European Journal of Clinical Pharmacology
Europ. J. clin. Pharmacol. 15, 367-371 (1979)
© by Springer-Verlag 1979
A Pharmacokinetic Study of Neostigndne in Man Using Gas Chromatography-Mass Spectrometry S.-M. Aquilonius, S.-A. Eckernfis, P. Hartvig 2, J. Hultman 1, B. Lindstr6m 3, and P. O. Osterman Departments of Neurology,AnaesthesiologyI and Pharmacy2, UniversityHospital, Uppsala, and Departmentof Drugs3, National Board of Health and Welfare, Uppsala, Sweden
Summary. To permit rational evaluation of the empirical pharmacotherapy of myasthenia with cholinesterase inhibitors, a sensitive and selective method for the determination of neostigmine has been developed. Analysis is based on ion-pair extraction of neostigmine into methylene chloride and determination by gas chromatography-mass spectrometry (chemical ionization). As neostigmine was found to be metabolized in plasma in vitro, deuterated (d6) neostigmine was immediately added to the plasma sample as the internal standard. The limit of quantitation of the method was about i ng/ml ( 3nmol/1). The kinetics following i. v. administration were studied in four patients, who received neostigmine 2.5-3.0mg iv to antagonize pancurone administered during anaesthesia. Elimination was rapid with a half-life tl/2 (/3-slope) of 0.89 + 0.05 h (mean _+ SE). The volume of distribution was 1.08 + 0.11 t/kg and plasma clearance was 0.84 +_ 0.041/ kg/h. In three fasting myasthenic patients plasma concentrations of neostigmine were followed for 5 h after a single oral dose of 30mg. Considerable interindividual differences in absorption were expressed in the peak concentrations, which occurred 1-2 h following drug ingestion. The bioavailability of neostigmine was estimated to be 1-2% of the ingested dose. Neostigmine concentration in plasma was found to differ considerably (up to forty-fold) between myasthenic patients on their ordinary doseschedules of cholinesterase inhibitors. Key words: myasthenia gravis, neostigmine; gas chromatography-mass spectrometry, pharmacokinetics Although quaternary cholinesterase inhibitors have been the basic drugs for treatment of myasthenia
gravis for the past 40 years (Remen, 1932; Walker, 1934), it is only recently that pharmacokinetic studies of them have been initiated in man. The first investigation of the basic pharmacokinetics were performed by Komfeld et al. (1970, 1971) with 14-Clabelled pyridostigmine. I.ater, two different gas chromatographic procedures for the assay of neostigmine and pyridostigmine were described (Chan et al., 1976; Cohan et al., 1976). None of the methods, however, seems to be sufficiently sensitive or selective to permit accurate determination of neostigmine in the plasma of myasthenic patients receiving oral treatment. In this paper a method for gas chromatographicmass spectrometric assay of neostigmine is described with use of deuterated drug as the internal standard. The method has been applied in a clinical study of the pharmacokinetics of neostigmine after its intravenous (iv) and oral administration.
Patients and Methods Reagents and Chemica& d6-neostigmine (3-hydroxyphenyltrimethyl-ammoniumbromide dimethyl (6-2H)-carbamate) was prepared from 3-(CH3)2-N-C 6 HsOCOCI in benzene and 30% (C2H3)2 Nit in water (Yanagisawa, 1951). The mixture was stirred for one hour under ice cooling. The benzene layer was dried and evaporated and the residue was treated overnight with a ten-fold excess of CH3Br. Glycine buffer 0.1 M prepared from glycine 7.5 g and sodium chloride 5.85 g was diluted to one litre with water. Glycine buffer with potassium iodide was made from potassium iodide 12.8 g, 0.1 M sodium hydroxide 4 ml and 0.1 M glycine buffer 6 ml. 0031-6970/79/0015/0367/$01.00
368
S,-M. Aquilonius et al.: Pharmacokineticsof Neostigmine
Ratio do/d6-10 O
3.5
Plasma sampie,5ng/ml Blank plasma
3.0
2.5 2,0
1.5 1.C
m/e 209
0,5
'
3'
,l
;
........
