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
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-
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.
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.
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
PB: higher doses caused proportionally greater inhibition (0.57 mg/kg yielded 43 percent inhibition).
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.
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
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-
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.
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
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.
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).
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,
creatine phosphokinase, and aspartate aminotransferase, these indices of myopathy (Hoffman et al., 1989) were unaccompanied by morphological evidence of PB-induced toxicity.
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.
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).
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).
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.
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
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).
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.
The authors speculated that the enhanced toxicity may result from interference with detoxification processes and raised the possibility of polymorphisms in esterases that play a determining role in toxic outcomes. Similarly, combinations of very large doses of PB, DEET and permethrin have been reported to increase the lethality of these agents in rats (McCain et al., 1997). The use of such large dosage levels in these studies makes interpretation of possible toxic synergisms problematic. A number of hypothetical mechanisms of interactions have been offered (Golomb, 1999).
There is no a priori reason to suspect that PB is capable of causing OPIDN. PB is not known to inhibit NTE, a hallmark of chemicals causing OPIDN. Although PB is known to inhibit butyrylcholinesterase (BuChE), it does not appear to be a substrate for paraoxonase;5 hence it seems biologically implausible that there is a genetic susceptibility for OPIDN to be induced by PB via this mechanism. Further, there are insufficient data to determine whether exposure to other chemicals, either before or after PB, enhances its potential to produce delayed neurotoxicity.
There are reports of other classes of chemicals interacting with PB to modify its toxicity. Chaney et al. (1997, 1998) noted that adrenergic agents varied in their ability to potentiate the toxicity of PB without apparent structure–activity relationships. The toxicity of PB was enhanced by certain beta-receptor agonists, and also by some alpha-receptor antagonists, whereas other adrenergic agents were without effect. Catecholamines (epinephrine and norepinephrine) were reported to be additive with PB toxicity. Both potentiation and addition were blocked by atropine, clearly pointing to the cholinergic link in this complex toxicity. In another study, the same authors (Chaney et al., 1999) noted that a toxic interaction of PB and DEET resulted in seizures, which—although resistant to conventional anticonvulsant drugs—were blocked by muscarinic antagonists such as atropine.
Keeler (1990) reviewed possible interactions between PB and drugs used in combat anesthesia and predicted the greatest potential for drug interaction to be with the neuromuscular blocking drugs. The author also recognized potential interactions leading to overstimulation of muscarinic receptors and, hence, to unwanted effects such as laryngospasm and bradycardia.
Available studies in which the toxicity of PB, alone or in combination with other chemicals, has been assessed in laboratory animals have been reviewed. Although data derived from laboratory investigations may have general applicability to humans, there are enough differences in toxic responses to the ChE inhibitors as a class to caution against making direct inferences to humans. For instance, although chickens are reported to be highly sensitive to OPIDN, this disorder is difficult to induce in laboratory rats, except at extremely high doses of organophosphates. Thus, although some general principles from laboratory
studies may be useful in determining human toxic responses, it is unsound to assume that toxic outcomes will be consistent across species.
Buchholz and colleagues (1997) examined the influence of PB on the CNS uptake of permethrin, at doses and durations relevant to human exposure, and found that oral PB actually reduced the amount of permethrin reaching the central nervous system. Such data, although limited, do not support a role for either decreased metabolism or enhanced penetration into the brain as the basis for enhanced toxicity. Little information regarding the influence of PB on absorption of other agents, or vice versa, is available; however, DEET has been used as a transdermal carrier for drug delivery (Hussain and Ritschel, 1988).
Interference with Metabolic Disposition
Simultaneous exposure to multiple therapeutic agents has repeatedly demonstrated the capacity for one drug to interfere with the metabolism of a second agent. This interaction may facilitate or impair biotransformation, the outcome of which is to diminish or exaggerate the pharmacological or toxicological actions of the chemical. Further, both pyrethroids and carbamates are known to rely to some extent on the cytochrome P-450 system for their metabolism, and possibilities exist for interactions to have toxic outcomes (Casida et al., 1983; Eiermann et al., 1993; Selim et al., 1995). However, direct experimental evidence for such interactions is lacking.
Penetration into the Brain
The brain is protected from many classes of xenobiotics by the blood– brain barrier (BBB), a layer of endothelial cells that prevents the movement of many chemicals from the circulation into the brain. Since the nervous system in the periphery is not afforded similar protection, PB would be expected to manifest its pharmacological and toxicological actions primarily in the peripheral nervous system.
The BBB is a specialized structure responsible for the maintenance of the central neuronal microenvironment, playing a pivotal role in tissue homeostasis, fibrinolysis and coagulation, vasotonus regulation, the vascularization of normal and neoplastic tissues, and blood cell activation and migration during physiological and pathological processes. Regulation of blood–tissue exchange is accomplished by individual endothelial cells. A pivotal function of endothelial cells is to regulate the selective transport and metabolism of substances from the blood to the brain. Because of the existence of tight junctions between adjacent endothelial cells, nonspecific paracellular ionic leakage across the BBB is thought to be minimal. It should be pointed out, however, that not all of the CNS
vasculature conforms to the above morphological description. Structural attributes of endothelial cells in nonbarrier areas (i.e., areas that lack the BBB) have been examined most systematically in the circumventricular organs. In contrast to the zonulae occludentes junctions of tight barrier areas, endothelial cells in the circumventricular organs exhibit maculae occludentes junctions, which only partially occlude the gaps between adjacent endothelial cells. Hence, the barrier is not as tight, and diffusion across the capillaries is more prevalent.
From the toxicological point of view, areas that lack a true BBB represent potential sites for the accumulation of neurotoxins, because their passage into the brain parenchyma is likely to be less restrictive. Circumventricular organs that lack the proper BBB are midline structures bordering the third and fourth ventricles. They include the pineal gland, median eminence, subfornical organ, area postrema, subcommissural organ, and organum vasculosum of the lamina terminalis (Aschner, 1998).
Friedman and colleagues (1996) raised the possibility that PB, combined with stress, may have enhanced potential to penetrate the BBB acting via a cholinergic mechanism. Experiments were conducted in which mice were stressed by forcing them to swim, a procedure reported to cause opening of the BBB (Sharma et al., 1991). Penetration of both Evans blue and AChE plasmid DNA including the cytomegalovirus promoter in the brain were increased tenfold in stressed mice, confirming that the procedure effectively produces breaches in the BBB. In stressed mice, the dose of PB required to obtain equivalent inhibition of brain AChE was reduced a hundredfold, suggesting enhanced penetration of PB. Doses of PB up to 1.0 mg/kg did not result in significant inhibition of brain AChE. The enhanced cholinergic stimulation resulting from the now-greater inhibition of ChE induced a cascade of c-Fos-mediated transcriptional responses. Importantly, similar increases in c-Fos mRNA could be elicited within minutes of administration of 2 mg/kg of PB alone.
Because PB is a positively charged carbamate, it is unlikely to gain access to the central nervous system. The observation that the acute symptoms associated with the use of PB are referable to actions on the peripheral nervous system supports this notion. However, the biologic plausibility of some degree of PB penetration into the CNS is suggested by reports of positive actions on behavioral measurements, as well as hypothalamic actions of PB modifying temperature regulation and release of growth hormone (see later discussion). The studies by Friedman and colleagues (1996) are significant in that they provide at least a hypothetical basis for enhanced brain penetration of PB. Although important, the interpretation of these findings must await successful replication and confirmation.
Anesthesiologists have long recognized that butyrylcholinesterase exists in more than one variant. A subpopulation of individuals given the neuromuscular-
blocking drug succinylcholine, which relies heavily on BuChE for its hydrolysis, exhibit unexpectedly prolonged paralysis of the respiratory muscles (succinylcholine apnea) because they possess an atypical BuChE. Individuals with atypical BuChE (i.e., aspartate rather than glycine at position 70) (McGuire et al., 1989) are incapable of metabolizing succinylcholine and are also less sensitive to some inhibitors (Neville et al., 1992). Atypical variants of BuChE are encountered in less than 5 percent of the general population, although this may vary in specific subpopulations (Ehrlich et al., 1994).
The possibility has been raised that individuals with atypical BuChE may be more susceptibile to PB toxicity (Loewenstein-Lichtenstein et al., 1995). This suggestion was based on observations of a single patient, homozygous for atypical BuChE, who manifested severe symptoms of toxicity during and immediately following the administration of PB. Since the serum BuChE in this individual was much less susceptible to inhibition by PB (and other carbamate ChE inhibitors), it was suggested that the decreased “buffering capacity” of atypical BuChE would lead to an abnormal accumulation of PB and signs of toxicity.
Hypothetically, the inhibition of plasma esterases by agents such as PB could also represent a loss of capacity to “scavenge” (hydrolyze) other xenobiotics that are esters, leading to their accumulation and toxic consequences (Loewenstein-Lichtenstein et al., 1995; Abou-Donia et al., 1996a,b; Shen, 1998). The possible importance of a scavenger role for BuChE in PB toxicity is suggested by the observation that Wistar rats, which constitutionally have lower levels of BuChE than the Sprague-Dawley strain, also exhibit more exaggerated acoustic startle responses following exposure to PB (Servatius et al., 1998). However, Lotti and Moretto (1995) question the importance of a scavenger role for BuChE as a major contributor to toxicity, noting that even the doses of PB employed in the treatment of myasthenic patients produce little inhibition of BuChE, and further argue that it would be unlikely to play a role in illnesses in Gulf War veterans. Examination of PB-exposed individuals who served in the Gulf War and reportedly are suffering from neurological problems failed to detect any differences between their BuChE activities and those of controls (Haley and Kurt, 1997).
Paraoxonase/Arylesterase 1 (PON1)
Polymorphisms are also known to occur in the PON1 gene. Three genotypes (Q, QR, and R) influence the catalytic activity of two alloenzymes, which—acting as paraoxonases/arylesterases—are capable of hydrolyzing organophosphates at different rates and with differing substrate specificities (Adkins et al., 1993; Humbert et al., 1993; Davies et al., 1996). PON1 activity is known to show considerable variation in humans (Mutch et al., 1992). Haley and colleagues (1999) have recently suggested a relationship between polymorphisms and neurological impairment in Gulf War veterans. The authors identified a study population of veterans with symptom complexes (Haley et al., 1997a,b) and characterized the alleles for both PON1 and BuChE in these individuals (Haley and Kurt, 1997). Among ill veterans, there was a greater tendency to
possess the R allele than in controls, and although the arylesterase activity was somewhat lower, the total paraoxonase activity was higher. Since PON1 does not appear to be involved in the metabolism of PB (Haley et al., 1999) the relationship between this polymorphism and PB toxicity is unclear. The authors suggest that PB, having inhibited BuChE, leaves individuals only PON1 as a defense against organophosphates and that this last line of defense is deficient in genetically predisposed individuals. Although this is an intriguing possibility, direct experimental evidence for a contributory role of polymorphisms in the combined toxicity of PB and organophosphates is lacking and requires additional investigation.
