T.N. Calvey

A quantitative gas-liquid chromatographic method for the determination of neostigmine and pyridostigmine in human plasma

A sensitive and selective analytical method was used to measure the concentration of neostigmine and pyridostigmine in human plasma. The procedure involved preliminary ion-pair extraction of the drugs into dichloromethane, followed by concentration and anlysis of the ion-pair complex using a gas-liquid chromatographic system fitted with a nitrogen detector. Using the peak area ratio technique, pyridostigmine bromide was used as the internal standard for the quantitation of neostigmine in plasma; neostigmine bromide was the internal marker for the determination of pyridostigmine. The method depends on the thermal dequaternisation of the quaternary amines, and can be used to detect 5 ng/ml in a 3-ml plasma sample. Accurate measurement can be made at levels of 50-1000 ng/ml. This assay procedure has been applied to the separate determination of the plasma concentration of neostigmine and pyridostigmine after single administration of intravenous doses in aneasthetised patients.

Plasma pyridostigmine levels in patients with myasthenia gravis

Plasma concentrations 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 myasthenic patients, the area under the plasma concentration‐time curve was relatively eonstant for 4 hr after the same oral dose of pyridostigmine (60 mg). Despite this similarity, there were in general considerable interindividual differences in the bioavailability of pyridostigmine in myasthenie patients. In 1 subject, 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 subject (4.25 hr) was almost three times as great as after intramuseular administration in a different patient (1.49 hr).

THE RELATIONSHIP BETWEEN THE PHARMACOKINETICS, CHOLINESTERASE INHIBITION AND FACILITATION OF TWITCH TENSION OF THE QUATERNARY AMMONIUM ANTICHOLINESTERASE DRUGS, NEOSTIGMINE, PYRIDOSTIGMINE, EDROPHONIUM AND 3-HYDROXYPHENYLTRIMETHYLAMMONIUM

1 The relationship between the concentration of drug in plasma, the inhibition of erythrocyte acetylcholinesterase and the facilitation of neuromuscular transmission has been studied in the rat after the administration of neostigmine, pyridostigmine, edrophonium and 3‐hydroxyphenyltrimethylammonium (3‐OH PTMA). 2 After the administration of neostigmine or pyridostigmine, acetylcholinesterase activity recovered only slowly due to the covalent nature of the inhibition. In contrast, recovery from the reversible inhibition caused by edrophonium or 3‐OH PTMA was rapid and showed a direct relationship to the plasma concentration of these drugs. 3 There was a statistically significant linear correlation between the logarithm of the plasma concentration of the drugs and the increase in the tibialis twitch tension. 4 The relationship between the inhibition of acetylcholinesterase and the facilitation of neuromuscular transmission was complex. When the enzyme was less than 85% inhibited no facilitation occurred. Between 85% and 98% inhibition, facilitation was linearly related to enzyme inhibition. Above 98% inhibition, facilitation was unrelated to inhibition of the enzyme.

Quantitative capillary column gas chromatographic method for the determination of glycopyrronium in human plasma.

A new sensitive and selective capillary column gas chromatographic method for the anti-cholinergic agent glycopyrronium bromide in human plasma is described. The procedure involves preliminary ion-pair extraction of the drug into dichloromethane, followed by concentration and analysis of the ion-pair complex by capillary column gas chromatography using a nitrogen-sensitive detector. The method depends on the thermal dequaternisation of the quaternary ammonium compound and can be used to detect 5 ng/ml in a 3-ml plasma sample. The assay procedure has been applied to the determination of the plasma concentration of glycopyrronium after intravenous administration to an anaesthetised patient.

PLASMA CLEARANCE OF NEOSTIGMINE AND PYRIDOSTIGMINE IN THE DOG

1 The pharmacokinetics of neostigmine and pyridostigmine was studied in conscious dogs by the use of a cross‐over design. 2 Both neostigmine and pyridostigmine were cleared from plasma in a biexponential manner. 3 The apparent volume of distribution of pyridostigmine was invariably greater than that of neostigmine, and its fast disposition half‐life was approximately three times longer. 4 The whole body clearance and the urinary elimination of pyridostigmine was approximately twice that of neostigmine. 5 The slow disposition half‐life of pyridostigmine was approximately three times longer than that of neostigmine, suggesting that the longer duration of action of pyridostigmine is related to the differential clearance of the two quaternary amines from plasma.

