Acetylcholinesterase (AChE) terminates the action of acetylcholine (ACh) at the junctions of the various cholinergic nerve endings with their effector organs or postsynaptic sites (seeChapter 8). Drugs that inhibit AChE are called anticholinesterase (anti-ChE) agents. They cause ACh to accumulate in the vicinity of cholinergic nerve terminals and thus are potentially capable of producing effects equivalent to excessive stimulation of cholinergic receptors throughout the central and peripheral nervous systems. The anti-ChE agents have received extensive application as toxic agents, in the form of agricultural insecticides, pesticides, and potential chemical warfare “nerve gases.” Nevertheless, some compounds of this class are used therapeutically for the treatment of Alzheimer disease.
History. Physostigmine, also called eserine, is an alkaloid obtained from the Calabar or ordeal bean, the dried, ripe seed of Physostigma venenosum, a West African perennial. The Calabar bean once was used by native tribes of West Africa as an “ordeal poison” in trials for witchcraft, in which guilt was judged by death from the poison, innocence by survival after ingestion of a bean. The recent suggestion that we apply this test to politicians was narrowly rejected on humanitarian grounds.
Prior to World War II, only the “reversible” anti-ChE agents were generally known, of which physostigmine is the prototype. Shortly before and during World War II, a new class of highly toxic chemicals, the organophosphates, was developed, first as agricultural insecticides and later as potential chemical warfare agents. The extreme toxicity of these compounds is due to their “irreversible” inactivation of AChE, which results in prolonged enzyme inhibition. Because the pharmacological actions of both the reversible and irreversible anti-ChE agents are qualitatively similar, they are discussed here as a group.
STRUCTURE OF ACETYLCHOLINESTERASE. AChE exists in 2 general molecular classes: simple homomeric oligomers of catalytic subunits and heteromeric associations of catalytic subunits with structural subunits. The homomeric forms are found as soluble species in the cell, presumably destined for export or for association with the outer membrane of the cell, typically through an attached glycophospholipid. One heteromeric form, largely found in neuronal synapses, is a tetramer of catalytic subunits disulfide-linked to a 20-kDa lipid-linked subunit and localized to the outer surface of the cell membrane. The other heteromeric form consists of tetramers of catalytic subunits, disulfide linked to each of 3 strands of a collagen-like structural subunit. This molecular species, whose molecular mass approaches 106 Da, is associated with the basal lamina of junctional areas of skeletal muscle. A separate, structurally related gene encodes butyrylcholinesterase, which is synthesized in the liver and is primarily found in plasma.
The active center of mammalian AChE is at the base of a 2 nm gorge, at the bottom of which lie the catalytic triad (Ser203, His447, and Glu334), an acyl pocket, and a choline subsite; a “peripheral” site lies at the mouth of the gorge. The interactions of ligands with AChE can be usefully considered by examining their interactions with these domains (see Figure 10–1). The catalytic mechanism resembles that of other hydrolases; the serine hydroxyl group is rendered highly nucleophilic through a charge-relay system involving the carboxylate anion from glutamate, the imidazole of histidine, and the hydroxyl of serine (Figure 10–1A). During enzymatic attack of ACh, an ester with trigonal geometry, a tetrahedral intermediate between enzyme and substrate is formed (see Figure 10–1A) that collapses to an acetyl enzyme conjugate with the concomitant release of choline. The acetyl enzyme is very labile to hydrolysis, which results in the formation of acetate and active enzyme. AChE is one of the most efficient enzymes known: 1 molecule of AChE can hydrolyze 6 × 105 ACh molecules/min; this yields a turnover time of 100 μsec.
Figure 10–1 Steps involved in the hydrolysis of acetylcholine by acetylcholinesterase and in the inhibition and reactivation of the enzyme. Only the 3 residues of the catalytic triad are depicted. The associations and reactions shown are: A. Acetylcholine (ACh) catalysis: binding of ACh, formation of a tetrahedral transition state, formation of the acetyl enzyme with liberation of choline, rapid hydrolysis of the acetyl enzyme with return to the original state. B. Reversible binding and inhibition by edrophonium. C. Neostigmine reaction with and inhibition of AChE: reversible binding of neostigmine, formation of the dimethyl carbamoyl enzyme, slow hydrolysis of the dimethyl carbamoyl enzyme. D. Diisopropyl fluorophosphate (DFP) reaction and inhibition of AChE: reversible binding of DFP, formation of the diisopropyl phosphoryl enzyme, formation of the aged monoisopropyl phosphoryl enzyme. Hydrolysis of the diisopropyl enzyme is very slow (not shown). The aged monoisopropyl phosphoryl enzyme is virtually resistant to hydrolysis and reactivation. The tetrahedral transition state of ACh hydrolysis resembles the conjugates formed by the tetrahedral phosphate inhibitors and accounts for their potency. Amide bond hydrogens from Gly121 and Gly122 stabilize the carbonyl and phosphoryl oxygens. E. Reactivation of the diisopropyl phosphoryl enzyme by pralidoxime (2-PAM). 2-PAM attack of the phosphorus on the phosphorylated enzyme will form a phospho-oxime with regeneration of active enzyme.