This section reviews what is known about the use of PB and potential adverse health outcomes from the literature on patients (clinical studies), healthy volunteers, and epidemiologic studies. In some cases, the patient or healthy-volunteer studies include populations of veterans, as do the epidemiologic studies reviewed by the committee. Several of the studies review general health outcomes, whereas others focus on specific organ systems.
There are a large number of clinical studies, principally related to the use of PB as a test of hypothalamic pituitary function or growth hormone response and in the treatment of myasthenia gravis. In addition, a smaller number of clinical studies (i.e., case reports and case series) are available that describe the effects of PB when used in patients. These studies are discussed below.
Clinical Studies of PB and Growth Hormone
Studies have been done on normal subjects and patients with a variety of chronic disorders who were given PB as a test of hypothalamic pituitary function, usually of growth hormone (GH) response to PB and one or more other GH-releasing stimuli. Typically, these have been acute studies using relatively low doses of PB, which offer the opportunity to investigate not only the pituitary responses to, but also the adverse effects of, small doses of PB in humans.
Insight into the acute hormonal effects of PB is available from an abundant literature that describes its use as a clinical test of pituitary GH reserve. Normally, the synthesis and secretion of growth hormone are regulated by two hypothalamic peptides: GH-releasing hormone (GHRH), which has a stimulating role, and somatostatin, which has an inhibitory role. In normal humans, GHRH and its analogues stimulate GH secretion in a dose-dependent fashion (Giustina et al., 1990; Cordido et al., 1993, 1995; Penalva et al., 1993; Arvat et al., 1995, 1997a,b), but its release is substantially modulated by cholinergic neurotrans-
mission (Ross et al., 1987; Ghigo et al., 1990a,b; Giustina et al., 1991; Bellone et al., 1992). Anticholinergic drugs abolish the GH response to certain physiological and pharmacological stimuli (Massara et al., 1986; Casanueva et al., 1990), while PB, an AChE inhibitor, stimulates GH secretion when administered alone (Ghigo et al., 1990b) and enhances the GH response when administered with GHRH or arginine (Giustina et al., 1990).
Mediated by a cholinergically provoked decrease in the hypothalamic release of somatostatin, PB substantially augments the dose–response relationship of GHRH to GH secretion in normal subjects and patients with a variety of pathological disorders (Giustina et al., 1990; Ghigo et al., 1990a, 1996a,b). These physiological effects of PB have been studied extensively in normal volunteers, and the drug has been widely used to test the hypothalamic–pituitary responses, particularly those of GH, of patients with many different disorders. In fact, combining GHRH with PB or arginine—substances that inhibit hypothalamic somatostatin release—is the most powerful means of testing the secretory capacity of human pituitary somatotrope cells (the GH response is always greater than 19 μg/L), which accounts for its wide clinical use (Ghigo et al., 1996b). Accordingly, there is considerable information about the acute administration of PB given as a diagnostic test of hypothalamic–pituitary function in normal volunteers and patients.
As a diagnostic test, PB is generally administered as a single 30–180-mg dose, usually with GHRH, which sometimes produces acute transient symptoms. Among several thousand patients, reported in numerous studies (e.g., Ross et al., 1987; Ghigo et al., 1990a,b,c, 1996a,b; Giustina et al., 1990, 1991; Murialdo et al., 1991,1993; Bellone et al., 1992; O’Keane et al., 1992, 1994; Cordido et al., 1995; Yang et al., 1995; Arvat et al., 1997a,b; Coiro et al., 1998) to whom PB was administered as a diagnostic test, the most common symptoms were muscarinic; abdominal or muscular symptoms usually appeared within 1 or 2 hours of ingesting PB and typically lasted 1–2 hours. Symptoms were monitored for several hours in most studies, and none reported long-term follow-up of symptoms. Abdominal symptoms were described as cramps, increased digestive sounds (due to the movement of gas in the intestines), pain, diarrhea, and nausea, and the principal nicotinic cholinergic symptoms were skeletal muscle and tongue fasciculations (sometimes accompanied by dysarthria).
No central nervous symptoms were reported following PB ingestion, which is particularly noteworthy since it was given to patients with mania (Dinan et al., 1994), depression (O’Keane et al., 1992; Thakore and Dinan, 1995; Coiro et al., 1998), dementia (Murialdo et al., 1991, 1993), schizophrenia (O’Keane et al., 1994), and alcoholism (Coiro and Vescovi, 1997).
A clear dose–response effect on symptoms was not apparent from a review of these studies, although none of the studies were designed to test this effect. However, there was a trend toward greater reporting of symptoms at higher PB dose ranges among subjects given several different doses in the same study. This point is particularly well documented in the results of a study by Yang and colleagues (1995) in which side effects were graded from 1 (mild) to 3 (severe): a
score of 1 in 17 percent (1/6) of subjects given 60 mg of PB; a score of 1 or 2 in 83 percent (5/6) of subjects given 120 mg of PB; and a score of 2 or 3 in 100 percent (6/6) of subjects given 180 mg of PB.
There was general agreement among authors of studies reviewed on several issues: PB is safe and effective when used as a diagnostic test to augment GH responses to GHRH; the test is perhaps the best means of identifying GH deficiency in adults and children; and PB is safe to use in patients with a wide variety of physical and mental disorders. Symptoms were always noted to be brief and tolerable, without requiring medical intervention, and by implication did not require long-term follow-up.
Thus, the main strength of these studies is the careful documentation of acute hormonal responses to PB in normal volunteers of all ages, including children, and in a variety of disease states. Although there is no evidence that PB causes long-term problems when used as a diagnostic test, this point has not been studied. The available studies uniformly show a relatively mild side-effect profile for PB; however, a major weakness is that none were specifically designed to test for adverse effects of PB and many simply ignored this aspect of the drug. Moreover, all of the observations were done over several hours, although several studies administered PB to normal volunteers or patients daily for 2 to 3 days. Nonetheless it is clear that patients with a variety of disease states—many causing severe stress such as thyrotoxicosis or Cushing’s disease—had relatively mild acute and transient side effects from PB, and no study reported major clinical problems or obvious acute CNS symptoms after PB administration.
Clinical Studies of PB and Myasthenia Gravis
Myasthenia gravis is an autoimmune disorder characterized by antibody blockade of the ACh receptor of the neuromuscular junction. This immune disturbance results in impaired transmission across the neuromuscular junction and fluctuating weakness in patients with the disease. Myasthenia gravis can affect individuals of all ages with variable and unpredictable severity.
Patients with mild cases may exhibit symptoms of blurred or double vision, while patients with more severe cases may exhibit generalized paralysis and respiratory failure. Some patients with myasthenia gravis also have been shown to have subtle changes in cognitive function related to ACh antibodies binding to ACh receptors in the central nervous system (Iwasaki et al., 1990). These changes may be reversed by treatments for myasthenia gravis, including plasmapheresis (Iwasaki et al., 1990), or with anticholinesterases (e.g., physostigmine) that easily cross the blood–brain barrier and enhance ACh activity (Tucker et al., 1998). Thus, the possible reversal of cognitive dysfunction with anticholinesterases in myasthenics may be an argument against adverse effects of PB on cognition in normal individuals unless there is a difference in the CNS effects of PB in these two populations.
PB (Mestinon) is a prostigmine analogue that has been used since the 1950s to control the myasthenic phenomenon without any major side effects reported (Schwarz, 1956). The usual oral daily dose prescribed to control muscle weakness in myasthenic patients ranges from 120 to 600 mg (Aquilonius et al., 1983), although oral doses may vary from 60 to 1,500 mg per day (Breyer-Pfaff et al., 1990). Despite a short half-life, the pre-dose plasma concentration is relatively stable (Aquilonius et al., 1983). Blood levels of PB do not correlate with the degree of clinical toxicity observed, although they are somewhat predictive of cholinergic crisis in myasthenia gravis patients (Breyer-Pfaff et al., 1990).
In a large series of myasthenic patients followed for 5 years, 34 percent of those receiving PB had one or more, mostly mild, side effects (Beekman et al., 1997). The most common effects were gastrointestinal (30 percent); infrequent effects were hypersalivation (6 percent), increased perspiration (4 percent), urinary urgency (3 percent), increased bronchial secretion (2 percent), rash (1 percent), and blurred vision (1 percent). Only 1 percent of the patients had to stop the drug because of stomach complaints.
A number of studies dating back to the 1950s consistently have shown PB to be safe and effective in the treatment of myasthenia gravis (Schwab and Timberlake, 1954; Schwab et al., 1957; Osserman et al., 1958). PB provides short-term benefit and is still used in the treatment of myasthenia gravis, despite the introduction of surgery, immunotherapy, and intravenous gamma globulin as therapeutic modalities.
Oral doses of 180 mg per day of PB are also used to treat the generalized fatigue and pain of patients with postpolio syndrome. Even though the drug is ineffective in improving the symptoms associated with postpolio syndrome, patients tolerate the medication well with minimal side effects (Trojan and Cashman, 1995; Trojan et al., 1999, described later in chapter).
In conclusion, the majority of studies in the clinical literature focus on the efficacy of PB in the treatment of myasthenia gravis; however, most of the studies reviewed were not designed to determine adverse health effects. The widespread use of this compound has not typically been associated with short-or long-term adverse effects in myasthenia gravis patients.
Clinical Studies of PB in Veterans and General Health Outcomes
Information on symptoms and health status of 41,650 soldiers (6.5 percent of whom were women) who received PB at the onset of combat during Operation Desert Storm has been described (Keeler et al., 1991). Thirty medical officers in close daily contact with the combat units they served were queried retrospectively about the general physiological response of soldiers to PB and potential adverse effects. The reported effects represent impressions of the unit officers and were based on the number of clinic visits, discontinuations of PB, hospitalizations, and evacuations attributed to PB that came to their attention. Based on these anecdotal reports, the authors concluded that soldiers taking PB under combat conditions performed at full effectiveness, but experienced more
minor gastrointestinal and urinary symptoms than expected. An estimated 1 percent of the soldiers had effects from PB for which they sought medical advice, and less than 0.1 percent had effects that warranted its discontinuation.
On their return from the Gulf War, two women sought counseling about their pregnancies because they realized they had been exposed, during their first trimester, to PB and anthrax vaccine (Sarno et al., 1991). PB has been used in pregnancy without producing fetal anomalies, but Sarno and colleagues (1991) note that there are no controlled studies of PB and reproductive risks in females.
Keeler and colleagues (1991) suggest that the higher proportion of personnel experiencing adverse physiological effects than reported in peacetime evaluations may result from the combined effects of anticipated combat, sleep deprivation, and life in the field. Due to the limitations of the retrospective and uncontrolled nature of the data, Neish and Carter (1991) challenged these conclusions.
