6
Pyridostigmine Bromide

Pyridostigmine bromide (PB) is a drug used during the Gulf War as a pretreatment to protect troops from the harmful effects of nerve agents. It has been used for more than 40 years in the routine treatment of myasthenia gravis and may be used following surgery in the reversal of neuromuscular blockade (Williams, 1984).

PB, a reversible cholinesterase (ChE) inhibitor, is a carbamate compound, specifically, the dimethylcarbamate ester of 3-hydroxy-1-methylpyridinium bromide. It was synthesized in 1945 by Hoffman-La Roche Laboratories in Switzerland and is sold under the trade name Mestinon bromide (Williams, 1984). PB is one of the quaternary ammonium anticholinesterase compounds, which generally do not penetrate cell membranes. Compounds in this category are poorly absorbed from the gastrointestinal tract and are excluded by the blood–brain barrier (Williams, 1984; Goodman et al., 1996).

Mestinon was approved by the Food and Drug Administration (FDA) in 1955 as safe for the treatment of myasthenia gravis.1 The FDA also approved an injectable form known as Regenol for reversing the effects of some anesthetic formulations (Rettig, 1999). In the treatment of myasthenia gravis, the average oral dose is 600 mg per day in divided doses; however, the size and frequency of the dose must be adjusted to the needs of the individual patient (Physicians’ Desk Reference, 2000). The drug is poorly absorbed after oral administration

1  

Myasthenia gravis is an autoimmune disorder characterized by antibody blockade of the acetylcholine (ACh) receptor at the neuromuscular junction.



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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines 6 Pyridostigmine Bromide Pyridostigmine bromide (PB) is a drug used during the Gulf War as a pretreatment to protect troops from the harmful effects of nerve agents. It has been used for more than 40 years in the routine treatment of myasthenia gravis and may be used following surgery in the reversal of neuromuscular blockade (Williams, 1984). PB, a reversible cholinesterase (ChE) inhibitor, is a carbamate compound, specifically, the dimethylcarbamate ester of 3-hydroxy-1-methylpyridinium bromide. It was synthesized in 1945 by Hoffman-La Roche Laboratories in Switzerland and is sold under the trade name Mestinon bromide (Williams, 1984). PB is one of the quaternary ammonium anticholinesterase compounds, which generally do not penetrate cell membranes. Compounds in this category are poorly absorbed from the gastrointestinal tract and are excluded by the blood–brain barrier (Williams, 1984; Goodman et al., 1996). Mestinon was approved by the Food and Drug Administration (FDA) in 1955 as safe for the treatment of myasthenia gravis.1 The FDA also approved an injectable form known as Regenol for reversing the effects of some anesthetic formulations (Rettig, 1999). In the treatment of myasthenia gravis, the average oral dose is 600 mg per day in divided doses; however, the size and frequency of the dose must be adjusted to the needs of the individual patient (Physicians’ Desk Reference, 2000). The drug is poorly absorbed after oral administration 1   Myasthenia gravis is an autoimmune disorder characterized by antibody blockade of the acetylcholine (ACh) receptor at the neuromuscular junction.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines and peak plasma levels occur at 2 to 3 hours after oral dosing. The drug is eliminated almost exclusively via the kidneys in the urine (Williams, 1984). Side effects of PB are generally related to the large doses given to myasthenics; in surgical patients, adverse reactions are controlled by simultaneous administration of atropine (Williams, 1984). Adverse reactions may be muscarinic or nicotinic (also see Chapter 5), both reactions are due to increased acetylcholine (ACh). Muscarinic reactions include nausea, vomiting, diarrhea, abdominal cramps, increased peristalsis, increased salivation, increased bronchial secretions, miosis, and heavy perspiration. Nicotinic effects are chiefly muscle cramps, fasciculations, and weakness (Williams, 1984). During the Gulf War, PB was used as a pretreatment for possible exposure to nerve agents because of its ability to reversibly bind to acetylcholinesterase (AChE).2 The bound fraction is thereby protected from subsequent exposure to nerve agents that would irreversibly bind to AChE. PB is not an antidote (i.e., it has no value when administered after nerve agent exposure) and is not a substitute for atropine or 2-pralidoxime chloride; rather, it enhances their efficacy (Madsen, 1998). PB was used as an investigational product during the Gulf War (Rettig, 1999) and was not recommended for routine use. The FDA, under a then newly enacted interim rule, had granted DoD a waiver from the requirement to obtain informed consent from service members taking this drug, but the rule did not address the record keeping that would ordinarily accompany the use of an investigational drug (FDA, 1990; Rettig, 1999). PB was manufactured for Duphar and Roche; it was produced by two different facilities outside the United States, specifically in the Netherlands for Duphar and in the United Kingdom for Roche. The Department of Defense (DoD) reported that 5,328,710 doses were fielded and estimated that approximately 250,000 personnel took at least some PB during the Gulf War.3 It was supplied as a 21-tablet blister pack, the dosage prescribed was one 30-mg tablet every 8 hours.4 Variation in use occurred, how- 2   AChE is an enzyme necessary to remove ACh. Acetylcholine transmits nerve signals at the cholinergic neuromuscular junction or synapses in the central nervous system. Anticholinesterase agents inhibit (inactivate) AChE, resulting in an accumulation of ACh. The accumulation repetitively activates the ACh receptors, resulting in exaggerated responses of the organ (e.g., excess salivation). 3   The number of doses fielded was obtained through a search of the Defense Personnel Support Center archived logistic records for Operations Desert Shield and Desert Storm and reflects the amount of product ordered and sent through supply channels. In most cases, only a review of each individual’s medical treatment record would provide the actual number of doses administered, and few records were maintained by individuals (Office of the Secretary of Defense Bernard Roskter, January 30, 1998 letter to the Honorable Arlen Specter, Chairman, Committee on Veterans’ Affairs, U.S. Senate, 1998). 4   PB was distributed as 30-mg tablets in a blister pack of 21 tablets within a sealed pouch. Each packet provided a 1-week supply of PB for one person. Military personnel were issued two blister packs each. Recommended long-term storage was at 2–80°C, and blister packs removed from refrigeration were to be used within 6 months (Madsen, 1998).

