The influence of pyridostigmine administration on human neuromuscular functions—Studies in healthy human subjects

FUNDAMENTAL AND APPLIED TOXICOLOGY 16,288-298 (1991)

The Influence of Pyridostigmine Functions-Studies

Administration on Human Neuromuscular in Healthy Human Subjects

M. GLIKSON, A. ACHIRON,* Z. RAM, A. AYALON,~ A. KARNI, I. SAROVA-PINCHAS,+ J. GLOVINSKI, AND M. REVAH Corps, Israel; *Department of Neurology, Beilinson Medical Center, Petah Tiqva and Faculty of Medicine, Tel Aviv University; tZinman College of Physical Education, Wingate Institute: and .$Department of Neurology, E. Wolfson Medical Center, Holon and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv Israel

Israel Defense Forces Medical Sackler

Received

March

12. 1990; accepted August

21, 1990

The Influence of Pyridostigmine Administration on Human Neuromuscular Functions-Studies in Healthy Human Subjects. GLIKSON, M., ACHIRON, A., RAM, 2.. AYALON, A., KARNI, A., SAROVA-PINCHAS, I., GLOVINSKI, J., AND REVAH, M. (1991). Fundam. Appl. Toxicol. 16, 288298. Pyridostigmine has a protective effect against organophosphate poisoning when given in a dosage of 30 mg three times daily causing 20-40s cholinesterase inhibition. To test its safety in the human neuromuscular system,a double-blind study on 35 subjects divided into two matched groups was performed. One group was treated with pyridostigmine in a dose of 30 mg three times daily and the other group was treated similarly with placebo, both for a IO-day period. The resultant average cholinesterase inhibition in the treatment group was 23%. Muscle strength and endurance were tested before, during (on the 8th day), and after treatment. Electrodiagnostic studies, including nerve conduction, electromyography, and response to repetitive stimulation, were carried out on four subjects of the treatment group and on two subjects of the placebo group, both before and during treatment (eighth day). Isometric handgrip strength, isokinetic elbow flexor, and extensor strength did not differ between groups as a result of the treatment. Knee flexor and extensor isokinetic strength showed a small (but statistically significant) trend to improve more during placebo treatment, whereas knee extensor endurance decreased slightly in the placebo group. Both these effects are probably due to large fluctuations in performance of the placebo group, whereas the treatment group performance was quite constant. They probably do not represent any adverse effect of pyridostigmine. No electrophysiological changes were found in any of the subjects during treatment. We conclude that pyridostigmine does not cause any significant neuromuscular effect in healthy subjects when taken in a dosage of 90 mg daily for 8 days, causing 20-30% inhibition of cholinesterase. o 1991 society of Toxicology.

Pyridostigmine, a carbamate, is a reversible inhibitor of acetylcholinesterase. It has been used for many years as a treatment for myasthenia gravis and for the reversal of neuromuscular blockade during anesthesia (Osserman et al., 1958; Lippmann and Rogoff, 1974). During the past 20 years it has been described as having a unique protective effect against organophosphate poisoning when given as a treatment before the intoxication 0272-0590/9

I $3.00

Copyright 0 1991 by the Society of Toxicology. All rights of reproduction in any form reserved.

(Berry and Davis, 1970; Deyi et al., 198 1; Dirnhuber et al., 1979; Gall, 198 1). This protection has been attributed to a temporary “masking” of the enzyme which prevents the irreversible binding of the organophosphate (Deyi et al., 1981; Lennox et al., 1985). Such protection could be achieved with a dosage of 30 mg three times daily, producing 20-40% blood cholinesterase inhibition (Gall, 198 1; Gordon et al., 1978; Lennox et al., 1985).