MECHANISM OF ACTION OF AChE INHIBITORS. Anti-ChE agents are divided into 3 classes whose interactions with AChE are depicted Figure 10–1: noncovalent “reversible’ inhibitors,” carbamoylating inhibitors, and organophosphate inhibitors.
• Reversible inhibitors, such as edrophonium and tacrine, bind to the choline subsite (Figure 10–1B). Additional reversible inhibitors include donepezil, propidium, and the peptidic snake toxinfasciculin.
• Carbamoylating inhibitors with a carbamoyl ester linkage, such as physostigmine and neostigmine, are hydrolyzed by AChE, generating the carbamoylated enzyme (Figure 10–1C). In contrast to the acetyl enzyme, methylcarbamoyl AChE and dimethylcarbamoyl AChE are far more stable (the t1/2 for hydrolysis of the dimethylcarbamoyl enzyme is 15-30 min). Sequestration of the enzyme in its carbamoylated form thus precludes the enzyme-catalyzed hydrolysis of ACh for extended periods of time. When administered systemically, the duration of inhibition is 3-4 h.
• Organophosphate inhibitors, such as diisopropyl fluorophosphate (DFP), form very stable conjugates with AChE, with the active center serine phosphorylated or phosphonylated (Figure 10–1D). If the alkyl groups in the phosphorylated enzyme are ethyl or methyl, spontaneous regeneration of active enzyme requires several hours. Secondary (as in DFP) or tertiary alkyl groups further enhance the stability of the phosphorylated enzyme, and significant regeneration of active enzyme usually is not observed. The stability of the phosphorylated enzyme is strengthened through “aging,” which results from the loss of 1 of the alkyl groups.
Thus, the terms reversible and irreversible as applied to the carbamoyl ester and organophosphate anti-ChE agents, respectively, reflect only quantitative differences in rates of decarbamoylation or dephosphorylation of the conjugated enzyme. Both chemical classes react covalently with the active center serine in essentially the same manner as does ACh.
ACTION AT EFFECTOR ORGANS. The characteristic pharmacological effects of the anti-ChE agents are due primarily to the prevention of hydrolysis of ACh by AChE at sites of cholinergic transmission. Transmitter thus accumulates, enhancing the response to released ACh. Virtually all acute effects of moderate doses of organophosphates are attributable to this action.
The consequences of enhanced concentrations of ACh at motor endplates are unique to these sites and are discussed later. The tertiary amine and particularly the quaternary ammonium anti-ChE compounds may have additional direct actions at certain cholinergic receptor sites (e.g., the effects of neostigmine on the spinal cord and neuromuscular junction are based on a combination of its anti-ChE activity and direct cholinergic stimulation).
CHEMISTRY AND STRUCTURE-ACTIVITY RELATIONSHIPS
NONCOVALENT INHIBITORS. While these agents interact by reversible and noncovalent association with the active site in AChE, they differ in their disposition in the body and their affinity for the enzyme.
Edrophonium, a quaternary drug whose activity is limited to peripheral nervous system synapses, has a moderate affinity for AChE (see Figure 10–1B). Its volume of distribution is limited and renal elimination is rapid, accounting for its short duration of action. By contrast, tacrine and donepezil have higher affinities for AChE, are more hydrophobic, and readily cross the blood-brain barrier to inhibit AChE in the CNS.
“REVERSIBLE” CARBAMATE INHIBITORS. Drugs of this class that are of therapeutic interest include physostigmine, neostigmine, and rivastigmine. Their interaction with AChE is depicted in Figure 10–1C.
The essential moiety of the physostigmine molecule is the methyl carbamate of an amine-substituted phenol. An increase in anti-ChE potency and duration of action can result from the linking of 2 quaternary ammonium moieties. An example is the miotic agent demecarium, which consists of 2 neostigmine molecules connected by a series of 10 methylene groups. The second quaternary group confers additional stability to the interaction. Carbamoylating inhibitors with high lipid solubilities (e.g., rivastigmine), which readily cross the blood-brain barrier and have longer durations of action, are approved or in clinical trial for the treatment of Alzheimer disease (see Chapter 22).
