Chapter 10: Anticholinesterase Agents

History. Physostigmine, also called eserine, is an alkaloid obtained from the Calabar or ordeal bean, the dried, ripe seed of Physostigma venenosum, a perennial plant found in tropical West Africa. 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. A pure alkaloid was isolated by Jobst and Hesse in 1864 and named physostigmine. The first therapeutic use of the drug was in 1877 by Laqueur, in the treatment of glaucoma, one of its clinical uses today. Accounts of the history of physostigmine have been presented by Karczmar (1970) and Holmstedt (2000).

After basic research elucidated the chemical basis of the activity of physostigmine, scientists began systematic investigations of a series of substituted aromatic esters of alkyl carbamic acids. Neostigmine was introduced into therapeutics in 1931 for its stimulant action on the GI tract and subsequently was reported to be effective in the symptomatic treatment of myasthenia gravis.

Remarkably, the first account of the synthesis of a highly potent organophosphorus anti-ChE, tetraethyl pyrophosphate (TEPP), was published by Clermont in 1854. It is even more remarkable that the investigator survived to report on the compound's taste; a few drops should have been lethal. Modern investigations of the organophosphorus compounds date from the 1932 publication of Lange and Krueger on the synthesis of dimethyl and diethyl phosphorofluoridates.

Upon synthesizing ∼2000 compounds, Schrader defined the structural requirements for insecticidal activity [(and, as learned subsequently, for anti-ChE) (discussed later) (Gallo and Lawryk, 1991)]. One compound in this early series, parathion (a phosphorothioate), later became the most widely used insecticide of this class. Malathion, which currently is used extensively, also contains the thionophosphorus bond found in parathion. Prior to and during World War II, the efforts of Schrader's group were directed toward the development of chemical warfare agents. The synthesis of several compounds of much greater toxicity than parathion, such as sarin, soman, and tabun, was kept secret by the German government. Investigators in the Allied countries also followed Lange and Krueger's lead in the search for potentially toxic compounds; diisopropyl phosphorofluoridate (diisopropyl fluorophosphate; DFP), synthesized by McCombie and Saunders, was studied most extensively by British and American scientists.

In the 1950s, a series of aromatic carbamates was synthesized and found to have substantial selective toxicity against insects and to be potent anti-ChE agents (Ecobichon, 2000).

Structure of Acetylcholinesterase. AChE exists in two general classes of molecular forms: simple homomeric oligomers of catalytic subunits (monomers, dimers, and tetramers) and heteromeric associations of catalytic subunits with structural subunits (Massoulié, 2000; Taylor et al., 2000). 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 20,000-Da 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 three 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.

Molecular cloning revealed that a single gene encodes vertebrate AChEs (Schumacher et al., 1986; Taylor et al., 2000). However, multiple gene products arise from alternative processing of the mRNA that differ only in their carboxyl-termini; the portion of the gene encoding the catalytic core of the enzyme is invariant. Hence, the individual AChE species can be expected to show identical substrate and inhibitor specificities.

A separate, structurally related gene encodes butyrylcholinesterase, which is synthesized in the liver and is primarily found in plasma (Lockridge et al., 1987). The cholinesterases define a superfamily of proteins whose structural motif is the α,β hydrolase-fold (Cygler et al., 1993). The family includes several esterases, other hydrolases not found in the nervous system, and surprisingly, proteins without hydrolase activity such as thyroglobulin and members of the tactin and neuroligin families of proteins (Taylor et al., 2000).

The three-dimensional structures of AChEs show the active center to be nearly centrosymmetric to each subunit, residing at the base of a narrow gorge ∼20 Å in depth (Bourne et al., 1995; Sussman et al., 1991). At the base of the gorge lie the residues of the catalytic triad: Ser203, His447, and Glu334 in mammals (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 10–2A).

During enzymatic attack of ACh, an ester with trigonal geometry, a tetrahedral intermediate between enzyme and substrate is formed (Figure 10–2A) 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 (Froede and Wilson, 1971; Rosenberry, 1975). AChE is one of the most efficient enzymes known: one molecule of AChE can hydrolyze 6 × 10⁵ ACh molecules per minute; this yields a turnover time of 100 microseconds.

