to the nicotinic receptor, it causes a change in the permeability of the membrane to allow passage of small cations Ca2, Na, and K. The physiological effect is to temporarily depolarize the end plate. results in muscular contraction at a neuromuscular junction or, as occurs in autonomic ganglia, continuation of the nerve impulse.

19. Neuromuscular nicotinic ACh receptors are of interest as targets for autoimmune antibodies in myasthenia gravis and for muscle relaxants used during the course of surgical procedures. Nicotinic receptors in autonomic ganglia, when blocked by drugs, can play a role in the control of hypertension.

20. NICOTINIC RECEPTOR SUBTYPES

21. Nicotinic receptors located in the neuromuscular junction differ from those on neurons, such as those in the CNS and autonomic ganglia, in that they have different ligand specificities. N1-Nicotinic receptors at the neuromuscular junction are blocked by: 1. succinylcholine, 2. d-tubocurarine, 3. Decamethonium Stimulated by: phenyltrimethylammonium

22. N2-nicotinic receptors are found in autonomic ganglia are blocked by: 1.hexamethonium 2.Trimethaphan Stimulated by: tetramethylammonium and dimethyl-4- phenylpiperazinium (DMPP)

23. Muscarinic Receptors

24. Muscarinic receptors play an essential role in regulating the functions of organs innervated by the autonomic nervous system to maintain homeostasis of the organism. The action of ACh on muscarinic receptors can result in stimulation or inhibition of the organ system affected. ACh stimulates secretions from salivary and sweat glands, secretions and contraction of the gut, and constriction of the airways of the respiratory tract. It inhibits contraction of the heart and relaxes smooth muscle of blood vessels. In 1980- it became apparent that a single muscarinic receptor type could not mediate the actions of ACh. Muscarinic receptors mediate their effects by activating guanosine triphosphate (GTP)-binding proteins (G proteins).

25. These receptors have seven protein helixes that transcend the plasma membrane, creating four extracellular domains and four intracellular domains:

26. MUSCARINIC RECEPTOR SUBTYPES

27. Located in the CNS and peripheral nervous system Five different cloned molecular mammalian muscarinic receptor subtypes: 1.M1 2.M2 3.M3 4.M4 5.M5

28. The nomenclature convention adopted for these receptors is that the pharmacologically defined subtypes M1, M2, and M3 correspond to the genetically defined subtypes m1, m2, and m3. The m4 gene-derived protein is referred to as the M4 subtype and has many pharmacological properties similar to those of the M2 subtype. The m5 receptor gene product does not have an equivalent pharmacological profile.

29. M1 Receptors defined as those with high affinity for pirenzepine and low affinity for compounds such as AF-DX 116 termed neural because of their distribution within particular brain structures located in exocrine glands and autonomic ganglia In humans, these receptors seem to affect arousal attention, rapid eye movement (REM) sleep, emotional responses, affective disorders including depression, and modulation of stress. believed to participate in higher brain functions, such as memory and learning. causes gastric secretion

30. McN-A343 or ((4-(m-Chlorophenylcarbamoyloxy)-2- butynyl)trimethylammonium chloride)- a selective agonist Pirenzepine Hydrochloride (HCl) - acts as an antagonist and has been used outside the United States for the treatment of peptic ulcer disease.

31. M2 Receptors Are identified by their high affinity for methoctramine, a polyamine, and by their low affinity for pirenzepine. also called cardiac muscarinic receptors (because they are located in the atria and conducting tissue of the heart) Causes a decrease in the strength and rate of cardiac muscle contraction These effects may be produced by affecting intracellular K and Ca2 levels in heart tissue. M2 receptors activate K channels to cause hyperpolarization of cardiac cells, resulting in bradycardia

32. Receptors may also act through an inhibitory G protein (Gi) to reduce adenylate cyclase activity and lower cyclic 3,5-adenosine monophosphate (cAMP) levels in cardiac cells. Lower cAMP levels decrease the amount of free Ca2 in cardiac cells and slow down the heart rate M2 receptors can also serve as autoreceptors on presynaptic terminals of postganglionic cholinergic nerves to inhibit ACh release. The balance of the effects of multiple muscarinic receptor subtypes determines the size of the airway of the smooth muscle in the bronchioles. Contraction is primarily the result of the action of ACh on M3 receptors (see on next page) following stimulation of the vagus. At the same time, ACh stimulates inhibitor M2 autoreceptors located on nerve endings to limit release of ACh. In asthmatics, neuronal M2 receptors in the lungs do not function normally.