The carbamate insecticides carbaryl (SEVIN), propoxur (BAYGON), and aldicarb (TEMIK), which are used extensively as garden insecticides, inhibit ChE in a fashion identical with other carbamoylating inhibitors. The symptoms of poisoning closely resemble those of the organophosphates.
ORGANOPHOSPHORUS COMPOUNDS. The general formula for this class of ChE inhibitors is shown at the top of Table 10–1. The group includes DFP, soman, malathion, and echothiophate.
Representative Organophosphorus Compounds
DFP produces virtually irreversible inactivation of AChE and other esterases by diisopropylphosphorylation followed by “aging” (conversion to the monoisopropylphosphoryl AChE (see Figure 10–1D). Its high lipid solubility, low molecular weight, and volatility facilitate inhalation, transdermal absorption, and penetration into the CNS. The “nerve gases”—tabun, sarin, and soman—are among the most potent synthetic toxins known; they are lethal to laboratory animals in nanogram doses. Insidious employment of these agents has occurred in warfare and terrorism attacks. Because of their low volatility and stability in aqueous solution, parathion and methylparathion were widely used as insecticides; however, acute and chronic toxicity has limited their use. These compounds are inactive in inhibiting AChE in vitro; paraoxon is the active metabolite produced in vivo via a substitution of phosphoryl oxygen for sulfur carried out by hepatic CYPs. This reaction also occurs in the insect, typically with more efficiency than in mammals and many other animals. Other insecticides possessing the phosphorothioate structure have been widely employed, including diazinon (SPECTRACIDE, others) and chlorpyrifos(DURSBAN, LORSBAN). Both of these agents have been placed under restricted use because of evidence of chronic toxicity in the newborn animal. They have been banned since 2005.
Malathion (CHEMATHION, MALA-SPRAY) also requires replacement of a sulfur atom with oxygen in vivo, conferring resistance in mammalian species. This insecticide can be detoxified by hydrolysis of the carboxyl ester linkage by plasma carboxylesterases, and plasma carboxylesterase activity dictates species resistance to malathion. The detoxification reaction is much more rapid in mammals and birds than in insects. Malathion has been employed in aerial spraying of relatively populous areas for control of citrus orchard-destructive Mediterranean fruit flies and mosquitoes that harbor and transmit viruses harmful to human beings (e.g., West Nile encephalitis virus). Evidence of acute toxicity from malathion arises only with suicide attempts or deliberate poisoning. The lethal dose in mammals is ~1 g/kg. Exposure to the skin results in a small fraction (<10%) of systemic absorption. Malathion is used topically in the treatment of pediculosis (lice) infestations.
Of the quaternary organophosphate AChE inhibitors (see Table 10–1, group D), only echothiophate is useful clinically and it is limited to ophthalmic administration. Being positively charged, it is not volatile and does not readily penetrate the skin.
Anti-ChE agents potentially can produce all the following effects:
• Stimulation of muscarinic receptor responses at autonomic effector organs
• Stimulation, followed by depression or paralysis, of all autonomic ganglia and skeletal muscle (nicotinic actions)
• Stimulation, with occasional subsequent depression, of cholinergic receptor sites in the CNS
In general, compounds containing a quaternary ammonium group do not penetrate cell membranes readily; hence, anti-ChE agents in this category are absorbed poorly from the GI tract or across the skin and are excluded from the CNS by the blood-brain barrier after moderate doses. However, such compounds act preferentially at the neuromuscular junctions of skeletal muscle, exerting their action both as anti-ChE agents and as direct agonists. They have comparatively less effect at autonomic effector sites and ganglia.
By contrast, the more lipid-soluble agents are well absorbed after oral administration, have ubiquitous effects at both peripheral and central cholinergic sites, and may be sequestered in lipids for long periods of time. Lipid-soluble organophosphorus agents also are well absorbed through the skin, and the volatile agents are transferred readily across the alveolar membrane.
The actions of anti-ChE agents where the receptors are largely of the muscarinic type are blocked by atropine. The sites of action of anti-ChE agents of therapeutic importance are the CNS, eye, intestine, and the neuromuscular junction of skeletal muscle; other actions are of toxicological consequence.