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Larger doses (i.e., 60 mg three times daily) may cause peak cholinesterase inhibitions of higher than 50%, which, although protective against organophosphates, may cause mild side effects in healthy subjects (our unpublished observations). In order to use the drug for protection in healthy persons, it must be proven to be free of any detrimental effects on human health or function, in the relevant dosage. Although there are reports involving large groups of patients receiving much larger doses of pyridostigmine for myasthenia gravis (Osserman et al., 1958) or for reversal of neuromuscular blockade (Lippmann and Rogoff, 1974), there is a lack of information concerning the adverse effects of such a low dose of pyridostigmine in healthy subjects. Studies of small animals reported changes in structure, ultrastructure, and electrophysiological properties of the neuromuscular synapse during and after repeated exposures to various carbamates, including pyridostigmine (Engel et al., 1973; Hudson et al., 1978; Hudson, 1985; Rash et al., 1985; Roberts and Thesleff, 1969; Schwartz et al., 1977; Tiedt et al., 1978; Ward et al., 1975). Another study reported a possible change in metabolism of the exercising muscle (Francesconi et al., 1984). We therefore decided to investigate possible detrimental effects of the drug on human neuromuscular function by studying large muscle strength and endurance. We also performed nerve conduction velocity studies, electromyography, and repetitive stimulation tests. These were performed during administration of the medication for 10 days in a dosage of 30 mg three times daily, with resultant whole blood cholinesterase inhibitions of 20-30%. METHODS Subjects Thirty-five healthy male volunteers between the ages of 18 and 20 years were studied. The subjects underwent an

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interview and a complete physical examination by a physician. Laboratory evaluation included complete blood count, liver and kidney function tests, basal whole blood cholinestemse activity, and lead-12 electrocardiography. No subject was allowed to participate in the study unless all the above-mentioned evaluations were normal. Upon his request, one of the subjects withdrew from the study after 4 days. Informed Consent All study protocols were reviewed and approved by the Joint Committee on Clinical Investigations of the Israel Defense Forces Medical Corps. Informed consent was obtained from all subjects in the study. Study Design The study was a double-blind placebo-controlled study. After a short training on the testing equipment, subjects were tested in order to obtain baseline values. On the basis of these results, the subjects were then divided into two matched groups (treatment group and placebo group), according to a multifactorial matching method. A combined muscle strength score, an endurance score, and a maximal oxygen consumption score were 3 of 11 variables contributing to the matching procedure. The other 8 variables were the scores of various other performance tests (mostly psychological tests), irrelevant to physiological function. Average weight of the subjects was 75.09 kg (SD, 13.07 kg) in the placebo group and 70.94 kg (SD, 12.75 kg) in the treatment group. No statistically significant differences existed between the groups in all the baseline values in spite of the removal of the baseline results of the subjects who left the study (Tables l-4). The matching procedure and division into two groups were carried out by independent persons who did not take any active part in the study. For the electrodiagnostic studies only six subjects were randomly chosen, four subjects from the treatment group and two subjects from the placebo group. The study was carried out in three stages: Stage I. The preparation stage lasted 1 week, during which medical examinations were carried out and the baseline measurements of the variables were taken. At the end of this week the matching procedure and division into two treatment groups was undertaken. Stage II. The treatment stage lasted 10 days during which drug (or placebo) were continuously administered, three times daily (as outlined under Treatment). Muscle strength and endurance in all the subjects and electrophysiological studies on six subjects were done on the 8th day of drug administration. Stage Ht. There was a recovery stage after discontinuation of medication. Muscle strength and endurance were

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measured again 5 days after discontinuation of the treatment.