33. M3 Receptors referred to as glandular muscarinic receptors located in exocrine glands and smooth muscle. The effect is mostly stimulatory Characteristic of M3 receptor activation 1.Glandular secretions from: lacrimal, 2.salivary, 3.bronchial, 4.pancreatic, and 5.mucosal cells in the GI tract Contraction of visceral smooth muscle is also a result of M3 receptor stimulation.

34. Contraction of visceral smooth muscle is also a result of M3 receptor stimulation. These stimulant effects are mediated through G protein activation of phospholipase C (PLC) to form the secondmessenger inositol triphosphate (IP3) and diacylglycerol (DAG).

35. As past studies have revealed that the endothelium can control the tone of vascular smooth muscle by the synthesis of a potent relaxant, endothelium-derived relaxing factor (EDRF), now identified as nitric oxide (NO), and a vasoconstrictor substance, endothelium-derived contracting factor (EDCF). The synthesis and release of these substances contribute to the tone of the vascular epithelium. M3 receptors, when activated in endothelial cells, cause the release of EDRF and contribute to vasodilation.

36. M4 Receptors like M2 receptors, act through Gi protein to inhibit adenylate cyclase. They also function by a direct regulatory action on K and Ca2 ion channels. M4 receptors in tracheal smooth muscle, when stimulated, inhibit the release of ACh19 in the same manner that M2 receptors do.

37. M5 Receptors A great deal of research remains to be performed on the M5 subclass of receptors. Because the M5 receptor messenger RNA (mRNA) is found in the substantia nigra, it has been suggested that M5 receptors may regulate dopamine release at terminals within the striatum.

38. BIOCHEMICAL EFFECTS OF MUSCARINIC RECEPTOR STIMULATION Transmission at the synapse involving second messengers is much slower, about 100 ms, compared with the few milliseconds at synapses where ion channels are activated directly. The delayed reaction to receptor stimulation is caused by a cascade of biochemical events that must occur to cause the pharmacological response. The sequence of events in these second-messenger systems begins with activation of the receptors by an agonist and involves the activation of G proteins that are bound to a portion of the intracellular domain of the muscarinic receptor.

39. G proteins G proteins are so called because of their interaction with the guanine nucleotides GTP and guanosine diphosphate (GDP). They translate drug–receptor interactions at the surface of the cell to components inside the cell to create the biological response. Three subunits: When the receptor is occupied, the subunit, which has enzymatic activity, catalyzes the conversion of GTP to GDP. The subunit bound with GTP is the active form of the G protein that can associate with various enzymes (i.e., PLC and adenylate cyclase) and ion channels (K and Ca2). G proteins are varied, and the subunit may cause activation (Gs) or inactivation (Gi) of the enzymes or channels. Recent studies suggest that B and y subunits also contribute to pharmacological effects.

40. Phosphoinositol System The phosphoinositol system requires the breakdown of membrane-bound phosphatidylinositol 4,5-diphosphate (PIP2) by PLC to IP3 and DAG, which serve as second messengers in the cell. IP3 mobilizes Ca2 from intracellular stores in the endoplasmic reticulum to elevate cytosolic free Ca2. The Ca2 activates Ca2- dependent kinases (e.g., troponin C in muscle) directly or binds to the Ca2-binding protein calmodulin, which activates calmodulin-dependent kinases. These kinases phosphorylate cell-specific enzymes to cause muscle contraction. DAG is lipidlike and acts in the plane of the membrane through activation of protein kinase C to cause the phosphorylation of cellular proteins, also leading to muscle contraction