EYE. When applied locally to the conjunctiva, anti-ChE agents cause conjunctival hyperemia and constriction of the pupillary sphincter muscle around the pupillary margin of the iris (miosis) and the ciliary muscle (block of accommodation reflex with resultant focusing to near vision). Miosis is apparent in a few minutes and can last several hours to days. The block of accommodation is more transient and generally disappears before termination of miosis. Intraocular pressure, when elevated, usually falls as the result of facilitation of outflow of the aqueous humor (see Chapter 64).
GI TRACT. In humans, neostigmine enhances gastric contractions and increases the secretion of gastric acid. The lower portion of the esophagus is stimulated by neostigmine; in patients with marked achalasia and dilation of the esophagus, the drug can cause a salutary increase in tone and peristalsis. Neostigmine also augments motor activity of the small and large bowel; the colon is particularly stimulated. Propulsive waves are increased in amplitude and frequency, and movement of intestinal contents is thus promoted. The effect of anti-ChE agents on intestinal motility probably represents a combination of actions at the ganglion cells of the myenteric (Auerbach) plexus and at the smooth muscle fibers (see Chapter 46).
NEUROMUSCULAR JUNCTION. Most of the effects of potent anti-ChE drugs on skeletal muscle can be explained adequately on the basis of their inhibition of AChE at neuromuscular junctions. However, there is good evidence for an accessory direct action of neostigmine and other quaternary ammonium anti-ChE agents on skeletal muscle.
The lifetime of free ACh within the nerve-muscle synapse (~200 μsec) is shorter than the decay of the endplate potential or the refractory period of the muscle. Therefore, each nerve impulse gives rise to a single wave of depolarization on the muscle fiber. After inhibition of AChE, the residence time of ACh in the synapse increases, allowing for lateral diffusion and interaction of the transmitter with multiple receptors. This successive stimulation of neighboring receptors site in the endplate results in a prolongation of the decay time of the endplate potential. Consequently, asynchronous excitation and fasciculations of muscle fibers occur. With sufficient inhibition of AChE, depolarization of the endplate predominates, and depolarization blockade ensues (see Chapter 11). When ACh persists in the synapse, it also may depolarize the axon terminal, resulting in antidromic firing of the motoneuron; this effect contributes to fasciculations that involve the entire motor unit.
The anti-ChE agents will reverse the antagonism caused by competitive neuromuscular blocking agents. Neostigmine is not effective against the skeletal muscle paralysis caused by succinylcholine; this agent also produces neuromuscular blockade by depolarization, and depolarization will be enhanced by neostigmine.
ACTIONS AT OTHER SITES. Secretory glands that are innervated by postganglionic cholinergic fibers include the bronchial, lacrimal, sweat, salivary, gastric (antral G cells and parietal cells), intestinal, and pancreatic acinar glands. Low doses of anti-ChE agents augment secretory responses to nerve stimulation, and higher doses actually produce an increase in the resting rate of secretion. Anti-ChE agents increase contraction of smooth muscle fibers of bronchioles and ureters, and ureters may show increased peristaltic activity.
The cardiovascular actions of anti-ChE agents are complex; they reflect both ganglionic and postganglionic effects of accumulated ACh on the heart and blood vessels and actions in the CNS. The predominant effect on the heart from the peripheral action of accumulated ACh is bradycardia, resulting in a fall in cardiac output. Higher doses usually cause a fall in blood pressure, often as a consequence of effects of anti-ChE agents on the medullary vasomotor centers of the CNS.
Anti-ChE agents augment vagal influences on the heart, thereby shortening the effective refractory period of atrial muscle fibers and increasing the refractory period and conduction time at the SA and AV nodes. At the ganglionic level, accumulating ACh initially is excitatory on nicotinic receptors, but at higher concentrations, ganglionic blockade ensues as a result of persistent depolarization. The excitatory action on the parasympathetic ganglion cells can reinforce the diminished cardiac output, whereas enhanced cardiac function would result from the action of ACh on sympathetic ganglion cells. ACh also elicits excitation followed by inhibition at the central medullary vasomotor and cardiac centers. These effects are complicated by the hypoxemia resulting from the bronchoconstrictor and secretory actions of increased ACh on the respiratory system; hypoxemia, in turn, can reinforce both sympathetic tone and ACh-induced discharge of epinephrine from the adrenal medulla. Hence, it is not surprising that an increase in heart rate is seen with severe ChE inhibitor poisoning. Hypoxemia is a major factor in the CNS depression after large doses of anti-ChE agents. The CNS-stimulant effects are antagonized by larger doses of atropine, although not as completely as are the muscarinic effects at peripheral autonomic effector sites.