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines ever, because it was self-administered and to be taken only when ordered by the unit commander (PAC, 1996). Thus, actual veterans’ exposures to PB are not known, and there are few examples of documentation in either individual health records or unit records, making it difficult to assess any potential contribution of this drug to the current unexplained illnesses (PAC, 1996). DoD noted that at the recommended dosage levels, soldiers reported acute but transient side effects. Keeler and colleagues (1991) conducted an uncontrolled retrospective survey of the medical officers of the XXVIII Airborne Corps. The unit’s 41,650 soldiers were instructed to take PB at the onset of Operation Desert Storm in January 1991. Usage varied from 1 to 21 tablets taken over 1–7 days; 34,000 soldiers reported taking the medication for 6–7 days. Reported side effects of PB were estimated to have been present in half the troops; they were not incapacitating, however, and were primarily gastrointestinal in nature. An estimated 1 percent of the soldiers believed they had symptoms that warranted medical attention, but less than 0.1 percent had effects sufficient to warrant discontinuation of the drug (Keeler et al., 1991). PB, alone and in combination with other exposures, has been suggested as one of several possible causative factors associated with illnesses in Gulf War veterans (Abou-Donia et al., 1996a,b; Chaney et al., 1997; Fukuda et al., 1998; Unwin et al., 1999). The remainder of this chapter examines the scientific literature on the potential adverse health effects of PB. The committee begins its review with a discussion of the toxicology and pharmacokinetics of PB, based primarily on findings from animal studies and other experimental test systems. The committee then turns its attention to studies in humans. These include clinical studies, related principally to the use of PB in the treatment of myasthenia gravis and its use as a test of hypothalamic pituitary function or growth hormone response. In addition to these clinical studies, the committee reviewed studies in healthy volunteers and epidemiologic studies. The healthy-volunteer studies were conducted among healthy military and nonmilitary populations to evaluate the tolerance of prophylactic doses of PB. Unfortunately, there is a paucity of epidemiologic studies on PB and adverse health effects in the peer-reviewed literature. Although there have been a number of descriptive epidemiologic studies of Gulf War veterans (see Chapter 2), those investigations generally sought to characterize the nature and frequency of the symptoms and illnesses reported by returning soldiers and did not examine the association of PB with the illnesses reported. Studies that attempted to evaluate the association of PB and symptoms among Gulf War veterans are reviewed. TOXICOLOGY There is an extensive toxicologic literature on PB, which was reviewed by the committee. The studies discussed below were designed to assess the pharmacologic and toxic properties of PB in animals and other test systems.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines Structure and Mechanism of Action There are several types of esterases in the body, all of which hydrolyze esters such as acetlycholine. Some of those in the plasma are nonspecific and hydrolyze many esters including ACh, whereas acetylcholinesterase, which is found at cholinergic synapses, is more specific for ACh. AChE is also found in erythrocytes, but its function in these cells is poorly understood. Inhibition of the plasma (or erythrocyte) esterase is without known consequence, whereas the inhibition of AChE at cholinergic synapses leads to a spectrum of toxicological effects. At cholinergic synapses, ACh released from nerve endings by action potentials activates the postjunctional receptors and thereby elicits responses. To prevent it from inappropriately reactivating the receptors, ACh is hydrolyzed to inactive products by the enzyme AChE in the synapse, thus ensuring that one action potential leads to a single response. Interference with the ability of AChE to hydrolyze ACh leads to accumulation of the latter in the synapse, and the excess neurotransmitter is then responsible for both the pharmacological and the toxicological manifestations of AChE inhibition. The toxicokinetics of PB are complex, and there is incomplete agreement on the fate of an ingested dose (Joiner and Kluwe, 1988; Golomb, 1999). The gastrointestinal tract erratically absorbs PB, leading to considerable variations in plasma concentration (Aquilonius et al., 1980). Absorbed PB is subject to first-pass metabolism by the liver (Barber et al., 1975), but since 60–85 percent of an administered dose is excreted unchanged via the kidney, the fraction of a dose undergoing hepatic biotransformation is not large. Hepatic biotransformation of neostigmine and pyridostigmine apparently gives rise to the metabolites 3-hydroxy-N-methylpyridinium, 3-hydroxyphenyltrimethylammonium, and edrophonium (Hennis et al., 1984); there is no evidence that these metabolites contribute to antagonism of neuromuscular blockade or that they are neurotoxic. The differences in duration and reversibility of cholinesterase inhibition by PB and organophosphate exposures provide the rationale for battlefield use of PB by the military. Although both PB and the organophosphate (OP) compounds employed as “nerve gases” inhibit AChE by binding to it, the OP–AChE bond is much stronger than the PB–AChE bond, making the former essentially irreversible. The differences in binding of carbamates and organophosphates to AChE have been exploited in the use of a reversible inhibitor of AChE (e.g., PB) to protect it against irreversible inhibitors such as the nerve gases (Gordon et al., 1978; Dirnhuber et al., 1979). In effect, protection results from “preinhibition” of the enzyme with a more readily reversible inhibitor. As noted, the prophylactic use of PB in military personnel calls for 30 mg to be taken three times a day. Since the plasma half-lives of orally administered PB are 120–195 minutes and the corresponding half-lives for reversal of erythrocyte AChE inhibition are in the same range (Kluwe et al., 1990), 8-hour intervals between doses are adequate to maintain constant levels of AChE inhibition and thus protection. Joiner and Kluwe (1988) found 30 percent inhibition of red blood cell (RBC) AChE in monkeys following oral administration of 0.28 mg/kg

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines PB: higher doses caused proportionally greater inhibition (0.57 mg/kg yielded 43 percent inhibition). Animal Studies Many neural and neuromuscular systems in the body employ ACh as a neurotransmitter, and still other organs are influenced by ACh. Given the numerous physiological functions influenced by ACh, it is not surprising that perturbations of its function, resulting from AChE inhibition by PB, have numerous and diverse toxicological consequences. There are many recently published and ongoing studies that may elucidate the nature of the mechanisms of PB toxicity; however, many of the studies have yet to be confirmed. The following sections briefly review the available information on PB toxicity, including those studies that await replication. Neuromuscular Effects The neuromuscular effects of PB are important for two reasons. First, impairment of neuromuscular function leads to muscle weakness. Second, experimental findings obtained from the readily accessible neuromuscular junction have long been interpreted to be applicable to other cholinergically innervated synapses in the central nervous system, which are much more difficult to access experimentally. Thus, events occurring at the neuromuscular junction have been thought to mirror those in the brain. PB, as a ChE inhibitor, modifies physiological function at the sites of innervation of all types of muscle: smooth, cardiac, and skeletal (or striated). The neuromuscular effects of PB have been described almost exclusively for skeletal muscle, while those in other types of muscle are relatively less studied. The effects of PB on the skeletal neuromuscular junction have recently been reviewed in detail (Golomb, 1999). Effects of PB on cardiac muscle have been reported (Glass-Marmor et al., 1996). Exposure to PB has pharmacological and/or toxicological consequences on neuromuscular function either by direct action of PB at low doses, acting as a weak agonist at the nicotinic ACh receptors (Sherby et al., 1984; Maelicke et al., 1993), or more importantly by accumulation of ACh resulting from inhibition of AChE. Acutely, PB leads to a facilitation (or augmentation) of the strength of contractile tension developed in skeletal muscle because ACh accumulation repetitively activates the contractile process. The relationship between the degree of ChE inhibition and the facilitation of twitch tension is complex. No twitch potentiation is seen until RBC AChE is at least 85 percent inhibited (Barber et al., 1979); this threshold is nearly identical to that noted for other ChE inhibitors. At inhibition levels of 85–98 percent there is a linear relationship between AChE inhibition and facilitation of twitch tension. Large doses of PB would normally be required to achieve these levels of RBC AChE inhibition, and it