41. Adenylate Cyclase a membrane enzyme, is another target of muscarinic receptor activation. The second- messenger cAMP is synthesized within the cell from adenosine triphosphate (ATP) by the action of adenylate cyclase. The regulatory effects of cAMP are many, as it can activate various protein kinases. Protein kinases catalyze the phosphorylation of enzymes and ion channels, altering the amount of calcium entering the cell and thus affecting muscle contraction. Muscarinic receptor activation causes lower levels of cAMP, reducing cAMP protein-dependent kinase activity, and a relaxation of muscle contraction. Some have suggested that a GTP-inhibitory protein (Gi) reduces the activity of adenylate cyclase, causing smooth muscle relaxation

42. Ion Channels In addition to the action of protein kinases that phosphorylate ion channels and modify ion conductance, G proteins are coupled directly to ion channels to regulate their action.24 The Ca2 channel on the cell membrane is activated by G proteins without the need of a second messenger to allow Ca2 to enter the cell. The subunit of the G protein in heart tissue acts directly to open the K channel, producing hyperpolarization of the membrane and slowing the heart rate.

43. CHOLINERGIC NEUROCHEMISTRY

44. - Cholinergic neurons synthesize, store, and release ACh. - The neurons also form choline acetyltransferase (ChAT) and AChE. These enzymes are synthesized in the soma of the neuron and distributed throughout the neuron by axoplasmic flow. -AChE is also located outside the neuron and is associated with the neuroglial cells in the synaptic cleft. -ACh is prepared in the nerve ending by the transfer of an acetyl group from acetyl-coenzyme -A (CoA) to choline. The reaction is catalyzed by ChAT. Cell fractionation studies show that much of the ACh is contained in synaptic vesicles in the nerve ending but that some is also free in the cytosol.

45. - Choline is the limiting substrate for the synthesis of ACh. Most choline for ACh synthesis comes from the hydrolysis of ACh in the synapse. Choline is recaptured by the presynaptic terminal as part of a high-affinity uptake system under the in- fluence of sodium ions25 to synthesize ACh. -Several quaternary ammonium bases act as competitive inhibitors of choline uptake. - Hemicholinium (HC-3), a bisquaternary cyclic hemiacetal, and the triethyl analog of choline, -2-hydroxyethyltriethylammonium, act at the presynaptic membrane to inhibit the high-affinity uptake of choline into the neuron. -These compounds cause a delayed paralysis at repetitively activated cholinergic synapses and can produce respiratory paralysis in test animals. The delayed block is caused by the depletion of stored ACh, which may be reversed by choline.

46. CHOLINERGIC AGONISTS

47. Cholinergic Stereochemistry Three techniques have been used to study the conformational properties of ACh and other cholinergic chemicals: x-ray crystallography, nuclear magnetic resonance (NMR), and molecular modeling by computation. each of these methods may report the spatial distribution of atoms in a molecule in terms of torsion angles. A torsion angle is defined as the angle formed between two planes, for example, by the O1MC5MC4MN atoms in ACh. The angle between the oxygen and nitrogen atoms is best depicted by means of Newman projections. A torsion angle has a positive sign when the bond of the front atom is rotated to the right to eclipse the bond of the rear atom. The spatial orientation of ACh is described by four torsion angles

49. The torsion angle determines the spatial orientation of the cationic head of ACh to the ester group. -X-ray diffraction studies have shown that the torsion angle on ACh has a value of 77°. Many com- pounds that are muscarinic receptor agonists containing a choline component (e.g., OMCMCMN [CH3]3) have a preferred synclinal (gauche) conformation, with r2 values ranging 68° to 89°.

50. The parasympathomimetic effects of muscarine were first reported in 1869 but its structure was not elucidated until 1957 Muscarine has four geometric isomers: muscarine, epimuscarine, allomuscarine, and epiallomuscarine

51. CHOLINERGIC RECEPTOR ANTAGONISTS Characterization of muscarinic receptors can now be ex- tended 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. M1 receptors have been identified as those with high affinity for pirenzepine and low affinity for a compound such as AF-DX 116.