;
ng/rnl
Fig. 1. Typical standard curve for the determination of neostigmine by gas chromatography-massspectrometry. Each point represents the mean of 2-4 determinations
m/e 215
Fig. 2. Typical chromatogramsof plasma containing (left)neostigmine 5 ng/ml and internal standard (d6-neostigmine),and of blank plasma (right)containing only d6-neostigmine The injector temperature was 200 ~ C. The flow of methane carrier gas was 15 ml/min. The mass spectrometer was focused at m/e = 209 for neostigmine and at m / e = 215 for d6-neostigmine.
initial coflc, lO
Determination of Plasma Neostigmine Concentration 50
\\ !
Hours
Fig. 3. Decline in neostigmineconcentration in plasma followingin vitro incubation of plasma and blood at different temperatures. O O plasma, 4° C; A . . . . /x plasma, 22° C; • • plasma, 37° C; A - - - • blood, 37° C
Gas Chromatography- Mass Spectrometry A Finnigan 4000 gas chromatograph-mass spectrometer with chemical ionization was used. The glass column (1.5 m × 2 mm) was filled with 3 % O V 225 on Gas C h r o m Q~ and was operated at 185 °C.
Venous blood samples were collected, immediately cooled and the plasma separated. Internal standard solution 0.1 ml (d6-neostigmine, 2 vg/ml) was added to 2 ml plasma and the sample was frozen until analysed. Extraction was performed essentially as described by Chan et al. (1976). For analysis, the sample was thawed and made alkaline with 5 M sodium hydroxide 20 ~tl. After washing twice with diethyl ether 5 ml, glycine buffer with potassium iodide 1 ml and methylene chloride 6 ml were added. The mixture was shaken for 10 rain and after centrifugation the methylene chloride phase was removed and dried over sodium sulphate. After evaporation of the organic solvent, the residue was reconstituted in methanol and 1 ~tt was taken for analysis by gas chromatography-mass spectrometry. A standard curve was prepared in parallel by adding known amounts of neostigmine to blank plasma and analysing samples according to the above procedure. T h e stability of neostigrnine was tested by adding known amounts (30-55 ng/ml) to plasma and blood.
S.-M. Aquilonius et al.: Pharmacokinetics of Neostigmine
369
200
200
A I00
100
50
50
200
100 5O
=--m
Fig. 4. Plasma concentrations of neostig-
mine followingintravenousinjectionin HOURS
The samples were incubated at 4-37 °C (Fig. 3). At various intervals samples were withdrawn, internal standard solution added, and analysis performed as above.
Patients The kinetics following iv administration were studied in four non-myasthenic patients, who received neostigmine 2.5-3.0 mg iv to antagonize the curare-like effect of pancurone used during anaesthesia (02/ N20, fentanyl) for general st~gery. In three myasthenic patients, who had fasted and had not taken any drugs overnight, the time course of the plasma concentration was studied following a single oral dose of neostigmine 30 mg. In four myasthenie patients on their ordinary dose schedule of chotinesterase inhibitors (neostigmine and pyfidostigmine; Fig. 6), plasma neostigmine concentrations were determined over a 6-h period. Results
Determination of Neostigmine Concentration The neostigmine concentration in plasma could be determined accurately at a level of I ng/ml (about
four patients (A, 42 gg/kg; B, 35 gg/kg; C, 48 9g/kg; D, 32 gg/kg) to antagonize pancurone used during anaesthesia
HDUHS
Table 1. Pharmacokinetic data for neostigmine following intravenous administration Patients
A
B
C
D
Dose, mg 3.0 2.5 3.0 3.0 Dose, ~tg × kg -1 42 35 48 32 AUC, hours × ng × m1-1 46.6 46.3 59.8 35.2 Volume of distribution 1 x kg -1 1.31 0.89 0.92 1 . 2 1 Plasma half-life, hours 1.01 0.82 0.79 0.92 Plasma clearance, t × kg -1 x hours -1 0.89 0.75 0.81 0.91
Mean +_ SE 1.08 _+ 0.11 0.89 _+ 0.05 0.84 _+ 0.04
AUC = area under curve
3 nmol/1), and the relative standard deviation of the method at the 20 ng/ml level was 3.7% (n = 7). Rectilinear standard curves for neostigmine were obtained in the range 1-500 ng (Fig. 1). No interference from endogenous compounds or drug metabolites was detected in the chromatograms (Fig. 2). As illustrated in Figure 3, a temperature-dependent breakdown of neostigmine took place in plasma in vitro. The effect of the in vitro metabolism, however, could be eliminated by rapid cooling and immediate addition of d6-neostigmine to the plasma sample, as described above.
