Plasma pyridostigmine levels in patients with myasthenia gravis Plasma eoneentrations of pyridostigmine were measured in 7 patients with myasthenia gravis. Six subjects on oral pyridostigmine bromide were stabilized on widely different doses of the drug (60 to 660 mg/day). Nevertheless, the eoneentration of the quaternary amine in plasma was maintained within a relativeiy narrow range (usually between 20 and 60 ng/ml). In 3 myasthenie patients, the area under the plasma eoneentration-time curve was relatively eonstant for 4 hr after the same oral dose of pyridostigmine (60 mg). Despite this similarity, there were in general eonsiderable interindividual differenees in the bioavailability of pyridostigmine in myasthenie patients. In f subjeet, the bioavailability of the quaternary amine was inereased sixfold by doubling the oral dose from 30 mg to 60 mg. After oral administration of pyridostigmine, the half-life of the drug in one subjeet (4.25 hr) was almost three times as great as after intramuseular administration in a different patient (f.49 hr).

T. N. Calvey, M.D., Ph.D., and K. Chan, Ph.D.* Liverpool, England

Department of Pharmaeology and Therapeutics, University of Liverpool

Pyridostigmine bromide (Mestinon) has been extensively used in the treatment of myasthenia gravis for almost 20 yr. Many patients with this rare disorder of neuromuscular function can be adequately controlled on oral pyridostigmine, although the dosage of the quaternary amine may vary from 30 to 2,000 mg/day.5 Individual differences in the dosage requirements of pyridostigmine are commonly attributed to varying severity in the course of the disease. The role of interindividual differences in drug Supported in part by the Muscular Dystrophy Group of Great Britain and the Peel Medical Research Trust. Received for publication July 6, 1976. Accepted for publication Aug. 24, 1976. Reprint requests 10: Dr. T. N. Calvey, Department of Pharmacol· ogy and Therapeutics, University ofLiverpool, Liverpool L69 3BX, England. *Present address: School of Pharmacy, Liverpool Polytechnic, Liverpool, England.

metabolism and excretion is a matter of conjecture since analytic methods that can be applied to the routine measurement of pyridostigmine concentrations in man have not been developed. In general, previous approaches to the determination of pyridostigmine in plasma and urine of myasthenia patients have relied on two distinct and mutually exclusive analytic techniques. In the first place, the clearance of 14C-pyridostigmine and its metabolites from myasthenie plasma and urine has been studied, using the radiolabeled quaternary amine. 6 , 9 AIthough values for the plasma concentration of the unchanged drug were not quoted, interpolation of the experimental data suggests that intravenous injection of 14C-pyridostigmine bromide (2.0 mg; approximately equivalent to 60 mg orally) results in maximal plasma concentrations of less than 1 ng/m1. 6 An alternative 187

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Table I. Daily dosage of pyridostigmine and other drugs in the 7 myasthenie patients Oral dose o{ pyridostigmine bromide

Patient

Age

1

2 3 4

40 43 40 64

F F F F

300 300 660 60

5

38

M

660 mg/day

6

40

F

480 mg/day

7

62

F

2 mg (intramuscularly)

Sex

analytie teehnique depends on the extraetion of pyridostigmine from plasma as an iodide ionpair eomplex into methylene diehloride; the eoneentration of the drug is then ealculated from the optieal density of the eomplex at two different wavelengths. 4 The limit of sensitivity of this speetrophotometrie method is relatively low (approximately 170 ng/ml), and it does not appear to separate pyridostigmine from its prineipal metabolites. After oral administration of pyridostigmine bromide (120 mg) to 4 myasthenie patients, the maximal plasma eoneentrations ranged from 220 to 420 ng/m1 (i.e., at least 200 to 400 times as great as eoneentrations produeed by approximately equivalent doses of the radiolabeled eompound). 4 In these two studies, pyridostigmine eoneentrations were measured after oral administration of two different dosage forms of the drug; one of these preparations was radiolabeled. Under these eonditions, differenees in the formulation of the labeled and nonlabeled drug might result in variations in the plasma eoneentration and biologie availability of the quaternary amine, but this eannot entirely aeeount for the large differenees in the plasma eoneentration of pyridostigmine obtained by two different methods. It was therefore deeided to develop a suitable method for the estimation of pyridostigmine in human plasma. Teehniques based on gas-liquid ehromatography have been used for the quantitative determination of other quaternary

mg/day mg/day mg/day mg/day

Other drugs concurrently administered None None None Bendrofluazide (10 mg/day) Chlorpheniramine (12 mg/day) Mec1ofenoxate (900 mg/day) Tocopheryl acetate (3 mg/day) Ampicillin (2,000 mg/day)

amines, and we have reeently developed a sensitive and speeifie analytie method for the measurement of pyridostigmine in plasma. 3 The present paper is eoneerned with the eoneentration of the drug in plasma and its c1earanee from the eireulation in 7 patients treated with oral or intramuseular pyridostigmine. Patients and methods