Three distinct domains on AChE constitute binding sites for inhibitory ligands and form the basis for specificity differences between AChE and butyrylcholinesterase: the acyl pocket of the active center, the choline subsite of the active center, and the peripheral anionic site (Reiner and Radic', 2000; Taylor and Radic', 1994). Reversible inhibitors, such as edrophonium and tacrine, bind to the choline subsite in the vicinity of Trp86 and Glu202 (Silman and Sussman, 2000) (Figure 10–2B). Edrophonium has a brief duration of action because its quaternary structure facilitates renal elimination and it binds reversibly to the AChE active center. Additional reversible inhibitors, such as donepezil, bind with higher affinity to the active center.

Other reversible inhibitors, such as propidium and the snake peptidic toxin fasciculin, bind to the peripheral anionic site on AChE. This site resides at the rim of the gorge and is defined by Try286 and Tyr72 and Tyr124 (Figure 10–1).

Drugs that have a carbamoyl ester linkage, such as physostigmine and neostigmine, are hydrolyzed by AChE, but much more slowly than is ACh. The quaternary amine neostigmine and the tertiary amine physostigmine exist as cations at physiological pH. By serving as alternate substrates to ACh (Figure 10–2C), attack by the active center serine generates the carbamoylated enzyme. The carbamoyl moiety resides in the acyl pocket outlined by Phe295 and Phe297. In contrast to the acetyl enzyme, methylcarbamoyl AChE and dimethylcarbamoyl AChE are far more stable (the t_1/2 for hydrolysis of the dimethylcarbamoyl enzyme is 15-30 minutes). 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 by the carbamoylating agents is 3-4 hours.

The organophosphate inhibitors, such as diisopropyl fluorophosphate (DFP), serve as true hemisubstrates, since the resultant conjugate with the active center serine phosphorylated or phosphonylated is extremely stable (Figure 10–2D). The organophosphorus inhibitors are tetrahedral in configuration, a configuration that resembles the transition state formed in carboxyl ester hydrolysis. Similar to the carboxyl esters, the phosphoryl oxygen binds within the oxyanion hole of the active center. 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 enhanced through "aging," which results from the loss of one of the alkyl groups. Hence, the return of AChE activity depends on synthesis of a new enzyme.

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.

The tertiary amine and particularly the quaternary ammonium anti-ChE compounds may have additional direct actions at certain cholinergic receptor sites. For example, 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.

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. Its volume of distribution is limited and renal elimination is rapid, accounting for its short duration of action. By contrast, tacrine and donepezil (Figure 10–3) have higher affinities for AChE, are more hydrophobic, and readily cross the blood-brain barrier to inhibit AChE in the CNS. Their partitioning into lipid and their higher affinities for AChE account for their longer durations of action.

"Reversible" Carbamate Inhibitors. Drugs of this class that are of therapeutic interest are shown in Figure 10–3. Early studies showed that the essential moiety of the physostigmine molecule was the methylcarbmate of an amine-substituted phenol. The quaternary ammonium derivative neostigmine is a compound of equal or greater potency. Pyridostigmine is a close congener that also is used to treat myasthenia gravis.

An increase in anti-ChE potency and duration of action can result from the linking of two quaternary ammonium moieties. One such example is the miotic agent demecarium, which consists of two neostigmine molecules connected by a series of ten methylene groups. The second quaternary group confers additional stability to the interaction by associating with a negatively charged amino side chain, Asn74, near the rim of the gorge. 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's disease (Cummings, 2004; Giacobini, 2000) (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 (Baron, 1991; Ecobichon, 2000). Carbaryl has a particularly low toxicity from dermal absorption. It is used topically for control of head lice in some countries. Not all carbamates in garden formulations are ChE inhibitors; the dithiocarbamates are fungicidal.

Organophosphorus Compounds. The general formula for this class of ChE inhibitors is presented in Table 10–1. A great variety of substituents is possible: R₁ and R₂ may be alkyl, alkoxy, aryloxy, amido, mercaptan, or other groups; and X, the leaving group, typically a conjugate base of a weak acid, is a halide, cyanide, thiocyanate, phenoxy, thiophenoxy, phosphate, thiocholine, or carboxylate group. For a compilation of the organophosphorus compounds and their toxicity, see Gallo and Lawryk (1991).