Acute poisoning with PB is uncommon, but Almog and colleagues (1991) report on nine cases of self-poisoning in men (n = 6) and women (n = 3) age 17–19 years associated with misuse of PB that was widely distributed as a prophylactic drug during the Gulf War. The doses ranged from 390 to 900 mg, but no CNS toxicity was observed in the nine patients. Mild to moderate cholinergic symptoms developed within several minutes after ingestion and lasted up to 24 hours. Two of the nine patients presented with muscarinic signs such as abdominal cramps, diarrhea, nausea, hypersalivation, vomiting, and urinary incontinence. One patient was observed to have transient nicotinic effects of fasciculations and weakness. The authors concluded that PB intoxication is self-limited and that PB is well tolerated by young adults (Almog et al., 1991). Thus, the various studies of PB exposure provide evidence that PB is not highly toxic, even at high doses or when taken during combat conditions.
Clinical Studies of PB and Neuromuscular Effects
A group of 17 patients with postpoliomyelitis syndrome (PPS) and 10 controls were studied for response to ChE inhibitors (Trojan et al., 1993). Patients with PPS suffer generalized weakness as well as signs and symptoms of muscle weakness thought to be due to neuromuscular junction transmitter defects. Patients responsive to the short-acting anticholinesterase edrophonium were subsequently treated with oral PB at 180 mg daily in divided doses for a period of a month. Side effects included intestinal cramps, diarrhea, muscle cramps, anxiety, blurred vision, and increase in urinary frequency. Using mobility and subjective fatigue as end points, clinical response to PB was measured before treatment and 1 month after treatment. Nine of the seventeen patients reported improvement in fatigue, including reduction in systemic fatigue as well as less muscle fatigability. One patient experienced reversible worsening of symptoms of fatigue. The nine responding patients continued the drug for a mean of 1.2 years despite associated mild gastrointestinal side effects. No other significant side effects were observed.
In a case series of six elderly patients (ages 65–73) suffering from frequent “drop attacks,” Braham (1994) reported on the efficacy of PB at 60 mg twice a day. None of the patients presented with any other cardiovascular or neurological diagnosis to explain these sudden falling episodes, and most benefited without suffering side effects from long-term therapy (the longest treatment period was 4 years at time of the report).
Thus, it appears that PB seems to be well tolerated and without significant neuromuscular side effects at the prescribed dose. However, as noted above, the number of patients observed for long periods with sensitive measures of motor function is insufficient to determine whether or not there is a long-term or latent effect of PB on the neuromuscular circuit.
Clinical Studies of PB and Behavioral or Cognitive Function
Molloy and Cape (1989) investigated the effects of PB on cognitive function in 15 elderly patients with Alzheimer’s disease. Their rationale for the study was the fact these patients have widespread dysfunction of central cholinergic systems necessary for memory and other higher functions. In addition, they noted that a number of previous studies with physostigmine had shown an improvement in the cognition of Alzheimer’s disease patients after treatment. Using a randomized, double-blind, crossover study design, seven elderly men and nine women (mean age 76 years) were treated with 240 mg of PB in divided doses over a 26-hour period. No significant difference in cognitive testing was found between subjects treated with PB and placebo. No side effects were observed. The authors suggested the possibility that doses were too low or the treatment period too short for observation of cognitive or untoward effects.
As noted earlier, patients with myasthenia gravis demonstrate subtle changes in cognitive function (Iwasaki et al., 1990) that are reversed with treatment, including plasmapheresis or anticholinesterases that cross the BBB (e.g., physostigmine) (Tucker et al., 1998).
Thus, these studies offer little evidence for long-term cognitive effects of PB in normal populations. The study of elderly patients with Alzheimer’s disease treated with 240 mg over 1 day showed no difference in cognitive testing between placebo and treatment subjects; no side effects were noted. However, Alzheimer’s patients would not necessarily be expected to reliably report untoward effects. Further, the possible reversal of cognitive dysfunction with anticholinesterases in myasthenics and the lack of evidence that the CNS of myasthenics responds differently to PB than that of the nonmyasthenic population might argue against adverse effects of PB on cognition in normal individuals.
Clinical Studies of PB and Cardiovascular-Related Effects
Because AChE inhibitors have been used for more than 50 years in the treatment of myasthenia gravis, clinical records may provide evidence of ad-
verse effects in this group of patients. From a pool of more than 1,000 patients with myasthenia gravis treated with ChE inhibitors, Arsura and colleagues (1987) present detailed clinical descriptions of drug-related hypotensive events in 12 patients (7 men, 5 women, mean age 62.6 years). Among these 12 case studies, 8 hypotensive events occurred after edrophonium, 2 after neostigmine, and 2 after PB. The proximal causes of documented syncopal or near-syncopal episodes were severe sinus bradycardia, junctional bradycardia, or complete atrioventricular (A-V) dissociation in nine patients and paradoxic sinus tachycardia in two others. None had obvious preceding signs of cholinergic excess, but all 12 patients had documented new exposure to or upward dose adjustment of their AChE-inhibiting medications, temporally consistent with the onset of the hypotensive episode. Also, the patients showed a strong tendency to respond to atropine and/or to reduce or discontinue use of the anticholinesterase drug, arguing against myasthenia gravis as the primary cause of the adverse event. Based on this review of more than 1,000 patients with myasthenia gravis treated with AChE inhibitors, the estimated frequency of such drug-related syncopal or presyncopal events is approximately 1 percent. The authors advise extreme caution in the use of this class of medications in all patients with pre-existing conduction defects and in the elderly, the groups that seem most prone to medication-precipitated hypotensive episodes.
Considerable clinical experience of adverse cardiovascular events also exists in relation to the use of AChE inhibitors for postanesthesia reversal of non-depolarizing muscle relaxants (e.g., d-tubocurare and pancuronium). Owens and colleagues (1978) studied 93 elderly patients (age > 65 years) undergoing general anesthesia for elective surgery. In this setting, 43 patients were treated with neostigmine and atropine and 50 with PB and atropine (all agents administered intravenously), and monitored for 90 minutes for the occurrence of postoperative cardiac dysrhythmias. Twenty-three percent of all patients experienced abnormal cardiac rhythms. Among those treated with neostigmine, 35 percent exhibited arrhythmias compared to 14 percent among the PB group, a statistically significant difference. The most commonly observed dysrhythmias were due to prolonged atrioventricular conduction resulting in bradycardia or A-V block. The authors concluded that the muscarinic effects of PB are associated with fewer cardiac side effects than those of neostigmine.
Arad and colleagues (1992a) explored the cardiovascular effects of PB on eight hypertensive patients treated with beta-adrenergic blockers (propranolol and atenolol). In this double-blind, crossover study, patients were treated with 30 mg of oral PB or placebo three times daily for 2 days. PB caused no significant effect on heart rate, plasma catecholamine levels, or resting blood pressure. Both systolic and diastolic blood pressure increased with exercise intensity, although a small but statistically significant decrease in diastolic blood pressure with exercise was noted during PB treatment. The authors attributed this to a mild decrease in peripheral vascular resistance (PVR) induced by the parasympathomimetic action of PB. Given the fact that patients with essential hypertension demonstrate increased PVR and that beta-blockers tend to further increase
PVR, this effect might be a beneficial one. No adverse reactions were observed. The investigators concluded that these results indicate the relative safety of the combination of PB and beta-blockers. However they acknowledged that such a small sample and brief period of treatment with PB do not rule out possible rare side effects, especially among people with significant cardiac conduction defects or congestive heart failure.
Teichman and colleagues (1985) described a beneficial interaction between PB and the ventricular antiarrhythmic agent disopyramide. Use of disopyramide is limited by its anticholinergic side effects (xerostomia, dryness of nose and eyes, urinary retention, constipation, abdominal pain, and blurred vision). To prevent or relieve these side effects, a sustained-release form of PB was administered to 27 of 106 disopyramide-treated patients referred for arrhythmia therapy. Doses varied from 90 mg every 12 hours (the usual dose) to as high as 180 mg every 8 hours. When PB was administered prophylactically, none of the patients receiving disopyramide developed anticholinergic side effects compared with 29 percent of those not treated with PB. Of the 10 patients treated at the onset of anticholinergic symptoms, 7 had complete resolution of their symptoms and 3 improved. There were no cardiac side effects attributable to PB, nor was there any evidence of decreased efficacy of disopyramide among PB-treated patients. The authors concluded that since PB caused no measurable decrease in disopyramide blood levels, the prevention or amelioration of anticholinergic side effects was related to its cholinomimetic activities and PB might be a useful agent for the treatment of disopyramide-related anticholinergic side effects.
In conclusion, studies of PB in patients with underlying medical problems are difficult to generalize to the normal healthy population because the disease state may affect the outcome of the response. The greatest experience with the cardiovascular side effects of PB is drawn from the clinical histories of patients with myasthenia gravis, usually at doses higher than those likely to have been used in the Gulf War. The incidence of drug-related cardiac arrhythmias appears to be approximately 1 percent and reversible with a decrease in dosage of PB. It is unlikely that the arrhythmias were due primarily to the underlying illness since myocardial involvement in myasthenia gravis is relatively uncommon. Older patients and those with pre-existing conduction abnormalities are at highest risk. It is of interest that studies of other patient groups also support the relationship between greater age and the risk of untoward cardiac events. This concordance among patient groups supports the association observed between age and cardiovascular side effects in PB-exposed individuals in the general population.
Other drug interactions have been described (see Box 6.1). In the literature reviewed above, no clinically significant side effects were noted when patients on beta-adrenergic blocking agents were treated with PB; although the study was brief and the number of subjects relatively small, no cardiac side effects of PB were observed.
SOURCE: Madsen (1998).
Clinical Studies of PB and Respiratory Effects
The respiratory effects of PB have been studied in clinical investigations of normal subjects, asthmatics, and individuals with myasthenia gravis. All of the
investigations of respiratory effects have been short term. The opposing effects of PB administration in myasthenics complicate respiratory studies in this population. For example, Ringqvist and Ringqvist (1971) studied respiratory effects of intravenous or intramuscular PB in 10 moderate to severe myasthenics aged 17–64 years. They found an increase in airway resistance (Re) and in maximum inspiratory (PEmax) or expiratory pressure (Pimax) in all subjects within 60 minutes of drug administration, but also observed an increase in vital capacity (VC). All of the subjects reported subjective improvement in respiration with PB administration. Pulmonary function parameters rapidly returned to normal with administration of a sympathomimetic drug.
In another study of 21 myasthenics, Shale and coworkers (1983) observed a decrease in airflow (FEV%) and an increase in airway resistance at 90 to 120 minutes following a dose of 60 or 120 mg PB given orally. The effect was completely blocked when ipratropium (a muscarinic blocker) was given simultaneously with PB. In a post hoc analysis, the increased airway resistance was found to be present only in subjects with airflow obstruction present at baseline.
De Troyer and Borenstein (1980) administered PB to myasthenics and normal controls and followed pulmonary function for 1–2 hours after drug administration. The control subjects (n = 4) were males aged 29–37 years. Following a 2-mg intramuscular dose there was no change in static lung volumes, conductance, or flow–volume curves. The PB dose was not titrated and there was no measurement of ChE level following administration.