370
S.-M. Aquilonius et al.: Pharmacokinetics of Neostigmine
Table 2. Pharmacokinetic data for neostigmine following oral administration Patients
E
F
G
Mean _+ SE
Dose, mg Dose, gg X kg - I AUC, hours x ng x m1-1 AUC, in per cent of expected mean i. v. value* Volume of distribution, 1 × kg -~ Plasma half-life, hours Plasma clearance, 1 X kg -1 X hours -1
30 492
30 357
30 476
oc
13.7
9.8
7.2
2.3
2.3
1.3
2.0
1.09
0.86
1.24
1.06 ± 0.11
0.91
0.71
1.0
0.87 ± 0.09
0.83
0.84
0.86
0.84 ± 0.01
± 0.33
* A linear relationship between the i. v. doses and the corresponding area under the curve (AUC) has been assumed. In that case the A U C corresponding to an i. v. neostigrnine dose of i ~tg/kg, as calculated from the data in Table 1, is 1.20 ± 0.05 h x ng x ml-a
0
2
I
HOURS
3
4
Fig. 5. Plasma concentrations of neostigmine in three myasthenic patients following an oral dose of 30 mg in the morning. The patients had fasted and had not any drugs overnight
100" 50
~°'°"°*'-°q~.°
...J. ""~IL..
....0 ''''° "°-..oql,.oo°'°
~tO" ==
5" ........
/\/'-..... "%.
I"..
,
0.!
.It,'l* I
0
/ ?,.,i
i
t,,__ .,t"
,f, "=1
A ,
I
I
I
1
2
"~
V
3 HOURS
, I
4
Pharmacokinetics of Neostigmine The time course of the neostigmine concentration in plasma in the four patients receiving an iv injection of the drug is shown in Figure 4, and the calculated pharmacokinetic data are summarized in Table 1. The interindividual differences in drug elimination were small and the mean plasma half-life was 0.89 + 0.05 h. The plasma concentrations of neostigmine following a single oral dose of 30mg are shown in Figure 5 and the corresponding pharmacokinetic data in Table 2. Considerable variation in absorption was found
S. I
5
I
6
Fig. 6. Plasma concentrations of neostigmine in four myasthenic patients on their ordinary dose schedules. The arrows~, Sand ;~represent oral doses of neostigmine ~5 mg plus pyridostigmine 60 mg anti,represents oral doses of neostigmine 150 mg plus pyridostigmine 60 mg. An asterisk beside a symbol indicates that the dose was not the first dose of the day
(Fig. 5), with peak concentrations (range 4-9 ng/ml) occurring between 1 and 2 h after intake of the drug. The rate of elimination of neostigmine, on the other hand, was similar in the three patients (Table 2) and corresponded to the rate following iv administration (Table 1). The plasma concentrations of neostigmine in the four myasthenic patients who were taking their ordinary medication of neostigmine and pyridostigmine are shown in Figure 6. Although the variations in daily dose were considerable in the three patients taking repeated doses of neostigmine 15 mg plus pyridostigmine 60 rag, the plasma concentrations of neostigmine 4 h after the first dose of the day were
371
S.-M. Aquilonius et al.: Pharmacokinetics of Neostigmine
fairly similar (about 3 ng/ml). In the patient who took repeated doses of neostigmine 150mg plus pyridostigmine 60 mg, the plasma concentration of neostigmine was about 10 times higher (20-40 ng/
ml). Discussion The analysis of plasma neostigmine was based on ion-pair extraction of the quaternary ammonium compound into methylene chloride (Chan et al., 1976) and its determination by gas chromatographymass spectrometry with chemical ionization. By the use of a deuterated internal standard, degradation and extraction losses of neostigmine during processing were compensated. In the gas-liquid chromatographic method recently described by Chan et al. (1976), pyridostigmine was used as internal standard in the neostigmine assay, which would hamper determinations in patients on combined treatment with the two cholinesterase inhibitors. Further, their method allows accurate measurements only at concentrations above 50 ng/ml. It is evident from Figure 6 that this level of sensitivity is too low. The method described by Cohan et al. (1976) for assay of pyridostigmine or neostigmine in plasma by conversion of the quaternary compounds to the iodide