PROCEDURES Strength and Endurance Tests Handgrip isometric strength test. Subjects were tested on handgrip isometric strength of the right arm with the use of a handgrip cable tensiometer from Pacific Scientific Co. (Anaheim, CA). Each subject was given three consecutive trials. Maximal isometric contractions were expected from the subjects. The best result of the three trials was used as the score of the test. Isokinetic strength measurements. Subjects were tested on a Cybex II isokinetic dynamometer from Cybex Division of Lumex, Inc. (New York). The dynamometer measured the torque produced by the subjects on the measuring device. This torque was recorded on the Cybex dual-channel recorder. The use of the Cybex II dynamometer for isokinetic strength determination is further described in the Cybex II handbook (1983). This method is in widespread use for assessment of various muscle group strength in orthopedic and rehabilitation practice as well as in physiological research (Borges, 1989; Nutter and Thorland, 1987). Maximal isokinetic strength of the left knee extensors/flexors. The subjects were tested in an upright sitting position. The test was performed at an angular velocity of 6O”/sec. Three consecutive trials were performed by each subject in both knee extension and flexion. The peak torque for both muscle groups was calculated. Maximal isokinetic strength of the right elbow extensors/flexors. Subjects were tested on the same Cybex II isokinetic device, described above, while reclining in a supine position on the UBTX table. The arm was supported by a supporting device in a horizontal position, leaving the forearm free for performance of the movement in the horizontal plane relative to the subject. Torque was measured and re-

ET AL.

corded as above, at an angular velocity of 60”/ sec. Three consecutive trials were performed by each subject in both elbow extension and flexion and the peak torque for both muscle groups was calculated. Isokinetic muscle endurance. The muscular endurance of the right knee extensors was measured on the same isokinetic device. The test was performed at an angular velocity of 18O”/sec. Thirty-three consecutive maximal knee extensions were performed by the subjects. The values of 30 consecutive trials, starting from the second contraction, were considered for calculation. The score for this test was the ratio between the mean score of the last five trials and the mean value of the score from the second to the sixth contraction (inclusive). Electrophysiologic

Tests

Electrophysiologic tests were carried out by two-channel DANTEC electromyograph, Neuromatic 2000 M/C. Motor nerve conduction studies, repetitive nerve stimulation, and electromyography were performed. These tests were carried out and interpreted by an experienced electromyographist (ISP), under the same conditions before and during drug administration, in terms of a double-blind study. Motor nerve conduction studies. Motor nerve conduction studies included: (a) Motor nerve conduction velocities evaluate the speed of propagation of an action potential along the fastest fibers of the examined motor nerve, and is expressed in meters per second. This test was performed to estimate the possibility of a conduction slowing or block in the stimulated nerve along its course. (b) Distal latency defines the time an impulse travels from the most distal site of the nerve to its innervated muscle and is measured in milliseconds. This test enables differentiation of a distal lesion of the peripheral nerve from a more proximal one.

PYRIDOSTIGMINE

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(c) F-wave latency measures the time in milliseconds an impulse travels along the proximal part of the nerve. When F-wave latency is prolonged it is suggestive of a proximal nerve lesion. (d) Amplitudes of the compound muscle action potentials of motor response and Fwave were measured in millivolts. A decrease in the amplitude in the presence of normal nerve conduction velocity suggests an axonal lesion of the peripheral nerve. (e) Durations of the compound muscle action potentials of the motor response and Fwave were measured in milliseconds. Changes in these parameters can signify neuronal or muscular lesion. (Kimura, 1983; Aminoff, 1987; Achiron and Sarova-Pinchas, 1984). Median, ulnar, and facial nerves were examined. The median and ulnar nerves were stimulated with a supramaximal pulse of 0.2 msec duration at the wrist and at the antecubital space and ulnar groove, respectively; Facial nerves were stimulated over the stylomastoid foramen. The responses were recorded using surface skin electrodes ( 13 K 60), consisting of two small silver plates, 6 X 12.5 X 1.5 mm, which were fastened to the skin with sticking plaster. The skin impedance was reduced with electrode paste. Repetitive nerve stimulation test (RST). To assess the integrity of the neuromuscular junction and to reveal a latent defect in neuromuscular transmission, RST was performed as described by Kimura (1983) and Aminoff (1987). The neuromuscular transmission was tested by a O.Zmsec square-pulse stimulation of 20 mA intensity with trains of 5 pulses, delivered at rates of 3 and 10 Hz. The ulnar nerve was stimulated at the wrist and isometric muscle responses were recorded on the hypothenar muscles. A response of amplitude decline above 10% was considered abnormal. After a 10-set voluntary muscle activity (of the examined muscles), postactivation facilitation and exhaustion were investigated in search of an increase or a decline of the motor