A clinical study of 12 normal and 13 asthmatic subjects by Ram and colleagues (1991) measured pulmonary function for 24 hours after PB administration. The subjects were all male nonsmokers. Normal and asthmatic subjects received 60 and 30 mg of oral PB, respectively. The mean decrease in ChE activity was 28.2 percent in normals and 23.3 percent in asthmatics. A small decrease in FEV1,6 but not FEV% or PEF (peak expiratory flow), was observed following PB at rest and postexertion in the normal subjects. The decrease correlated with ChE depression and was statistically, but not clinically, significant. Among the asthmatic subjects there was an exercise-induced increase in airway resistance, but no effect of PB at rest or with exercise.
In summary, the literature on respiratory effects of PB is sparse and inconsistent. Taken together, the existing studies suggest that mild increases in airflow obstruction may occur within 1–2 hours of PB administration, but the effects are subclinical and rapidly reversible. Asthmatics may experience a small increase in exercise-induced airway resistance following PB administration.
A number of studies were conducted in healthy military and nonmilitary volunteers to evaluate the tolerance of prophylactic doses of PB that might be
used in combat to protect troops in the event of exposure to chemical warfare agents. These studies were reviewed for reports of general health outcomes and adverse effects on different organ systems.
Neuromuscular Studies in Healthy Volunteers
In a double-blind, placebo-controlled crossover study, Graham and Cook (1984) evaluated the effects of PB on 24 healthy male volunteers between the ages of 21 and 35, measuring a wide variety of performance measures including neuromuscular strength by grip testing and perceived level of exertion. Thirty milligrams of oral PB or placebo was administered three times daily for 5 days. No significant difference in strength or perceived exertion between PB and placebo-treated subjects was observed. The PB regimen produced the expected mean level of inhibition of plasma ChE, although large variation in inhibition between individuals was observed. No evidence of adverse health effects was found.
In another double-blind, placebo-controlled study of 35 healthy volunteers, Glikson and colleagues (1991) measured the effects of oral dosing of PB on direct tests of muscle strength and endurance. Subjects received either PB as 30-mg tablets or a placebo every 8 hours for 10 days. Before and during treatment, four subjects in the PB group and two in the placebo group also underwent electrodiagnostic studies (e.g., electromyography [EMG], nerve conduction velocity, and repetitive strength testing) of neuromuscular function. Subjects (16 placebo and 19 PB treated) underwent baseline tests of these parameters and were retested after 8 days of treatment and again 5 days after the cessation of treatment. Blood AChE levels showed a mean decrement of 23 percent, compared to baseline, among PB-treated subjects during therapy. Posttherapy AChE levels were identical to baseline.
Test parameters were analyzed as the percentage change from baseline for every variable in each subject. Measurements of handgrip strength as well as isokinetic elbow flexor and extensor strength did not differ between the placebo and PB groups. Electrodiagnostic studies of subjects treated with PB showed no significant change from baseline during or after the treatment. Some direct, standardized measurements of muscle strength (knee flexor and isokinetic strength) showed a small but statistically significant improvement on day 8 among placebo- but not PB-treated subjects. Conversely, knee extensor endurance showed a slight but statistically significant decrease on day 8 in the placebo group, whereas this variable remained unchanged in PB-treated subjects. Posttreatment strength measurements were not significantly different from baseline in either group.
The authors attributed the differences in some measures of muscle strength and endurance to large fluctuations in performance among the placebo group. It is possible that the improvement in knee flexor and extensor muscle strength at day 8 among placebo-treated subjects reflected only a training effect that was prevented by PB in the treatment group. However this would not explain the return to baseline 5 days posttreatment. The slight but statistically significant
advantage in day 8 muscle endurance for PB-treated subjects also was not observed posttreatment, giving additional credibility to the authors’ conclusions and arguing against the clinical significance of both of these findings. The authors’ overall conclusion is that treatment of healthy subjects with PB at oral daily doses of 90 mg for 8 days caused no significant neuromuscular effect.
In summary, two studies of healthy volunteers treated with PB, at doses and for a time comparable to those for some military personnel during the Gulf War, found no evidence of significant, clinical neuromuscular abnormality. One study (Graham and Cook, 1984) of men between the ages of 21 and 35 tested grip strength and perceived exertion, and found no significant PB-related changes.
The second study (Glikson et al., 1991) included more detailed strength testing as well as electrodiagnostic studies, but was limited by the extreme youth of its subjects (18–20-year-old males). Any age-related propensity to develop the symptoms seen in Gulf War veterans, which has been reported, would not be apparent in this study. The follow-up period for both studies was very brief—only 1 to 2 weeks after the initiation of therapy—making it difficult to determine whether abnormalities might occur at a later time. It should also be noted that one set of parameters, the electrodiagnostic tests, were performed on only four treated subjects, limiting the ability to rule out the potential for EMG, nerve conduction velocity, and repetitive strength testing abnormalities, which might have been detected by studying a larger, more diverse group for a longer period of time.
Neurobehavioral and Cognitive Function in Healthy Volunteers
A number of controlled studies have shown subtle neurobehavioral changes in subjects exposed to low doses of PB. Cognitive tests such as visuomotor coordination, dynamic visual acuity, reaction time, digit symbol, critical flicker fusion, and mood have been used to assess the effect of PB on performance. Individual performance on these tests was not significantly affected, although when the data were pooled, visual–motor coordination decreased (Borland et al., 1985). One study showed that in addition, perceptual speed and reaction time were impaired by heat and exercise rather than by PB (Arad et al., 1992b). Four subjects exposed to military doses of PB did not show compromised visual performance when tested for low-contrast acuity with dim illumination, a demanding task used to assess aviators’ visual ability (Wiley et al., 1992). Flight performance was not impaired by four doses of 30 mg PB in subjects tested on the A-4 simulator (Izraeli et al., 1990).
A 1984 study of psychomotor performance by Graham and Cook (1984) evaluated the effect of PB on multiple parameters of performance including psychophysiological indices, psychomotor performance, cognitive function, and other central processing functions. In addition, multiple task performance measurements were included in order to assess whether the drug had any impact on conditions of increased workload, where a subject was required to perform two tasks simultaneously (dual-task performance). Finally, subjective measures of symptoms including fatigue, perceived workload, and depression were applied.
Twenty-four healthy men between the ages of 21 and 35 participated in this placebo-controlled, double-blind, crossover study. An oral dose of 30 mg PB or a placebo was administered three times daily for 5 days with crossover after a 1-week period of “washout.” Testing of study variables was performed on days 2, 4, and 5 of treatment and 3 days after cessation of each treatment.
On day 2 of PB intake, performance was worse on a visual probability monitoring task (a test of perception and reaction time). On days 4 and 5, PB treatment was associated with decrements in dual-task performance. For example, when a visual tracking task was performed simultaneously with a memory search task, there was a tendency for the memory task to be more disrupted under PB treatment than under placebo conditions. Interestingly, PB significantly improved tests of hand steadiness on days 4 and 5. No adverse health effects were observed, and measures of subjective state and daily work activities failed to distinguish a difference between PB and placebo treatment. The authors concluded that PB, in the doses used, is well tolerated by healthy young men. However, the subtle decrements in cognitive function were considered noteworthy since they related to complex functions, which might be of particular importance in military operations. The rapid and precise actions required of an F-16 pilot were cited as an example of such performance demands (Graham and Cook, 1984).
Thomas and colleagues (1990) studied the effects of PB on cognitive performance of ten U.S. Navy divers during extended heat and warm water exposures. After prolonged pre-dive heat exposure, followed by a 3-hour dive, tests of short-term memory, learning acquisition, vision, and coordination were administered. For 2 days prior to and during one of the test exposures, subjects were treated with oral PB. Before and during another exposure, placebo was orally administered. The dose of PB is not stated. Short-term memory and measures of learning were impaired after heat exposure dives. No differences between PB and placebo-treated subjects were observed for any of the study parameters. The authors interpreted the findings as revealing that heat stress has a clear effect on the performance of complex cognitive tasks, but that PB had no such effect on behavioral or psychophysiological performance when administered either alone or in combination with heat stress (Thomas et al., 1990).
In summary, the 1984 study by Graham and Cook (1984) of the effects of PB on multiple parameters of performance provides a remarkably comprehensive and subtle assessment of psychomotor and cognitive functions in volunteers treated with doses of PB likely to have been used in the Gulf War. Although tests of many psychomotor and cognitive functions are unaffected by PB, there appears to be a trend for decrements in performance of complex tasks involving rapid shifts of attention. The study suggests subtle effects of this drug on cognition, reaction time, and complex performance that are not, however, supported by the prevailing concepts regarding the inability of PB to penetrate the blood– brain barrier. Although there was no evidence of persistent effects 3 days after discontinuation of the drug, suggesting that these are short-term effects, no long-term follow-up was reported.
The study of navy divers (Thomas et al., 1990) under heat stress conditions showed no effect of PB on neurobehavioral parameters. Unfortunately, the dose of PB is not stated, making these results difficult to interpret. Again, no long-term follow-up was reported. Thus, these studies offer little evidence with which to predict possible long-term effects of PB on normal patients.
Cardiovascular-Related Studies in Healthy Volunteers
It has been suggested that acute or chronic low-dose exposure to anticholinesterase compounds may cause subtle, subclinical effects that might be masked by tolerance or by CNS compensatory mechanisms. Izraeli and colleagues (1991) proposed that pharmacological challenge with a cholinomimetic agent (atropine) could prove useful in unmasking the latent muscarinic effects of AChE inhibitors such as PB.
To this end, eight healthy male subjects (mean age 29) were enrolled in a placebo-controlled, single-blind crossover study in which a 30-mg oral dose of PB or a placebo was administered every 8 hours for a total of four doses. Dosing began 24 hours before the commencement of noninvasive cardiopulmonary monitoring, with the fourth and final dose given 75 minutes before the monitoring session. After baseline recording of electrocardiogram (ECG), respiratory rate, and cardiac power spectra, these parameters were measured for 7 minutes after each of nine consecutive increasing intravenous doses of atropine. Volunteers reported no symptoms; no changes in mean heart rate or heart rate power spectrum were noted after PB treatment alone. The usual bimodal effect of atropine on heart rate (low doses ordinarily cause bradycardia, whereas higher doses cause tachycardia) occurred, but PB at the higher doses of atropine blunted this expected effect. The expected inverse effect on the respiratory peak (mediated mainly by parasympathetic input) was also observed, with the respiratory peak increased at the low dose of atropine and decreasing incrementally at higher atropine doses. Moreover, analogous to the findings for heart rate, this expected atropinic effect was attenuated by pretreatment with PB. These results indicate that PB-mediated cardiac rate effects are “unmasked” by interaction with atropine and that such effects are present even at asymptomatic dose levels of PB.
