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The metabolism of the 14 C-niflumic acid † specific activity 16 mc/mM has been studied in the rat, after intraperitoneal administration. With doses of 50 mg/kg, the blood level is of 75 μg/ml at the 1st hr, and of 6 μg/ml at the 24th hr. The product is nearly all bound to the plasma proteins. The liver retains, after 1 hr, 4 per cent of the injected activity and the stomach 0.5 per cent only traces (0.08 per cent) have been detected in the brain. The technique of autoradiography in the mouse, after administration of the product per os and i.p. confirms these results. About 5 per cent of 14 CO 2 is found with respect to the injected activity, after 12 hr. The radioactivity eliminated in the urine is about 2 per cent of the injected activity after 1 hr, reaches 30 per cent at the 12th hr, is practically completed after 24 hr. The radioactivity found in the faeces is generally of the order of 0.01 per cent. Five catabolites were found by paper chromatography in the urine. Niflumic acid is not eliminated unchanged, and the greatest quantity appears to be conjugated glucuronide. One of the catabolites, after paper Chromatographie study in various systems, corresponds to 2-aminonicotinic acid. Possible metabolic pathways of the Niflumic acid discussed.

The metabolic pathways of imipramine and its metabolites by rat liver microsomes have been studied with the aim to obtain a comprehensive picture of the metabolism of imipramine and further insight into the action of the microsomal drug-metabolizing enzyme system. Paper- and thin-layer chromatography was used for detection and determination of metabolites; demethylation was additionally determined by formaldehyde assay. Imipramine is extensively metabolized and in addition to the known major metabolites, desmethylimipramine and 2-hydroxy-imipramine, all ten hitherto described metabolites of imipramine were found to be formed in the rat, nine of them in vitro. Time course-studies and the separate investigation of the metabolism of imipramine metabolites allow conclusions as to the sequence of metabolite formation, and primary, secondary and tertiary metabolites of imipramine can be distinguished. A total of sixteen metabolic pathways were detected: demethylations, aromatic hydroxylations, side chain dealkylations, N-oxidation, N-oxide reduction and conjugations. Based on these results an attempt was made to establish a comprehensive scheme of the metabolism of imipramine in the rat and to estimate relative conversion rates for the metabolic pathways.

The structures of the water-soluble derivatives of 3 H-labelled 7,12-dimethyl- benz[a]anthracene that are formed by rat-liver homogenates have been investigated. Paper chromatography resolved the mixture of metabolites into two regions of high radioactivity. The products of the alkaline hydrolyses of the material eluted from both of these regions contained cysteine, glycine and glutamic acid, indicating that GSH conjugates were present. The material forming the faster moving region appeared to consist of a single compound that was identified as S -(5,6-dihydro-6-hydroxy-7,12-dimethyl-benz[a]anthracen-5-yl)glutathione by comparisons with the synthetic conjugate. The slower-moving region of radioactivity may contain a mixture of conjugates since the dihydrodiols that are formed during the metabolism of 7,12-dimethylbenz[a]anthracene by rat-liver homogenates are all converted by further metabolism into water-soluble derivatives that are located in this region of the chromatograms. The possible structures of these conjugates is discussed.

Hematological and toxicological studies revealed a strong resistance of the rabbit to the folic acid antagonist methotrexate. Investigation of its metabolism led to the isolation and purification of a drug metabolite from urine and liver incubation samples, based on solubility characteristics of the compound. The metabolite was characterized by u.v. absorption spectrum, pattern of crystallization, and paper chromatography in phosphate buffers. Elementary analysis was compatible with the 4,7-dihydroxy derivative of methotrexate. Toxicity studies in mice indicated that the metabolite is a product of detoxification with reduced but apparently not fully obliterated antifolic acid activity.

Analytical procedures for the characterization and quantitative estimation of phosphonic acids derived from Soman, based upon paper chromatography and partition between isobutanol-benzene and an aqueous phase, were developed. Utilization of 32P-labeled phosphonic acids permitted the determination of as little as 10 −2 μmole of acid. Application of these methods to the enzymatic hydrolysis products of Soman suggests that the principal, if not the only, metabolite of Soman incubated with rat plasma or liver tissue in vitro is pinacolyl methylphosphonic acid.