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines would be anticipated that numerous other incapacitating consequences of ChE inhibition, particularly muscarinic (e.g., salivation, sweating), would be apparent before neuromuscular effects became manifest. Failure of neuromuscular transmission, whereby nerve signals no longer evoke muscle contraction, is also thought to be an extension of the effects of accumulation of ACh. Prolonged depolarization leads to a desensitization of the postjunctional receptor; high doses of ChE inhibitors may further directly block ACh receptors, adding to the desensitization (Maselli and Leung, 1993a,b). Neuromuscular blockade by this mechanism would require very large doses of PB. It has long been known that inhibition of AChE at the neuromuscular junction results in both pre- and postjunctional morphological alterations, and the effects of PB exposure are no exception (Hudson et al., 1986; Matthew et al., 1998). Alterations in the prejunctional apparatus of the neuromuscular junction (i.e., the nerve ending), most often associated with denervation phenomena, are not usual sequelae of PB intoxication; rather most evidence of exposure occurs postsynaptically. Microscopic examination of the postsynaptic and myofibrillar structures following exposure to PB reveals that most damage occurs in the vicinity of the neuromuscular junction; Z-lines are blurred and electron microscopy reveals swollen mitochondria, suggestive of a disruption in calcium homeostasis (Gebbers et al., 1986). Myopathic changes decrease with distance from the postjunctional region (Adler et al., 1992), and normal myofibers occur within distances of 12–14 microns. Studies in which myopathic changes were observed employed large doses of PB (20–98 mg/kg per day), which yielded inhibition of AChE in excess of 50 percent (Hudson et al., 1985; Bowman et al., 1989), greater than the inhibition seen in humans following PB administration. When blood ChE inhibition was reduced to levels expected (about 30 percent) by reducing the PB dose, neither acute nor subchronic (4-week) exposure produced neuromuscular lesions (Matthew et al., 1998). The susceptibility of neuromuscular junctions to neural and/or myofibrillar damage does not appear related to fiber type, being observed in muscles with substantially different fiber type compositions (Hudson et al., 1985). Despite the initial appearance of pathological alterations at the neuromuscular junction during continuous administration of PB, these alterations (principally myopathic) reversed by the second week of daily exposure to 90 mg of PB (Bowman et al., 1989; Matthew et al., 1990). Similar patterns of myopathic lesions (i.e., initial appearance of lesions which subsequently resolve) are observed with exposure to other carbamate and to organophosphorus ChE inhibitors (reviewed recently, Golomb, 1999). Acetylcholine-associated myopathy is not a new observation (Fenichel et al., 1974). All ChE inhibitors cause cholinergic toxicity as a result of the accumulation of excess amounts of ACh; hence they induce similar toxicities (generally referred to as acute toxic or cholinergic effects). In addition to acute toxicity, certain ChE inhibitors, particularly the organophosphorus compounds, produce other neuro- and myopathic effects, which are apparently unrelated to ChE inhibition and are described as intermediate and delayed neurotoxicity (or organo-

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines phosphate-induced delayed neuropathy [OPIDN]). Intermediate syndrome (intermediate in onset between the acute toxic effects following exposure to a ChE inhibitor and the delayed neuropathic actions associated with certain OP-type cholinesterase inhibition) is a toxic syndrome associated with muscle weakness. It typically occurs 24 hours after exposure and is characterized by weakness or paralysis involving neck flexors, cranial nerves, proximal limb muscles, and respiratory muscles (Leon et al., 1996). The intermediate syndrome usually resolves over time, and although it has been associated with exposure to a variety of ChE inhibitors, PB has not been implicated (Golomb, 1999). Clinically, OPIDN (see below and Chapter 5) is a delayed neuropathy, its symptoms becoming manifest some 2–3 weeks after exposure to certain organophosphate ChE inhibitors. A detailed description of the disorder has been given recently (Golomb, 1999). Like the intermediate syndrome, OPIDN has not been associated with exposure to PB. PB administration also results in enhanced expression of AChE in skeletal muscle, evident even after the enzyme is no longer inhibited (Lintern et al., 1997a,b). Further, repeated administration of PB over a period of weeks produces a dose-related increase in the expression of beta-endorphin and beta-endorphin 30-31 (glycylglutamine), both of which are derived from the same precursor protein pro-opiomelanocortin (POMC); the endorphins are thought to lead to the augmented AChE (Amos and Smith, 1998). POMC levels are also increased by nerve section (Edwards et al., 1986), as well as by other neurotoxicants including iminodipropionitrile (IDPN), acrylamide, and organophosphates (Hughes et al., 1992, 1995; Amos and Smith, 1998). Thus, increased expression of POMC and the consequent increase in AChE levels are probably obligatory components of an injury response, regardless of whether the injury is physical or chemical. Neurobehavioral Toxicology Compared to other carbamates, particularly physostigmine, or to organophosphate cholinesterase inhibitors, PB has had limited investigation for its potential neurobehavioral effects. Based on its reported lack of access to the central nervous system (CNS), PB has historically been used as a negative control in behavioral studies of other ChE inhibitors or as an agent to selectively produce peripheral nervous system actions of ChE inhibitors. PB is a carbamate possessing a positively charged quaternary group that restricts its penetration of the blood–brain barrier (Xia et al., 1981). Doses of PB employed in these studies, often in the range of 200 μg/kg, failed to produce observable effects on the behavioral paradigm under examination (McMaster and Finger, 1989; Wolthius et al., 1995); thus, PB has traditionally been thought to be devoid of CNS action. The use of PB as a preventive measure against the effects of chemical warfare agents, coupled with the emerging understanding of the importance of a cholinergic link in Alzheimer’s disease, has led to a reexamination of the action of PB, particularly of chronic dosing schedules, on behaviors in both humans and laboratory animals. Low doses of PB have been reported to have behavioral

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines consequences after acute administration. Two-way shuttle box avoidance learning, open-field behavior, and complex coordinated movements in rats were interrupted by PB at doses of about 0.27 mg/kg, which neither produced overt symptoms nor affected running speed and coordinated locomotion (Wolthius and Vanwersch, 1984). Shih and colleagues (1991) examined a wider range of PB doses on lever pressing of rats maintained under a multiple fixed-ratio (FR 20) time-out schedule of reinforcement for water reward. They noted that doses greater than 6 mg/kg disrupted responding but there were no overt signs of peripheral neurotoxicity until doses in excess of 12 mg/kg were administered. Liu (1991) confirmed that doses of 3–12 mg/kg interfered with responding but did not cause overt toxicity. PB has been tested in primates (Macaca mulatta) for its effects on the ability of subjects to perform compensatory tracking on an equilibrium platform (Blick et al., 1994). Of the doses of PB tested, only the highest dose interfered with performance of the task (Murphy et al., 1989). Plasma ChE inhibition at this dose was 83 percent. Thus, it appears that PB, particularly at higher doses, is capable of modifying experimentally measured behavioral end points. This might suggest some degree of entry of PB into the CNS. Gastrointestinal Effects Many aspects of gastrointestinal function are mediated or influenced by ACh, and PB would be predicted to cause gastrointestinal disturbance, especially if administered orally. Thus, the most common complaints of troops taking the prescribed dosage of PB (3 × 30 mg per day—the equivalent of 0.4 mg/kg every 8 hours) included nausea, vomiting, diarrhea, abdominal cramping, increased salivation, bronchial secretions, miosis, and diaphoresis—symptoms referable to a parasympathetic (and peripheral) predominance. Human symptoms are in accord with observations made in laboratory animals. In beagles, the threshold dosage for gastrointestinal effects of PB is as low as 0.05 mg/kg; this dose causes significant inhibition of both plasma butyrylcholinesterase and RBC AChE (Kluwe et al., 1990). Higher doses of PB result in proportionally greater effects. Species differences in responsiveness to toxicities of PB have been noted (Levine et al., 1991). 180-Day Exposure One 180-day subchronic oral toxicity study of PB has been reported (Morgan et al., 1990). Rats were administered 0–10 mg PB/kg, 5–7 days per week, for 180 days. During the dosing phase of the study, ChE inhibition was up to 63 percent in plasma and 49 percent in erythrocytes. An extensive battery of tests (including hematological and serum analyses) were performed 30 days after the last dose of PB, at which time ChE levels had returned to control values. Although serum chemistry revealed elevations in lactate dehydrogenase,