Another study of healthy volunteers by Nobrega and colleagues (1996) was designed to explore the effects of PB on cardiac cholinergic responses. Eight healthy volunteers (five men and three women, mean age 27) participated in a randomized, double-blind crossover trial comparing cholinergic effects of PB as a single 30-mg oral dose to placebo. Each subject underwent a 12-lead EKG, and three noninvasive cardiovascular maneuvers (respiratory sinus arrhythmia, Valsalva maneuver, and 4-second exercise test) were performed before and 2 hours after taking PB or placebo. PB was found to have a negative chronotropic cardiac effect (i.e., slowing of the heart rate) as evidenced by increased R-R intervals7 at rest and during the three autonomic tests. However, the drug had no
effect on compensatory reflex changes in heart rate as determined by the magnitude of the ratio of the longest to the shortest R–R intervals with each of the maneuvers. The authors concluded that despite tonic, dynamic reflex responses remain intact, suggesting a possible cardioprotective role for PB on post-myocardial infarction patients.
Harriman and colleagues (1990) designed a study to assess the possibility of PB treatment-related decrements in psychophysiological performance. Their preliminary publication dealt with the measurement of physiological parameters, including cardiovascular effects. A double-blind placebo-controlled crossover study of 24 male, trained A-10 pilots (mean age = 29) was carried out in flight simulators (with 12 pilots wearing chemical defense garments) after administration of either placebo or the standard PB regimen (30 mg every 8 hours) for 3 days. The pilots were first screened by measurement of plasma ChE inhibition after 30 mg of oral PB. Although there was marked individual variation of levels, in none of the pilots was ChE inhibition greater than 40 percent. Respiratory rate was not affected by PB. Heart rate and heart rate ratio (beats per minute/respirations per minute) were both decreased by PB. PB treatment also led to reports of 27 symptoms among 12 (50 percent) of the pilots. In contrast, placebo treatment resulted in only five (20 percent) pilots reporting six symptoms. The most common symptoms among PB-treated subjects were gastrointestinal upset, fatigue, confusion or giddiness, and headache. The authors concluded that the standard chemical warfare pretreatment regimen is safe for personnel who are prescreened for PB sensitivity.
In summary, in one small study (Izraeli et al., 1991), using relatively low doses (30 mg every 8 hours) of PB and causing no symptoms in healthy volunteers, subtle changes in heart rate and its normal respiratory fluctuations were observed. Another small study (Nobrega et al., 1996) of healthy subjects revealed slight negative cardiac chronotropic effects shortly after a single 30-mg oral dose of PB. However, this finding did not seem to be clinically significant because it was asymptomatic and autonomic reflex mechanisms easily compensated for the mild slowing in heart rate. The study of mission-ready pilots (Harriman et al., 1990) treated with the standard chemical warfare pretreatment regimen demonstrated gastrointestinal upset, fatigue, confusion or giddiness, headache, and decreases in heart rate and in the ratio of heart rate to respiratory rate without clinically significant cardiovascular symptoms.
These studies in healthy volunteers are limited because of the small number of participants and the brief duration of therapy without follow-up. It is, therefore, difficult to determine the significance of the findings or to compare their results to individuals with chronic illnesses or with genetically determined variations in cholinesterase activity.
Respiratory Studies in Healthy Volunteers
A study of FEV1 was done on six, healthy male volunteers with no history of asthma following intravenous (i.v.) administration of PB (0.143 mg/kg,
maximum 10 mg) and atropine (0.0143 mg/ kg, maximum 1 mg) (Feldt-Rasmussen et al., 1985). A maximum decrease of 27 ± 5 percent in ChE was observed 5 minutes after injection, but no decrease in FEV1 was observed during 90 minutes of follow-up. The study demonstrated that the muscarinic effects of PB are completely blocked by atropine.
Gouge and colleagues (1994) reported on pulmonary function changes following PB administration among soldiers during the Gulf War. Ten asthmatics and six healthy normal soldiers received 30 mg PB orally and had a vital capacity measurement and lung exam every 2 hours for 8 hours. No consistent change in VC was observed, but seven of the ten asthmatics reported increased chest tightness 2–6 hours after PB. No respiratory symptoms were reported by the normal controls or by any of the 264 other members of the unit who received a 30-mg PB dose. There was no measurement of airflow obstruction in this clinical study, and the reported symptoms cannot be interpreted. The study was not blinded or controlled (with a placebo), symptoms may have been due to other etiologies (e.g., stress), and may not have been correlated with airflow obstruction.
In summary, there are few studies in normal subjects on the respiratory effects of PB. Respiratory function is of interest because the muscarinic effects of PB overdosage include increased bronchial secretions and possibly smooth muscle contraction, which can increase airflow obstruction. Nicotinic side effects include muscular weakness, which can also impair respiratory function. However, as noted above, the existing clinical studies suggest that mild increases in airflow obstruction may occur within 1–2 hours of PB administration, but the effects are subclinical and rapidly reversible.
Thermoregulation in Healthy Volunteers
Seidman and Epstein (1989) published a review of the existing literature on the thermoregulatory effects of anticholinesterase agents in 1989 (the earliest of the six reviewed studies). Case studies of acute organophosphate poisoning in humans are noteworthy for the frequent finding of severe hyperthermia, which often occurred late (more than 24 hours after initial presentation) and lasted for several days in patients surviving the initial toxicities (reviewed in Seidman and Epstein, 1989). Less commonly, OP-poisoned individuals presented with hypothermia, probably due to a combination of effects including hypothalamic dysregulation, excessive sweating, and muscle paralysis leading to obliteration of the shivering response.
In an investigation of heat stress and PB effects on thermoregulatory indices, eight heat-acclimated healthy young men were subjected to repeated bouts of exercise in a hot–humid environment after receiving four oral doses of either PB or placebo in a double-blind crossover fashion (Seidman and Epstein, 1989). No significant effects of PB could be demonstrated on physiological parameters, including final rectal temperature, amount of heat stored in the body, dry heat exchange, sweat excretion, and sweat efficiency.
Other studies of small numbers of healthy male volunteers of comparable design have yielded similar results. In one such study (Epstein et al., 1990a), a slight but statistically significant decrease in heart rate was observed but was asymptomatic. In this study the mean age of volunteers was 23.5 years and the eight subjects were preacclimatized.
Epstein and colleagues (1990b) also studied heat exercise performance in eight subjects (mean age 23.5 years) under the added stress of wearing chemical protective clothing. Using a study design similar to those previously described, the volunteers were subjected to 170 minutes of exercise heat stress in protective clothing 4 hours after the fourth 30-mg oral dose of PB. Heart rate, heat storage, and sweat rates were similar in PB- and placebo-treated subjects. Nonevaporative (dry) heat exchange was significantly greater for PB-treated subjects than for controls. The authors concluded that heat stress in subjects wearing chemical warfare (CW) protective garments could lead to severe increases in body heat after 2 hours, but pretreatment with PB did not further decrease exercise performance beyond the limitations presented by heat, exercise, and CW protective clothing.
Wenger and colleagues (1993) studied thermoregulatory effects of dry heat, exercise, and PB treatment in a 7-day, double-blind, crossover study. Seven subjects (mean age 22) received 30 mg PB or placebo every 8 hours and exercised on a treadmill in a dry heat environment. PB increased sweating and evaporative water loss and lowered skin temperature during exercise compared to placebo. PB had no significant effect on rectal (core) temperature, oxygen uptake, or fluid balance. Although PB alone had no significant effect on heart rate, there was an interaction between the day of study and PB treatment on heart rate such that heart rate changed from a decrease of 0.4 beats per minute on day 1 to a decrease of 7.9 beats per minute on day 4. Similar temporal interactions occurred with regard to lowering of skin temperature with exercise. An interaction between increased sweating and treatment day also occurred such that the effect of PB changed from an increase of 0.1 liter of sweat on day 1 to 0.3 liter on day 7. The authors concluded, however, that standard CW prophylactic doses of PB had no clinically significant effect on thermoregulatory response in subjects exposed to exercise and dry heat.
Two studies of PB on thermoregulation during cold exposure were reviewed. Prusaczyk and Sawka (1991) studied the effects of a single 30-mg dose of PB on thermoregulatory responses in six men (mean age 21.8 years) subjected to cold water immersion for up to 180 minutes 2 hours after ingestion of PB or placebo. Cold exposure increased metabolic rate, ventilatory volume, and respiratory rate similarly in PB- and placebo-treated subjects. PB had no significant effects on rectal temperature, mean body temperature, subjective thermal sensations, plasma cortisol levels, or plasma volume. However, severe, but transient, abdominal discomfort caused termination of cold exposure in three of six PB experiments. Investigators concluded that PB did not increase susceptibility to hypothermia but could result in severe abdominal cramping that might limit cold tolerance.
Roberts and colleagues (1994) studied thermoregulatory responses in subjects exposed to exercise and cold air. Seven healthy volunteers (mean age 20)
participated in a 14-day, double-blind, placebo-controlled, crossover study with 7 days of PB treatment (30 mg three times a day and 7 days of placebo), during which exercise and cold testing were performed on days 2 and 3 and again on days 6 and 7. PB and control treatments resulted in similar metabolic rates, body temperatures, and regional heat concentrations. No differences were noted between earlier and later measurements with regard to any of the thermoregulatory and metabolic parameters. It was concluded that the study showed no “acute or chronic” effects of PB treatment on thermoregulation and metabolism during exercise in cold air.
In summary, review of studies of the effects of PB on healthy male volunteers are in general agreement with the conclusion that PB (at doses similar to those taken by troops during the Gulf War) results in no clinically significant perturbation of thermoregulatory homeostasis.
It should be noted, however, that some physiological parameters in some of the studies showed statistically significant differences for PB treatment compared to placebo. These PB-related findings include the following: mild, asymptomatic decrease in heart rate among heat- and exercise-stressed subjects (Epstein et al., 1990a); increase in nonevaporative heat exchange during heat stress with CW-protective clothing (Epstein et al., 1990b); increased sweating, increased evaporative water loss, and lowered skin temperature with dry heat stress and exertion, with an increased change in exercise-induced sweating and drop in skin temperature and heart rate on later days of treatment (Wenger et al., 1993); and episodes of self-limited but severe abdominal cramping in cold-exposed subjects (Prusaczyk and Sawka, 1991). These findings are of unknown clinical significance, but because of the rapid return to baseline, these PB-associated changes are not likely to be harbingers of long-term effects.
It is also important to notice that of the five studies (36 subjects) in which average weight and body surface area are stated,8 the measurements are similar to those expected for an average healthy male (the standard “70-kg man”). Smaller men and most women are substantially different from these subjects with regard to weight and body surface area (e.g., an average fit young American woman weighs approximately 55–65 kg and has a body surface area of about 1.5–1.75 m2). Since CW preexposure PB dosages are fixed (30 mg every 8 hours), the experimental findings in these studies could underestimate drug effects on many personnel.
Finally, these studies are limited by the small numbers of subjects and high degree of fitness among participants. Further, none of the studies were chronic in duration, nor did any study report long-term follow-up of experimental subjects. Hence, delayed effects of PB on thermoregulation, although unlikely, require further study.