DFP produces virtually irreversible inactivation of AChE and other esterases by alkylphosphorylation. Its high lipid solubility, low molecular weight, and volatility facilitate inhalation, transdermal absorption, and penetration into the CNS. After desulfuration, the insecticides in current use form the dimethoxy or diethoxyphosphoryl enzyme.

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 (Nozaki and Aikawa, 1995).

Because of their low volatility and stability in aqueous solution, parathion and methylparathion were widely used as insecticides. Acute and chronic toxicity has limited their use, and potentially less hazardous compounds have replaced them for home and garden use, now largely throughout the world. These compounds are inactive in inhibiting AChE in vitro; paraoxon is the active metabolite. The phosphoryl oxygen for sulfur substitution is carried out predominantly by hepatic CYPs. This reaction also occurs in the insect, typically with more efficiency. Other insecticides possessing the phosphorothioate structure have been widely employed for home, garden, and agricultural use. These include 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 from indoor and outdoor residential use since 2005.

Malathion (chemathion, mala-spray) also requires replacement of a sulfur atom with oxygen in vivo, conferring resistance to mammalian species. Also, 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 (Costa et al., 2003). In recent years, 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, such as the 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.

Among the quaternary ammonium organophosphorus compounds (group D in Table 10–1), only echothiophate is useful clinically and is limited to ophthalmic administration. Being positively charged, it is not volatile and does not readily penetrate the skin.

Metrifonate is a low-molecular-weight organophosphate that is spontaneously converted to the active phosphoryl ester, dimethyl 2,2-dichlorovinyl phosphate (DDVP, dichlorvos). Both metrifonate and DDVP readily cross the blood-brain barrier to inhibit AChE in the CNS. Metrifonate originally was developed for the treatment of schistosomiasis (Chapter 51). Its capacity to inhibit AChE in the CNS and its reported low toxicity led to its clinical trial in treatment of Alzheimer's disease (Cummings, 2004); a low incidence of skeletal muscle paralysis has limited its acceptance.

Neostigmine also augments motor activity of the small and large bowel; the colon is particularly stimulated. Atony produced by muscarinic receptor antagonists or prior surgical intervention may be overcome, propulsive waves are increased in amplitude and frequency, and movement of intestinal contents is thus promoted. The total effect of anti-ChE agents on intestinal motility probably represents a combination of actions at the ganglion cells of Auerbach's plexus and at the smooth muscle fibers, as a result of the preservation of ACh released by the cholinergic preganglionic and postganglionic fibers, respectively (Chapter 46).

Normally, a single nerve impulse in a terminal motor-axon branch liberates enough ACh to produce a localized depolarization (end-plate potential) of sufficient magnitude to initiate a propagated muscle action potential. The ACh released is rapidly hydrolyzed by AChE, such that the lifetime of free ACh within the nerve-muscle synapse (∼200 μsec) is shorter than the decay of the end-plate potential or the refractory period of the muscle. Therefore, each nerve impulse gives rise to a single wave of depolarization. After inhibition of AChE, the residence time of ACh in the synapse increases, allowing for lateral diffusion and rebinding of the transmitter to multiple receptors. Successive stimulation of neighboring receptors to the release site in the end plate results in a prolongation of the decay time of the end-plate potential. Quanta released by individual nerve impulses are no longer isolated. This action destroys the synchrony between end-plate depolarizations and the development of the action potentials. Consequently, asynchronous excitation and fasciculations of muscle fibers occur. With sufficient inhibition of AChE, depolarization of the end plate predominates, and blockade owing to depolarization ensues (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.

Anti-ChE agents increase contraction of smooth muscle fibers of the bronchioles and ureters, and the ureters may show increased peristaltic activity.

The cardiovascular actions of anti-ChE agents are complex, since 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. This shortens the effective refractory period of atrial muscle fibers and increases 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 of the cell membrane. The excitatory action on the parasympathetic ganglion cells would tend to reinforce the diminished cardiac output, whereas the opposite sequence would result from the action of ACh on sympathetic ganglion cells. Excitation followed by inhibition also is elicited by ACh at the central medullary vasomotor and cardiac centers. All of these effects are complicated further 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 probably is a major factor in the CNS depression that appears 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.

