Eur J Appl Physiol (1990) 61:128-132

Applied Physiology European Journal of

and Occupational Physiology @ Springer-Verlag I990

Effect of pyridostigmine on the exercise-heat response of man Y. Epstein, R. Arnon, D. Moran, D. S. Seidman, and Y. Danon Heller Institute of Medical Research Sheba Medical Center, Tel-Hashomer 52621, Israel Accepted March 20, 1990

Summary. The effect of pyridostigmine on thermoregulatory responses was evaluated during exercise and heat stress. Eight heat acclimated, young adult male subjects received four doses of pyridostigmine (30 rag) or identical placebo tablets every 8 h, in a double blind, randomized, cross-over trial. A 30.3%, SD 4.6% inhibition of the circulating cholinesterase (ChE) activity was induced in the pyridostigmine-treated group. The subjects were exposed to 170-rain exercise and heat-stress (dry bulb temperature, 33 ° C; relative humidity 60%) consisting of 60 min in a sitting position and two bouts of 50-min walking (1.39m-s -1, 5% gradient) which were separated by 10-min rest periods. No differences were found between treatments in the physiological responses and heat balance parameters at the end of exposure: heart rate (fc) was 141 beats.min -1, SD 16 and 150 beats-min -1, SD 12, rectal temperature (Tre) was 38.5 ° C, SD 0.4 ° and 38.6 ° C, SD 0.3 °, heat storage was 6 0 W . m -2, SD 16 and 5 9 W . m -2, SD 15 and sweat rate was 678 g . h -1, SD 184 and 661 g-h -1, SD 133, in the pyridostigmine and placebo treatments, respectively. The changes in Tre and fo over the heat-exercise period were parallel in both study and control groups. Pyridostigmine caused a slight slowing of fc (5 beats-min -1) which was consistent throughout the entire exposure (P< 0.001) but was of no clinical significance. The overall change in fo was similar for both groups. We have concluded that pyridostigmine administration, in a dose sufficient to induce a moderate degree of ChE inhibition, does not significantly affect performance of exercise in the heat. Key words: Pyridostigmine - Carbamates - Exerciseheat stress - Thermoregulation - Thermal stress

Introduction

Pyridostigmine bromide is a quaternary carbamate Offprint requests to: Y. Epstein

which reversibly inhibits cholinesterase (ChE) (Taylor 1985). It has been extensively used in the treatment of myasthenia gravis for almost 30 years (Drachman 1978). At therapeutic dosage levels (200-1400 mg. day-1, orally), pyridostigmine stimulates gastro-intestinal mobility, induces miosis, facilitates skeletal muscle contraction, stimulates salivary and sweat gland secretion, and causes bradycardia (Taylor 1985). By increasing sweat output, pyridostigmine could modify the state of hydration and thereby influence thermoregulation when working in a hot environment. Its effects on heart rate (fo) and skeletal muscle could affect strength and/or endurance. Acute administration of pyridostigmine to rats, in doses that inhibited plasma ChE by 65%, resulted in significant decrements in physical performance in the heat, adverse thermoregulatory effects, and exacerbated increases in indices of heat exercise injury (Francesconi et al. 1984). In a subsequent report (Francesconi et al. 1986), chronic oral administration of pyridostigmine in rats, in doses that inhibited plasma ChE by less than 40%, elicited no marked effects on thermoregulation and performance in the heat. Yet, in a recent report, the same group of investigators showed that acute i.v. administration of pyridostigmine, in doses that inhibited ChE by 40%, resulted in reduced endurance and an increased rate of increase in core temperature of exercising rats (Matthew et al. 1988). Avlonitau and Elizondo (1988) found an acute increase in effective sweating in Patas monkeys receiving pyridostigmine orally which induced a significant 25%-30% inhibition of serum ChE activity. Currently, pyridostigmine is considered as the only effective therapeutic agent against intoxication with the potent organophosphate (OP) soman (pinacolyl methylphosphonofluoridate) (Dunn and Sidell 1989; Maxwell et al. 1988). As a reversible ChE inhibitor, pyridostigmine prevents OP from phosphorylating and thus irreversibly inactivating the active site of ChE. Gall (1981) has suggested that pyridostigmine could be used as a pretreatment against OP poisoning. He proposed that oral administration of 30-rag pyridostigmine at 8-h intervals would preserve a proportion of CHE activity,