salt and subsequent detection of released methyl iodide by gas chromatography was claimed to be extremely sensitive. The selectivity of this technique, however, can be expected to be imperfect, due to formation of methyl iodide from other drugs or endogenous quaternary compounds. Gas chromatography-mass spectrometry as applied in the present investigation seems to be the most selective method available at present. The interindividual differences in absorption (Fig. 5) are not unexpected in view of the low penetrance of quaternary compounds across biological membranes (Goldstein et al., 1974). The elimination of neostigmine, however, was very similar in all patients studied. The quantitative role of the probable enzymatic degradation of neostigmine in plasma (Fig. 3) remains to be evaluated. The plasma concentrations of neostigmine following oral administration of the drug were not studied in the same individuals nor under identical experimental conditions, as after iv injection. Nonetheless, a reasonable estimate of the biological availability of neostigmine (1-2%) was obtained by comparing the area under the curves in Figure 4 and
5, following adjustment for differences in dose per kg body weight. A similar low availability has been reported for another quaternary drug, emepronium, studied in humans (Sundwall et. al., 1973). Of special interest is the finding that myasthenic patients taking their routine medication may have up to a forty-fold difference in the plasma neostigmine concentration (1-40 ng/ml). So far nothing is known about the relationship between plasma concentration and therapeutic effect. Further, the effect of combined treatment with neostigmine and pyridostigmine lacks clinico-pharmacological documentation. Studies of such problems are now in progress in our laboratories, using the method described in this report, as well as a similar procedure for simultaneous determination of pyridostigmine in plasma.
Acknowledgements. This study was partly supported by the Swedish Medical Research Council (project no. 4373). The authors are indebted to Miss Susanne Floberg for skilful technical assistance. References Chan, R., Williams, N. E., Baty, J. D., Calvey, T. N.: A quantitative gas-liquid chromatographic method for the determination of neostigmine and pyridostigmine in human plasma. J. Chromatogr. 120, 349-58 (1976) Cohan, S. L., Pohlmann, L. W., Mikszewski, J., O'Doherty, D. S.: The pharmacokinetics of pyridostigmine. Neurology 26, 536--39 (1976) Goldstein, A., Aronow, L., Kalman, M.: Principles of drug action. New York: John Wiley 1974 Kornfeld, P., Samuels, A.J., Wolf, R.L., Osserman, K.E.: Metabolism of 14C-labeled pyridostigmine in myasthenia gravis. Neurology 20, 634-41 (1970) Kornfeld, P., Wolf, R.L., Samuels, A. J., Osserman, K.E.: The effect of chronic pyridostigmine administration on pyridostigmine-a4C metabolism in myasthenia gravis. Neurology 21, 550-52 (1971) Remen, L.: Zur Pathogenese und Therapie der Myasthenia gravis pseudoparalytica. Dtsch. Z. Nervenheilk. 128, 66-78 (1932) Sundwall, A., Vessman, J., Strindberg, B.: Fate of emepronium in man in relation to its pharmacological effects. Europ. J. clin. Pharmacol. 6, 191-95 (1973) Walker, M.B.: Treatment of myasthenia gravis with physostigmine. Lancet 1934/1, 1200 Yanagisawa, S.: Patent Japan 1951:3071 (June 13). Cited from Chem. Abstr. 47, 4908 (1953) Received: October 12, 1978 accepted in revised form: January 23, 1979 Dr. S.-M. Aquilonius Department of Neurology University Hospital S-750 14 Uppsala, Sweden
Responsible for the text: Prof. Dr. H. J. Dengler, Bonn and Prof. Dr. F. Gross, Heidelberg Responsible for advertisements: L. Siegel, M. Olle, Kurffirstendamm 237, D-1000 Berlin 15 Springer-Verlag Berlin-Heidelberg-New York. Printed in Germany by aprinta, Wemding Copyright © by Springer-Verlag Berlin • Heidelberg 1979 DieseAusgabeenthalt eine eingehefteteBeilagevom SpringerVerlag,Berlin,Heidelberg,New York.