The organophosphate anti-ChE agents are hydrolyzed by two families of enzymes: the carboxylesterases and the paraoxonases (A-esterases). These enzymes are found in the plasma and liver and scavenge or hydrolyze a large number of organophosphates by cleaving the phosphoester, anhydride, PF, or PCN bonds. The paraoxonases are low-molecular-weight enzymes, requiring Ca²⁺ for catalysis, whose natural substrate may be lactones. Some of the isozymes are associated with high density lipoproteins, and in addition to their capacity to hydrolyze organophosphates, they may control low density lipoprotein oxidation, thereby exerting a protective effect in atherosclerosis (Harel et al., 2004; Mackness et al., 2004). Genetic polymorphisms that govern organophosphate substrate specificity and possible susceptibility to atherosclerosis have been found (Costa et al., 2003; Mackness et al., 2004). Wide variations in paraoxonase activity exist among animal species. Young animals are deficient in carboxylesterases and paraoxonases, which may account for age-related toxicities seen in newborn animals and suspected to be a basis for toxicity in human beings (Padilla et al., 2004).

Plasma and hepatic carboxylesterases (aliesterases) and plasma butyrylcholinesterase are inhibited irreversibly by organophosphates (Lockridge and Masson, 2000); their scavenging capacity for organophosphates can afford 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 two organophosphorus insecticides can be synergistic.

In the U.S., the Environmental Protection Agency (EPA), by virtue of revised risk assessments and the Food Quality Protection Act of 1996, has placed several organophosphate insecticides, including diazinon and chlorpyrifos, on restricted use and phase-out status in consumer products for home and garden use. A primary concern relates to children, since the developing nervous system may be particularly susceptible to certain of these agents (Eaton et al., 2008). The Office of Pesticide Programs of the EPA provides continuous reviews of the status of organophosphate pesticides, their tolerance reassessments, and revisions of risk assessments through their Web site (www.epa.gov/pesticides/op/).

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 fatigability and generalized weakness, involuntary twitchings, scattered fasciculations, and eventually severe weakness and paralysis. The most serious consequence is paralysis of the respiratory muscles. Knockout mice lacking the gene encoding AChE can survive under highly supportive conditions and with a special diet, but they exhibit continuous tremors and are stunted in growth (Xie et al., 2000). Mice that selectively lack AChE expression in skeletal muscle but have normal or near normal expression in brain and organs innervated by the autonomic nervous system can reproduce, but have continuous tremors and severe compromise of skeletal muscle strength. By contrast, mice with selective reductions of CNS AChE by elimination of the exons encoding alternative splices or expression of the structural subunits influencing expression in brain yield no obvious phenotype. This arises from large compensatory reductions of acetylcholine synthesis and storage and receptor responses (Camp et al., 2008; Dobbertin et al., 2009). These studies show that cholinergic systems in the CNS adapt in development to chronically diminished hydrolytic capacity for AChE.

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.

The time of death after a single acute exposure may range from < 5 minutes to nearly 24 hours, depending on the dose, route, agent, and other factors. The cause of death primarily is respiratory failure, usually accompanied by a secondary cardiovascular component. Peripheral muscarinic and nicotinic as well as central actions all contribute to respiratory compromise; effects include laryngospasm, bronchoconstriction, increased tracheobronchial and salivary secretions, compromised voluntary control of the diaphragm and intercostal muscles, and central respiratory depression. Blood pressure may fall to alarmingly low levels and cardiac arrhythmias intervene. These effects usually result from hypoxemia and often are reversed by assisted pulmonary ventilation.

Delayed symptoms appearing after 1-4 days and marked by persistent low blood ChE and severe muscle weakness are termed the intermediate syndrome (Lotti, 2002). Delayed neurotoxicity also may be evident after severe intoxication (discussed later).

In moderate or severe intoxication with an organophosphorus anti-ChE agent, the recommended adult dose of pralidoxime is 1-2 g, infused intravenously over not < 5 minutes. If weakness is not relieved or if it recurs after 20-60 minutes, 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. Many of the alkylphosphates are extremely lipid soluble, and if extensive partitioning into body fat has occurred and desulfuration is required for inhibition of AChE, toxicity will persist and symptoms may recur after initial treatment. With severe toxicities from the lipid-soluble agents, it is necessary to continue treatment with atropine and pralidoxime for a week or longer.