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response. Postactivation facilitation was examined 3 set, while postactivation exhaustion was examined 30 set, after a period of 10 set of maximal voluntary activity. RST and responses of postactivation facilitation, followed by depression with abnormal decrement, can elicit disorders in the neuromuscular junction and/or block neuromuscular transmission even when no clinical involvement occurs. Electromyography (EMG). Electromyographic studies were performed to detect neurogenic or myopathic lesions of the neuromuscular system (Kimura, 1983; Aminoff, 1987). The electromyographic examination was performed at rest and at voluntary muscle activity. To record the muscle electrical activity, coaxial needle electrodes of 0.45 mm diameter and 30 mm length were used. The examined muscles were the first dorsal interossei of the hands and the orbicularis oculi. Insertion activity was recorded and reported as normal (if it ceased in 2-3 set) or prolonged. During rest, in fully relaxed muscle, no electrical activity was expected in a normal muscle; if electrical activity was recorded, it was considered as spontaneous activity and the potentials were characterized as fibrillation, positive sharp waves, or fasciculations, according to their amplitude and duration. During voluntary activity, recruitment patterns of the muscle action potentials were examined at weak and maximal effort. During graded voluntary muscle contractions, the recruitment frequency and the amplitude, as well as the duration and the shape, of the individual motor units were measured. Treatment Pyridostigmine bromide 30 mg tablets (Duphar, Holland) were used. Placebo tablets were of similar appearance and were prepared by the same manufacturer. Medications were administered three times daily (at 8 AM, 4 PM, and midnight). Administration was observed by supervising physicians to ensure that the treatment protocol was strictly followed.

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Cholinesterase Determination

TABLE WHOLE

BLOOD

I

CHOLINESTERASE

ACTIVITY

Whole blood cholinesterase activity was Treatment group carried out according to Johnson and Russel Placebo group (n = 16) (n = 18) Day (1975) once during the preparation stage (baseline values), on the sixth and ninth days Baseline 100 100 of treatment, and on the fifth day after dis- 6” 100 * 2 II IL 2 continuation. Samples were drawn 2 hr fol- 9” 100, 1 7-l zk 2 Postb 101 + 2 lowing the morning dose of pyridostigmine, 100 +_ 2 at about the same hour when most of the Note. Results are given as percentage of baseline choabove-described tests were performed on the linesterase activity (baseline = 100%) + SEM. eighth day. Results for each subject were cala 6, 9-sixth and ninth days of treatment. culated as percentages of baseline cholinesterb post, fifth day after discontinuation of treatment. ase activity. Statistical Methods

Muscle Strength

The results of the muscle strength tests are Mean scores of the two treatment groups presented in Tables 2 and 3. The two groups for the various strength and endurance tests were not significantly different in the absolute were compared using Student’s t test, after strength values. In isometric handgrip (Table proving normal and equal distribution of val- 3), elbow extensors (Table 2), and elbow flexues in both groups (by Shapiro-Wilk test and ors (Table 2), no significant differences beF test, respectively). If abnormal or unequal tween treatment groups were found in average distribution was found, aparametric Wilcoxon changes from baseline as well. ranked sum test was used for comparison. Comparison between the groups of average Since final matching between groups was not changes of knee extensors and flexors isokiperfect (due to withdrawal of one subject) and netic strength revealed a statistically significant in order to control for training effect from one difference between groups. For knee extensors, test session to another, the percentage change an average improvement of 8% from baseline from baseline for every variable in every sub- values to the eighth day of treatment was ject was calculated. Average percentage change found in the placebo group and only 0.7% improvement was found in the treatment group. from baseline for each group was calculated This difference is statistically significant. For and compared between groups, using the same the knee flexors, an average improvement in statistical methods. p < 0.05 was defined as performance of about 2 1% in the placebo significant. group between baseline and the eighth day of treatment was found, whereas only 5.1% imRESULTS provement was found in the treatment group. This difference was also statistically significant. Cholinesterase Activity Both values of placebo group performance Results are presented in Table 1. Baseline were much closer to baseline after cessation cholinesterase values were within the normal of treatment. range in all subjects. Seventy-seven percent activity (23% inhibition) was observed on the Muscle Endurance sixth and ninth days of treatment in the pyrResults are presented in Table 4. idostigmine group, with normal activity values No significant difference was found between 5 days following cessation of treatment. the groups in the absolute values of the en-