Europ. J. clin. Pharmacol. 15, 367-371 (1979)
© by Springer-Verlag 1979
A Pharmacokinetic Study of Neostigndne in Man Using Gas Chromatography-Mass Spectrometry S.-M. Aquilonius, S.-A. Eckernfis, P. Hartvig 2, J. Hultman 1, B. Lindstr6m 3, and P. O. Osterman Departments of Neurology,AnaesthesiologyI and Pharmacy2, UniversityHospital, Uppsala, and Departmentof Drugs3, National Board of Health and Welfare, Uppsala, Sweden
Summary. To permit rational evaluation of the empirical pharmacotherapy of myasthenia with cholinesterase inhibitors, a sensitive and selective method for the determination of neostigmine has been developed. Analysis is based on ion-pair extraction of neostigmine into methylene chloride and determination by gas chromatography-mass spectrometry (chemical ionization). As neostigmine was found to be metabolized in plasma in vitro, deuterated (d6) neostigmine was immediately added to the plasma sample as the internal standard. The limit of quantitation of the method was about i ng/ml ( 3nmol/1). The kinetics following i. v. administration were studied in four patients, who received neostigmine 2.5-3.0mg iv to antagonize pancurone administered during anaesthesia. Elimination was rapid with a half-life tl/2 (/3-slope) of 0.89 + 0.05 h (mean _+ SE). The volume of distribution was 1.08 + 0.11 t/kg and plasma clearance was 0.84 +_ 0.041/ kg/h. In three fasting myasthenic patients plasma concentrations of neostigmine were followed for 5 h after a single oral dose of 30mg. Considerable interindividual differences in absorption were expressed in the peak concentrations, which occurred 1-2 h following drug ingestion. The bioavailability of neostigmine was estimated to be 1-2% of the ingested dose. Neostigmine concentration in plasma was found to differ considerably (up to forty-fold) between myasthenic patients on their ordinary doseschedules of cholinesterase inhibitors. Key words: myasthenia gravis, neostigmine; gas chromatography-mass spectrometry, pharmacokinetics Although quaternary cholinesterase inhibitors have been the basic drugs for treatment of myasthenia
gravis for the past 40 years (Remen, 1932; Walker, 1934), it is only recently that pharmacokinetic studies of them have been initiated in man. The first investigation of the basic pharmacokinetics were performed by Komfeld et al. (1970, 1971) with 14-Clabelled pyridostigmine. I.ater, two different gas chromatographic procedures for the assay of neostigmine and pyridostigmine were described (Chan et al., 1976; Cohan et al., 1976). None of the methods, however, seems to be sufficiently sensitive or selective to permit accurate determination of neostigmine in the plasma of myasthenic patients receiving oral treatment. In this paper a method for gas chromatographicmass spectrometric assay of neostigmine is described with use of deuterated drug as the internal standard. The method has been applied in a clinical study of the pharmacokinetics of neostigmine after its intravenous (iv) and oral administration.
Patients and Methods Reagents and Chemica& d6-neostigmine (3-hydroxyphenyltrimethyl-ammoniumbromide dimethyl (6-2H)-carbamate) was prepared from 3-(CH3)2-N-C 6 HsOCOCI in benzene and 30% (C2H3)2 Nit in water (Yanagisawa, 1951). The mixture was stirred for one hour under ice cooling. The benzene layer was dried and evaporated and the residue was treated overnight with a ten-fold excess of CH3Br. Glycine buffer 0.1 M prepared from glycine 7.5 g and sodium chloride 5.85 g was diluted to one litre with water. Glycine buffer with potassium iodide was made from potassium iodide 12.8 g, 0.1 M sodium hydroxide 4 ml and 0.1 M glycine buffer 6 ml. 0031-6970/79/0015/0367/$01.00
368
S,-M. Aquilonius et al.: Pharmacokineticsof Neostigmine
Ratio do/d6-10 O
3.5
Plasma sampie,5ng/ml Blank plasma
3.0
2.5 2,0
1.5 1.C
m/e 209
0,5
'
3'
,l
;
........