ADME. Physostigmine is absorbed readily from the GI tract, subcutaneous tissues, and mucous membranes. The conjunctival instillation of solutions of the drug may result in systemic effects if measures (e.g., pressure on the inner canthus) are not taken to prevent absorption from the nasal mucosa. Parenterally administered physostigmine is largely destroyed within 2-3 h by plasma esterases; renal excretion plays only a minor role in its elimination. Neostigmine and pyridostigmine are absorbed poorly after oral administration; the effective parenteral dose of neostigmine is 0.5-2 mg whereas the equivalent oral dose may be 15-30 mg or more. Neostigmine and pyridostigmine are destroyed by plasma esterases, and the quaternary aromatic alcohols and parent compounds are excreted in the urine; the half-lives of these drugs are 1-2 h.
Organophosphate anti-ChE agents with the highest risk of toxicity are highly lipid-soluble liquids; many have high vapor pressures. The less volatile agents that are commonly used as agricultural insecticides (e.g., diazinon, malathion) generally are dispersed as aerosols or as dusts adsorbed to an inert, finely particulate material. Consequently, the compounds are absorbed rapidly through the skin and mucous membranes following contact with moisture, by the lungs after inhalation, and by the GI tract after ingestion.
Following their absorption, most organophosphates are hydrolyzed by plasma and liver esterases; the hydrolysis products are excreted in the urine. Plasma and liver esterases are responsible for hydrolysis to the corresponding phosphoric and phosphonic acids. However, the CYPs are responsible for converting the inactive phosphorothioates containing a phosphorus-sulfur (thiono) bond to phosphorates with a phosphorus-oxygen bond, resulting in their activation. These enzymes also play a role in the inactivation of certain organophosphorus agents, and allelic differences are known to affect rates of metabolism. Plasma and hepatic carboxylesterases (aliesterases) and plasma butyrylcholinesterase are inhibited irreversibly by organophosphates; their scavenging capacity for organophosphates affords partial protection against inhibition of AChE in the nervous system. The carboxylesterases also catalyze hydrolysis of malathion and other organophosphates that contain carboxyl-ester linkages, rendering them less active or inactive. Since carboxylesterases are inhibited by organophosphates, toxicity from simultaneous exposure to 2 organophosphorus insecticides can be synergistic.
The toxicological aspects of the anti-ChE agents are of practical importance to clinicians. In addition to cases of accidental intoxication from the use and manufacture of organophosphorus compounds as agricultural insecticides, these agents have been used frequently for homicidal and suicidal purposes. Organophosphates account for as many as 80% of pesticide-related hospital admissions. Pesticide toxicity is a widespread global problem associated with >200,000 deaths a year; most poisonings occur in Southeast Asia. Occupational exposure occurs most commonly by the dermal and pulmonary routes, while oral ingestion is most common in cases of nonoccupational poisoning.
ACUTE INTOXICATION. The effects of acute intoxication by anti-ChE agents are manifested by muscarinic and nicotinic signs and symptoms, and, except for compounds of extremely low lipid solubility, by signs referable to the CNS. The broad spectrum of effects of acute AChE inhibition on the CNS includes confusion, ataxia, slurred speech, loss of reflexes, Cheyne-Stokes respiration, generalized convulsions, coma, and central respiratory paralysis. Actions on the vasomotor and other cardiovascular centers in the medulla oblongata lead to hypotension.
Systemic effects appear within minutes after inhalation of vapors or aerosols. The onset of symptoms is delayed after GI and percutaneous absorption. The duration of toxic symptoms is determined largely by the properties of the compound: its lipid solubility, whether it must be activated to form the oxon, the stability of the organophosphate-AChE bond, and whether “aging” of the phosphorylated enzyme has occurred.
After local exposure to vapors or aerosols or after their inhalation, ocular and respiratory effects generally appear first. Ocular manifestations include marked miosis, ocular pain, conjunctival congestion, diminished vision, ciliary spasm, and brow ache. With acute systemic absorption, miosis may not be evident due to sympathetic discharge in response to hypotension. In addition to rhinorrhea and hyperemia of the upper respiratory tract, respiratory responses consist of tightness in the chest and wheezing respiration, caused by the combination of bronchoconstriction and increased bronchial secretion. GI symptoms occur earliest after ingestion and include anorexia, nausea and vomiting, abdominal cramps, and diarrhea.
With percutaneous absorption of liquid, localized sweating and muscle fasciculations in the immediate vicinity are generally the earliest symptoms. Severe intoxication is manifested by extreme salivation, involuntary defecation and urination, sweating, lacrimation, penile erection, bradycardia, and hypotension.