52. 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 also exhibit affinity for M1 and M2 receptors.21 Tropicamide has been reported to be a putative M4 receptor antagonist.

54. 1. 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 myocardial contractions. -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.

55. -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.

56. 2. Methacholine Chloride, United States Pharmacopeia (USP). - Methacholine chloride, acetyl-B-methylcholine chloride or (2- hydroxypropyl)trimethylammonium chloride acetate, is the acetyl ester of B-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

57. -Methacholine chloride occurs as colorless or white crystal 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.

58. 3. 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 anti-cholinesterase activity. -Carbachol forms a carbamyl 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.

59. -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.

60. 4. Bethanechol Chloride, USP. -Bethanechol, B-methyl- choline chloride carbamate, (2-hydroxypropyl)trimethy- lammonium chloride carbamate, carbamylmethylcholine chloride (Urecholine), is nonspecific in its action on mus- carinic 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 B- 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.

61. 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.

62. 5. Pilocarpine Hydrochloride, USP. -Pilocarpine monohyrochloride is the hydrochloride of an alkaloid obtained from the dried leaflets of Pilocarpus jaborandi or P. microphyllus, in which it occurs to the extent of about 0.5% together with other alkaloids. -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 sol- vent at 25°C. -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. Both routes result in loss of pharmacological activity. -Pilocarpine is a nonselective agonist on the muscarinic receptors.

63. -Despite this, it reportedly acts on M3 receptors in smooth muscle to cause contractions in the gut, trachea, and eye. - 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.

64. 6. 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.

65. 7. Cevimeline Hydrochloride. - Cevimeline (Evoxac) is cis-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.

66. Cholinesterase Inhibitors

67. -There are two types of cholinesterases in humans Acetylcholinesterase (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 du- ration 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. -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 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.

70. Reversible Inhibitors

71. 1. Physostigmine, USP. -Physostigmine is an alkaloid obtained from the dried ripe seed of Physostigma venenosum. It occurs as a white, odorless, microcrystalline powder that is slightly soluble in water and freely soluble in alcohol, chloroform, and the fixed oils. -The alkaloid, as the free base, is quite sensitive to heat, light, moisture, and bases, undergoing rapid decomposition. -In solution, it is hydrolyzed to methyl carbamic acid and eseroline, neither of which inhibits AChE. -Eseroline is oxidized to a red compound, rubreserine, and then further decomposed to eserine blue and eserine brown. -Addition of sulfite or ascorbic acid prevents oxidation of the phenol, eseroline, to rubreserine. Hydrolysis does take place, however, and the physostigmine is inactivated. Solutions are most stable at pH 6 and should never be sterilized by heat.

73. -Physostigmine is a relatively poor carbamylating agent of AChE and is often considered a reversible inhibitor of the enzyme. -Its cholinesterase-inhibiting properties vary with the pH of the medium. - Physostigmine was used first as a topical application in the treatment of glaucoma. - Its lipid solubility properties permit adequate absorption from ointment bases. -It is used systemically as an antidote for atropine poisoning and other anticholinergic drugs by increasing the duration of action of ACh at cholinergic sites through inhibition of AChE. -Physostigmine, along with other cholinomimetic drugs act- ing in the CNS, has been studied for use in the treatment of Alzheimer disease. -Cholinomimetics that are currently used or which have been recently evaluated in the treatment of Alzheimer disease include donepezil, galantamine, metri- fonate, rivastigmine, and tacrine. It is anticipated that this list will continue to grow as the etiology of this disease be- comes better understood.