Cholinesterase Reactivators. Although the phosphorylated esteratic site of AChE undergoes hydrolytic regeneration at a slow or negligible rate, nucleophilic agents, such as hydroxylamine (NH₂OH), hydroxamic acids (RCONH—OH), and oximes (RCH=NOH), reactivate the enzyme more rapidly than does spontaneous hydrolysis. Researchers reasoned that selective reactivation could be achieved by a site-directed nucleophile, wherein interaction of a quaternary nitrogen with the negative subsite of the active center would place the nucleophile in close apposition to the phosphorus. This goal was achieved to a remarkable degree with pyridine-2-aldoxime methyl chloride (pralidoxime); reactivation with this compound occurs at a million times the rate of that with hydroxylamine. The oxime is oriented proximally to exert a nucleophilic attack on the phosphorus; a phosphoryloxime is formed, leaving the regenerated enzyme (Figure 10–2E).

Several bis-quaternary oximes are even more potent as reactivators for insecticide and nerve gas poisoning (described later); an example is HI-6, which is used in Europe as an antidote.

The velocity of reactivation of phosphorylated AChE by oximes depends on their accessibility to the impacted active center serine (Wong et al., 2000). Furthermore, certain phosphorylated AChEs can undergo a fairly rapid process of "aging," so that within the course of minutes or hours they become completely resistant to the reactivators. "Aging" is due to the loss of one alkoxy group, leaving a much more stable monoalkyl- or monoalkoxy-phosphoryl-AChE (Figure 10–2D and E). Organophosphorus compounds containing tertiary alkoxy groups are more prone to "aging" than are congeners containing the secondary or primary alkoxy groups. The oximes are not effective in antagonizing the toxicity of the more rapidly hydrolyzing carbamoyl ester inhibitors; since pralidoxime itself has weak anti-ChE activity, it is not recommended for the treatment of overdosage with neostigmine or physostigmine or poisoning with carbamoylating insecticides such as carbaryl.

Pharmacology, Toxicology, and Disposition. 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 (Eddleston et al., 2009)

Although high doses or accumulation of oximes can inhibit AChE and cause neuromuscular blockade, they 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.

Parenterally administered human butyrylcholinesterase is under development as an antidote, to scavenge the AChE inhibitor in the plasma before it reaches peripheral and central tissue sites (Cerasoli et al., 2005). Because this effect of butyrylcholinesterase is stoichiometric rather than catalytic, large quantities are required.

Delayed Neurotoxicity of Organophosphorus Compounds. Certain fluorine-containing organophosphorus anti-ChE agents (e.g., DFP, mipafox) have the property of inducing delayed neurotoxicity, a property they share with the triarylphosphates, of which triorthocresyl phosphate (TOCP) is the classical example. This syndrome first received wide-spread attention following the demonstration that TOCP, an adulterant of Jamaica ginger, was responsible for an outbreak of thousands of cases of paralysis that occurred in the U.S. during Prohibition.

The clinical picture is that of a severe polyneuropathy manifested initially by mild sensory disturbances, ataxia, weakness, muscle fatigue and twitching, reduced tendon reflexes, and tenderness to palpation. In severe cases, the weakness may progress to flaccid paralysis and muscle wasting. Recovery may require several years and may be incomplete.

Toxicity from this organophosphate-induced delayed polyneuropathy is not dependent upon inhibition of cholinesterases; instead a distinct esterase, termed neurotoxic esterase, is linked to the lesions (Johnson, 1993). This enzyme has a specificity for hydrophobic esters, but its natural substrate and function remain unknown (Glynn, 2000). Myopathies that result in generalized necrotic lesions and changes in end-plate cytostructure also are found in experimental animals after long-term exposure to organophosphates (De Bleecker et al., 1992).

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's 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.

Neostigmine is used for the relief of abdominal distension and acute colonic pseudo-obstruction from a variety of medical and surgical causes (Ponec et al., 1999). The usual subcutaneous dose of neostigmine methylsulfate for postoperative paralytic ileus is 0.5 mg, given as needed. Peristaltic activity commences 10-30 minutes after parenteral administration, whereas 2-4 hours 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.