Ocular or Visual Effects in Healthy Volunteers
Graham and Cook (1984) studied the effects of PB on multiple psychomotor parameters (see earlier discussions of neuromuscular effects and behavioral and cognitive function) as well as visual performance in 24 healthy male volunteers. In a double-blind, placebo-controlled, crossover study comparing 5 days of treatment with 30 mg of oral PB or placebo three times daily, investigators tested a number of visual function parameters, including spatial resolution by contrast sensitivity; neural transit time by steady-state visual evoked response; visual acuity by Snellen eye chart; and depth perception by biopter test. Testing was performed on days 4 and 5 of treatment and again 3 days after cessation of treatment. Interestingly, on days 4 and 5, subjects receiving PB had significant improvement in tests of depth perception compared to subjects on placebo. No significant drug-related effect on stationary visual acuity, contrast sensitivity, or stereopsis was noted. The drug regimen produced the expected level of inhibition of plasma ChE. However large differences between individuals in inhibition (range = −21.7 to +8.3 percent) were observed. Regression analyses were performed to determine the relationship between individual differences in ChE inhibition and study variables. These analyses indicated that as the level of enzyme inhibition increased, performance on tests of visual acuity decreased while depth perception improved.
Borland and colleagues (1985) investigated the effect of 3 days of oral PB at 30 mg every 8 hours on visual function and visual–motor coordination. Four healthy men between the ages of 19 and 27 participated in this double-blind, placebo-controlled, crossover study. After PB treatment, the mean critical flicker fusion threshold was significantly raised, an improvement in performance compared to that of subjects on placebo. PB-treated subjects performed dynamic visual acuity tasks with fewer missed responses than those on placebo. Visual– motor coordination was impaired with PB. No effect of PB was seen on pupillary diameter, static visual acuity, macular threshold, or kinetic perimetry. The authors suggest that the mild but statistically significant improvement in some visual performance tests was due to an increase in the level of arousal caused by increased stimulation of CNS cholinoceptive sites. The mild decrease in visual–motor coordination was attributed to either the peripheral anticholinesterase activity of the drug or a direct nicotinic action on the neuromuscular junction.
Also using a placebo-controlled, double-blind, crossover design, Kay and Morrison (1988) studied the effects on vision of a single 60-mg oral dose of PB in 14 male volunteers 18–40 years of age. In the first of a two-part experiment, contrast sensitivity to stationary oscilloscope-generated gratings showed a small but significant increase of 7 percent, which was attributed to a small reduction in pupillary diameter. Secondly, contrast sensitivity to laser interference fringes was tested, and with this method, which bypasses the optic media, no effect of PB was found. The authors concluded that PB could be used as a pretreatment for chemical warfare agents without a deleterious effect on stationary visual function.
Wiley and colleagues (1992) investigated the effects of PB on a variety of measures of visual performance. Four healthy male pilots-in-training (mean age 23) were treated with 30 mg oral PB every 8 hours for 3 days. On the day prior to beginning treatment (day 1), subjects underwent measurements of spatial resolution ability, dark adaptation, refractive error, and oculomotor function. Testing was repeated on each of 3 days of treatment (days 2, 3, and 4) and then again on day 5 during which no drug was administered. Mean contrast sensitivity thresholds showed no significant change during PB treatment. Six tests of oculomotor function were performed, and PB did not affect four of these tests. Fusional divergence, however, did show an effect of PB with test distance (6 m versus 70 cm), a finding the authors thought could not be due to PB (although no alternative explanation was offered). Pupillary diameter and refractive error both demonstrated significant PB-associated changes with relative miosis and myopia, respectively. The authors concluded that at the study dosage, PB would not adversely affect an aviator’s visual ability. The investigators do acknowledge that longer periods of treatment might have increased effects on vision.
Alhalel and colleagues (1995) studied the use of 30 mg PB three times a day as an agent to prevent the ocular anticholinergic effects of double-dose transdermal hyoscine patches. The hyoscine (scopolamine) patch has been commonly used against motion sickness, but its use is limited by the anticholinergic symptoms of impairment of near vision and accommodation as well as mydriasis. Investigators studied 47 healthy men (ages 18 to 21) in a placebo-controlled double-blind fashion. Subjects were treated with either double-strength or placebo hyoscine patches and PB tablets or placebo. Treatment duration was 2 days. PB significantly ameliorated hyoscine-induced impairment of near vision and accommodation but did not prevent the mydriatic effect of the patch. The authors concluded that PB at this dose was an effective antagonist of some ocular anticholinergic effects (impaired near vision and accommodation) of the hyoscine anti-motion sickness patch.
In summary, studies of the effects of PB on ocular or visual function in healthy volunteers reveal at least mild effects on visual function, however, the specific effects, their mechanism of action, and their significance are unclear.
No severe PB-related changes in stationary visual acuity were observed in most studies, although some findings are suggestive of such an effect. Graham and Cook (1984) noted that visual acuity decreased as the percentage of plasma AChE inhibition increased, while an opposite relationship occurred with depth perception, which improved as AChE inhibition was decreased by PB treatment. Therefore, PB treatment seemed to augment depth perception while having a negative effect on visual acuity. The latter effect might be due to drug-related induction of mild myopia (nearsightedness), which was indirectly observed by Wiley and colleagues (1992), who found that PB prevented the hyperopia (farsightedness) caused by the hyoscine motion sickness patch.
With the usual CW pre-exposure dose and schedule, some studies found miosis (i.e., decrease in pupillary diameter) (Wiley et al., 1992). PB might also
improve accommodation as evidenced by its prevention of hyoscine-induced impairment of accommodation (Alhalel et al., 1995).
Borland et al. (1985) described PB-induced improvement in the threshold for the fusion of flickering light and augmentation of dynamic visual acuity, findings the authors attributed to stimulation of CNS cholinergic actions. If these findings were related to CNS action, it would argue against the more generally accepted idea that PB cannot significantly penetrate the blood–brain barrier (see earlier discussion). These findings are important for the issue of possible long-term PB effects but currently are preliminary and unconfirmed. Other authors conclude that any observed effects on visual function are due to the peripheral action of PB on the pupillary sphincter, ciliary muscle, or oculomotor muscles. In general, the visual function effects of PB at routine CW pre-exposure doses are mild and reversible. However, the possibility of long-term or latent effects is difficult to assess since the studies reviewed were all of short duration.
Gastrointestinal Effects in Healthy Volunteers
Oigaard (1975) examined and compared the effects of PB and metoclopramide on upper gastrointestinal activity in 40 normal volunteers and 8 postoperative laparotomy patients. Using simultaneous recordings of electrical action potentials and intraluminal pressure, investigators compared the effects of the two drugs on upper gastrointestinal activity in healthy and surgical subjects. Measurements were taken after an oral dose of 40 mg of metoclopramide or an intravenous dose of 1.5 mg of PB. Both drugs had significant excitatory effects, with metoclopramide being the stronger stimulant of gut activity (possibly because the experimental dose of PB was relatively less potent than that chosen for metoclopramide).
A 1967 report of pharmacologic regulation of salivary gland secretion assessed PB’s effects on the saliva flow rate (Mandel et al., 1967). Both neostigmine and PB increased salivary flow, PB to a lesser degree than neostigmine.
In conclusion, studies of PB administration to normal volunteers demonstrate its expected short-term stimulatory effects on gastrointestinal motility and salivation, without any noted adverse effects.
Overall Performance in Healthy Volunteers
A number of studies have been carried out to assess overall measurements of both physiological and behavioral or cognitive parameters in healthy subjects treated with PB.
Izraeli and colleagues (1990) examined the effect of four doses of PB (30 mg every 8 hours) on flight skills in a placebo-controlled, double-blind, crossover study. The technical performance of 10 healthy male pilots (21–33 years) was tested in a flight simulator. No decrement in flight performance was observed among subjects treated with PB compared to placebo. Although they were mild in quality, slightly more symptoms were noted in PB-treated subjects.
A similarly designed study by Gawron and colleagues (1990) of 21 healthy male pilots found no significant decrements in performance in a flight simulator aircraft after standard treatment with PB. Pilots were unable to subjectively determine whether they received PB or placebo. However, another study of similar design (Brooks et al., 1992) of 24 A-10 pilots performing complex maneuvers in a flight simulator did not reveal a significant difference except in regard to one task. In a simulation of low-level penetration into a target area, pilots on PB were “killed” by surface-to-air missiles (SAMs) 25 percent more frequently; this finding was statistically significant. The authors attributed this effect to the “simulation factor” without explaining why simulation would preferentially exert nonpharmacological effects on PB-treated subjects.
Other investigators addressed the issue of PB effects on performance in combination with other physiological stressors. Forster and colleagues (1994) studied the effects of gravitational acceleration (G-force) stress and the usual CW pre-exposure PB regimen on overall performance in five healthy male volunteers (average age 26). This placebo-controlled, double-blind study assessed physiological and cognitive parameters. No statistically significant difference between PB- and placebo-treated subjects was noted for pulmonary function, heart rate, QT intervals,9 PR intervals,10 handgrip strength, or tolerance for acceleration. Cognitive performance was not systematically statistically affected by PB. Likewise, a similarly designed study of 10 Navy divers examining exercise, immersion, and warm temperature stresses (Doubt et al., 1991), showed no drug-related changes in heart rate, minute respiration, oxygen consumption, tidal volume, handgrip strength, or perceived exertion. Another study of like design measured the effects of standard doses of PB combined with heat–exercise stress and wearing CW protective clothing on eight healthy male volunteers (Arad et al., 1992b). PB had no significant effect on cognitive and physiological parameters but a statistically significant increase was found in “shortness of breath” (p < .005) in the PB-treated group. The authors concluded that in light of the absence of objective changes in respiratory function, this symptom is not likely to be of clinical significance. Cook and colleagues (1992) studied the simultaneous effects of heat, exercise, and standard PB doses for 7 days on seven healthy male subjects (mean age 21.4 years). PB was associated with a 4-mm decrease in resting diastolic blood pressure, a 0.050-mm decrease in pupillary diameter, a 3 percent decrease in handgrip strength, and a 0.1°C higher final rectal temperature. Although statistically significant, effects of this magnitude are unlikely to be clinically important. Subjects were unable to distinguish between days on placebo and days on PB.
In summary, the preponderance of evidence in studies reviewed above supports minimal effects of PB on overall motor, physiological, and cognitive functioning when normal healthy subjects are tested with complex tasks or physical
stressors. However, this statement must be understood to carry some important caveats:
Most subjects were extremely young males (<30 years) in exemplary physical condition (flight-ready pilots, Navy divers, medically screened volunteers). Whether the mild and/or equivocal performance effects (such as possible decrease in ability of pilots to avoid SAM attack in one simulation study) or the slight physiological changes or symptoms noted in others are clinically important in older or less well-screened individuals remains to be determined.