Nicotinic actions at the neuromuscular junctions of skeletal muscle usually consist of generalized weakness, involuntary twitchings, scattered fasciculations, and eventually severe weakness and paralysis. The most serious consequence is paralysis of the respiratory muscles. The time of death after a single acute exposure may range from <5 min to nearly 24 h, depending on the dose, route, agent, and other factors. The cause of death primarily is respiratory failure, usually accompanied by a secondary cardiovascular component. Delayed symptoms appearing after 1-4 days and marked by persistent low blood ChE and severe muscle weakness are termed the intermediate syndrome. Delayed neurotoxicity also may be evident after severe intoxication.
DIAGNOSIS AND TREATMENT. In cases of mild or chronic intoxication, determination of the ChE activities in erythrocytes and plasma generally will establish the diagnosis. Although these values vary considerably in the normal population, they usually are depressed well below the normal range before symptoms are evident. Atropine in sufficient dosage effectively antagonizes the actions at muscarinic receptor sites, including increased tracheobronchial and salivary secretion, bronchoconstriction, bradycardia, and to a moderate extent, peripheral ganglionic and central actions.
Atropine should be given in doses sufficient to cross the blood-brain barrier. Following an initial injection of 2-4 mg, given intravenously if possible, otherwise intramuscularly, 2 mg should be given every 5-10 min until muscarinic symptoms disappear, if they reappear, or until signs of atropine toxicity appear. More than 200 mg may be required on the first day. A mild degree of atropine block then should be maintained for as long as symptoms are evident. The AChE reactivators can be of great benefit in the therapy of anti-ChE intoxication, but their use is supplemental to the administration of atropine.
Atropine is ineffective against the peripheral neuromuscular compromise caused by moderate or severe intoxication with an organophosphorus anti-ChE agent; the condition can be reversed by pralidoxime (2-PAM), a cholinesterase reactivator.
The recommended adult dose of pralidoxime is 1-2 g, infused intravenously over ≥5 min. If weakness is not relieved or if it recurs after 20-60 min, the dose should be repeated. Early treatment is very important to assure that the oxime reaches the phosphorylated AChE while the latter still can be reactivated. With severe toxicities from the lipid-soluble agents, it is necessary to continue treatment with atropine and pralidoxime for a week or longer.
General supportive measures also are important, including:
• Termination of exposure, by removal of the patient or application of a gas mask; removal of contaminated clothing; copious washing of contaminated skin or mucous membranes with water; gastric lavage, if indicated
• Maintenance of a patent airway, including endobronchial aspiration
• Artificial respiration; administration of O2, if required
• Alleviation of persistent convulsions with diazepam (5-10 mg, intravenously)
• Treatment of shock
CHOLINESTERASE REACTIVATORS. The phosphorylated esteratic site of AChE undergoes hydrolytic regeneration at a slow or negligible rate, but nucleophilic agents, such as hydroxylamine (NH2OH), hydroxamic acids (RCONH—OH), and oximes (RCH=NOH), reactivate the enzyme more rapidly than does spontaneous hydrolysis. Reactivation with pralidoxime occurs at a million times the rate of that with hydroxylamine. The oxime is oriented proximally to exert a nucleophilic attack on the phosphorus, forming a phosphoryloxime and regenerating the enzyme (Figure 10–1E). Several bis-quaternary oximes are more potent as reactivators for insecticide and nerve gas poisoning (e.g., HI-6, used in Europe as an antidote).
The reactivating action of oximes in vivo is most marked at the skeletal neuromuscular junction. Following a dose of an organophosphorus compound that produces total blockade of transmission, the intravenous injection of an oxime can restore the response to stimulation of the motor nerve within a few minutes. Antidotal effects are less striking at autonomic effector sites, and the quaternary ammonium group restricts entry into the CNS. Although high doses or accumulation of oximes can inhibit AChE and cause neuromuscular blockade, oximes should be given until one can be assured of clearance of the offending organophosphate. Many organophosphates partition into lipid and are released slowly as the active entity. Current antidotal therapy for organophosphate exposure resulting from warfare or terrorism includes parenteral atropine, an oxime (2-PAM or HI-6), and a benzodiazepine as an anticonvulsant. The oximes and their metabolites are readily eliminated by the kidney.