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TABLE 2 ISOK~NETICSTRENGTH TESTS Test Day Baseline

8’

Post’

Group Placebo (n = Treatment (n p absoluten,’ Placebo (n = Treatment (n p absolute”’ p changed,b Placebo (n = Treatment (n p absoluten,b p changed,b

16) = 18) 16) = 18) 16) = 18)

Left knee extensors

Left knee flexors

Right elbow extensors

Right elbow flexors

180.93 + 9.15 187.94 k 7.61 NS 195.06 f 10.71 189.80 + 11.2 NS <0.05 181.62 f 11.20 179.00 + 8.50 NS NS

101.69 + 4.33 110.28 + 5.17 NS 124.39 f 8.25 114.67 A 4.90 NS <0.05 110.12 f 6.04 117.70 f 5.80 NS NS

44.94 % 2.35 47.61 i 3.07 NS 50.44 + 3.38 49.94 + 3.70 NS NS 48.69 f 3.20 47.21 iz 2.60 NS NS

50.00 -+ 3.34 47.61 + 2.86 NS 43.94 f 2.95 42.60 + 1.80 NS NS 44.25 k 3.23 42.47 f 2.05 NS NS

Note. Results are given in Newton meters (mean f SEM). 0 p absolute, significance of difference between absolute values. ’ NS, not significant. c Eighth day of treatment. dpchange, significance of difference between changes of values from baseline values. ’ Post, 5 days after discontinuation of treatment.

durance test, but the placebo group showed an 8% reduction in endurance from baseline to the eighth day of treatment, whereas the treatment group showed only a 1% reduction. This difference between groups was statistically significant. The placebo group value after ces-

sation of treatment was again much closer to baseline value. Nerve Conduction Velocity

Nerve conduction velocity and time (Fwave and distal latencies) were in the normal

TABLE 3 MAXIMAL ISOMETRIC HANDGRIP STRENGTH

Day

Baseline 8d Post’

Placebo group (n = 16)

Treatment group (n = 18)

pa.b (absolute)

(change)

33.94 + 2.35 36.90 i 2.32 34.63 + 2.32

29.87 + 1.32 32.41 i 1.36 29.85 k 1.58

NS NS NS

NS NS

Note. Results are given in kilograms (mean + SEM). ’ p (absolute), significance of difference between absolute values of groups. b NS, not significant. cp (change), significance of difference between changes of values from baseline values. d Eighth treatment day. e Post, 5 days after discontinuation of treatment.

pc.b

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TABLE 4 MUSCULARISOKINETICENDURANCE-RIGHTKNEEEXTENSORS

Day

Baseline 8d PostP

pc.b

Placebo group (n = 16)

Treatment group (n = 18)

(absolute)

(change)

58.06 f 1.67 50.88 + 1.66 56.00 + 1.90

55.00 f 1.40 52.65 + 1.12 55.90 f 2.10

NS NS NS

<0.05 NS

pa.b

Note. Results are given in percentages (mean f SEM)-see text for further details. np (absolute), significance of difference between absolute values of groups. b NS, not significant. ‘p (change), significance of difference between changes of values from baseline values. d Eighth treatment day. ’ Post, 5 days after discontinuation of treatment.

range in all the subjects before and during treatment. Results for individual components after treatment were not different from control values. No changes were found between the subjects who received pyridostigmine and those who received placebo.

ment. No changes were found for either each group or each individual. There were no fasciculations and no evidence of increased motor unit irritability was observed.