The level of male rat liver glutathione (GSH) rapidly increased following intraperitoneal injection of the strong heptocarcinogen, 3'-methyl-4-dimethylamino-azobenzene (3'-MeDAB). A more gradual, less-pronounced increase resulted from injection of the weaker hepatocarcinogen, 4'-methyl-4-dimethylaminoazobenzene (4'-MeDAB). On the contrary, 24 hr after injection of the non-carcinogenic azo dye, 2-methyl-4-dimethylaminoazobenzene, the level of rat liver GSH dropped below normal. Paper chromatography of trichloracetic acid extracts of 3'-MeDAB-treated liver showed that besides the increase in GSH, other changes occurred including an increase in phosphoethanolamine (PE). The pattern of changes was markedly similar to that observed by other workers in extracts of regenerating rat liver. The only marked change with the weak carcinogen 4'-MeDAB was the increase in GSH. A survey is given of substances capable of increasing liver GSH levels. These include the carcinogen 3,4:5,6-dibenzcarbazole, the anti-tumour agent, nitromin and two anti-inflammatory agents salicylic acid and antipyrine which are not known to be carcinogenic. Attention is directed to the metal-chelating agent, 8-hydroxy-quinoline which has both anti-inflammatory properties and carcinogenic activity. It is suggested that anti-inflammatory substances should be carefully screened for carcinogenic and/or cocarcinogenic activity.

Nicotine-1′-oxide, prepared by the oxidation of (−)-nicotine with hydrogen peroxide, has been resolved into two optically active stereoisomers by fractional precipitation with ammonium reineckate. (−)-Nicotine is enzymically oxidized into both isomers of nicotine-1′-oxide in vitro. These enzyme systems are stereospecific since guinea-pig and rabbit liver form similar amounts of each stereoisomer white BALB/c mouse and hamster liver and guinea-pig lung synthesize chiefly laevo-rotatory nicotine-1′-oxide. Both stereoisomers of nicotine-1′-oxide were identified in the urine of cigarette smokers.

Liver, brain and urine extracts of normal rats and of rats treated with β,β′-iminodipropionitrile (IDPN) have been analysed by separation on Dowex 50-H + resin and by paper electrophoresis and chromatography. The possibility that IDPN could be exerting its toxic effects by metabolism into 3,3′-diaminopropylamine (DAPA) with alteration in the physiological activity of spermine and spermidine was investigated. It has been shown that DAPA does not accumulate in the liver or brain following IDPN administration, and that gross alteration in the polyamine content of these tissues does not occur. The presence of an unknown ninhydrin-reacting substance was detected in the IDPN-treated rat brains. Three metabolites of IDPN were identified in the urine following IDPN treatment, namely cyanoacetic acid, β-alanine and β-aminopropionitrile. Prevention of the symptoms of IDPN poisoning with ethionine did not inhibit the formation of these metabolites or of thiocyanate.

In rats diethyl maleate (DEM, 0.7 ml/kg i.p.) decreased the hepatic glutathione level to one tenth of the control value. Owing to the low glutathione level the conjugation of bromsulphthalein (BSP) with glutathione was markedly depressed. DEM-treated rats were given BSP and a glutathione conjugate of BSP (BSP-GSH) intravenously at various dose levels, and their biliary excretion and tissue concentrations were determined. No significant difference between the hepatic transport maxima for BSP (673 μg/min/kg) and for BSP-GSH (689 μg/min/kg) was found. BSP-GSH increased the biliary flow, BSP diminished it. Depending on the dose, 52–83 per cent of the BSP administered was taken up by the liver in 45 min, whereas the BSP-GSH predominantly appeared in extra-hepatic tissues. The half saturation doses for transport maxima were 75 mg/kg for BSP and 31 mg/ kg for BSP-GSH. After administration of these doses the hepatic concentration of BSP was approximately ten times as high as the hepatic concentration of BSP-GSH.

Rats given m -methyl orange and methyl orange intraperitoneally and intravenously excrete the monomethylated and completely demethylated dyes in urine and in bile. Mouse liver microsomes also demethylate methyl orange. The results indicate that factors other than lipid solubility are important in the microsomal metabolism of drugs.