Physostigmine salicylate (ANTILIRIUM) is available for injection. Physostigmine sulfate ophthalmic ointment and physostigmine salicylate ophthalmic solution also are available. Pyridostigmine bromide is available for oral (MESTINON) or parenteral (REGONOL, MESTINON) use. Neostigmine bromide (PROSTIGMIN) is available for oral use. Neostigmine methylsulfate (PROSTIGMIN) is marketed for parenteral injection. Ambenonium chloride (MYTELASE) is available for oral use. Tacrine (COGNEX), donepezil (ARICEPT), rivastigmine (EXELON), and galantamine (REMINYL) have been approved for the treatment of Alzheimer disease. Pralidoxime chloride (PROTOPAM CHLORIDE) is the only AChE reactivator currently available in the U.S. and can be obtained in a parenteral formulation. HI-6 is available in several European and Near Eastern countries.
USES. Current use of anti-AChE agents is limited to 4 conditions in the periphery:
• Atony of the smooth muscle of the intestinal tract and urinary bladder
• Myasthenia gravis
• Reversal of the paralysis of competitive neuromuscular blocking drugs
and 1 in the CNS:
• Treatment of dementia symptoms of Alzheimer disease
PARALYTIC ILEUS AND ATONY OF THE URINARY BLADDER. Neostigmine generally is preferred among the anti-ChE agents in the treatment of these conditions. Directly acting muscarinic agonists (see Chapter 9) are employed for the same purposes. The usual subcutaneous dose of neostigmine methylsulfate for postoperative paralytic ileus is 0.5 mg, given as needed. Peristaltic activity commences 10-30 min after parenteral administration, whereas 2-4 h are required after oral administration of neostigmine bromide (15-30 mg). It may be necessary to assist evacuation with a small low enema or gas with a rectal tube. A similar dose of neostigmine is used for the treatment of atony of the detrusor muscle of the urinary bladder. Neostigmine should not be used when the intestine or urinary bladder is obstructed, when peritonitis is present, when the viability of the bowel is doubtful, or when bowel dysfunction results from inflammatory bowel disease.
GLAUCOMA AND OTHER OPHTHALMOLOGIC INDICATIONS. See Chapter 64.
MYASTHENIA GRAVIS. Myasthenia gravis is a neuromuscular disease characterized by weakness and marked fatigability of skeletal muscle; exacerbations and partial remissions occur frequently. The defect in myasthenia gravis is in synaptic transmission at the neuromuscular junction such that mechanical responses to nerve stimulation are not well sustained. Myasthenia gravis is caused by an autoimmune response primarily to the ACh receptor at the postjunctional endplate. Antireceptor antibodies are detectable in sera of 90% of patients with the disease. Immune complexes along with marked ultrastructural abnormalities appear in the synaptic cleft and enhance receptor degradation through complement-mediated lysis in the endplate. In ~10% of patients presenting with a myasthenic syndrome, muscle weakness has a congenital rather than an autoimmune basis, with mutations in the ACh receptor that affect ligand-binding and channel-opening kinetics, or mutations in the form of AChE that contains the collagen-like tail unit. Administration of anti-ChE agents does not result in subjective improvement in most congenital myasthenic patients.
Diagnosis. Although the diagnosis of autoimmune myasthenia gravis usually can be made from the history, signs, and symptoms, its differentiation from certain neurasthenic, infectious, endocrine, congenital, neoplastic, and degenerative neuromuscular diseases can be challenging. Myasthenia gravis is the only condition in which muscular weakness can be improved dramatically by anti-ChE medication. The edrophonium test for evaluation of possible myasthenia gravis is performed by rapid intravenous injection of 2 mg of edrophonium chloride, followed 45 sec later by an additional 8 mg if the first dose is without effect; a positive response consists of brief improvement in strength, unaccompanied by lingual fasciculation (which generally occurs in nonmyasthenic patients).
An excessive dose of an anti-ChE drug results in a cholinergic crisis, a condition characterized by weakness resulting from generalized depolarization of the motor endplate; other features result from overstimulation of muscarinic receptors. The weakness resulting from depolarization blockade may resemble myasthenic weakness, which is manifest when anti-ChE medication is insufficient. The distinction is of obvious practical importance, because the former is treated by withholding, and the latter by administering, the anti-ChE agent. When the edrophonium test is performed cautiously (limiting the dose to 2 mg and with facilities for respiratory resuscitation available) a further decrease in strength indicates cholinergic crisis, while improvement signifies myasthenic weakness. Atropine sulfate, 0.4-0.6 mg or more intravenously, should be given immediately if a severe muscarinic reaction ensues. Detection of antireceptor antibodies in muscle biopsies or plasma is now widely employed to establish the diagnosis.