When neostigmine is used for the treatment of atony of the detrusor muscle of the urinary bladder, postoperative dysuria is relieved, and the time interval between operation and spontaneous urination is shortened. The drug is used in a similar dose and manner as in the management of paralytic ileus. 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.

Narrow-angle glaucoma is nearly always a medical emergency in which drugs are essential in controlling the acute attack, but the long-range management is often surgical (e.g., peripheral or complete iridectomy). Wide-angle glaucoma, on the other hand, has a gradual, insidious onset and is not generally amenable to surgical improvement; in this type, control of intraocular pressure usually is dependent upon continuous drug therapy.

Since the cholinergic agonists and ChE inhibitors also block accommodation and induce myopia, these agents produce transient blurring of far vision, limited visual acuity in low light, and loss of vision at the margin when instilled in the eye. With long-term administration of the cholinergic agonists and anti-ChE agents, the compromise of vision diminishes. Nevertheless, other agents without these side effects, such as β adrenergic receptor antagonists, prostaglandin analogs, or carbonic anhydrase inhibitors, have become the primary topical therapies for open-angle glaucoma (Alward, 1998), with AChE inhibitors held in reserve for the chronic conditions when patients become refractory to the above agents. Topical treatment with long-acting ChE inhibitors such as echothiophate gives rise to symptoms characteristic of systemic ChE inhibition. Echothiophate treatment in advanced glaucoma may be associated with the production of cataracts (Alward, 1998).

Anti-ChE agents have been employed locally in the treatment of a variety of other less common ophthalmologic conditions, including accommodative esotropia and myasthenia gravis confined to the extraocular and eyelid muscles. Adie (or tonic pupil) syndrome results from dysfunction of the ciliary body, perhaps because of local nerve degeneration. Low concentrations of physostigmine are reported to decrease the blurred vision and pain associated with this condition. In alternation with a mydriatic drug such as atropine, short-acting anti-ChE agents have proven useful for breaking adhesions between the iris and the lens or cornea. (For a complete account of the use of anti-ChE agents in ocular therapy, see Chapter 64).

The defect in myasthenia gravis is in synaptic transmission at the neuromuscular junction. When a motor nerve of a normal subject is stimulated at 25 Hz, electrical and mechanical responses are well sustained. A suitable margin of safety exists for maintenance of neuromuscular transmission. Initial responses in the myasthenic patient may be normal, but they diminish rapidly, consistent with the rapid muscle fatigue seen in patients.

The relative importance of prejunctional and postjunctional defects in myasthenia gravis was a matter of considerable debate until Patrick and Lindstrom found that rabbits immunized with the nicotinic receptor purified from electric eels slowly developed muscular weakness and respiratory difficulties that resembled the symptoms of myasthenia gravis. The rabbits also exhibited decremental responses following repetitive nerve stimulation, enhanced sensitivity to curare, and following the administration of anti-AChE agents, symptomatic and electrophysiological improvement of neuromuscular transmission. This animal model prompted intense investigation into whether the natural disease represented an autoimmune response directed toward the ACh receptor. Anti-receptor antibodies are detectable in sera of 90% of patients with the disease, although the clinical status of the patient does not correlate precisely with antibody titers (Drachman, 1994; Lindstrom, 2000). Sequences and the structural location in the α1 subunit constituting the main immunogenic region are well defined (Lindstrom 2008).

The picture that emerges is that myasthenia gravis is caused by an autoimmune response primarily to the ACh receptor at the post-junctional end plate. These antibodies reduce the number of receptors detectable either by snake α-neurotoxin-binding assays (Fambrough et al., 1973) or by electrophysiological measurements of ACh sensitivity (Drachman, 1994). Immune complexes along with marked ultrastructural abnormalities appear in the synaptic cleft and enhance receptor degradation through complement-mediated lysis in the end plate. A related disease that also compromises neuromuscular transmission is Lambert-Eaton syndrome. Here, antibodies are directed against Ca²⁺ channels that are necessary for pre-synaptic release of ACh (Lang et al., 1998).