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines creatine phosphokinase, and aspartate aminotransferase, these indices of myopathy (Hoffman et al., 1989) were unaccompanied by morphological evidence of PB-induced toxicity. Reproductive Effects A single study in rats has been reviewed that reports the reproductive toxicity of PB (Levine et al., 1989; Levine and Parker, 1991). No teratogenic effects were noted even after 90 days’ exposure to doses ranging up to 60 mg/kg per day. Although there was a suggestion of postimplantation loss at the highest dose tested (which also resulted in 10 percent mortality), other fertility indices and offspring were unchanged in perinatal and postnatal studies. Contact Dermatitis In skin sensitization studies, 44 percent of guinea pigs exposed to 50 percent PB alone exhibited positive responses. Addition of dermal penetration enhancers (surfactants) such as sodium lauryl sulfate increased the incidence of positive responses to greater than 80 percent (Harris and Maibach, 1989). Bronchial Asthma Excess ACh resulting from ChE inhibition might be expected to exacerbate bronchial asthma by causing increased respiratory secretions and bronchoconstriction. Dogs administered PB doses of 2–5 mg/kg exhibited dose-dependent increases in airway resistance and decreases in tidal volume (Caldwell et al., 1989). Since these doses are much higher than those given to humans, no complications of PB administration in asthmatics were predicted. However, there have been reports of human studies (discussed later in this chapter) and anecdotal reports suggesting a possible dose-dependent outcome in asthmatics (Ram et al., 1991; Gouge et al., 1994). Cardiac Dysrhythmias and Cardiomyopathy The autonomic, parasympathetic innervation of the heart is concerned principally with the regulation of heart rate and atrioventricular conduction and exerts this influence via cholinergic synapses much like those found elsewhere in the nervous system. Inhibition of AChE at these synapses results in prolonged residence time of ACh, leading to slowing of the sinoatrial firing rate (bradycardia), along with prolongation of phase four conduction parameters. Recent studies in cats indicate that the magnitude of the bradycardia resulting from PB does not correlate with the degree of AChE inhibition, but rather reflects the extent of muscarinic agonist actions (Yamamoto et al., 1996; Stein et al., 1997).

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines Scattered reports (Kato et al., 1989; Glass-Marmor et al., 1996) suggest that ACh accumulation in cardiac muscle compromises mitochondrial function and thus impairs myocardial energetics in a manner similar to that observed in skeletal muscle (see discussion of neuromuscular effects). The doses of PB employed in these studies were 20 and 60 mg/kg, respectively. Thermoregulation Like other ChE inhibitors, PB is capable of altering cholinergically mediated thermoregulatory processes in the hypothalamus. The cholinergic component is demonstrated by the ability of atropine to block PB alterations in thermoregulation (Matthew et al., 1988). Compromised temperature regulation is most prominent at higher ambient temperatures. In rats, acute administration of PB produces hyperthermia, whereas chronic administration elicits much less elevation in body temperature, indicating the relatively rapid emergence of adaptive processes akin to heat acclimation during prolonged exposure (Matthew et al., 1994). In contrast, mice given 0.2 mg/kg PB have been reported to be hypothermic (Kaufer et al., 1999). It appears that hyperthermia (and the extent of debilitation) is correlated with the degree of brain ChE inhibition (and presumably with the extent of penetration of the ChE inhibitor through the blood–brain barrier) and, further, there are no significant effects on temperature regulation when plasma ChE inhibition is less than 30 percent (Francesconi et al. 1984, 1986; Matthew et al., 1988, 1994). This also appears to be true for humans exposed to PB (Seidman and Epstein, 1989). The impact of physical conditions such as heat, alone and in combination with pharmacological agents, on task performance has been evaluated in both animals and military personnel. PB is of particular interest in this regard since sweating is under autonomic, muscarinic control. In monkeys, doses of PB that produce a 25–30 percent inhibition of serum ChE levels result in only transient alterations in physiological parameters (Avlonitou and Elizondo, 1988). Francesconi and colleagues (1986) reported that chronic (14-day) inhibition of ChE in rats to levels as high as 39 percent is without effect on thermoregulation or exercise performance. In human volunteers, single doses of 30 mg are without effect on psychomotor performance or thermoregulation (Wenger and Latzka, 1992) as were multiple doses (Seidman and Epstein, 1989; Izraeli et al., 1990; Arad et al., 1992a,b). Chronic administration of PB does not appear to alter thermoregulation at cold ambient temperatures (Sawka et al., 1994). It has been reported (Sharma et al., 1992) that moderate heat stress (38°C for 4 hours) enhances the entry of tracers such as Evans blue into the brains of rats, adding to earlier evidence supporting the notion that stress augments the permeability of the blood–brain barrier (Belova and Jonsson, 1982; Ben-Nathan et al., 1991). Subsequent studies have failed to confirm these findings. Lallement and colleagues (1998) administered tritiated PB to guinea pigs maintained at an ambient temperature of 42.6°C for 2 hours and noted that even though those given PB succumbed to heat stress and exhibited high levels of plasma