The distribution and metabolic fate of [3H]harmaline-HCl were studied in rats. Thirty min after subcutaneous injection, high radioactivity was found in the small intestine, liver, adrenals, kidneys and lungs. A rapid turnover and elimination was evident after the first hour, as most of the tissues, except the liver, kidneys and intestines, had decreased nearly 50 per cent in levels of radioactivity. About 40 per cent of the harmaline was bound to human serum or rat serum proteins in vitro. The blood levels, however, were low at all times in vivo. The peak concentration in the brain occurred at 1 hr postinjection. The major route of excretion of harmaline and its metabolites was through the kidneys; a total of 62 per cent of the injected dose was excreted in the urine during 96 hr as compared to only 11·5 per cent in the feces over the same period. The major fate of harmaline in rats was demethylation to form harmalol, which was predominantly excreted as the glucuronide conjugate. Six to 10 per cent of the radioactivity was identified as the sulfate conjugate of harmol, which was formed by the dehydrogenation of harmalol. During the first 8 hr, unchanged harmaline in the urine amounted to about 25 per cent; however, this decreased to only 7 per cent during the 8–24 hr period.

Metabolic pathways responsible for the demethylation, N -oxidation and 2-hydroxylation of imipramine were studied in rat liver in vitro . Isotope-trapping experiments have revealed that imipramine- N -oxide is not involved as an intermediate in the formation of desmethylimipramine from imipramine. The enzyme systems for demethylation and 2-hydroxylation are induced by phenobarbital, but that for N -oxidation is not induced. All available evidence indicates that the demethylation, N -oxidation and 2-hydroxylation reactions may be independent of one another.

Pretreatment with either pyrogallol, tropolone or 8-hydroxyquinoline enhanced markedly the mean stereotyped behavior scores after apomorphine treatment in the rat. Experiments in vitro , using rat liver or brain catechol- O -methyltransferase (COMT) preparations and 14 C-methyl- S -adenosyl- l -methionine, demonstrated that apomorphine was methylated by this enzyme system. The apparent K m values for dopamine and apomorphine were 2.6 × 10 −4 M and 1.4 × 10 −3 M, respectively, for liver COMT. Pyrogallol and tropolone inhibited the methylation of apomorphine in vitro competitively when the apomorphine concentration was varied. These results suggest that methylation by COMT may represent an important metabolic pathway for the deactivation of apomorphine in vivo .

The metabolism of two tertiary aromatic phosphines, 3-dimethylaminopropyldiphenylphosphine (a CNS depressant) and diphenylmethylphosphine, was studied. These compounds were incubated with subcellular fractions of rat liver homogenates and found to be enzymatically converted to the corresponding phosphine oxides; the aminophosphine gave rise to an N , P -dioxide. Enzymatic activity was localized in the microsomal fraction and shown to be associated with the cytochrome P-450 mixed function oxidases. The substrates were also found to undergo a facile non-enzymatic reaction with thiols, a factor which portends their possible chronic toxicity.

The metabolism of 5-hydroxytryptamine-3'- 14 C (5-HT, serotonin) was studied in isolated perfused rat livers when the monoamine oxidase pathway was inhibited by β-phenylisopropylhydrazine (JB 516) or the glucuronyl transferase pathway was deficient (Gunn rat livers). In all cases, 5-HT was cleared rapidly from the plasma by liver and platelets. In bile and in plasma in control perfusions (Sprague-Dawley livers), monoamine oxidase metabolites [chiefly conjugates of 5-hydroxyindoleacetic acid (5-HIAA) and 5-hydroxytryptophol (5-HTOH)] accounted for 29 per cent of the dose; conjugates of N -acetyl-hydroxytryptamine accounted for 12 per cent of the dose and 5-hydroxytryptamine glucuronide for 11 per cent. On addition of 2 mg JB 516 to the perfusate, the amounts of conjugates of 5-HT and N -acetyl-5-hydroxytryptamine increased and no radioactive 5-HTOH or 5-HIAA could be detected. Gunn rat livers produced less glucuronides, more sulfate conjugates and more free 5-HIAA compared to perfusion of Sprague-Dawley livers without JB516. The total amount of 5-HT metabolized was not reduced when a major metabolic pathway was either deficient or inhibited, because compensatory increases occurred in the amount metabolized by alternate pathways.