129 as j u d g e d b y the i n h i b i t i o n o f b l o o d e n z y m e activity b y a b o u t 30%. To date, sparse i n f o r m a t i o n is a v a i l a b l e c o n c e r n i n g the effects o f p y r i d o s t i g m i n e a d m i n i s t r a t i o n o n therm o r e g u l a t o r y a n d p h y s i o l o g i c a l r e s p o n s e s d u r i n g exercise in the h e a t i n h u m a n subjects. O n e dose o f 30-mg p y r i d o s t i g m i n e t a k e n b y n u d e subjects h a d o n l y m i n o r effects o n t o l e r a n c e to m o d e r a t e exercise heat-stress a n d did n o t aggravate the strain o f h y p o h y d r a t i o n ( W e n g e r a n d Latzka 1989). F o l l o w i n g G a l l ' s s u g g e s t i o n that p y r i d o s t i g m i n e tablets c o u l d be t a k e n for e x t e n d e d p e r i o d s o f time ( G a l l 1981) a n d the reports o f adverse effects o n t h e r m o r e g u l a t i o n r e p o r t e d i n a n i m a l m o d e l s ( A v l o n i t o u a n d E l i z o n d o 1988; M a t t h e w et al, 1988), the p r e s e n t s t u d y was u n d e r t a k e n to investigate the effect of c h r o n i c p y r i d o s t i g m i n e i n g e s t i o n b y h u m a n subjects o n t h e r m o r e g u l a t i o n .

Methods Subjects. Eight male volunteers participated in this study. Their mean age was 23.5 years, SD 1.1, mass 67.4 kg, SD 6.3, body surface area (AD) 1.81 m 2, SD 0,08 and maximal aerobic power (I?O2max) 51.1 m l - k g - l - m i n -1, SD 2.9. Prior to participation the subjects underwent a thorough medical examination and their baseline ChE activity was determined in whole blood. Subjects considered to be at risk from cardiovascular or heat intolerance were excluded. Participants were informed of the purpose and potential risks of the study and signed a statement of informed consent. During the experimental period subjects abstained from alcohol and both prescribed and non-prescribed medications. All parts of the study were approved by the Institution's Ethics Committee.

Protocol. Prior to experimental testing each subject's I?O2m~ was determined by a treadmill running test. The protocol was progressive in intensity and continuous in nature. Running velocity was 3.13 re.s-l; the initial treadmill gradient was zero and was increased by 2.5% increments every 2 rain. Established criteria were employed for determination of VO2m,x (Taylor et al. 1955). All testing was conducted during the summer (August-September) while the subjects were naturally acclimatized. In addition, subjects were heat acclimated to the specific experimental climatic conditions by walking on a level treadmill at 1.39 m-s-~ for two 50-min exercise bouts separated by 10-min rest, for 5 consecutive days in a hot-humid environment [ambient temperature (T,) = 35° C; relative humidity (r.h.)= 60%]. Additional heat acclimation sessions were completed on days between experimental testing. During acclimation, subjects wore shorts and tennis shoes. Following acclimation each subject completed two experimental exercise-heat stress tests in a controlled h0t-humid environment (T,=33°C; r.h.=60%). The tests were separated by 3 days of rest. A double blind, cross-over study was conducted. Py, ridostigmine bromide tablets (30 mg, Duphar) or a placebo of identical form were taken orally. To maintain a steady-state 30% inhibition of ChE activity, tablets were taken every 8 h (Gall 1981: Glickson et al. 1986); at 25 h, 17 h, 9 h and 1 h before heat exposure. Each exposure to heat lasted 180 min, consisting of 60-min rest, in a sitting position, followed by two bouts of 50-rain walking separated by a 10-rain rest. Exercise consisted of a treadmill walk at a speed of 1.39 m - s - ~ and 5% gradient. During the experimental exposures subjects wore standard cotton fatigues (thermal insulation - 0.15° C- m 2. W - 1, 0.95 clo; moister permeability index (im) -- 0.58). Measurements. Electrocardiograms were obtained with chest electrodes (CM5 placement) and radiotelemetered to an oscilloscope-