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines cortisol (an indicator of stress), there was no inhibition of brain AChE. There was also no autoradiographic evidence that PB had entered the CNS. Hence, stress in guinea pigs fails to enhance PB penetration, while in mice (Friedman et al., 1996) and rats (Sharma et al., 1992), stress appears to permit entry of the drug. Whether these differences stem from species differences, age of the animals used, or other variables remains to be determined. Interactions with Other Agents Interactions between agents present during the Gulf War have been hypothesized or suspected to contribute to the illnesses of Gulf War veterans (IOM, 1995; PAC, 1996, 1997). Several mechanisms exist whereby other chemical compounds may influence the pharmacological and toxicological actions of PB. These may occur through pharmacological antagonism, synergism, addition, and so forth, presumably by actions on entirely different receptor types. Alternatively, the presence of other chemical(s) may enhance absorption or interfere with detoxification processes, leading in either case to an exaggeration of the pharmacological and/or toxicological effects of PB. The possible outcomes of the interactions of several relevant chemicals have recently been discussed (Golomb, 1999). Pharmacological Interactions There have been limited studies of possible toxic interactions between PB and other agents to which there was reported or putative exposure during the Gulf War. Co-exposure of hens to total cumulative doses of 200, 400, and 20,000 mg/kg of PB, chlorpyrifos, and N,N-diethyl-m-toluamide (DEET) respectively, over 2 months resulted in increased indices of toxicity (Abou-Donia et al., 1996a). Neurological indices of dysfunction were more severe in birds receiving the combined exposures, paralleling perhaps the more prominent neuropathology observed in the sciatic nerve and spinal cord and the greater degree of inhibition of plasma ChE and brain AChE. It is noteworthy that the pathology and the neurological impairment are hallmarks of OPIDN, but symptoms of this neurotoxic disorder are not consistent with those reported in Gulf War veterans’ illnesses. Also, in this study (Abou-Donia et al., 1996a), neither PB nor DEET inhibited NTE (neuropathy target esterase), consistent with observations that neither produces OPIDN. Chlorpyrifos, an organophosphate pesticide used in the Gulf War, does inhibit NTE, but only by 27–29 percent, which is below the threshold of inhibition required to precipitate OPIDN. An analogous study by the same authors (Abou-Donia et al., 1996b) exposed hens to combinations of PB and DEET, but with administration of permethrin (total dose 20,000 mg/kg) rather than chloropyrifos. Again, combinations of the agents proved more toxic than single exposures, and with the substitution of permethrin for chlorpyrifos, weaker inhibition of ChE was noted.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines Dirnhuber P, French MC, Green DM, Leadbeater L, Stratton JA. 1979. The protection of primates against soman poisoning by pretreatment with pyridostigmine. J Pharm Pharmacol 31(5):295–299. Doubt TJ, Roberts JR, Taylor NA, Weinberg RP, Holmes NE. 1991. Pyridostigmine and Warm Water Diving Protocol 90-05. 4. Physical Performance. Available from the National Technical Information Service. AD-A231 431–8. Edwards PM, Kuiters RR, Boer GJ, Gispen WH. 1986. Recovery from peripheral nerve transection is accelerated by local application of alpha-MSH by means of microporous Accurel polypropylene tubes. J Neurol Sci 74(2–3):171–176. Ehrlich G, Ginzberg D, Loewenstein Y, Glick D, Kerem B, Ben-Ari S, Zakut H, Soreq H. 1994. Population diversity and distinct haplotype frequencies associated with ACHE and BCHE genes of Israeli Jews from trans-Caucasian Georgia and from Europe. Genomics 22(2):288–295. Eiermann B, Sommer N, Winne D, Schumm F, Maier U, Breyer-Pfaff U. 1993. Renal clearance of pyridostigmine in myasthenic patients and volunteers under the influence of ranitidine and pirenzepine. Xenobiotica 23(11):1263–1275. Epstein Y, Arnon R, Moran D, Seidman DS, Danon Y. 1990a. Effect of pyridostigmine on the exercise–heat response of man. Eur J Appl Physiol 61(1–2):128–132. Epstein Y, Seidman DS, Moran D, Arnon R, Arad M, Varssano D. 1990b. Heat–exercise performance of pyridostigmine-treated subjects wearing chemical protective clothing. Aviat Space Environ Med 61(4):310–313. FDA (Food and Drug Administration). 1990. Informed consent for human drugs and biologics: Determination that informed consent is not feasible; Interim rule and opportunity for public comment (21 CFR Part 50). Federal Register 55:52814–52816. Feldt-Rasmussen BF, Gefke K, Mosbech H, Hanel HK. 1985. Effect of a mixture of pyridostigmine and atropine on forced expiratory volume (FEV1), and serum cholinesterase activity in normal subjects. Br J Anaesth 57(2):204–207. Fenichel GM, Dettbarn WD, Newman TM. 1974. An experimental myopathy secondary to excessive acetylcholine release. Neurology 24(1):41–45. Forster EM, Barber JA, Parker FR Jr, Whinnery JE, Burton RR, Boll P. 1994. Effect of pyridostigmine bromide on acceleration tolerance and performance. Aviat Space Environ Med 65(2):110–116. Francesconi R, Hubbard R, Mager M. 1984. Effects of pyridostigmine on ability of rats to work in the heat. J Appl Physiol 56(4):891–895. Francesconi R, Hubbard R, Matthew C, Leva N, Young J, Pease V. 1986. Oral pyridostigmine administration in rats: Effects on thermoregulation, clinical chemistry, and performance in the heat. Pharmacol Biochem Behav 25(5):1071–1075. Friedman A, Kaufer D, Shemer J, Hendler I, Soreq H, Tur-Kaspa I. 1996. Pyridostigmine brain penetration under stress enhances neuronal excitability and induces early immediate transcriptional response. Nat Med 2(12):1382–1385. Fukuda K, Nisenbaum R, Stewart G, Thompson WW, Robin L, Washko RM, Noah DL, Barrett DH, Randall B, Herwaldt BL, Mawle AC, Reeves WC. 1998. Chronic multisymptom illness affecting Air Force veterans of the Gulf War. JAMA 280(11):981–988. Gawron VJ, Schiflett SG, Miller JC, Slater T, Ball JF. 1990. Effects of pyridostigmine bromide on in-flight aircrew performance. Hum Factors 32(1):79–94. Gebbers JO, Lotscher M, Kobel W, Portmann R, Laissue JA. 1986. Acute toxicity of pyridostigmine in rats: Histological findings. Arch Toxicol 58(4):271–275.