Repetitive Nerve Stimulation

In this study we tried to find out whether pyridostigmine, in a small dosage, intended to inhibit cholinesterase activity by 20-40% and thus protect healthy subjects from the effect of organophosphate poisoning, could cause any detrimental effects on neuromuscular functions. This was based on observations, in animals, of various adverse effects of carbamate exposure on the neuromuscular junction, which usually occurred following exposure to relatively large doses of carbamates, as is discussed later.

Test

There were no findings suggesting neuromuscular block. After voluntary contraction, no postactivation facilitation or exhaustion was found. In one subject of the placebo group and in three of the treated group, repetitive stimulations were normal both before and during treatment. One subject of the treated group showed a decremental response of 7% at slow rates, before and during treatment. This patient response was considered normal (Desmedt and Borenstein, 1970). One subject of the placebo group showed a decremental response of 40% before and during the study period without any clinical complaints. Electromyography Electromyographic examination was normal in all subjects before and during treat-

DISCUSSION

Cholinesterase Activity Technical difficulties prevented blood sampling during the muscle strength and endurance tests. However, cholinesterase was determined while the subjects were on the same drug regime 2 days before and 1 day after the muscle and electrophysiologic tests, at the same hour. Since pyridostigmine achieves steady-state kinetics on the third day of treat-

PYRIDOSTIGMINE

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ment (Gall, 1981) we can assume similar cholinesterase activities at the time of the tests. The average cholinesterase inhibition in our series is somewhat lower than the value described by Gall (198 l), but higher than that described by Graham et al. (1984). Our inhibition values were in the desirable range of 20-40%, expected to protect against organophosphate poisoning without causing any side effects (Gall, 198 1). Muscle Strength and Endurance A very subtle trend toward a difference between the groups, favoring the placebo group, has been shown in two of five muscle groups by comparing changes from baseline, but not absolute values. The significance of differences between treatment groups in both knee flexors and extensors arises from the especially high values of performance of the placebo group on the eighth day of treatment, while 5 days after cessation of treatment values were again much closer to baseline. On the other hand, the performance in the treatment group tended to be very constant. The physiological significance of this improvement in the placebo group is unclear and it may probably represent only incidental intragroup variability between days. Therefore, the difference between groups probably does not represent a true detrimental effect of pyridostigmine. The same is true regarding results of the endurance tests. While minimal significant advantage to the pyridostigmine group was found in muscle endurance, this significance arose from an extremely low performance of the placebo group on the eighth day of treatment, the reason for which is unclear. Pyridostigmine group performance was much more consistent throughout baseline, treatment, and posttreatment time. Therefore, all differences between groups did not involve absolute values but only changes from baseline. They are clinically in-

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significant and probably arise from large fluctuations in performance in the placebo group and not from pyridostigmine effect, although such an effect cannot be absolutely excluded. Graham et al. (1984) examined the influence of pyridostigmine administered in a similar dosage, 30 mg three times daily, for 5 days on various functions including handgrip strength. Cholinesterase inhibitions were lower than those in our series and measured only 8%. They found a statistically insignificant trend toward decreased performance in this test under pyridostigmine treatment. No further strength tests were performed by them and there are no other works done on healthy subjects that examine this issue. In the present study, a small difference in muscle endurance was found between the groups, which is probably unrelated to pyridostigmine. It is interesting that neostigmine, an analogue of pyridostigmine, has been found to improve the force generated by fatigued diaphragms in anesthetized dogs (Howell et al., 1985). On the other hand, Francesconi et al. (1984) found a decrease in the ability of rats treated by pyridostigmine to work in heat with increased plasma lactate, probably resulting from increased production of lactate by exercising muscles. This change probably reflected altered exercising muscle metabolism under the influence of pyridostigmine. It is noteworthy that they achieved a much higher cholinesterase inhibition (average 64%). In a later work, no such effects were observed when cholinesterase inhibitions were in the range of 23-39% (Francesconi et al., 1986). Electrophysiologic

Tests

The electrophysiologic examination is a very useful method for early detection of various types of neuromuscular disorders as well as for monitoring neuromuscular transmission and function during exposure to various medications and toxins (De Jesus et al., 1973; Donat and Donat, 1981; Hazelwood et al., 1979).