cardiotachometer unit (Lifescope, Nihon Koiden, Tokyo, Japan). Metabolic parameters [02 uptake, CO2 production and respiratory exchange ratio (RER)] were determined by open circuit spirometry from expired gases; they were measured toward the end of each bout of exercise. During the 1102maxtest and the experimental tests, the metabolic measurements were determined using an automated metabolic system (MMC Horizon, SensorMedics, Anaheim, Ca.). Rectal temperature (Tre) was obtained from a rectal thermistor probe (YSI no. 401, Yellow Springs Instrument, Yellow Springs, Ohio, USA) inserted N 10 cm beyond the anal sphincter. Skin tempertures were measured at three locations (chest, calf, forearm) using skin thermistor probes (YSI no._ 409, Yellow Springs Instrument). Weighted skin temperature (T~k) was calculated according to Burton (1935). Nude body mass (m) was determined on an electronic balance (accuracy ___10 g), before and after the exposures. Total body sweat rate (rhsw) was calculated from m loss adjusted for water intake and urine output; respiratory and metabolic losses of mass were considered negligible and were not taken into account (Kamon and Avellini 1976). Venous blood samples ( - 5 ml each) were drawn using heparinized Vacutainers (Becton Dikinson, Rutherford, NJ, USA). Three samples from each subject, on both testing days, were taken in a sitting position; immediately before entering the climatic chamber (60 min after the last tablet), after 60 min in the climatic chamber, and immediately after exercise had terminated. Blood was kept refrigerated and assayed within 2 h for ChE activity. The ChE activity was measured in whole blood using a radiometric method as described by Johnson and Ressel (1975). A reduction in ChE activity was presented as ChE inhibition using each subject's baseline values to represent 100% activity.

Calculations. Metabolic energy production (~Q) was calculated from direct 02 measurements and based on the appropriate RER. Heat storage (AS) was calculated as follows: AS=0.965.m-(0.8 Tre+0.2 Tsk)/AD; (W.m -2)

(1)

where: 0.965=specific heat of the body ( W . h . k g - l . ° C - 1 ) ; m (kg); At) (m2). The radiative and convective heat exchange with the environment and the evaporative cooling power needed to maintain thermal equilibrium (Ereq) were calculated as originally described by Givoni and Goldman (1972) using the measured T~kand M. Sweat evaporation from the skin (Esk) was then calculated as:

gsk~Eteq-AS" (W.m -2)

(2)

Sweat efficiency 01sw) - the ratio between evaporated and total sweat loss was calculated as: E~k ~sw = h~-~

(3)

where hsw=O.68rh~w/Ao (W.m -2) [0.68-heat of vaporization (W.h.g-1)l.

Statistical analys&. Paired t-tests and two-way analyses of variance with repeated measures were used, where appropriate, to Table 1. Blood cholinesterase inhibition during the exercise-heat test after ingestion of four doses of pyridostigmine or placebo Time (rain) 0 (60)* 60 (120) 180 (240)

Pyridostigmine

Placebo

%

SD

%

SD

30.3 32.9 28.9

4.6 7.2 3.0

3.0 2.0 1.1

3.3 2.2 2.4

* In brackets, time elapsed (min) from ingestion of last tablet

130

160

Table 2. Physiological responses and heat balance parameters after 180 rain of heat exposure Pyridostigmine

T~(f) (o C) T~k(f) (o C) fc(f) (beats-min -~) A S ( W - m -2) (R + C) (W. m z) E~k ( W . m -z) rh~w ( g . h -1) r/~w (%)

~'b-140

Placebo

•~m~'~120 4__4._._./jL.~~),,[ "~ '-t--Plaoeb° ~ ~

mean

SD

mean

SD

38.5 36.3 141 60 - 20.4 186 678 73.7

0.4 0.4 16 16 3.3 8 184 17.6

38.6 36.5 150 59 - 22.4 184 661 73.0

0.3 0.7 12 15 3.7 11 133 13.3

60 I

og.o ...... Pyridoetigrnine P acebo

cc 88,0 HI n LtJ F- 87.5 d I

,

, ,

I

I

I

I

.