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines Ghigo E, Arvat E, Mazza E, Mondardini A, Cappa M, Muller EE, Cammani F. 1990a. Failure of pyridostigmine to increase both basal and GHRH-induced GH secretion in the night. Acta Endocrinol (Copenh) 122(1):37–40. Ghigo E, Bellone J, Imperiale E, Arvat E, Mazza E, Valetto MR, Boffano GM, Cappa M, Loche S, De Sanctis C, et al. 1990b. Pyridostigmine potentiates L-dopa- but not arginine- and galanin-induced growth hormone secretion in children. Neuroendocrinology 52(1):42–45. Ghigo E, Imperiale E, Boffano GM, Mazza E, Bellone J, Arvat E, Procopio M, Goffi S, Barreca A, Chiabotto P, et al. 1990c. A new test for the diagnosis of growth hormone deficiency due to primary pituitary impairment: Combined administration of pyridostigmine and growth hormone-releasing hormone. J Endocrinol Invest 13(4): 307–316. Ghigo E, Aimaretti G, Gianotti L, Bellone J, Arvat E, Camanni F. 1996a. New approach to the diagnosis of growth hormone deficiency in adults. Eur J Endocrinol 134(3): 352–356. Ghigo E, Bellone J, Aimaretti G, Bellone S, Loche S, Cappa M, Bartolotta E, Dammacco F, Camanni F. 1996b. Reliability of provocative tests to assess growth hormone secretory status. Study in 472 normally growing children. J Clin Endocrinol Metab 81(9):3323–3327. Giustina A, Bodini C, Bossoni S, Doga M, Girelli A, Pizzocolo G, Wehrenberg WB. 1990. Effects of calcitonin on GH response to pyridostigmine in combination with hGHRH (1–29)NH2 in normal adult subjects. Clin Endocrinol (Oxf) 33(3):375–380. Giustina A, Bossoni S, Bodini C, Doga M, Girelli A, Buffoli MG, Schettino M, Wehrenberg WB. 1991. The role of cholinergic tone in modulating the growth hormone response to growth hormone-releasing hormone in normal man. Metabolism 40(5): 519–523. Glass-Marmor L, Chen-Zion M, Beitner R. 1996. Effects of carbamylcholine and pyridostigmine on cytoskeleton-bound and cytosolic phosphofructokinase and ATP levels in different rat tissues. Gen Pharmacol 27(7):1241–1246. Glikson M, Achiron A, Ram Z, Ayalon A, Karni A, Sarova-Pinchas I, Glovinski J, Revah M. 1991. The influence of pyridostigmine administration on human neuromuscular functions—studies in healthy human subjects. Fundam Appl Toxicol 16(2):288–298. Golomb BA. 1999. A Review of the Scientific Literature as It Pertains to Gulf War Illnesses. Santa Monica, CA: RAND. Goodman LS, Gilman A, Hardman JG, Limbird LE. 1996. Goodman & Gilman’s The Pharmacological Basis of Therapeutics. 9th edition. New York: McGraw-Hill. Gordon JJ, Leadbetter L, Maidment MP. 1978. The protection of animals against organophosphate poisoning by pretreatment with a carbamate. Toxicol Appl Pharmacol 43(1):207–216. Gordon V, Schlesinger N, Engel CC Jr, Jing Z, Hyams KC, Wignall FS, Amato AA, Jackson C, McVey A, Gots RE, Schwartz SL, Hershkowitz N, Chaudhry V, Vogel RL, Kaires P, Haley RW, Kurt TL, Wolfe GI, Bryan WW, Hom J, Roland PS, Van Ness PC, Bonte FJ, Devous MD Sr, Mathews D, Fleckenstein JL, Wians FH, Landrigan PJ. 1997. Identification of Gulf War syndrome: Methodological issues and medical illnesses. JAMA 278(5):383–387. Gouge SF, Daniels DJ, Smith CE. 1994. Exacerbation of asthma after pyridostigmine during Operation Desert Storm. Mil Med 159(2):108–111. Graham C, Cook M. 1984. Effects of Pyridostigmine on Psychomotor and Visual Performance. Air Force Aerospace Medical Research Laboratory. Available from the National Technical Information Service. AD-A148 553.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines Gray GC, Knoke JD, Berg SW, Wignall FS, Barrett-Connor E. 1998. Counterpoint: Responding to suppositions and misunderstandings. Am J Epidemiol 148(4):328–333; discussion 334–338. Haley RW. 1997. Dr. Haley replies to Wegman et al.’s invited commentary on Gulf War syndrome. Am J Epidemiol 146(9):712. Haley RW, Kurt TL. 1997. Self-reported exposure to neurotoxic chemical combinations in the Gulf War. A cross-sectional epidemiologic study. JAMA 277(3):231–237. Haley RW, Hom J, Roland PS, Bryan WW, Van Ness PC, Bonte FJ, Devous MD, Mathews D, Fleckenstein JL, Wians FH Jr, Wolfe GI, Kurt TL. 1997a. Evaluation of neurologic function in Gulf War veterans: A blinded case-control study. JAMA 277(3):223–230. Haley RW, Kurt TL, Hom J. 1997b. Is there a Gulf War syndrome? Searching for syndromes by factor analysis of symptoms. JAMA 277(3):215–222. Haley RW, Billecke S, La Du BN. 1999. Association of low PON1 type Q (type A) arylesterase activity with neurologic symptom complexes in Gulf War veterans. Toxicol Appl Pharmacol 157(3):227–233. Harriman AE, Hubbard DC, Brooks RB, Woodruff RR. 1990. Effects of Pyridostigmine Bromide on A-10 Pilots During Execution of a Simulated Mission: Physiology. Available from the National Technical Information Service. AD-A221222-3. Harris GL, Maibach HI. 1989. Allergic contact dermatitis potential of 3 pyridostigmine bromide transdermal drug delivery formulations. Contact Dermatitis 21(3):189–193. Hennis PJ, Cronnelly R, Sharma M, Fisher DM, Miller RD. 1984. Metabolites of neostigmine and pyridostigmine do not contribute to antagonism of neuromuscular blockade in the dog. Anesthesiology 61(5):534–539. Hoffman WE, Kramer J, Main AR, Torres JL. 1989. Clinical enzymology. In: Loeb WF, Quinby FW, eds. The Clinical Chemistry of Laboratory Animals, 1st edition. New York: Pergamon Press. Pp. 237–278. Hudson CS, Foster RE, Kahng MW. 1985. Neuromuscular toxicity of pyridostigmine bromide in the diaphragm, extensor digitorum longus, and soleus muscles of the rat. Fundam Appl Toxicol 5(6 part 2):S260–S269. Hudson CS, Foster RE, Kahng MW. 1986. Ultrastructural effects of pyridostigmine on neuromuscular junctions in rat diaphragm. Neurotoxicology 7(1):167–185. Hughes S, Smith ME, Simpson MG, Allen SL. 1992. Effect of IDPN on the expression of POMC-derived peptides in rat motoneurones. Peptides 13(5):1021–1023. Hughes S, Child T, Simpson MG, Smith ME, Ferry CB. 1995. Upregulation of the proopiomelanocortin (POMC) gene in motoneurones after acrylamide administration in mice. Human Exp Toxicol 14:374. Humbert R, Adler DA, Disteche CM, Hassett C, Omiecinski CJ, Furlong CE. 1993. The molecular basis of the human serum paraoxonase activity polymorphism. Nat Genet 3(1):73–76. Hussain AS, Ritschel WA. 1988. Influence of dimethylacetamide, N-N-diethyl-m-toluamide and 1-odecylazacycloheptan-2-one on ex vivo permeation of phosphonoformic acid through rat skin. Methods Find Exp Clin Pharmacol 10(11):691–694. IOM (Institute of Medicine). 1995. Health Consequences of Service During the Persian Gulf War: Initial Findings and Recommendations for Immediate Action. Washington, DC: National Academy Press. Iwasaki Y, Kinoshita M, Ikeda K, Takamiya K, Shiojima T. 1990. Cognitive dysfunction in myasthenia gravis. Intern J Neuroscience 54:29–33.