74. 2. Physostigmine Salicylate, USP. -The salicylate of physostigmine (eserine salicylate) may be prepared by neutralizing an ethereal solution of the alkaloid with an ethereal solution of salicylic acid. -Excess salicylic acid is removed from the precipitated product by washing it with ether. The salicylate is less deliquescent than the sulfate. -Physostigmine salicylate occurs as a white, shining, odorless crystal or white powder that is soluble in water (1:75), alcohol (1:16), or chloroform (1:6) but much less soluble in ether (1:250). On prolonged exposure to air and light, the crystals turn red. - The red may be removed by washing the crystals with alcohol, although this causes loss of the compound as well. Aqueous solutions are neutral or slightly acidic and take on a red coloration after a period. -The coloration may be taken as an index of the loss of activity of physostigmine solutions. -Solutions of physostigmine salicylate are incompatible with the usual reagents that precipitate alkaloids (alkalies) and with iron salts. Incompatibility also occurs with benzalkonium chloride and related wetting agents because of the salicylate ion.

76. 3. Neostigmine bromide -(m-hy- droxyphenyl)trimethylammonium bromide dimethylcarba- mate or the dimethylcarbamic ester of 3-hydroxyphenyl- trimethylammonium bromide (Prostigmin bromide), -is used as an antidote to nondepolarizing neuromuscular blocking drugs and in the treatment of myasthenia gravis. - It occurs as a bitter, odorless, white, crystalline powder. It is soluble in water and alcohol. -The crystals are much less hygroscopic than those of neostigmine methylsulfate and thus may be used in tablets. Solutions are stable and may be sterilized by boiling. Aqueous solutions are neutral to litmus.

77. 4. Neostigmine Methylsulfate. - Neostigmine methylsul- fate, (m- hydroxyphenyl)trimethylammonium methylsulfate dimethylcarbamate or the dimethylcarbamic ester of 3-hydro- xyphenyltrimethylammonium methylsulfate (Prostigmin methylsulfate), is a bitter, odorless, white, crystalline powder. -It is very soluble in water and soluble in alcohol. Solutions are stable and can be sterilized by boiling. -The compound is too hygroscopic for use in a solid form and thus is always used as an injection. Aqueous solutions are neutral to litmus. -The methylsulfate salt is used postoperatively as a uri- nary stimulant and in the diagnosis and treatment of myas- thenia gravis.

78. 5. Pyridostigmine Bromide, USP. - Pyridostigmine bromide, 3-hydroxy-1-methylpyridinium bromide dimethylcarbamate or pyridostigmine bromide (Mestinon), occurs as a white, hygroscopic, crystalline powder with an agreeable, characteristic odor. It is freely soluble in water, alcohol, and chloroform. -Pyridostigmine bromide is about one fifth as toxic as neostigmine. - It appears to function in a manner similar to that of neostigmine and is the most widely used anti- cholinesterase agent for treating myasthenia gravis. -The liver enzymes and plasma cholinesterase metabolize the drug. The principal metabolite is 3-hydroxy-N-methylpyridinium. -Orally administered pyridostigmine has a half-life of 90 minutes and a duration of action of between 3 and 6 hours.

80. 6. Ambenonium Chloride. -Ambenonium chloride, [oxa- lylbis(iminoethylene)]bis[(o-chlorobenzyl)diethylammo- nium] dichloride (Mytelase chloride), is a white, odorless powder, soluble in water and alcohol, slightly soluble in chloroform, and practically insoluble in ether and acetone. - is used for the treatment of myasthenia gravis in patients who do not respond satisfactorily to neostigmine or pyridostigmine. -This drug acts by suppressing the activity of AChE. It possesses a relatively prolonged duration of action and causes fewer side effects in the GI tract than the other anti- cholinesterase agents. The dosage requirements vary considerably, and the dosage must be individualized according to the response and tolerance of the patient. Because of its quaternary ammonium structure, ambenonium chloride is absorbed poorly from the GI tract. - In moderate doses, the drug does not cross the blood-brain barrier. Ambenonium chloride is not hydrolyzed by cholinesterases.