,

,

.exercise -----7

0

80

I

GO

,

exeroise i

90 110120 TIME (rain)

,

,

,

180

,

i

i 80

i

I

I

170

Fig. 1. The changes in rectal temperature (° C) in heat stressed subjects treated with pyridostigmine or a placebo

i

"

.... "'' ....

.

~ - exerc, se -----] eo

eo TIME

Blood ChE inhibition after ingestion of four doses of 30-mg pyridostigmine at 8-h intervals averaged 30% (Table 1). No significant differences in the level of inhibition were found in the three samples taken during the tests, indicating that a steady-state was present. No inhibition in ChE activity was demonstrated following the placebo treatment. Physiological responses to exercise and heat stress and calculated heat balance parameters with pyridostigmine and placebo treatments are summarized in Table 2. No significant differences were observed in any of the parameters listed. Under both treatments the final Tre was similar, (38.5 ° C, SD 0.4 ° ande 38.6 ° C, SD 0.3 ° in the pyridostigmine and placebo groups, respectively). This is reflected in the equivalent amounts of

I

i

o

Results

0 s7.o LIJ DC 8G.6

/

T

search for significant differences. If a significant F value was found, critical differences were analysed by Tukey's procedure to locate the significant m e a n difference. Statistical significance was accepted at P < 0 . 0 5 level. All data have been presented as m e a n and SD.

1~288.6 D

~

re 100 l-et < LU 80 ..... ." "'-'.---'---'""

* A S was calculated for 120 min of the exercise phase. T~, Rectal temperature; T~k, Skin temperature;f~, Heart rate; AS, Heat storage; (R ÷ C), Radiative and convective heat exchange with the environment; E~k, Sweat evaporation from the skin; Thaw,Total body sweat rate; ~/~w, sweat efficiency; f, Final measurement

0

..... Pyridostigmine]

[

11o 12o

i

exerc, se ----] I I I ! 1so

17o

(min)

Fig. 2. Changes in heart rate (beats. rain-1) in heat stressed subjects treated with pyridostigmine or a placebo