Treatment. Pyridostigmine, neostigmine, and ambenonium are the standard anti-ChE drugs used in the symptomatic treatment of myasthenia gravis. All can increase the response of myasthenic muscle to repetitive nerve impulses, primarily by the preservation of endogenous ACh. The optimal single oral dose of an anti-ChE agent can be determined empirically. Baseline recordings are made for grip strength, vital capacity, and a number of signs and symptoms that reflect the strength of various muscle groups. The patient then is given an oral dose of pyridostigmine (30-60 mg), neostigmine (7.5-15 mg), or ambenonium (2.5-5 mg). The improvement in muscle strength and changes in other signs and symptoms are noted at frequent intervals until there is a return to the basal state. After an hour or longer in the basal state, the drug is given again, with the dose increased to one and one-half times the initial amount, and the same observations are repeated. This sequence is continued, with increasing increments of one-half the initial dose, until an optimal response is obtained. The interval between oral doses required to maintain muscle strength usually is 2-4 h for neostigmine, 3-6 h for pyridostigmine, or 3-8 h for ambenonium. However, the dose required may vary from day to day; physical or emotional stress, intercurrent infections, and menstruation usually necessitate an increase in the frequency or size of the dose. Unpredictable exacerbations and remissions of the myasthenic state may require adjustment of dosage.
Pyridostigmine is available in sustained-release tablets containing a total of 180 mg, of which 60 mg is released immediately and 120 mg over several hours; this preparation is of value in maintaining patients for 6–8-h periods, but should be limited to use at bedtime. Muscarinic cardiovascular and GI side effects of anti-ChE agents generally can be controlled by atropine or other anticholinergic drugs (see Chapter 9). However, these anticholinergic drugs mask many side effects of an excessive dose of an anti-ChE agent. In most patients, tolerance develops eventually to the muscarinic effects.
Several drugs, including curariform agents and certain antibiotics and general anesthetics, interfere with neuromuscular transmission (see Chapter 11); their administration to patients with myasthenia gravis requires proper adjustment of anti-ChE dosage and other precautions. Other therapeutic measures are essential elements in the management of this disease. Glucocorticoids promote clinical improvement in a high percentage of patients. Initiation of steroid treatment augments muscle weakness; however, as the patient improves with continued administration of steroids, doses of anti-ChE drugs can be reduced. Immunosuppressive agents such as azathioprine and cyclosporine have been beneficial in more advanced cases (see Chapter 35).
ALZHEIMER DISEASE. A deficiency of intact cholinergic neurons, particularly those extending from subcortical areas such as the nucleus basalis of Meynert, has been observed in patients with progressive dementia of the Alzheimer type (see Chapter 22). Using a rationale similar to that in other CNS degenerative diseases, therapy for enhancing concentrations of cholinergic neurotransmitters in the CNS has been used in mild to moderate Alzheimer disease. Long-acting and hydrophobic ChE inhibitors are the only inhibitors with well-documented, albeit limited, efficacy.
Donepezil may improve cognition and global clinical function and delay symptomatic progression of the disease. Side effects are largely attributable to excessive cholinergic stimulation, with nausea, diarrhea, and vomiting being most frequently reported. The drug is well tolerated in single daily doses. Usually, 5-mg doses are administered at night; if this dose is well tolerated, the dose can be increased to 10 mg daily. Rivastigmine, a long-acting carbamoylating inhibitor, has efficacy, tolerability, and side effects similar to those of donepezil. Galantamine has a side-effect profile similar to those of donepezil and rivastigmine. These 3 cholinesterase inhibitors, which have good affinity, sufficient hydrophobicity to cross the blood-brain barrier, and prolonged durations of action constitute current modes of therapy, along with an excitatory amino acid transmitter mimic, memantine.
INTOXICATION BY ANTICHOLINERGIC DRUGS. In addition to atropine and other muscarinic agents, many other drugs, such as the phenothiazines, antihistamines, and tricyclic antidepressants, have central and peripheral anticholinergic activity. The effectiveness of physostigmine in reversing the anticholinergic effects of these agents has been clearly documented (Physostigmine, a tertiary amine, crosses the blood-brain barrier, in contrast to the quaternary anti-AChE drugs). However, other toxic effects of the tricyclic antidepressants and phenothiazines (see Chapters 15 and 16), such as intraventricular conduction deficits and ventricular arrhythmias, are not reversed by physostigmine. In addition, physostigmine may precipitate seizures; hence, its usually small potential benefit must be weighed against this risk. The initial intravenous or intramuscular dose of physostigmine is 2 mg, with additional doses given as necessary.