Subjects. Studies were earried out on 7 adult patients with myasthenia gravis. Six patients were stabilized on varying amounts of oral pyridostigmine bromide (60 to 660 mg/day; Table I) in divided doses. All of these patients were ambulant. The seventh patient was treated in an intensive eare unit with pyridostigmine bromide (2.0 mg, intramuscularly) as required. Three myasthenie patients were treated with pyridostigmine alone; the other 4 reeeived other drugs (Table I). Procedure. The dosage sehedule of eaeh patient was not modified in any way. After the initial morning dose of pyridostigmine (30 mg, 60 mg, or 120 mg orally, or 2.0 mg intramuseularly), blood sampies were removed by venipuneture at half-hourly or hourly intervals for at least 4 hours; subsequent speeimens were usually obtained every 2 hr. Blood (8 to 10 ml) was added to tubes eontaining lithium heparin; plasma was promptly obtained by eentrifugation and stored at -200 C. Measurement of plasma pyridostigmine. The eoneentration of pyridostigmine in plasma

Pyridostigmine levels in myasthenia

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was determined by a gas-liquid chromatographie method. The proeedure depends on the ion-pair extraetion of pyridostigmine followed by pyrolysis to the dequaternized tertiary amine, and this has been deseribed in detail in a previous publication. 3 In this study, the standard solutions of pyridostigmine used eontained eoneentrations of the quaternary amine ranging from 10 to 200 ng/ml. Results

Oral pyridostigmine. Three of the myasthenie patients studied were stabilized on intermittent dosage with single tablets (60 mg) of pyridostigmine bromide. In these patients, the maximum plasma eoneentration after the initial morning dose of pyridostigmine ranged from 44.2 to 54.5 ng/ml, and the area under the plasma eoneentration-time eurve during the first 4 hr was relatively eonstant (6,404 to 7,419 ng/ ml . min; Table 11). The maximum eoneentration of pyridostigmine in plasma was reaehed between 1 and 3 hr after oral administration of the quaternary amine (Fig. 1). In 2 patients, administration of a subsequent dose of pyridostigmine bromide (60 mg) indueed differential effeets on the eoneentration of the drug in plasma. In 1 subjeet, the seeond dose of pyridostigmine inereased the eoneentration of the drug in plasma from 15.9 ng/ml to 54.9 ng/ml, whereas little or no change in plasma eoneentration was observed in the seeond patient (Fig. 1). In another patient, the plasma eoneentration of pyridostigmine was measured far 9 hr after a single oral dose of the drug; the eoneentration of pyridostigmine deereased from 54.5 ng/ml to 20.5 ng/mI during a 6-hr period, eorresponding to a plasma half-life of 4.25 hr (Fig. 1). Three other myasthenie patients were aB stabilized on larger doses of oral pyridostigmine (480 to 660 mg/day). In 2 patients, the morning dose of pyridostigmine bromide (120 mg) resulted in maximum plasma eoneentrations of 54.7 and 77.0 ng/ml; the area under the plasma eoneentration-time eurve during the first 4 hr ~as 7,563 ng/ml' min and 10,320 ng/ ml . min, respeetively (Table II). In the third patient, a larger morning dose of pyridostigmine (180 mg) resulted in a lower peak plasma

189

Table 11. Bioavailability of pyridostigmine 0-4 hr after oral and intramuseular administration

Patient

I 2 3 4 5 6 7

Maximum concenDose of pyridostigmine tration bromide (ng/ml)

60 60 120 60 120 180 2

mg mg mg mg mg mg mg

orally orally orally orally orally orally im

44.2 58.6 54.7 54.5 77.0 53.0 62.4

Area under plasma conr:entration -time cun'e (ng/mi . min)

7,419 6,404 7,563 7,401 10,320 5,955 5,755

Table 111. Plasma eoneentrations of pyridostigmine after oral administration of two different doses (30 mg and 60 mg) in the same myasthenie patient

Time (hr)