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines Izraeli S, Avgar D, Almog S, Shochat I, Tochner Z, Tamir A, Ribak J. 1990. The effect of repeated doses of 30 mg pyridostigmine bromide on pilot performance in an A-4 flight simulator. Aviat Space Environ Med 61(5):430–432. Izraeli S, Alcalay M, Benjamini Y, Wallach-Kapon R, Tochner Z, Akselrod S. 1991. Modulation of the dose-dependent effects of atropine by low-dose pyridostigmine: Quantification by spectral analysis of heart rate fluctuations in healthy human beings. Pharmacol Biochem Behav 39(3):613–617. Joiner R, Kluwe W. 1988. Multiple Animal Studies for Medical Chemical Defense Program in Soldier/Patient Decontamination and Drug Development. Task 85-18: Conduct of Pralidoxime Chloride, Atropine in Citrate Buffer and Pyridostigmine Bromide Pharmacokinetic Studies, and Comparative Evaluation of the Efficacy of Pyridostigmine Plus Atropine. Available from the National Technical Information Service. ADB127309. Kato T, Sugiyama S, Hanaki Y, Fukushima A, Akiyama N, Ito T, Ozawa T. 1989. Role of acetylcholine in pyridostigmine-induced myocardial injury: Possible involvement of parasympathetic nervous system in the genesis of cardiomyopathy. Arch Toxicol 63(2):137–143. Kaufer D, Friedman A, Soreq H. 1999. The vicious circle of stress and anticholinesterase responses. Neuroscientist 5(3):173–183. Kay CD, Morrison JD. 1988. The effects of ingestion of 60 mg pyridostigmine bromide on contrast sensitivity in man. Hum Toxicol 7(4):347–352. Keeler JR. 1990. Interactions between nerve agent pretreatment and drugs commonly used in combat anesthesia. Mil Med 155(11):527–533. Keeler JR, Hurst CG, Dunn MA. 1991. Pyridostigmine used as a nerve agent pretreatment under wartime conditions. JAMA 266(5):693–695. Kluwe WM, Page JG, Toft JD, Ridder WE, Chung H. 1990. Pharmacological and toxicological evaluation of orally administered pyridostigmine in dogs. Fundam Appl Toxicol 14(1):40–53. Lallement G, Foquin A, Baubichon D, Burckhart M-F, Carpentier P, Canini F. 1998. Heat stress, even extreme, does not induce penetration of pyridostigmine into the brain of guinea pigs. Neurotoxicology 19(6):759–766. Landrigan PJ. 1997. Illness in Gulf War veterans. Causes and consequences. JAMA 277(3):259–261. Leon FE, Pradilla G, Vesga E. 1996. Neurological effects of organophosphate pesticides. BMJ 313(7058):690–691. Levine BS, Parker RM. 1991. Reproductive and developmental toxicity studies of pyridostigmine bromide in rats. Toxicology 69(3):291–300. Levine BS, Waller DP, Long R, Parker R, Denny K, Chung H. 1989. Subchronic and reproductive toxicity studies on pyridostigmine bromide. J Am Coll Toxicol 8(6): 1209. Levine BS, Long R, Chung H. 1991. Subchronic oral toxicity of pyridostigmine bromide in rats. Biomed Environ Sci 4(3):283–289. Lintern MC, Smith ME, Ferry CB. 1997a. Effects of pyridostigmine on acetylcholinesterase in different muscles of the mouse. Hum Exp Toxicol 16(1):18–24. Lintern MC, Smith ME, Ferry CB. 1997b. Effect of repeated treatment with pyridostigmine on acetylcholinesterase in mouse muscles. Hum Exp Toxicol 16(3):158–165. Liu WF. 1991. Cholinolytic antagonism to the disruptive effects of oral low doses of pyridostigmine on simple discrimination performance in rats. Pharmacol Biochem Behav 40(4):745–749.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines Loewenstein-Lichtenstein Y, Schwarz M, Glick D, Norgaard-Pedersen B, Zakut H, Soreq H. 1995. Genetic predisposition to adverse consequences of anti-cholinesterases in “atypical” BCHE carriers. Nat Med 1(10):1082–1085. Lotti M, Moretto A. 1995. Cholinergic symptoms and Gulf War syndrome. Nat Med 1(12):1225–1226. Madsen JM. 1998. Clinical Considerations in the Use of Pyridostigmine Bromide as Pretreatment for Nerve-Agent Exposure. Aberdeen Proving Ground, MD: Army Medical Research Institute of Chemical Defense. Available from the National Technical Information Service. NTIS/AD-A353931. Maelicke A, Coban T, Schrattenholz A, Schroder B, Reinhardt-Maelicke S, Storch A, Godovac-Zimmermann J, Methfessel C, Pereira EF, Albuquerque EX. 1993. Physostigmine and neuromuscular transmission. Ann NY Acad Sci 681:140–154. Mandel ID, Katz R, Zengo A, Kutscher AH, Greenberg RA, Katz S, Scharf R, Pintoff A. 1967. The effect of pharmacologic agents on salivary secretion and composition in man. I. Pilocarpine, atropine and anticholinesterases. J Oral Ther Pharmacol 4(3): 192–199. Marino MT, Schuster BG, Brueckner RP, Lin E, Kaminskis A, Lasseter KC. 1998. Population pharmacokinetics and pharmacodynamics of pyridostigmine bromide for prophylaxis against nerve agents in humans. J Clin Pharmacol 38(3):227–235. Maselli RA, Leung C. 1993a. Analysis of anticholinesterase-induced neuromuscular transmission failure. Muscle Nerve 16(5):548–553. Maselli RA, Leung C. 1993b. Analysis of neuromuscular transmission failure induced by anticholinesterases. Ann NY Acad Sci 681:402–404. Massara F, Ghigo E, Demislis K, Tangolo D, Mazza E, Locatelli V, Muller EE, Molinatti GM, Camanni F. 1986. Cholinergic involvement in the growth hormone releasing hormone-induced growth hormone release: Studies in normal and acromegalic subjects. Neuroendocrinology 43(6):670–675. Matthew CB, Hubbard RW, Francesconi RP, Thomas GJ. 1988. Carbamates, atropine, and diazepam: Effects on performance in the running rat. Life Sci 42(20):1925–1931 . Matthew CB, Francesconi RP, Bowers WD, Hubbard RW. 1990. Chronic vs. acute carbamate administration in exercising rats. Life Sci 47(4):335–343. Matthew CB, Glenn JF, Bowers WD Jr, Navara DK. 1994. Cholinergic drug interactions and heat tolerance. Life Sci 54(17):1237–1245. Matthew CB, Bowers WD, Sils IV, Francesconi RP. 1998. Thermoregulatory, Endurance and Ultrastructural Effects of Acute and Subchronic Pyridostigmine Bromide Administration in the Exercising Rat. Available from the National Technical Information Service. AD-A339 025-9. McCain WC, Lee R, Johnson MS, Whaley JE, Ferguson JW, Beall P, Leach G. 1997. Acute oral toxicity study of pyridostigmine bromide, permethrin, and DEET in the laboratory rat. J Toxicol Environ Health 50(2):113–124. McGuire MC, Nogueira CP, Bartels CF, Lightstone H, Hajra A, Van der Spek AF, Lockridge O, La Du BN. 1989. Identification of the structural mutation responsible for the dibucaine-resistant (atypical) variant form of human serum cholinesterase. Proc Natl Acad Sci 86(3):953–957. McMaster SB, Finger AV. 1989. Effects of exercise on behavioral sensitivity to carbamate cholinesterase inhibitors. Pharmacol Biochem Behav 33(4):811–813. Molloy DW, Cape RD. 1989. Acute effects of oral pyridostigmine on memory and cognitive function in SDAT. Neurobiol Aging 10(2):199–204.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines Morgan EW, Zaucha GM, Waring PP, LeTellier Y, Seewald JB. 1990. One Hundred Eighty Day Subchronic Oral Toxicity Study of Pyridostigmine Bromide in Rats. Volume 1. Available from the National Technical Information Service. ADA224450. Murialdo G, Zerbi F, Filippi U, Tosca P, Fonzi S, Di Paolo E, Costelli P, Porro S, Polleri A, Savoldi F. 1991. Cholinergic modulation of growth hormone-releasing hormone effects on growth hormone secretion in dementia. Neuropsychobiology 24(3):129–134. Murialdo G, Fonzi S, Torre F, Costelli P, Solinas G, Tosca P, Di Paolo E, Porro S, Zerbi F, Polleri A. 1993. Effects of pyridostigmine, corticotropin-releasing hormone and growth hormone-releasing hormone on the pituitary–adrenal axis and on growth hormone secretion in dementia. Neuropsychobiology 28(4):177–183. Murphy MR, Blick DW, Brown GC. 1989. Effects of Hazardous Environments on Animal Performance. USAF School of Aerospace Medical Technical Report 88–40. Mutch E, Blain PG, Williams FM. 1992. Interindividual variations in enzymes controlling organophosphate toxicity in man. Hum Exp Toxicol 11(2):109–116. Neish SR, Carter B. 1991. More on Desert Storm. JAMA 266(23):3282–3283. Neville LF, Gnatt A, Loewenstein Y, Seidman S, Ehrlich