82. 7. Demecarium Bromide, USP. -Demecarium bromide, (m-hydroxyphenyl)trimethylammonium bromide, decame- thylenebis[methylcarbamate] (Humorsol), is the diester of (m- hydroxyphenyl)trimethylammonium bromide with decamethylene-bis- (methylcarbamic acid) and thus is comparable to a bis-prostigmine molecule. -It occurs as a slightly hygroscopic powder that is freely soluble in water or alcohol. -Ophthalmic solutions of the drug have a pH of 5 to 7.5. Aqueous solutions are stable and may be sterilized by heat. Its efficacy and toxicity are comparable to those of other potent anticholinesterase inhibitor drugs. -It is a long-acting miotic used to treat wide-angle glaucoma and accommodative esotropia. Maximal effect occurs hours after administration, and the effect may persist for days.

84. 8. Donepezil. -Donepezil(+/-)-2,3-dihydro-5,6-dimethoxy- 2-[[1- (phenylmethyl)-4-piperidinyl]methyl]-1H-inden-1- one (Aricept), commonly referred to in the literature as E2020, is a reversible inhibitor of AChE. It is indicated for the treatment of symptoms of mild-to-moderate Alzheimer disease. -Donepezil is approximately 96% bound to plasma proteins, with an elimination half-life of 70 hours. It is metabolized principally by the 2D6 and 3A4 isozymes of the P450 system.

85. 9. Edrophonium Chloride, USP. -Edrophonium chloride, ethyl(m-hydroxyphenyl)dimethylammonium chloride (Tensilon), is a reversible anticholinesterase agent. -It is bitter and very soluble in water and alcohol. Edrophonium chloride injection has a pH of 5.2 to 5.5. On parenteral ad- ministration, edrophonium has a more rapid onset and shorter duration of action than neostigmine, pyridostig- mine, or ambenonium. - It is a specific anticurare agent and acts within 1 minute to alleviate overdose of d- tubocu- rarine, dimethyl d-tubocurarine, or gallamine triethiodide. -The drug is also used to terminate the action of any one of these drugs when the physician so desires. - It is of no value, however, in terminating the action of the depolarizing (i.e., noncompetitive) blocking agents, such as decamethonium and succinylcholine. - In addition to inhibiting AChE, edro- phonium chloride has a direct cholinomimetic effect on skeletal muscle, which is greater than that of most other anticholinesterase drugs.

86. -Edrophonium chloride is structurally related to neostigmine methylsulfate and has been used as a potential diagnostic agent for myasthenia gravis. - This is the only degenerative neuromuscular disease that can be temporarily improved by administration of an anticholinesterase agent. Edrophonium chloride brings about a rapid increase in muscle strength without significant side effects.

87. 10. Galantamine -Galantamine, 4a,5,9,10,11,12-hexahydro- 3- methoxy-11-methyl-6H-benzofuro-[3a,3,2,ef][2]- benzazepin-6-ol (Nivalin, Reminyl), is an alkaloid extracted from the tuberous plant Leucojum aestivum (L.) belonging to the Amaryllidaceae family and from the bulbs of the daffodil, Narcissus pseudonarcissus. - It is a reversible cholinesterase inhibitor that appears to have no effect on bu- tyrylcholinesterase. -In addition, it acts at allosteric nicotinic sites, further enhancing its cholinergic activity. -Galantamine undergoes slow and minor biotransformation with approxi- mately 5% to 6% undergoing demethylation. It is primarily excreted in the urine.

88. 11.Metrifonate -Metrifonate is an organophosphate that was originally developed to treat schistosomiasis under the trade name Bilarcil. - It is an irreversible cholinesterase inhibitor with some selectivity for BuChE over AChE. It achieves sustained cholinesterase inhibition by its non-enzymatic metabolite dichlorvos (DDVP), a long-acting organophosphate. - Its use in mild-to-moderate Alzheimer disease was suspended recently because of adverse effects experienced by several patients during the clinical evaluation of this product. -Toxicity at the neuromuscular junction is probably attributable to the inhibition by the drug of neurotoxic esterase, a common feature of organophosphates.

89. 12. Rivastigmine -Rivastigmine (Exelon, EA 713) is a pseudoirreversible noncompetitive carbamate inhibitor of AChE. Although the half-life is approximately 2 hours, the inhibitory properties of this agent last for 10 hours be- cause of the slow dissociation of the drug from the enzyme. -The Food and Drug Administration (FDA) approved its use in mild-to-moderate Alzheimer disease in April 2000. -In July 2007, rivastigmine was granted approval for use in managing mild-to- moderate dementia associated with Parkinson disease.