heat stored in the body (AS = 60 W. m-2). Dry heat exchange with the environment was - 2 0 . 4 W.m -2, SD 3.3 for pyridostigmine-treated subjects and - 2 2 . 4 W - m -z, SD 3.7 for placebo-treated subjects, with no significant difference between them. Final fo was lower, though not significantly so following pyridostigmine compared to placebo (141 beats, min- a, SD 16 and 150 beats.min -1, SD 12, respectively). No effect of pyridostigmine, on sweat excretion or qsw was demonstrated. Sweat loss was 678 g-h -1, SD 184 with pyridostigmine treatment and 661g-h -a, SD 133 with placebo treatment. Similarly, r/sw was 73% with both treatments. Figure 1 exhibits the changes in Tre throughout the entire exposure, with both treatments. The pattern of change in Tre was the same for each and no effect of the drug was demonstrated. The time course of changes in fc are plotted in Fig. 2. Baseline fc was slightly lower (N 5 beats.min -a) with pyridostigmine treatment and remained so throughout the entire exposure (P__50%) and its effect on body heat regulation, via dopamine levels in the CNS (Mount and Oehme, 1981). In contrast, Lomax et al. (1985) reported that in rats injected with soman, hypothalamic ChE activity was reduced to approximately 30% of the normal level, concomitantly with a significant decrease in body tempera 7 ture. There is controversy about the effect of ChE inhibition on temperature regulation in humans. Some investigators have found an increase in body temperature (Namba et al. 1971), while others have observed hypothermia in cases of OP insecticide poisoning (Willems et al. 1971). Noteworthy in all the above investigations was the influence on thermoregulation attributed to ChE inhibition in the CNS. It should be recalled, that pyridostigmine is a quaternary carbamate and is, therefore, unable to cross the blood brain barrier (Taylor 1985). Even with very large doses (50% LDso) of pyridostigmine, brain ChE is only slightly inhibited (17%) (Deyi et al. 1981). The major effect of the drug is its action on the nicotinic acetylcholine receptors at the neuromuscular junction (Taylor 1985). It exerts its function by forming covalent linkages between its carbamyl portion and ChE (Wilson et al. 1960). The resulting carbamylated enzyme complex decomposes rapidly and enzyme activity is restored in a relatively short period (Reiner 1971). In addition to the primary effects on the nicotinic cholinergic receptor, the quaternary ammonium anti-ChE compounds have a direct action at some choline receptor sites, either cholinomimetic or cholinergic (Taylor 1985). The predominant effect on the heart from the peripheral action of accumulated acetylcholine is bradycardia (Willems et al. 1971). In our results following pyridostigmine a slight slowing of the pulse by about 5 beats-rain-1 was found, both at rest and during exercise (Fig. 2). This observation, described also by Gall (1981), was of no practical clinical significance. Investigations in rats showed that pyridostigmine may reduce endurance capacity, compromise thermoregulatory efficiency and exacerbate effects on several pathochemical indices of heat-exercise injury. Francesconi et al. (1986) implied from their experiments using rats that the degree of inhibition of ChE activity in the blood by pyridostigmine and the duration of drug administration may be responsible for the debilitating effect of pyridostigmine on thermoregulation and circulatory indices of heat-exercise injury. They showed that a high level of inhibition (64%) of circulating ChE resulted in a marked decrement in the ability of rats to work in the heat (Francesconi et al. 1986). A more moderate inhibition of ChE (40%), induced by acute administration of pyridostigmine to rats exercising in a moderate environment, also reduced endurance and increased the rate of increase in core temperature (Matthew 1988). However, when similar levels (39%) of ChE inhibition in the blood were elicited by "chronic" (14 days) ingestion of pyridostigmine by rats, no effect was

found on physical endurance and thermoregulation during exercise in the heat (Francesconi et al. 1986). This discrepancy was attributed to the different route of administration. Both the duration and toxicity of blood ChE inhibition have been found to be strongly dependent on the route of administration of pyridostigmine (Francesconi et al. 1986; Gall 1981). This is of significance, since protection by carbamates against intoxication by OP is through partial inhibition and is thus related to the extent of blood ChE inhibition (Heyl et al. 1980). The biological availability of this carbamate in humans after oral administration is low (7.6%) and its half-life in plasma is about 1.78 h (Aquilonius et al. 1980; BreyerPfaff et al. 1985). The drug should, therefore, be approximately 95% eliminated within 7-8 h after dosage. The blood levels of pyridostigmine and ChE inhibition have been shown to run closely parallel; after a single oral dose of 30 mg, a maximum level of 17 mg.m1-1 pyridostigmine, and 30% inhibition in ChE activity were found at 3-4 h (Gall 1981). A 2-day period of 30nag pyridostigmine every 8-h produced very similar curves. No cumulative inhibition of ChE beyond 40% was found even after 4 weeks of repeated doses (Gall 1981). Our data supports previous reports by Glickson et al. (1986) and Gall (1981), who showed a stable level of ChE inhibition, (30%-40%) after four doses of 30-nag pyridostigmine every 8 h. Furthermore, our results showed that the ChE inhibition levels are not influenced by hot environmental conditions or physical exercise. In summary, the present investigation demonstrated that physical performance and thermoregulation under exercise-heat stress is not adversely effected by moderate ChE inhibition which has been induced by repeated oral doses of pyridostigmine. References