Long-term studies are unavailable at this time. In the longest of the above-cited investigations (Cook et al., 1992), a trend for decrease in hand strength became significant only after the full 7 days of PB treatment. The possibility exists that this change represents a downregulation of the ACh receptor after excessive stimulation by PB block of AChE. Long-term follow-up is needed to determine the possibility of a chronic adverse neuromuscular effect.
The number of subjects in each study is small and may not identify a sensitive subpopulation of individuals who might respond with greater adverse effects due to genetic variability (e.g., variants of BuChE activity).
Genetic and Population Pharmacokinetics and Pharmacodynamic Studies
Clinical Research Services, Inc. (1996), studied the safety, tolerance, and pharmacokinetics or pharmacodynamics (PK/PD) of PB in 45 male and 45 female healthy volunteer subjects in a randomized, placebo-controlled study. Subjects were divided into six groups by gender and weight (low, medium, and high weights for both sexes), with 15 subjects in each group. Within each group, 10 subjects received 30 mg of PB three times daily for 21 days and 5 subjects received placebo on the same schedule and for the same duration. Side effects were mild and similar between PB- and placebo-treated subjects except for mild gastrointestinal muscarinic symptoms that occurred with greater frequency among the PB-treated group. Blood chemistry, hematology, hormone levels, and electrocardiography showed no significant changes between those receiving PB and those on placebo. Plasma concentration of PB showed gender and weight effects early in the study, and maximum concentration of PB was significantly associated with weight (lower weights associated with higher serum concentrations).
Marino and colleagues (1998) describe a 1998 population PK/PD analysis of PB: 60 healthy men and women between 18 and 45 years of age received either 30 mg PB or placebo orally every 8 hours for 21 days (30 PB subjects and 30 placebo subjects). The PK model that best fit plasma PB levels was a two-compartment open model with first-order absorption and first-order elimination from the central compartment. The PD model that best fit RBC AChE activity was an inhibitory Emax model with an effect compartment linked to the central compartment. The results of this study show that the pharmacokinetics of PB are both gender and weight dependent. Due to wide individual variations, 30 percent
of the population may not achieve CW protective red blood cell AChE inhibition levels (>10 percent inhibition) on this standard regimen.
Loewenstein-Lichtenstein and colleagues (1995) presented a case study of an Israeli soldier who suffered from severe symptoms of nausea, insomnia, weight loss, fatigue, and depression while receiving standard doses of PB during the Gulf War as CW pre-exposure prophylaxis. The symptoms resolved some weeks after PB was discontinued. His past medical history was noteworthy for an earlier episode of postanesthesia apnea (succinylcholine used as paralytic agent). The authors analyzed the soldier’s BuChE activity spectrophotometrically and also delineated his (and his family’s) genotype by recombinant expression in Xenopus oocytes. These studies documented the subject to be homozygous for the most common variant allele of BuChE, with enzyme serum activity about one-third that associated with the usual genotype. The authors noted that homozygous carriers of this particular allele comprise about 0.04 percent of people of European ancestry but may be as high as 0.6 percent in certain subsets of this population. They posited that such atypical homozygotes and possibly even heterozygotes could be at risk for severe symptoms from PB due to the relative deficiency of PB-scavenging effective BuChE. They further speculated that anticholinesterase exposure might lead to long-term adverse consequences with symptoms that are not incompatible with those of Gulf War veterans, and that combined exposures to other ChE inhibitors might increase the risk of such outcomes.
In summary, individuals within a healthy population differ widely in their rates of absorption, distribution, elimination (PK), and enzymatic inhibition (PD) related to PB. PK is affected by weight and gender, whereas PD may vary considerably between normal individuals. Genotypic variations of BuChE may cause adverse (conceivably, long term) effects in some populations. Further studies to identify the relationship between such variants and the risk of both acute and chronic health outcomes is warranted (see Chapter 8).
A number of clinical and human volunteer studies have investigated a range of potential adverse responses associated with PB exposure. These are described above and essentially indicate minimal toxicity of PB with no irreversible side effects.
Although there have been several descriptive epidemiologic studies of Gulf War veterans, these investigations sought to characterize the nature and frequency of the illnesses reported by returning soldiers and did not examine the association of PB with these illnesses.
There are no analytic epidemiologic studies of the association of PB and adverse health effects in humans. Such studies would optimally have to include both exposed and nonexposed individuals as well as deployed and nondeployed soldiers to control for the environmental conditions associated with combat. A series of reports published by Haley and colleagues attempt to evaluate illnesses in Gulf War veterans (referred to by the authors as Gulf War syndrome) and
specific exposures associated with this syndrome (see Chapter 2). Because these studies used an incomplete study design (i.e., lacked an unexposed comparison group) and demonstrated other weaknesses, they have been strongly criticized on methodological grounds (Cowan et al., 1996; Gordon et al., 1997; Landrigan, 1997; Gray et al., 1998; Wolfe et al., 1998). This section reviews the Haley studies and other epidemiologic investigations of the association between Gulf War veterans’ symptoms and PB exposure.
In an initial survey designed to search for syndromes characteristic of Gulf War veterans, Haley et al. (1997b) studied the questionnaire responses of 249 men (41 percent of 606 males from a reserve naval mobile construction battalion [i.e., Seabees]) living in five southeastern states. All respondents had been called to active duty during the Gulf War, and there were no survey responses from nondeployed personnel. Characteristics of the participants (n = 249) and non-participants11 (n = 357) of the 24th Reserve Naval Mobile Construction Battalion (RNMCB-24) indicate that members of the battalion were, on average, older than most deployed forces, with a mean age of 41 years for participants and 37 years for nonparticipants. Participants and nonparticipants were similar in race or ethnicity, education, active reserve status, wartime military rank, and wartime job ranking (Haley et al., 1997b). However, large differences between participants and nonparticipants, respectively, were noted for percentage reporting serious health problems since the war (70 percent versus 43 percent) and percentage unemployed at the time of the survey (11 percent versus 3 percent). Of 249 individuals who responded to the survey several years after deployment, 145 (58 percent) had retired from the military, and the rest were still serving in the battalion. Symptoms included in the survey were those commonly associated with post–Gulf War illness in clinical examinations performed by teams of DoD and Department of Veterans Affairs physicians. From survey responses, the authors used factor analysis12 to identify six clinical syndromes: (1) impaired cognition, (2) confusion–ataxia, (3) arthromyoneuropathy, (4) phobia–apraxia, (5) fever–adenopathy, and (6) weakness–incontinence.
Psychological testing indicated that veterans with any of the six syndromes had the same psychological profile, which differed only in clinical severity but did not represent posttraumatic stress disorder. Those with syndromes 2 (confusion– ataxia) and 4 (phobia–apraxia) had increased self-reported occupational disability compared to the others. The low participation rate (41 percent) of veterans in the battalion suggests that results may have been affected by selection bias, in that
participants were older, had more illnesses, and were more likely to be unemployed than nonparticipants. The authors believe that such biases were avoided because participants were demographically representative of the entire battalion and because retired veterans were included in the study. Nevertheless, nonparticipants were less likely to report having had a serious illness since the war and were more likely to be employed. Moreover, the average age of this group of Seabees was greater than that of most active duty units, suggesting that study subjects might not accurately reflect the nature of illnesses in other military units.
At least 25 percent of ill veterans in the battalion studied had symptoms that the authors believe suggested generalized neurological injuries, mainly combinations of damage to the brain or brain stem (e.g., cognitive and vestibular dysfunction), the spinal cord and peripheral nervous system (e.g., paresthesias of the extremities, muscle pain and weakness, joint pain, urinary incontinence), and the autonomic nervous system (e.g., chronic diarrhea).
There is concern that the survey sample used by Haley and colleagues was small, increasing the potential to generate spurious results (Gray et al., 1998). Another potential source of bias is the numerous medical examinations and media contacts of study subjects before the survey was conducted and the reliance on self-reports of symptoms and adverse responses to PB that occurred many years earlier (Gray et al., 1998). The study population was a reserve naval command, whose members were often employed full-time in nonmilitary careers, with occupational exposures and subsequent confounding health risks, and thus may not be representative of the general population of Gulf War veterans.
The most important of the Haley reports with regard to an association with PB exposure is described as “a cross-sectional epidemiologic study” (Haley and Kurt, 1997). This study of the association between self-reported wartime exposures and self-reported symptoms in a small proportion (41 percent) of the 606 members from the RNMCB-24 relies heavily on the syndromes developed by factor analysis of symptoms reported by these same veterans (Haley et al., 1997b). The survey instrument used by Haley and colleagues to elicit self-reported exposures and symptoms in participating members of the battalion was developed by Haley and colleagues and pretested on five Gulf War veterans. After revision, the survey instrument (exposure and symptom booklet) was again pretested on five additional Gulf War veterans. It is important to note that associations reported by Haley and Kurt (1997) are based on comparisons of responses by ill and non-ill Gulf War veterans and do not include comparisons of responses from nondeployed veterans.
The authors report that the prevalence of syndrome 1 (impaired cognition) was greater among veterans who reported wearing flea collars during the war (5 of 25, 20 percent) than in those who never wore them (7 of 229, 3 percent; RR [relative risk] 9.7 [3.0–24.7], p < .001). Syndrome 1 was not associated with subjects having taken PB or reporting adverse effects from PB.
The prevalence of syndrome 2 (confusion–ataxia) was eight times greater among veterans who reported having experienced a likely CW attack. The prevalence of syndrome 2 was not higher in people who reported having taken a
larger number of PB tablets but was higher in soldiers who recalled having experienced more severe adverse effects from PB (χ2 for trend, p < .001). Syndrome 2 was more prevalent among veterans who believed they had been involved in a chemical weapons exposure (18 of 108, 17 percent) than those who did not believe they were so involved (3 of 141, 2 percent; RR 7.8 [2.3–25.9], p < .001) and was higher in veterans who reported having been in a sector of far northeastern Saudi Arabia (a site allegedly exposed to CW agents) on the fourth day of the air war (6 of 21, 29 percent) than those who did not report having been there (15 of 228, 7 percent; RR 4.3 [1.9–10.0] p < .004). Effects of perceived chemical weapons exposure and perceived advanced adverse effects from PB were synergistic using a Rothman S test.
The study found that the prevalence of syndrome 3 (arthomyoneuropathy”) increased with the frequency of reporting certain adverse effects for PB and reporting the use of large amounts of government-issued insect repellent containing DEET (p < .001 for both).13 Each of the three syndromes was associated with a different set of risk factors. Haley and Kurt conclude that the risk factor associations observed in their study suggest that these three syndromes may represent variants of OPIDN due to varying degrees of exposure to organophosphate nerve agents potentiated by interactions with other chemical exposures and older age.
A major limitation of the design of this cross-sectional study, and its subsequent interpretation, is the lack of comparable symptom and exposure data for a nondeployed or non–Gulf-deployed military population. Based on discussions with the committee, Dr. Haley is aware of this limitation and plans to pursue studies of comparable phenomena in nondeployed military populations.