In a subset of ∼10% of patients presenting with a myasthenic syndrome, muscle weakness has a congenital rather than an autoimmune basis. Characterization of biochemical and genetic bases of the congenital condition has shown mutations to occur in the acetylcholine receptor which affect ligand-binding and channel-opening kinetics and durations (Engel et al., 2008; Sine and Engel, 2006). Other mutations occur as a deficiency in the form of AChE that contains the collagen-like tail unit. As expected, following administration of anti-ChE agents (see following discussion), subjective improvement is not seen in most congenital myasthenic patients, although some of the above channel syndromes can be ameliorated pharmacologically.

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. However, myasthenia gravis is the only condition in which the aforementioned deficiencies 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 seconds 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 non-myasthenic patients).

An excessive dose of an anti-ChE drug results in a cholinergic crisis. The condition is characterized by weakness resulting from generalized depolarization of the motor end plate; 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, since 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 (for complete details, see Drachman, 1994; Osserman et al., 1972). Detection of anti-receptor antibodies in muscle biopsies or plasma is now widely employed to establish the diagnosis.

When the diagnosis of myasthenia gravis has been established, 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 duration of action of these drugs is such that the interval between oral doses required to maintain muscle strength usually is 2-4 hours for neostigmine, 3-6 hours for pyridostigmine, or 3-8 hours 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-hour 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 (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 (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. However, when treatment with steroids is continued over prolonged periods, a high incidence of side effects may result (Chapter 42). Gradual lowering of maintenance doses and alternate-day regimens of short-acting steroids are used to minimize side effects. 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 (Drachman, 1994). Other immunosuppressive agents such as azathioprine and cyclosporine also have been beneficial in more advanced cases (Chapter 35).

Thymectomy should be considered in myasthenia associated with a thymoma or when the disease is not controlled adequately by anti-ChE agents and steroids. The relative risks and benefits of the surgical procedure versus anti-ChE and glucocorticoid treatment require careful assessment. Since the thymus contains myoid cells with nicotinic receptors (Schluep et al., 1987), and a predominance of patients have thymic abnormalities, the thymus may be responsible for the initial pathogenesis. It also is the source of autoreactive T-helper cells.

In keeping with the presumed autoimmune etiology of myasthenia gravis, plasmapheresis and immune therapy have proven beneficial in patients who have remained disabled in the face of other treatments (Drachman, 1994, 1996). Improvement in muscle strength correlates with the reduction of the titer of antibody directed against the nicotinic ACh receptor.

Prophylaxis in Cholinesterase Inhibitor Poisoning. Studies in experimental animals have shown that pretreatment with pyridostigmine reduces the incapacitation and mortality associated with nerve agent poisoning, particularly for agents such as soman that show rapid aging. The first large-scale administration of pyridostigmine to humans occurred in 1990 in anticipation of nerve-agent attack in the first Persian Gulf War. At an oral dose of 30 mg every 8 hours, the incidence of side effects was around 1%, but fewer than 0.1% of the subjects had responses sufficient to warrant discontinuing the drug in the setting of military action (Keeler et al., 1991). Long-term follow-up indicates that veterans of the Persian Gulf War who received pyridostigmine showed a low incidence of a neurologic syndrome, now termed the Persian Gulf War syndrome. It is characterized by impaired cognition, ataxia, confusion, myoneuropathy, adenopathy, weakness, and incontinence (Haley et al., 1997; Institute of Medicine, 2003). While pyridostigmine has been implicated by some as the causative agent, the absence of similar neuropathies in pyridostigmine-treated myasthenic patients makes it far more likely that a combination of agents, including combusted organophosphates and insect repellents in addition to pyridostigmine, contributed to this persisting syndrome. It also is difficult to distinguish residual chemical toxicity from post-traumatic stress experienced after combat action. Pyridostigmine is FDA-approved for prophylaxis against soman, an organophosphate that rapidly "ages" following inhibition of cholinesterases.

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. Physostigmine is potentially useful in reversing the central anticholinergic syndrome produced by overdosage or an unusual reaction to these drugs (Nilsson, 1982). The effectiveness of physostigmine in reversing the anticholinergic effects of these agents has been clearly documented. However, other toxic effects of the tricyclic antidepressants and phenothiazines (Chapters 15 and Chapter 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. Physostigmine, a tertiary amine, crosses the blood-brain barrier, in contrast to the quaternary anti-AChE drugs. The use of anti-ChE agents to reverse the effects of competitive neuromuscular blocking agents is discussed in Chapter 11.