Aquilonius S, Eckernas SA, Hartvig P, LindstromB, Osterman PO (1980) Pharmacokinetics and oral bio-availabilityof pyridostigmine in man. Eur J Clin Pharmacol 18:423-428 Avlonitou E, Elizondo R (1988) Effectof atropine and pyridostigmine in heat-stressed paras monkeys. Aviat Space Environ Med 59:544-548 Breyer-PfaffU, Maier U, BrinkmannAM, SchumrnF (1985) Pyridostigmine kinetics in healthy subjects and patients with myasthenia gravis. Clin Pharmacol Ther 37:495-501 Burton AC (1935) Human calorimetry.II. The average temperature of the tissues of the body. J Nutr 9:261-280 Deyi X, Wang L, Shuqiu P (1981) The inhibitionand protection of cholinesterase by physiostigmineand pyridostigmineagainst soman poisoning in vivo. Fundam Appl Toxicol 1:217-221 Drachman DB (1978) Myasthenia gravis. New Engl J Med 298:186-192 Dunn MA, Sidell FR (1989) Progress in medical defense against nerve agents. JAMA 262:649-652 Francesconi R, Hubbard R, Mager M (1984) Effectsof pyridostigmine on ability of rats to work in the heat. J Appl Physiol 56:891-895 Francesconi R, Hubbard R, Matthew C, Leva N, Young J, Pease V (1986) Oral pyridostigmineadministrationin rats: effect of thermoregulation, clinical chemistry, and performance in the heat. Pharmacol Biochem Behav 25:1071-1075

132 Gall D (1981) The use of therapeutic mixtures in the treatment of cholinesterase inhibition. Fundam Appl Toxicol 1:214-216 Givoni B, Goldman RF (1972) Predicting rectal temperature response to work, environment, and clothing. J Appl Physiol 32:812-822 Glickson M, Karni A, Ram Z (1986) No hyperglycemia after pyridostigmine administration. J Appl Physiol 60:344 Heyl WC, Harris LW, Stitcher DL (1980) Effects of carbamates on whole blood cholinesterase activity: chemical protection against soman. Drug Chem Toxicol 3:319-332 Johnson CD, Ressel RL (1975) A rapid simple radiometric assay for cholinesterase, suitable for multiple determinations. Anal Biochem 64:229-233 Kamon E, Avellini B (1976) Physiologic limits to work in the heat and evaporative coefficient for women. J Appl Physiol 41:7176 Lomax P, Kokka N, Lee RJ (1985) Acetylcholinesterase inhibitors and thermoregulation. In: Cooper KE, Lomax P, Schonbaum E, Veale WL (eds) Homeostasis and thermal stress. Karger, Basle, pp 108-112 Matthew CB, Hubbard RW, Francesconi RP, Glenn JT (1988) Carbamates, atropine, and diazepam: effects on performance in the running rat. Life Sci 42:1925-1931 Maxwell DM, Brecht KM, Lenz DE, O'Neill BL (1988) Effect of

carboxylase inhibition on carbamate protection against soman toxicity. J Pharmacol Exp Ther 246:986-991 Mount ME, Oehme FW (1981) Diagnostic criteria for carbaryl poisoning in sheep. Arch Environ Contain Toxicol 10:483495 Namba T, Nolte CT, Jackrel J, Grob D (1971) Poisoning due to organophosphate insecticides. Am J Med 50:475-492 Reiner E (1971) Spontaneous reactivation of phosphorylated and carbamylated cholinesterases. Bull WHO 44:109-112 Taylor HL, Buskirk E, Henschel A (1955) Maximal oxygen intake as an objective measure of cardiorespiratory performance. J Appl Physiol 8:73-80 Taylor P (1985) Anticholinesterase agents. In: Gilman AG, Goodman LS, Rail TW, Murad F (eds) The Pharmacological basis of therapeutics, 7th edn. Macmillan, New York, pp 110-129 Wenger CB, Latzka A (1989) Effect of pyridostigmine pretreatment on physiological responses to heat, exercise, and hypohydration. Proc Med Def Biosci Rev pp 841-844 Willems J, Vermeire P, Rolly G (1971) Some observations on severe human poisoning with organophosphate pesticides. Arch Toxicol 28:182-191 Wilson LB, Hatch MA, Ginsburg S (1960) Carbamylation of acetylcholinesterase. J Biol Chem 235:2312-2315