;
ng/rnl
Fig. 1. Typical standard curve for the determination of neostigmine by gas chromatography-massspectrometry. Each point represents the mean of 2-4 determinations
m/e 215
Fig. 2. Typical chromatogramsof plasma containing (left)neostigmine 5 ng/ml and internal standard (d6-neostigmine),and of blank plasma (right)containing only d6-neostigmine The injector temperature was 200 ~ C. The flow of methane carrier gas was 15 ml/min. The mass spectrometer was focused at m/e = 209 for neostigmine and at m / e = 215 for d6-neostigmine.
initial coflc, lO
Determination of Plasma Neostigmine Concentration 50
\\ !
Hours
Fig. 3. Decline in neostigmineconcentration in plasma followingin vitro incubation of plasma and blood at different temperatures. O O plasma, 4° C; A . . . . /x plasma, 22° C; • • plasma, 37° C; A - - - • blood, 37° C
Gas Chromatography- Mass Spectrometry A Finnigan 4000 gas chromatograph-mass spectrometer with chemical ionization was used. The glass column (1.5 m × 2 mm) was filled with 3 % O V 225 on Gas C h r o m Q~ and was operated at 185 °C.
Venous blood samples were collected, immediately cooled and the plasma separated. Internal standard solution 0.1 ml (d6-neostigmine, 2 vg/ml) was added to 2 ml plasma and the sample was frozen until analysed. Extraction was performed essentially as described by Chan et al. (1976). For analysis, the sample was thawed and made alkaline with 5 M sodium hydroxide 20 ~tl. After washing twice with diethyl ether 5 ml, glycine buffer with potassium iodide 1 ml and methylene chloride 6 ml were added. The mixture was shaken for 10 rain and after centrifugation the methylene chloride phase was removed and dried over sodium sulphate. After evaporation of the organic solvent, the residue was reconstituted in methanol and 1 ~tt was taken for analysis by gas chromatography-mass spectrometry. A standard curve was prepared in parallel by adding known amounts of neostigmine to blank plasma and analysing samples according to the above procedure. T h e stability of neostigrnine was tested by adding known amounts (30-55 ng/ml) to plasma and blood.
S.-M. Aquilonius et al.: Pharmacokinetics of Neostigmine
369
200
200
A I00
100
50
50
200
100 5O
=--m
Fig. 4. Plasma concentrations of neostig-
mine followingintravenousinjectionin HOURS
The samples were incubated at 4-37 °C (Fig. 3). At various intervals samples were withdrawn, internal standard solution added, and analysis performed as above.
Patients The kinetics following iv administration were studied in four non-myasthenic patients, who received neostigmine 2.5-3.0 mg iv to antagonize the curare-like effect of pancurone used during anaesthesia (02/ N20, fentanyl) for general st~gery. In three myasthenic patients, who had fasted and had not taken any drugs overnight, the time course of the plasma concentration was studied following a single oral dose of neostigmine 30 mg. In four myasthenie patients on their ordinary dose schedule of chotinesterase inhibitors (neostigmine and pyfidostigmine; Fig. 6), plasma neostigmine concentrations were determined over a 6-h period. Results
Determination of Neostigmine Concentration The neostigmine concentration in plasma could be determined accurately at a level of I ng/ml (about
four patients (A, 42 gg/kg; B, 35 gg/kg; C, 48 9g/kg; D, 32 gg/kg) to antagonize pancurone used during anaesthesia
HDUHS
Table 1. Pharmacokinetic data for neostigmine following intravenous administration Patients
A
B
C
D
Dose, mg 3.0 2.5 3.0 3.0 Dose, ~tg × kg -1 42 35 48 32 AUC, hours × ng × m1-1 46.6 46.3 59.8 35.2 Volume of distribution 1 x kg -1 1.31 0.89 0.92 1 . 2 1 Plasma half-life, hours 1.01 0.82 0.79 0.92 Plasma clearance, t × kg -1 x hours -1 0.89 0.75 0.81 0.91
Mean +_ SE 1.08 _+ 0.11 0.89 _+ 0.05 0.84 _+ 0.04
AUC = area under curve
3 nmol/1), and the relative standard deviation of the method at the 20 ng/ml level was 3.7% (n = 7). Rectilinear standard curves for neostigmine were obtained in the range 1-500 ng (Fig. 1). No interference from endogenous compounds or drug metabolites was detected in the chromatograms (Fig. 2). As illustrated in Figure 3, a temperature-dependent breakdown of neostigmine took place in plasma in vitro. The effect of the in vitro metabolism, however, could be eliminated by rapid cooling and immediate addition of d6-neostigmine to the plasma sample, as described above.