90. 13. Tacrine Hydrochloride -Tacrine hydrochloride, 1,2,3,4-tetrahydro-9-aminoacridine hydrochloride (THA, Cognex), is a reversible cholinesterase inhibitor that has been used in the treatment of Alzheimer disease for several years. - The drug has been used to increase the levels of ACh in these patients on the basis of observations from autopsies that concentrations of ChAT and AChE are markedly re- duced in the brain, whereas the number of muscarinic recep- tors is almost normal. - The use of the drug is not without controversy, as conflicting results on efficacy have been re- ported.58,59 The drug has been used in mild-to- moderate Alzheimer dementia.

92. Irreversible Inhibitors

93. -Both AChE and BuChE are inhibited irreversibly by a group of phosphate esters that are highly toxic (LD50 for humans is 0.1–0.001 mg/kg). -These chemicals are nerve poisons and have been used in warfare, in bioterrorism, and as agricul- tural insecticides. They permit ACh to accumulate at nerve endings and exacerbate ACh like actions. The compounds belong to a class of organophosphorus esters. - A is usually oxygen or sulfur but may also be selenium. When A is other than oxygen, biological activation is required before the compound becomes effective as an inhibitor of cholinesterases. - Phosphorothionates [R1R2P(S)X] have much poorer electrophilic character than their oxygen analogs and are much weaker hydrogen bond-forming molecules because of the sulfur atom.

95. - Cholinesterases that have been exposed to phosphorylating agents (e.g., sarin) become refractory to reactivation by cholinesterase reactivators. -The process is called aging and occurs both in vivo and in vitro with AChE and BuChE. Aging occurs by partial hydrolysis of the phosphorylated moiety that is attached to the serine residue at the esteratic site of the enzyme.

96. 1. Isofluorphate, USP -Isofluorphate, diisopropylphos- phorofluoridate (Floropryl), is a colorless liquid soluble in water to the extent of 1.54% at 25°C, which decomposes to give a pH of 2.5. -It is soluble in alcohol and to some extent in peanut oil. It is stable in peanut oil for a period of 1 year but decomposes in water in a few days. Solutions in peanut oil can be sterilized by autoclaving. -The compound should be stored in hard glass containers. Continued contact with soft glass is said to hasten decomposition, as evidenced by discoloration. -Isofluorphate must be handled with extreme caution. Contact with eyes, nose, mouth, and even skin should be avoided because it can be absorbed readily through intact epidermis and more so through mucous tissues.Because isofluorphate irreversibly62 inhibits cholinesterase, its activity lasts for days or even weeks. During this period, new cholinesterase may be synthesized in plasma, erythrocytes, and other cells.

97. -A combination of atropine sulfate and magnesium sul- fate protects rabbits against the toxic effects of isofluorphate. -Atropine sulfate counteracts the muscarinic effect, and magnesium sulfate counteracts the nicotinic effect of the drug. Isofluorphate has been used in the treatment of glaucoma.

98. 2. Echothiophate Iodide, USP - Echothiophate iodide, (2-mercaptoethyl)trimethylammonium iodide, S- ester with O,O-diethylphosphorothioate (Phospholine Iodide), oc- curs as a white, crystalline, hygroscopic solid that has a slight mercaptan-like odor. - It is soluble in water (1:1) and dehydrated alcohol (1:25); aqueous solutions have a pH of about 4 and are stable at room temperature for about 1 month. Echothiophate iodide is a long-lasting cholinesterase inhibitor of the irreversible type, as is isofluorphate. -Unlike the latter, however, it is a quaternary salt, and when applied locally, its distribution in tissues is limited, which can be very desirable. -It is used as a long-acting anticholinesterase agent in the treatment of glaucoma.