G, Soreq H. 1992. Intramolecular relationships in cholinesterases revealed by oocyte expression of site-directed and natural variants of human BCHE. EMBO J 11(4):1641–1649. Nobrega AC, Carvalho AC, Bastos BG. 1996. Resting and reflex heart rate responses during cholinergic stimulation with pyridostigmine in humans. Braz J Med Biol Res 29(11):1461–1465. O’Keane V, O’Flynn K, Lucey J, Dinan TG. 1992. Pyridostigmine-induced growth hormone responses in healthy and depressed subjects: Evidence for cholinergic supersensitivity in depression. Psychol Med 22(1):55–60. O’Keane V, Abel K, Murray RM. 1994. Growth hormone responses to pyridostigmine in schizophrenia: Evidence for cholinergic dysfunction. Biol Psychiatry 36(9):582–588. Oigaard A. 1975. The motor-stimulating effect of metoclopramide and pyridostigmine bromide in normal man and laparotomized patients. A combined study of duodenal electric and motor activity. Scand J Gastroenterol 10(1):65–71. Osserman K, Kornfeld P, Cohen E, Genkins G, Mendelow H, Goldberg H, Windsley H, Kaplan L. 1958. Studies in myasthenia gravis: Review of two hundred eighty-two cases at the Mount Sinai Hospital, New York City. Arch Intern Med 102:72–81. Owens WD, Waldbaum LS, Stephen CR. 1978. Cardiac dysrhythmia following reversal of neuromuscular blocking agents in geriatric patients. Anesth Analg 57(2):186–190. PAC (Presidential Advisory Committee on Gulf War Veterans’ Illnesses). 1996. Presidential Advisory Committee on Gulf War Veterans’ Illnesses: Final Report. Washington, DC: U.S. Government Printing Office. PAC (Presidential Advisory Committee on Gulf War Veterans’ Illnesses). 1997. Presidential Advisory Committee on Gulf War Veterans’ Illnesses: Special Report. Washington, DC: U.S. Government Printing Office. Penalva A, Carballo A, Pombo M, Casanueva FF, Dieguez C. 1993. Effect of growth hormone (GH)-releasing hormone (GHRH), atropine, pyridostigmine, or hypoglycemia on GHRP-6-induced GH secretion in man. J Clin Endocrinol Metab 76(1): 168–171. Physicians’ Desk Reference. 2000. 54th edition. Montvale, NJ: Medical Economics Company. Prusaczyk WK, Sawka MN. 1991. Effects of pyridostigmine bromide on human thermoregulation during cold water immersion. J Appl Physiol 71(2):432–437.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines Ram Z, Molcho M, Danon YL, Almog S, Baniel J, Karni A, Shemer J. 1991. The effect of pyridostigmine on respiratory function in healthy and asthmatic volunteers. Isr J Med Sci 27(11–12):664–668. Rettig RA. 1999. Military Use of Drugs Not Yet Approved by the FDA for CW/BW Defense. Santa Monica, CA: RAND. Ringqvist I, Ringqvist T. 1971. Changes in respiratory mechanics in myasthenia gravis with therapy. Acta Med Scand 190:509–518. Roberts DE, Sawka MN, Young AJ, Freund BJ. 1994. Pyridostigmine bromide does not alter thermoregulation during exercise in cold air. Can J Physiol Pharmacol 72(7): 788–793. Ross RJ, Tsagarakis S, Grossman A, Nhagafoong L, Touzel RJ, Rees LH, Besser GM. 1987. GH feedback occurs through modulation of hypothalamic somatostatin under cholinergic control: Studies with pyridostigmine and GHRH. Clin Endocrinol (Oxf) 27(6):727–733. Rostker B. 1998. Letter to Arlen Specter, Chairman Committee on Veterans’ Affairs, U.S. Senate, January 30. Sarno AP, Neish SR, Carter B, Keeler JR, Hurst CG, Dunn MA. 1991. More on Desert Storm (side effects of anti-nerve gas agent pyridostigmine bromide, possibility of teratogenicity). JAMA 266(23):3282–3283. Sawka MN, Young AJ, Freund BJ, Roberts DE. 1994. Pyridostigmine bromide does not alter thermoregulation during exercise in cold air. Med Sci Sports Exerc 26 (5 Suppl):S3. Schwab RS, Timberlake WH. 1954. Pyridostigmin (mestinon) in the treatment of myasthenia gravis. N Engl J Med 251(7):271–272. Schwab R, Osserman K, Tether J. 1957. Prolonged action with multiple-dose tablets of neostigmine bromide and mestinon bromide. JAMA 165(6):671–674. Schwarz H. 1956. Mestinon (pyridostigmine bromide) in myasthenia gravis. Can Med Assoc J 75:98–100. Seidman DS, Epstein Y. 1989. Thermoregulation in man under pyridostigmine induced cholinesterase inhibition. Thermal Physiology. Proceedings of the International Symposium on Thermal Physiology. Pp. 273–277. Selim S, Hartnagel RE Jr, Osimitz TG, Gabriel KL, Schoenig GP. 1995. Absorption, metabolism, and excretion of N,N-diethyl-m-toluamide following dermal application to human volunteers. Fundam Appl Toxicol 25(1):95–100. Servatius R, Ottenweller JE, Beldowicz D, Guo W, Zhu G, Natelson BH. 1998. Persistently exaggerated startle responses in rats treated with pyridostigmine bromide. J Pharmacol Exp Ther 287(3):1020–1028. Shale DJ, Lane DJ, Davis CJ. 1983. Air-flow limitation in myasthenia gravis. The effect of acetylcholinesterase inhibitor therapy on air-flow limitation. Am Rev Respir Dis 128(4):618–621. Sharma HS, Cervos-Navarro J, Dey PK. 1991. Increased blood–brain barrier permeability following acute short-term swimming exercise in conscious normotensive young rats. Neurosci Res 10(3):211–221. Sharma HS, Nyberg F, Cervos-Navarro J, Dey PK. 1992. Histamine modulates heat stress-induced changes in blood–brain barrier permeability, cerebral blood flow, brain oedema and serotonin levels: An experimental study in conscious young rats. Neuroscience 50(2):445–454. Shen ZX. 1998. Pyridostigmine bromide and Gulf War syndrome. Med Hypotheses 51(3):235–237.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines Xia DY, Wang LX, Pei SQ. 1981. The inhibition and protection of cholinesterase by physostigmine and pyridostigmine against soman poisoning in vivo. Fundam Appl Toxicol 1(2):217–221. Yamamoto K, Shimizu M, Ohtani H, Hayashi M, Sawada Y, Iga T. 1996. Toxicodynamic analysis of cardiac effects induced by four cholinesterase inhibitors in rats. J Pharm Pharmacol 48(9):935–939. Yang I, Woo J, Kim S, Kim J, Kim Y, Choi Y. 1995. Combined pyridostigmine–thyrotrophin-releasing hormone test for the evaluation of hypothalamic somatostatinergic activity in healthy normal men. Eur J Endocrinol 133(4):457–462.

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Gulf War and Health: Volume 1. Depleted Uranium, Sarin, Pyridostigmine Bromide, Vaccines CONTENTS      ISSUES IN IDENTIFYING ADVERSE EFFECTS   268      Surveillance,   269      Difficulties in Detecting Adverse Events Due to Vaccination in Humans,   270      Difficulties in Detecting Adverse Events Due to Vaccination in Animals,   271      ANTHRAX VACCINE   272      Toxicology,   275      Human Studies,   280      BOTULINUM TOXOID   287      Toxicology,   289      Human Studies,   291      MULTIPLE VACCINATIONS   294      Toxicology,   296      Human Studies,   299      SQUALENE   307      Dietary Intake, Absorption, Distribution, and Metabolism,   307      Animal Studies,   308      Use of Squalene as a Vaccine Adjuvant,   309      Gulf War Issues,   311      Future Research Directions Regarding Squalene,   312      CONCLUSIONS   312      Anthrax Vaccine,   312      Botulinum Toxoid,   313      Multiple Vaccinations,   314      REFERENCES   314