1.0 1.5 2.0 3.0 4.0 5.0 7.0 9.0 Bioavailability

Plasma concentration of pyridostigmine (ng /m!) Dose = 30 mg

Dose = 60 mg

16.2 18.3 11.0 7.0 9.0 8.0 Not detectable Not detectable

11.2

45.6 32.6 20.5

2,976 ng/ ml' min

18,168 ng/ ml' min

32.9 54.5

eoneentration (53.0 ng/ml) and redueed bioavailability (5,955 ng/ml . min; Table II); however, after a further oral dose of pyridostigmine (60 mg) the plasma level rose to 84 ng/ml (Fig. 2). In 2 of these myasthenie patients, the initial rise in the plasma eoneentration of pyridostigmine was followed by a seeond peak 4 to 6 hr later (Fig. 2) whieh was not related to subsequent drug administration. In 1 subjeet, the bioavailability of pyridostigmine (as measured by the area under the plasma eoneentration-time eurve) was compared after different oral doses of the drug (30 mg and 60 mg). After 60 mg pyridostigmine bromide, the bioavailability of the quaternary

190

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Clinical Pharmacology and Therapeutics

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amine (18,168 ng/ ml . min) was more than 6 times as great as after an oral dose of 30 mg (Table III). Intramuscular pyridostigmine. After intramuseular injeetion of pyridostigmine bromide (2.0 mg), the eoneentration of the drug in plasma was maximal after 30 min. Subsequently, the plasma level rapidly fell sharply (from 62.4 ng/ml at 30 min to 33.7 ng/ml after 1 hr) and then declined more slowly (Fig. 3). During the first 3 hr, the area under the plasma eoneentration-time eurve was 5,10 1 ng/ ml . min, and the plasma half-life of the drug was 1.49 hr. Discussion

In this study, the 6 myasthenie patients on oral pyridostigmine were stabilized on widely different doses of the drug (60 to 660 mg/day). Nevertheless, the eoneentration of the quaternary amine in plasma was maintained within a relatively narrow range; during the period of investigation, the peak plasma eoneentration

ranged only from 44.2 to 84.0 ng/ml, in spite of the ll-fold differenee in daily dosage. In most instanees, the plasma eoneentration was maintained between 20 and 60 ng/ml, and the area under the plasma eoneentration-time eurve within 4 hr of oral administration was relatively eonstant (5,955 to 10,320 ng/ml . min) irrespeetive of the dose. These observations suggest that the differential dosage requirements of pyridostigmine in myasthenie patients may be partially related to interindividual differenees in drug absorption, metabolism, or exeretion, as weIl as to the evaneseent and variable nature of the disease. These proeesses mayaiso be modified by the eoneurrent administration of other drugs. Thus, pyridostigmine is eliminated in urine by renal tubular seeretion as weIl as glomerular filtration, 2 and the clearanee of the drug is redueed by eoneurrent administration of some tertiary amines; these eompounds are partially present as eations at a physiologie pB, and may therefore eompete with pyridostigmine for renal

Pyridostigmine levels in myasthenia

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tubular transport. 2 The 2 myasthenie patients with the highest peak plasma eoncentrations of pyridostigmine (Patients 5 and 6) were receiving eoneurrent therapy with tertiary amines. The possible signifieanee of this phenomenon is a matter of eonjeeture; drug interaetions between pyridostigmine and other drugs in patients with myasthenia gravis have not been previously reported. The presenee and signifieanee of interindividual differenees in the absorption of pyridostigmine from the gut are diffieult to assess. In 3 myasthenie patients, the area under the plasma eoneentration-time eurve was maintained within a narrow range (i.e., 6,404 to 7,419 ng/ml . min) 4 hr after the same oral dose (60 mg), suggesting that absorption of the drug from the gut is relatively eonstant in different subjeets. On the other hand, in I subjeet the

bioavailability of the drug was inereased 6-fold (from 2,976 ng/ml' min to 18,168 ng/ ml . min) by doubling the dose of pyridostigmine bromide (from 30 mg to 60 mg). These observations suggest that absorption of the drug from the gut does not obey first-order kineties; the quaternary amine may undergo a signifieant "first-pass effeet" in the hepatie sinusoids. The former explanation is eonsistent with physieoehemieal eonsiderations, whieh suggest that pyridostigmine will be poorly and unpredietably absorbed from the gut. Quaternary amines are permanently ionized irrespeetive of pH, and therefore eannot readily penetrate lipophilie eellular membranes in the small intestine. Absorption of pyridostigmine is presumably greatest in the duodenum, whieh has a relatively large surfaee area; in our experiments, the plasma eoneentration of pyridostig-

192

Calvey and Chan

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Clinical Pharmacology and Therapeutics