100. 3. Hexaethyltetraphosphate (HETP) and Tetraethyl pyrophosphate (TEPP) - HETP and TEPP are compounds that also show anticholinesterase activity. -HETP was devel- oped by the Germans during World War II and is used as an insecticide against aphids. -When used as insecticides, these compounds have the advantage of being hydrolyzed rapidly to the relatively nontoxic, water-soluble compounds phosphoric acid and ethyl alcohol. -Fruit trees or vegetables sprayed with this type of compound retain no harmful residue after a period of a few days or weeks, depending on the weather conditions. -Workers spraying with these agents should use extreme caution so that the vapors are not breathed and none of the vapor or liquid comes in contact with the eyes or skin.

102. 4. Malathion -Malathion, 2-[(dimethoxyphosphinothioyl) thio]butanedioic acid diethyl ester, is a water-insoluble phosphodithioate ester that has been used as an agricultural insecticide. -Malathion is a poor inhibitor of cholinesterases. Its effectiveness as a safe insecticide is a result of the differ- ent rates at which humans and insects metabolize the chem- ical. - Microsomal oxidation, which causes desulfuration, occurs slowly to form the phosphothioate (malaoxon), which is 10,000 times more active than the phosphodithioate (malathion) as a cholinesterase inhibitor. - Insects detoxify the phosphothioate by a phosphatase, forming dimethyl phosphorothioate, which is inactive as an inhibitor. Humans, however, can rapidly hydrolyze malathion by a carboxyesterase enzyme, yielding malathion acid, a still poorer inhibitor of AChE. Phosphatases and carboxyesterases further metabolize malathion acid to dimethylphosphothioate.

104. 5. Parathion -Parathion, O,O-diethyl O-p-nitrophenyl phosphorothioate (Thiophos), is a yellow liquid that is freely soluble in aromatic hydrocarbons, ethers, ketones, esters, and alcohols but practically insoluble in water, petroleum ether, kerosene, and the usual spray oils. It is decomposed at a pH above 7.5. Parathion is used as an agricultural insecticide. -It is a relatively weak inhibitor of cholinesterase;however, enzymes present in liver microsomes and insect tissues convert parathion (pI50 8). -Parathion is also metabolized by liver microsomes to yield p-nitrophenol and diethylphosphate; the latter is inactive as an irreversible cholinesterase inhibitor.

106. 6. Schradan - Schradan, octamethyl pyrophosphoramide (OMPA), bis[bisdimethylaminophosphonous] anhydride (Pestox III), is a viscous liquid that is miscible with water and soluble in most organic solvents. It is not hydrolyzed by alkalies or water but is hydrolyzed by acids. -Schradan is used as a systemic insecticide for plants, being absorbed by the plants without appreciable injury. Insects feeding on the plant are incapacitated. -Schradan is a weak inhibitor of cholinesterases in vitro. - In vivo, it is metabolized to the very strong inhibitor hydroxymethyl OMPA. Hydroxymethyl OMPA is not stable and is metabolized further to the N-methoxide, which is a weak inhibitor of cholinesterase.

107. 7. Pralidoxime Chloride, USP. -Pralidoxime chloride, 2- formyl-1-methylpyridinium chloride oxime, 2-PAM chlo- ride, or 2-pyridine aldoxime methyl chloride (Protopam chloride), is a white, nonhygroscopic, crystalline powder that is soluble in water, 1 g in less than 1 mL. -Pralidoxime chloride is used as an antidote for poisoning by parathion and related pesticides. It may be effective against some phosphates that have a quaternary nitrogen. It is also an effective antagonist for some carbamates, such as neostigmine methylsulfate and pyridostigmine bromide.

108. -The biological half-life of pralidoxime chloride in hu- mans is about 2 hours, and its effectiveness is a function of its concentration in plasma, which reaches a maximum 2 to 3 hours after oral administration. -Pralidoxime chloride, a quaternary ammonium com- pound, is most effective by intramuscular, subcutaneous, or intravenous administration. -Treatment of poisoning by an anticholinesterase will be most effective if given within a few hours. Little will be accomplished if the drug is used more than 36 hours after parathion poisoning has occurred.