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Neostigmine has been increasingly used in clinical practice during the past twenty-five years. A number of problems have been encountered in its use as a diagnostic and therapeutic agent for patients with myasthenia gravis and as an antagonist to tubocurarine. For example, the daily requirement of myasthenic patients varies considerably and many can tolerate very large doses without experiencing unpleasant muscarinic effects. Neostigmine resistance or failure to respond to a normal therapeutic dose of neostigmine has been described by physicians in the diagnosis of myasthenia gravis and by anaesthetists in reversing the effects of tubocurarine.Little information is available about its absorption, metabolism and excretion; an exhaustive search of the literature has shown that only two groups of research workers have studied the fate of neostigmine. Goldstein and his colleagues deduced the fate of the drug by measuring its inhibitory effect on the cholinesterase activity of serum (Krayer, Goldstein & Plachte, 1944;Goldstein, Krayer, Root, Acheson & Doherty, 1949). When they infused neostigmine intravenously into dogs they found that the method of clearance depended on the concentration infused. At low concentrations most of the drug was destroyed by the plasma and a little was excreted by the kidneys; whilst at higher concentrations renal excretion by glomerular filtration was considered to play a predominant role.Nowell, Scott & Wilson (1962a) developed a method of measuring directly the amount of-neostigmine in urine by coupling the drug with the dye bromophenol blue which afforded a colorimetric method of quantitative analysis. They reported that, in patients with myasthenia gravis who were given doses of neostigmine by intramuscular injection, up to 67% of the daily dose was excreted in the urine in 24 hr. By contrast, after oral administration of the drug less than 5% of the daily dose was excreted. They also reported the presence of two metabolic products of neostigmine in extracts of urine from patients who had been given oral neostigmine (Scott, Nowell & Wilson, 1962). Later work by Nowell and his colleagues provided the first evidence that in myasthenic patients up to 50%1. of an intramuscular dose of neostigmine is excreted in the urine within 2 hr (Scott, 1962). The colorimetric method described by Nowell et al. (1962a) cannot be used for the estimation of the two metabolic products described by them since these compounds form a weak colour complex with bromophenol blue. It was decided therefore to use a radioactive form of neostigmine for further investigation of the drug. Since all the evidence regarding the nature of the metabolic products pointed in the direction of hydrolysis of the carbamic

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Observations of myasthenic patients given oral pyridostigmine and 14C‐pyridostigmine by intramuscular injection were designed to determine the excretion in urine of unchanged pyridostigmine and its metabolites. Paper chromatography of urine revealed the presence of three metabolites in addition to pyridostigmine. The unchanged drug and its major metabolite were isolated from the urine of a patient given oral doses with the use of the combined techniques of ion‐exchange chromatography, electrophoresis, and paper chromatography. Pyridostigmine and 3‐hydroxy‐N“methyl pyridinium thus isolated were characterized by ultraviolet spectra and mass spectra and estimated by a reverse isotope‐dilution method. It is suggested that the problems of chronic overdosage with pyridostigmine are related to the possible accumulation of some of these substances in patients who are treated for prolonged periods with high doses of pyridostigmine.

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