Plasma concentration of edrophonium in man

The plasma concentration of edrophonium was measured in man after intravenous administration. In 5 patients, the clearance of edrophonium from the circulation during the 1‐hr period of sampling was invariably resolved into 2 exponential components. An initial rapid phase of elimination from plasma (T/2 = 0.54 to 1.92 min) was followed by a much slower decline (T/2 = 24.23 to 45.00 min), corresponding to the fall in concentration between 10 and 60 min. In parallel experiments in the rat, the clearance of 14C‐edrophonium was resolved into 3 exponential components, although the final component could not be reliably defined until 1 to 3 hr after intravenous injection. It is suggested that the rapid fall in the plasma concentration of edrophonium in both species is not dependent on metabolism or excretion, but is due to the rapid uptake of the drug by the liver and kidneys.

Excretion of 14C‐edrophonium and its metabolites in bile: role of the liver cell and the peribiliary vascular plexus

1 The metabolism and biliary excretion of 14C‐edrophonium chloride was studied in Wistar rats. 2 Approximately 5% of the dose was recovered from bile in 6 hours. Most of the radioactivity was eliminated as 14C‐edrophonium glucuronide. Small amounts of the unchanged drug were also detected in bile, particularly during the first hour after administration of the drug. 3 The concentration of 14C‐edrophonium glucuronide in bile was approximately 15–20 times its concentration in plasma. 4 In contrast, the concentration of unchanged 14C‐edrophonium was similar in bile and plasma. 5 Evidence is presented that unchanged 14C‐edrophonium is transferred from plasma to bile via the peribiliary vascular plexus.

THE PHARMACOKINETICS OF PYRIDOSTIGMINE AND 3‐HYDROXY‐N‐METHYLPYRIDINIUM IN THE RAT: DOSE‐DEPENDENT EFFECTS AFTER PORTAL VEIN ADMINISTRATION

1 The elimination kinetics of [14C]‐pyridostigmine iodide and [14C‐methyl]‐3‐hydroxypyridinium bromide (3‐OH NMP) have been studied in the rat. 2 For pyridostigmine, at a given dose level, the fraction of the dose eliminated unchanged was reduced and the metabolite fraction was increased after portal vein administration when compared to jugular vein administration. This indicates that pyridostigmine is subject to metabolism during the first passage through the liver. 3 When doses of pyridostigmine 1.25 μmol/kg and higher were injected via the portal vein, the proportion excreted in urine as unchanged drug remained constant; in contrast, the percentage of the dose eliminated as the metabolite was significantly reduced. This indicates that a dose‐dependent process is involved in the urinary excretion of 3‐OH NMP. 4 This conclusion was supported by studies involving the portal and systemic venous injection of 3‐OH NMP at different dose levels. After 4 h, approximately 85% of the lowest dose was eliminated unchanged in urine; in contrast, only 63% of the higher dose was excreted during this period. The proportion of the dose eliminated in urine was not related to the route of administration. 5 After the injection of pyridostigmine into the jugular vein, the initial rate of drug excretion fell rapidly for approximately 10 min; in contrast, after injection into the portal vein, the rate of excretion of the drug rose to a maximum at 30 minutes. This suggests that the hepatoportal system behaves as a distinct region during the distribution of this drug.

Plasma concentration of pyridostigmine and effects in myasthenia gravis

The relation between the plasma concentration of pyridostigmine and its effects was studied in 5 patients with myasthenia gravis. In 4 patients with typical electromyographic decrement in the adductor pollicis, there was a positive correlation between the concentration of pyridostigmine in plasma and the effect on neuromuscular transmission. The plasma concentration of pyridostigmine required to restore transmission to normal (as calculated from the regression line relating plasma concentration to neuromuscular function) varied over a 5‐fold range, reflecting the variable severity of the disease. In another myasthenic patient with purely ocular symptoms, there was a significant correlation between the plasma concentration of the drug and the diameter of the palpebral fissure. It is suggested that the routine measurement of the plasma concentration of pyridostigmine may be of value in the management of myasthenia gravis. A method to calculate the optimal daily dose of pyridostigmine in individual myasthenic patients is described.

The effect of edrophonium on erythrocyte acetylcholinesterase and neuromuscular function in the rat.

1 The relation between the concentration of edrophonium in plasma, inhibition of red cell acetylcholinesterase, and neuromuscular transmission was studied in the rat. 2 In both in vivo and in vitro conditions, red cell acetylcholinesterase activity was predictably related to the concentration of the quaternary amine. 3 After low doses of edrophonium (1.0 μmol/kg) there was a significant correlation between the monophasic potentiation of twitch tension and the plasma concentration of the drug. In contrast, with higher doses of edrophonium (4.0 or 10.0 μmol/kg) a biphasic potentiation of twitch tension was observed; this was only correlated with the plasma concentration of the drug during the secondary decline in neuromuscular facilitation. Subsequent recovery of normal neuromuscular transmission invariably occurred at a constant plasma concentration of edrophonium.