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mine invariably increased within 1 to 2 hr of oral administration of the drug. Nevertheless, there may be some absorption throughout the gastrointestinal tract, since the difference between equieffective oral (60 mg) and intramuscular (2 mg) doses suggests that most of the drug is eliminated in feces after oral administration. In the 6 myasthenic patients on oral pyridostigmine, there were frequent f1uctuations in the plasma concentration which were apparently not related to drug administration. These f1uctuations are not observed after intravenous administration of the quaternary amine. 3 In particular, 2 patients on large doses of pyridostigmine had a secondary rise in the plasma concentration of the drug 4 to 5 hr after an oral dose. Some quaternary amines are known to form complexes with intestinal mucin or bile salts that are readily extracted into organic solvents,7, 8 and the secondary rise may be re-

lated to the formation and absorption of such lipophilic complexes in the gut. After oral administration of pyridostigmine, the half-life of the drug in 1 subject (4.25 hr) was almost three times as great as after intramuscular administration in a different patient (1.49 hr). Although this difference could be due to some other aspect of individual variability, the longer half-life after oral administration may reflect continued absorption of the drug from the gut. In experimental animals with ligated renal pedicles, the half-life of pyridostigmine is 53.3 ± 7.0 min (mean ± standard error of the mean).l This is not far from the half-life after intramuscular administration of the drug in one of our myasthenic patients. In clinical practice, intramuscular pyridostigmine bromide (2 mg) and an oral tablet of the drug (60 mg) are generally considered to be equieffective. The results obtained in so me subjects suggest that in certain conditions the

Valurne 21 Nurnber 2

bioavailability of these two preparations is similar, as assessed by the maximum plasma eoneentration or the area under the plasma eoneentration-time eurve. In 3 patients, the maximum plasma eoneentration after the initial morning dose of pyridostigmine bromide (60 mg) ranged from 44.2 to 58.6 ng/ml, and the area under the plasma eoneentration-time eurve during the first 4 hr was relatively eonstant 6,404 to 7,419 ng/ml . min). After intramuseular administration of pyridostigmine (2 mg), the maximum plasma eoneentration (62.4 ng/ml) was aehieved within 30 min. The area under the eoneentration-time eurve during the first three hr was 5,101 ng/ml . min. These results suggest that the bioavailability of the two preparations (whether assessed by the peak plasma eoneentration or the area under the plasma eoneentration-time eurve) is elosely eomparable in some subjeets. The eomparison may not be universally applieable due to interindividual differenees in the biologie availability of pyridostigmine. In our experiments, the plasma eoneentration of pyridostigmine usuallY ranged from 20 to 60 ng/ ml. These results are not eonsistent with values obtained by other workers4 , 6 using different analytie teehniques. We have no explanation for these differenees. Clearly, further experienee with the present method is required to assess its value in the clinieal management of patients with myasthenia gravis. We are indebted to Drs. R. Finn, D. M. Freeman, M. Hayward, and R. R. Hughes, for allowing us to

Pyridostigmine levels in myasthenia

193

study patients under their care, and to the patients for their ready cooperation.

References I. Burdfield, P. A., and Calvey, T. N.: Plasma clearance of neostigmine and pyridostigmine in rats with ligated renal pedicles, Eur. J. Pharmacol. 24:252-255, 1973. 2. Chan, K., and Calvey, T. N.: Renal clearance of pyridostigmine in patients with myasthenia gravis, Eur. Neurol. (In press.) 3. Chan, K., Williams, N. E., Baty, J. 0., and Calvey, T. N.: A quantitative gas-liquid chromatographie method for the determination of neostigmine and pyridostigmine in human plasma, J. Chromatogr. 120:349-358, 1976. 4. Coper, H., Deyhle, G., and Dross, K.: Studies on the absorption of pyridostigmine: The application of a spectrophotometric method for the determination of pyridostigmine in plasma, Z. Klin. Chem. Klin. Biochem. 12:273-275, 1974. 5. Greene, R.: Medical treatment, in Greene, R., editor: Myasthenia gravis, London, 1969, William Heinemann, Ltd., pp. 135-137. 6. Kornfeld, P., Samuels, A. J., Wolf, R. L., and üsserman, K. E.: Metabolism of 14C-labeled pyridostigmine in myasthenia gravis, Neurology (Minneap.) 20:634-641, 1970. 7. Levine, R. M., and Clark, B. B.: The physiological disposition of oxyphenonium bromide (Antrenyl) and related compounds, J. Pharmacol. Exp. Ther. 121:63-70, 1957. 8. Schanker, L. S., and Solomon, H. M.: Active transport of quaternary ammonium compounds into bile, Am. J. Physiol. 204:829-832, 1963. 9. Somani, S. M., Roberts, J. B., and Wilson, A.: Pyridostigmine metabolism in man, CLIN. PHARMACOL. THER. 13:393-399, 1972.