A further study by Haley et al. (1997a) evaluated neurological function in 23 Gulf War veterans with symptoms (cases) and 20 Gulf War controls (10 deployed and 10 not deployed, from the same battalion) in a nested case-control study. This study found impairment in ill Gulf War veterans compared to controls on each of two global measurements of brain dysfunction (the Hallstead Impairment Index, p = .01, and the General Neuropsychological Deficit Scale, p = .05. There were significant differences on 20 of 89 tests with endpoints that did not depend on volitional action by subjects (e.g., evoked potentials); cases were more impaired on 18 tests and controls on 2 tests (p <0.001). By contrast, there were significant differences on 15 of 76 tests with end points depending on volitional action; cases were more impaired on 9 and controls on 6 (p = .30).
Cases with impaired cognition (syndrome 1) were more impaired on brainstem auditory evoked potentials; those with confusion–ataxia (syndrome 2) were more impaired on the Halstead Impairment Index, asymmetry of saccadic velocity, and somatosensory evoked potentials; and those with arthromyoneuropathy (syndrome 3) were more impaired on caloric stimulation than the controls. An independent examination by six neurologists blinded to the case-
control status of the subjects indicated that about two-thirds of all veterans (cases and controls) had at least one abnormal neurological finding (most frequently reduced strength of the lower extremities); however, there were no significant differences in the frequency of neurological findings in the cases and controls. The examining neurologists and study investigators reviewed the findings on each subject individually and concluded that the clinical and laboratory findings were nonspecific and not sufficient to diagnose any known neurologic syndrome in any subgroup of subjects (Haley et al, 1997a). Nevertheless, the authors expressed the view that the neuropsychological abnormalities seen in Gulf War veterans are likely the result of neurotoxic exposures associated with service in the Gulf War.
This study also has come under criticism. For example, despite the assertion of neurological damage in the 23 most symptomatic subjects, there were no statistically significant differences on neurological examination between cases and controls, and a panel of neurologists concluded that the confusion–ataxia syndrome was not specifically defined by their standard clinical neurological exams. Furthermore, the changes noted on evoked potentials and saccadic velocity were nonspecific compared to larger control populations. Moreover, evoked potentials have limited utility in evaluating patients with possible neurological disorders; their major purpose is to look for evidence of demyelination, which would not be useful in persons suspected of having OPIDN—classically, a distal axonal neuropathy, not a permanently demyelinating disorder of the brain stem and spinal cord. Although a statistically significant difference in interpeak latencies related to PB was reported on the somatosensory evoked potentials in the 23 cases compared with the 20 healthy controls, there were no significant differences between the results in veterans and the authors’ own laboratory’s established normative values from healthy people. Some “statistically significant” differences are expected by chance alone when a large number of tests are performed on a small number of patients, and in this instance, no adjustment was made for “multiple comparisons.”
Some findings had uncertain clinical relevance (e.g., asymmetric nystagmus velocities following caloric stimulation in patients complaining of arthralgia, myalgia, and weakness). Electromyography shows abnormalities in individuals with OPIDN, but single-fiber EMG results were normal in the Haley study. Thus, the clinical data presented provide little evidence with which to conclude that these patients had a neuropathic process, and it is difficult to interpret the findings of a battery of neurological tests under these conditions. For example, five veterans with confusion–ataxia and three with arthromyoneuropathy had peripheral neurophysiological tests (nerve conduction studies, electromyography, single-fiber EMG, and quantitative sensory tests) that were normal, whereas most of the veterans with syndrome 3 (symptoms consistent with peripheral neuropathy) had no findings consistent with this peripheral neuropathy, which calls into question the validity of the symptoms as a measure of neurological damage. The principal limitation of this nested case-control study, in
addition to the limitations of the Seabees study population described above, is the lack of clinical validity of measures used to infer neurological damage.
A more recent study (Haley et al., 1999) investigated genetic polymorphisms of PON1 and BuChE in 45 subjects (cases and controls), who had been reported by Haley and colleagues in the previously described case-control study. The authors measured serum activity levels of various allozymes to test the hypothesis that differences in allozyme levels might have put some Gulf War veterans at higher risk of neurological damage from exposure to environmental chemicals that require these enzymes for detoxification. The study found that ill veterans with the neurological symptom complexes described were more likely to have the R allele of the PON1 gene (heterozygous QR or homozygous R) than to be homozygous Q. They found that low activity of the PON1-type Q arylesterase allozyme distinguished ill veterans from controls better than the PON1 genotype alone or the activity levels of the type R arylesterase allozyme, total arylesterase, total paraoxonase, or BuChE. The authors found that a history of advanced acute toxicity after taking PB was also correlated with low PON1-type Q arylesterase activity.
A major criticism of this study is that type Q, the allozyme of PON1 that most efficiently hydrolyzes several organophosphates (sarin, soman, and diazinon), does not hydrolyze PB (unpublished data from their laboratories cited by the authors); thus, the interpretation of lowered levels of this enzyme is uncertain insofar as it relates to PB use in ill Gulf War veterans.
A study of U.K. servicemen who served in the Gulf War (Unwin et al., 1999) addressed PB exposure. This cross-sectional postal survey of Gulf War veterans (n = 4,248), Bosnia conflict veterans (n = 4,250), and those serving during the Gulf War but not deployed there (n = 4,246) found a greater perception of worse physical health in the Gulf War cohort. Gulf War veterans reported all symptoms and disorders more commonly than the comparison cohort. All of the self-reported exposures showed associations with all outcome measures in the three cohorts. Three exposures were reported more frequently by Gulf War veterans than by the other cohorts: exposure to burning oil-well smoke, vaccination against biological warfare agents, and CW protection measures. Very small proportions of the Bosnia and nondeployed Gulf War era veterans reported exposure to PB, 1.9 percent and 5.2 percent, respectively, compared to 81.6 percent of deployed Gulf War veterans. Nevertheless, the association of symptoms with self-reported PB use was not different between the three cohorts with elevated odds ratios observed for the three principal health outcome measures in all three cohorts.
A large number of clinical studies report acute transient cholinergic effects in normal volunteers and patients with a wide variety of clinical disorders given PB as a diagnostic test of hypothalamic pituitary function and patients with my-
asthenia gravis treated with the drug for extended periods. As a diagnostic test, PB is generally administered as a single oral 30–180-mg dose, which produces acute transient cholinergic symptoms in a minority of patients and normal volunteers. Within 1 or 2 hours after ingesting PB, about 25 percent of subjects experience abdominal symptoms, and about 10 percent have muscular symptoms that typically last 1–2 hours. The abdominal symptoms are cramps, increased digestive sounds, pain, diarrhea, and nausea, and the principal nicotinic cholinergic symptoms are skeletal muscle and tongue fasciculations sometimes accompanied by dysarthria. The symptoms are characteristically mild, transient, and tolerable, without requiring medical intervention, and are not accompanied by central nervous system symptoms. Although a clear dose–response effect on symptoms was not apparent from a review of the studies summarized in this report, none were designed to demonstrate this effect. There is, however, a trend toward a greater rate of symptoms at higher PB dose ranges among subjects given several different doses in the same study.
PB is used to control muscle weakness in myasthenic patients, and the daily dose of PB usually ranges from 120 to 600 mg. Studies indicate that about 34 percent of those receiving PB have one or more, mostly mild, side effects, usually gastrointestinal, although a few patients experience other cholinergic symptoms such as hypersalivation, increased perspiration, urinary urgency, increased bronchial secretion, and blurred vision. Patients rarely have to stop the drug because of abdominal complaints.
During the Gulf War, acute accidental poisoning with PB in doses ranging from 390 to 900 mg resulted in mild to moderate cholinergic symptoms within several minutes of ingestion, which lasted up to 24 hours. Patients typically developed muscarinic effects such as abdominal cramps, diarrhea, nausea, hypersalivation, vomiting, and urinary incontinence. The effects are self-limited and well tolerated by young adults.
As noted above, the most extensive information available on the acute effects of PB comes from studies of its use for diagnosis of growth hormone deficiency in adults and children and its therapeutic use in the treatment of myasthenia gravis. These studies, of doses higher than those used for prophylaxis during the Gulf War, consistently indicate that PB is safe and effective in clinical applications. Side effects noted are predominantly gastrointestinal and muscular, and are of a short duration with no long-term residual effects.
Results from other human studies of both clinical and healthy volunteer populations, report the same gastrointestinal and muscular side effects, which are transient and characteristically mild. A small number of idiosyncratic reactions are noted.
The committee concludes that there is sufficient evidence of an association between PB and transient acute cholinergic effects in doses normally used in treatment and for diagnostic purposes.
No reports of chronic toxicity related to human PB exposure in clinical or military populations are available. The suggestions by Haley and Kurt, 1997 of a unique manifestation of organophosphate-induced delayed polyneuropathy associated with PB exposure alone or in combination with other wartime exposures, in the absence of acute symptoms of organophosphate toxicity, requires further investigation.
Although Haley and colleagues provide evidence that chronic neurological changes are present in a small number of ill Gulf War veterans compared to a small number of well veterans from the same unit, the validity and causal nature of this association are uncertain due to the large potential for selection and information biases in this study population and the lack of a nondeployed comparison group.
In addition, the evidence that some types of chronic neuropsychological changes may be linked to acute responses to administration of PB, also suggested by Haley and Kurt (1997), is limited by the lack of consistency with results from toxicological and clinical studies; uncertainty about the selection, administration, and interpretation of the neuropsychological tests employed; the highly select nature of the small number of Gulf War veterans studied; and the lack of comparable studies in a nondeployed comparison group.
The epidemiologic data do not provide evidence of a link between PB and chronic illness in Gulf War veterans. Most epidemiologic studies of Gulf War veterans focused on whether a unique Gulf War syndrome exists and on defining its characteristics. Only two epidemiologic studies specifically investigated the possible association of PB use and chronic symptoms among Gulf War veterans (Haley and Kurt, 1997; Unwin et al., 1999). The limitations of the small, selected population studied by Haley have been noted. With regard to PB, Haley’s factor-derived syndromes were not associated with taking PB or with the dose of PB. Two of the three syndromes showed an association with self-reported symptoms that are consistent with adverse effects of PB. This finding may result from reporting bias for adverse health syndromes and adverse effects of PB, and provides an inadequate basis for concluding that an association exists. The other epidemiologic study was of U.K. servicemen (Unwin et al., 1999), and all exposures studied (PB, diesel or petrochemical fumes, oil fire smoke, viewing dismembered bodies, etc.) showed an association of similar magnitude with adverse symptoms. Recall and reporting bias may also explain this finding. Thus, neither of these two studies provides a basis for determining that a specific association between PB and chronic adverse health effects exists.
The committee concludes that there is inadequate/insufficient evidence to determine whether an association does or does not exist between PB and long-term adverse health effects.
The available evidence is of insufficient quality, consistency, or statistical power to permit a conclusion regarding the presence or absence of an association in humans. This is true particularly when PB exposures occur in combination with other combat exposures.
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