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In the present study we found that an 8-day treatment with pyridostigmine, 30 mg three times daily, did not cause motor neuropathy. The mean values of nerve conduction velocity and distal and F-wave latencies were not altered by treatment and there was no decrease of the amplitude of the evoked potentials. The findings of unchanged nerve conduction velocity measurements after treatment with carbamates were also shown in dogs treated with neostigmine (Hazelwood et al., 1979). It is worthwhile to note that nerve conduction velocity was not expected to change under the conditions of the experiment, since large diameter fibers are not affected in such a short time and at such low doses of drug. We did not observe electrophysiologically myopathic or neurogenic changes in the examined muscles. No fibrillation potentials or high frequency discharges were detected; afterdischarge activity was not enhanced by pyridostigmine. Hazelwood et al. ( 1979) found increased after-discharge activity and fasciculations in animals treated with neostigmine. The differences in results can be related to different carbamates, to different dosages and routes of administration, and to the fact that the dogs were also anesthetized and atropinized during the electromyographic examination. Repetitive stimulation tests in our study demonstrate no changes in three of four subjects treated with pyridostigmine. The fourth subject was considered normal since the decremental response was below 10% during treatment. The occasional finding of a decremental response of 40% in the placebo-treated subject can imply subclinical myasthenia gravis. Considering the results of the electrodiagnostic tests, we conclude that pyridostigmine, in a dosage of 30 mg three times daily for 8 days, causing 20-30% cholinesterase inhibition, neither affected neuromuscular function nor produced any electrophysiological abnormalities. Numerous investigations, carried out on rats, dealt with adverse effects of carbamate

ET AL.

treatment (mainly neostigmine) on electrophysiological properties of the neuromuscular junction. These effects included a decrease in miniature end-plate potential attributed mostly to presynaptic changes (Engel et al., 1973; Tiedt et al., 1978; Ward et al., 1975); decreases in the quanta1 content of end-plate potentials and the number of quanta available for release, resulting in decreased acetylcholine release per nerve stimulus (Chang et al., 1973; Roberts and Thesleff, 1969; Tiedt et al., 1978); and a decrease in the number of acetylcholine receptors in the postsynaptic area (Chang et al., 1973). Although still incompletely understood, many of these changes can be explained by the inhibition of synaptic acetylcholinesterase causing prolonged accumulation of acetylcholine in the synaptic cleft with desensitization and down regulation of acetylcholine receptors as well as possible negative feedback inhibition of acetylcholine release and synthesis. All these experiments involved relatively large doses of carbamates which probably resulted in acetylcholine inhibition higher than that in our study (although they were not measured in most of these experiments). Changes were also described in myasthenic patients treated with pyridostigmine for prolonged periods. Yet, these changes could not be distinctly differentiated from those caused by the disease (Fenichel, 1966; Fenichel and Shy, 1963; Fenichel et al., 1972). In our study we were not able to demonstrate any changes in the electrophysiologic or muscle function parameters. Although minute changes in neuromuscular synapse structure or function in our subjects cannot be excluded, we assume that no significant change occurred due to the lower inhibition of blood (and therefore synaptic) cholinesterase activity in comparison with the animal studies. Higher cholinesterase inhibition or longer exposure times are probably required in order to initiate the assumed sequence of events terminating in significant effects on neuromuscular synapse function.

PYRIDOSTIGMINE

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We, therefore, conclude that in healthy young men given pyridostigmine in a dosage of 30 mg three times daily for 8 days, causing 20-30% cholinesterase inhibition, no significant neuromuscular adverse effects occurred. REFERENCES ACHIRON, A., ANDSAROVA-PINCHAS,~.(~ 984). Thevalue of F wave response in Bell’s palsy-a study of F wave response in the facial muscles. Electromyogr. Clin. Neurophysiol. 24, 99-106. AMINOFF, M. J. (1987). Electromyography in Clinical Practice: Disease,

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