The isolated perfused rat liver was used to study the effect of an inhibitor of l -aromatic amino acid decarboxylase, N-( d,l -seryl)-N′-(2,3,4-trihydroxybenzyl)-hydra-zine (RO 4-4602), on hepatic metabolism of l -3,4-dihydroxyphenylalanine ( l -dopa). When inhibitor was added to the perfusate before addition of l -dopa-3-14C, the amount of radioactivity in the liver was less. Rapid uptake of l -dopa by erythrocytes in the perfusate removed 28 per cent of it from plasma in control perfusions, and this was unaffected by RO 4-4602. However, radioactivity in erythrocytes decreased to 7 per cent of the dose during control perfusions but only to 19 per cent when RO 4-4602 had been added. In all perfusions, about half of the 14C disappeared from plasma in 5 min. In control perfusions, there was a continued decrease in 14C in plasma during the first hour, to 35 per cent of the dose, but when RO 4-4602 had been added the decrease after the first 5 min was negligible. Between 5 and 120 min after injection, the half-life of l -dopa in plasma was 27 min in control perfusions and 57 min if RO 4-4602 had been added. However, between 120 and 300 min after dopa injection, when comparatively small amounts of it were present in plasma, the disappearance was more rapid in the presence of RO 4-4602. Excreted bile contained 44 per cent of the dose in control perfusions but only 8 per cent if RO 4-4602 had been added to the perfusate. When RO 4-4602 was added, the formation of conjugates of acidic and neutral metabolites of dopamine was decreased, especially the glucuronides of N-acetyldopamine and N-acetyl-3-O-ethyldopamine, the major metabolites in bile. However, there was an almost compensatory increase in the formation of 3-methoxy-4-hydroxyphenylalanine by direct O-methylation of l -dopa. The effect of RO 4-4602 did not appear to be prolonged because it was less obvious when the inhibitor was added 30–60 min before the l -dopa.

The metabolic disposition of allopurinol [4-hydroxypyrazolo(3,4-d)-pyrimidine) was determined in mice, dogs, and human subjects. The drug is a substrate for, as well as an inhibitor of, xanthine oxidase and is converted in all species to the corresponding xanthine analog, alloxanthine, which is its major metabolite. Neither is bound to human plasma proteins, and both are distributed more or less equally in total body water in the mouse. Both analogs are cleared rapidly by the mouse and dog kidney. In the human subject allopurinol is cleared rapidly, but alloxanthine resembles uric acid in having a slow clearance responsive to probenecid. The accumulation of alloxanthine during prolonged therapy with allopurinol may contribute significantly to the therapeutic effects of the drug in the control of hyperuricemias.

The effect of phenobarbital (PB) pretreatment of young male rats on the metabolism in vivo and in vitro of the carcinogen N -2-fluorenylacetamide (FAA) was studied. PB increased the urinary excretion of 14 C from labeled FAA, mainly as met-abolites conjugated with glucuronic acid. There was a small drop in excretion of sulfuric acid conjugates. In the glucosiduronic acid fraction, there were increases in the N - and 7-hydroxy derivatives of FAA and a decrease in the 5-hydroxy compound, while the 3-hydroxy metabolite was virtually unchanged. The concentration of metabolites of FAA in the liver was considerably lower, as was the amount of isotope bound to DNA. This important finding, correlated with a reduced carcinogenicity of FAA in rats given PB, is ascribed to increased conjugation with glucuronic acid and lesser formation of sulfate esters. The microsomal fraction in vitro from the livers of young rats yielded metabolites of FAA, in decreasing order, hydroxylated at 7-, 5-, N - and 3-. Carbon monoxide significantly inhibited formation of the 7-hydroxy metabolite but elevated somewhat that of the N -hydroxy compound. Pretreatment with PB increased cytochrome P-450 and hydroxylation at all positions 2- to 4-fold, the greatest effect being with N -hydroxy-FAA. With microsomes from PB-treated rat livers, carbon monoxide depressed hydroxylation at the 3-position, while N -hydroxylation was least affected. Because hydroxylation of FAA occurs at several well-defined ring positions and on the amido nitrogen, FAA is a good substrate to explore the mechanisms of hydroxylation reactions. The data obtained suggest that these metabolic reactions are performed by a family of related enzyme systems.

After intraperitoneal injection of the title compounds into rats the major metabolite found in the urine was 2-(-4-hydroxyphenylazo-)-1-naphthol-4 or 5-sulphonate and the corresponding glucuronide. No reduction of the dye was found to take place. This was confirmed using 2-phenylazo-14C-1-naphthol-5- sulphonate. Radioactivity was quantitatively excreted in the urine after 48 hr and was associated only with whole dye metabolites. Some enterophepatic circulation of the dyes took place but did not lead to reduction of the dyes in the gastro-intestinal tract. The reasons for the failure of the dye to be reduced in vivo are discussed. The hydroxylation of these compounds appears to be the first example of such a reaction in vivo with a non-lipid soluble molecule and has been shown to take place in the microsomal fraction of rat liver.