370
S.-M. Aquilonius et al.: Pharmacokinetics of Neostigmine
Table 2. Pharmacokinetic data for neostigmine following oral administration Patients
E
F
G
Mean _+ SE
Dose, mg Dose, gg X kg - I AUC, hours x ng x m1-1 AUC, in per cent of expected mean i. v. value* Volume of distribution, 1 × kg -~ Plasma half-life, hours Plasma clearance, 1 X kg -1 X hours -1
30 492
30 357
30 476
oc
13.7
9.8
7.2
2.3
2.3
1.3
2.0
1.09
0.86
1.24
1.06 ± 0.11
0.91
0.71
1.0
0.87 ± 0.09
0.83
0.84
0.86
0.84 ± 0.01
± 0.33
* A linear relationship between the i. v. doses and the corresponding area under the curve (AUC) has been assumed. In that case the A U C corresponding to an i. v. neostigrnine dose of i ~tg/kg, as calculated from the data in Table 1, is 1.20 ± 0.05 h x ng x ml-a
0
2
I
HOURS
3
4
Fig. 5. Plasma concentrations of neostigmine in three myasthenic patients following an oral dose of 30 mg in the morning. The patients had fasted and had not any drugs overnight
100" 50
~°'°"°*'-°q~.°
...J. ""~IL..
....0 ''''° "°-..oql,.oo°'°
~tO" ==
5" ........
/\/'-..... "%.
I"..
,
0.!
.It,'l* I
0
/ ?,.,i
i
t,,__ .,t"
,f, "=1
A ,
I
I
I
1
2
"~
V
3 HOURS
, I
4
Pharmacokinetics of Neostigmine The time course of the neostigmine concentration in plasma in the four patients receiving an iv injection of the drug is shown in Figure 4, and the calculated pharmacokinetic data are summarized in Table 1. The interindividual differences in drug elimination were small and the mean plasma half-life was 0.89 + 0.05 h. The plasma concentrations of neostigmine following a single oral dose of 30mg are shown in Figure 5 and the corresponding pharmacokinetic data in Table 2. Considerable variation in absorption was found
S. I
5
I
6
Fig. 6. Plasma concentrations of neostigmine in four myasthenic patients on their ordinary dose schedules. The arrows~, Sand ;~represent oral doses of neostigmine ~5 mg plus pyridostigmine 60 mg anti,represents oral doses of neostigmine 150 mg plus pyridostigmine 60 mg. An asterisk beside a symbol indicates that the dose was not the first dose of the day
(Fig. 5), with peak concentrations (range 4-9 ng/ml) occurring between 1 and 2 h after intake of the drug. The rate of elimination of neostigmine, on the other hand, was similar in the three patients (Table 2) and corresponded to the rate following iv administration (Table 1). The plasma concentrations of neostigmine in the four myasthenic patients who were taking their ordinary medication of neostigmine and pyridostigmine are shown in Figure 6. Although the variations in daily dose were considerable in the three patients taking repeated doses of neostigmine 15 mg plus pyridostigmine 60 rag, the plasma concentrations of neostigmine 4 h after the first dose of the day were
371
S.-M. Aquilonius et al.: Pharmacokinetics of Neostigmine
fairly similar (about 3 ng/ml). In the patient who took repeated doses of neostigmine 150mg plus pyridostigmine 60 mg, the plasma concentration of neostigmine was about 10 times higher (20-40 ng/
ml). Discussion The analysis of plasma neostigmine was based on ion-pair extraction of the quaternary ammonium compound into methylene chloride (Chan et al., 1976) and its determination by gas chromatographymass spectrometry with chemical ionization. By the use of a deuterated internal standard, degradation and extraction losses of neostigmine during processing were compensated. In the gas-liquid chromatographic method recently described by Chan et al. (1976), pyridostigmine was used as internal standard in the neostigmine assay, which would hamper determinations in patients on combined treatment with the two cholinesterase inhibitors. Further, their method allows accurate measurements only at concentrations above 50 ng/ml. It is evident from Figure 6 that this level of