METABOLISM OF [¹⁴C]‐NEOSTIGMINE IN THE RAT

Abstract

When neostigmine is administered orally to patients with myasthenia gravis two metabolites are excreted in the urine one of which has been identified as m-hydroxyphenyltrimethylammonium (Scott, Nowell & Wilson, 1962). It was later reported by these investigators that after prolonged in vitro incubation of neostigmine with human plasma two metabolites were identified; these were not present when plasma had been previously incubated with dyflos (Nowell, Scott & Wilson, 1962b). The fact that no metabolites could be detected in the urine of myasthenic patients who had been given neostigmine by intramuscular injection (Nowell, Scott & Wilson, 1962a) and that the hydrolysis of neostigmine in vitro was very slow suggested that some tissue other than plasma might be concerned in the metabolism of the drug.Recent work in this laboratory has shown that in the rat, after intramuscular injection of [14C]-neostigmine, radioactivity is rapidly concentrated in the liver and slowly declines to negligible amounts within 24 hr (Roberts, Thomas & Wilson, 1965). This evidence strongly suggests that the liver is an important organ in the metabolism of neostigmine and is supported by the results described in this paper which is concerned with the estimation of unchanged drug and metabolite in the urine and liver of the rat after intramuscular injection of [14C]-neostigmine. METHODS[14C]-Neostigmine iodide, supplied by the Radiochemical Centre, Amersham, had a specific activity of 15 pc/mg and was used to examine its excretion in the urine and distribution in the liver of the rat. Male rats weighing 150 to 200 g were hydrated and injected intramuscularly into the hind-limb with a standard dose of 25 Hg of [14C]-neostigmine in 0.1 ml. of water; urine was collected and the liver was extracted and estimated for total radioactivity by the methods previously described (Roberts et al., 1965). In the later experiments with SKF 525-A (2-diethylaminoethyl-3,3-diphenylpropylacetate) the method for extraction from liver was modified by evaporating the extract to 1 ml. instead of 10 ml. and then adjusting the volume to 10 ml. with absolute ethanol. After settling, the supernatant fluid was used to estimate metabolite and neostigmine.Estimation of neostigmine and metabolite. Although the metabolite has not been separated and unequivocally identified as m-hydroxyphenyltrimethylammonium, there is fairly strong evidence from its RF on paper chromatography and its mobility on paper electrophoresis to support this assumption . To separate the metabolite from neostigmine in urine and liver extracts a paper electrophoresis method was developed.Specimens of urine were applied as a spot, and extracts of liver as a line, to Whatman 541 paper (33 x 4 cm) at a point 7.5 cm from one end and dried by a current of hot air (Nowell et al., 1962a) or with an infrared lamp placed underneath the paper. The liver extracts were applied to the paper through a fine polyethylene

Ligated Renal Pedicles and Duration of Action of Neostigmine and Pyridostigmine

“…In addition to a difference in species, the problems of equating intensity of pharmacological action with plasma concentrations of drugs are well known (Koch-Weser, 1972). The importance of renal excretion to the elimination of both neostigmine and pyridostigmine is suggested by the fact that 40-60% of both neostigmine and pyridostigmine are excreted in the urine (Nowell, Scott & Wilson, 1962;Roberts, Thomas & Wilson, 1965;Birtley, Roberts, Thomas & Wilson 1966). Our results of prolonged duration of action of both drugs therefore are not surprising.…”

Metabolism and excretion of 3‐hydroxyphenyltrimethylammonium and neostigmine

“…3-OH PTMA and glucuronide in liver after administration of neostigmine It has previously been shown that after intramuscular administration of neostigmine more than 90% of the radioactivity in liver is present as metabolite (Roberts et a., 1965b). In view of the results currently reported, it was considered important to determine to what extent the metabolite in liver is present as free and conjugated 3-OH PTMA.…”

The excretion and metabolism of oral 14C‐pyridostigmine in the rat

“…Estimation of pyridostigmine and its metabolite Urine and extracts of blood and liver were estimated for total radioactivity and for radioactive metabolite, using paper electrophoresis as described by Roberts et al (1965b). The radioactivity separated into two distinct bands; counts recorded between 10 and 13 cm from the origin are a measure of pyridostigmine and those recorded between 5 and 9 cm are a measure of metabolite (Birtley et al, 1966).…”