sensitivity is too low. The method described by Cohan et al. (1976) for assay of pyridostigmine or neostigmine in plasma by conversion of the quaternary compounds to the iodide salt and subsequent detection of released methyl iodide by gas chromatography was claimed to be extremely sensitive. The selectivity of this technique, however, can be expected to be imperfect, due to formation of methyl iodide from other drugs or endogenous quaternary compounds. Gas chromatography-mass spectrometry as applied in the present investigation seems to be the most selective method available at present. The interindividual differences in absorption (Fig. 5) are not unexpected in view of the low penetrance of quaternary compounds across biological membranes (Goldstein et al., 1974). The elimination of neostigmine, however, was very similar in all patients studied. The quantitative role of the probable enzymatic degradation of neostigmine in plasma (Fig. 3) remains to be evaluated. The plasma concentrations of neostigmine following oral administration of the drug were not studied in the same individuals nor under identical experimental conditions, as after iv injection. Nonetheless, a reasonable estimate of the biological availability of neostigmine (1-2%) was obtained by comparing the area under the curves in Figure 4 and
5, following adjustment for differences in dose per kg body weight. A similar low availability has been reported for another quaternary drug, emepronium, studied in humans (Sundwall et. al., 1973). Of special interest is the finding that myasthenic patients taking their routine medication may have up to a forty-fold difference in the plasma neostigmine concentration (1-40 ng/ml). So far nothing is known about the relationship between plasma concentration and therapeutic effect. Further, the effect of combined treatment with neostigmine and pyridostigmine lacks clinico-pharmacological documentation. Studies of such problems are now in progress in our laboratories, using the method described in this report, as well as a similar procedure for simultaneous determination of pyridostigmine in plasma.
Acknowledgements. This study was partly supported by the Swedish Medical Research Council (project no. 4373). The authors are indebted to Miss Susanne Floberg for skilful technical assistance. References Chan, R., Williams, N. E., Baty, J. D., Calvey, T. N.: A quantitative gas-liquid chromatographic method for the determination of neostigmine and pyridostigmine in human plasma. J. Chromatogr. 120, 349-58 (1976) Cohan, S. L., Pohlmann, L. W., Mikszewski, J., O'Doherty, D. S.: The pharmacokinetics of pyridostigmine. Neurology 26, 536--39 (1976) Goldstein, A., Aronow, L., Kalman, M.: Principles of drug action. New York: John Wiley 1974 Kornfeld, P., Samuels, A.J., Wolf, R.L., Osserman, K.E.: Metabolism of 14C-labeled pyridostigmine in myasthenia gravis. Neurology 20, 634-41 (1970) Kornfeld, P., Wolf, R.L., Samuels, A. J., Osserman, K.E.: The effect of chronic pyridostigmine administration on pyridostigmine-a4C metabolism in myasthenia gravis. Neurology 21, 550-52 (1971) Remen, L.: Zur Pathogenese und Therapie der Myasthenia gravis pseudoparalytica. Dtsch. Z. Nervenheilk. 128, 66-78 (1932) Sundwall, A., Vessman, J., Strindberg, B.: Fate of emepronium in man in relation to its pharmacological effects. Europ. J. clin. Pharmacol. 6, 191-95 (1973) Walker, M.B.: Treatment of myasthenia gravis with physostigmine. Lancet 1934/1, 1200 Yanagisawa, S.: Patent Japan 1951:3071 (June 13). Cited from Chem. Abstr. 47, 4908 (1953) Received: October 12, 1978 accepted in revised form: January 23, 1979 Dr. S.-M. Aquilonius Department of Neurology University Hospital S-750 14 Uppsala, Sweden
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