When sodium 35S-sulphide is administered to rats either intraperitoneally, orally or intravenously it is oxidized primarily to inorganic 35S-sulphate which is eventually excreted in the urine. Intravenously administered sulphide has a transient existence in blood and the lack of an accumulative effect of sulphide poisoning is due to the rapid rate of oxidation. The blood is not the principle site of sulphide oxidation in vivo. Sulphide detoxication occurs in both plasma and red cells in vitro by binding to proteins.

Rats and rabbits have been dosed with cyclopentyl, cyclohexyl and cyclo-heptyl bromide and also the corresponding cycloalkenes and cycloalkene epoxides. The excretion of glucosiduronic acids and ethereal sulphates by rabbits dosed with the above compounds has been measured, the extent of glucosiduronic acid formation from cycloalkyl bromides and cycloalkenes being found to increase with increasing ring size. The above cycloalkyl bromides, cycloalkenes and cycloalkene epoxides were fed to rats, and the level of total liver glutathione measured 1, 2 and 4 hr after dosing, cycloalkenes of a series being found to give the greatest reduction in liver glutathione, and cycloalkyl bromides the least. The excretion of mercapturic acids has been measured in both rabbit and rat, the 3-hydroxymercapturic acids being the major metabolites from the cycloalkyl bromides and cycloalkenes, while the epoxides only form the 2-hydroxy isomers. These mercapturic acids have been identified by mass spectrometry and comparison with synthetic compounds. Evidence is presented to show that these hydroxylations are probably carried out by a system involving cytochrome P-450.

As an evidence for the hydroxylation of l -tryptophan (Try) to 5-hydroxytryptophan (5-HTP) in the brain, it was demonstrated that intracerebrally injected (14C)- l -Try led to the appearance of (14C)-5-hydroxytryptamine (5-HT) in the brains of pigeons and rats. Under identical conditions, the injection of the d -isomer did not give labeled 5-HT, but it decreased the 5-HT level in the brain. Intraperitoneal injection of(14C)-Try in amounts equal to or 17 times larger than those injected i.e. did not lead to the appearance of labeled 5-HT in the central nervous system. Identification of label in 5-HT was proved by column, paper chromatography, enzymatic conversion to 5-hydroxyindoleacetic acid, and by recovery of (14C)-5-HT from its picrate salt. Results of the studies of the efflux and turnover of Try, 5-HTP, and 5-HT are also presented.

It is known that epoxides are intermediates in the conversion of aromatic hydrocarbons into dihydrodiols, phenolic metabolites and GSH conjugates. The effect of oestradiol, in vitro , on the metabolic conversion of 7,12-dimethylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene and 12-hydroxymethyl-7-methylbenz[a]-anthracene into these derivatives by a system containing NADPH, GSH and the microsomal and dialysed soluble fractions of female rat-liver has been investigated. The amounts of all three types of metabolites formed are much reduced by the presence of oestradiol and it is likely that the steroid acts by reducing the amounts of epoxide intermediates formed rather than by inhibiting the various reactions concerned in their further metabolism. This is supported by the finding that oestradiol also reduces the total amount of each substrate metabolized. Cyclohexene oxide, an inhibitor of the conversion of epoxides into dihydrodiols by the enzyme “epoxide hydrase”, greatly reduces the amount of dihydrodiols formed, but is without effect on the amounts of substrate metabolized. Furthermore, although the omission of GSH and soluble liver fraction from reaction mixtures prevents the synthesis of GSH conjugates, it does not reduce the amount of substrate metabolized, an effect that differs from that produced by oestradiol.

Diaryl o -( α -hydroxy)tolyl phosphates are cyclized with liberation of an aryl group to yield aryl saligenin cyclic phosphates, which usually are potent antiesterase agents. This cyclization is catalyzed by various plasmata, but not by liver homogenate. The cyclizing activity of plasma is associated with the albumin component when this fraction is prepared either by salting-out with ammonium sulfate or by using Cohn's method and ethanol (fraction V). The activity of the cyclizing enzyme could not be separated from albumin either by Sephadex G-100 or by DEAE-cellulose column chromatography. p -Nitrophenyl acetate, which is also hydrolyzed by the albumin fraction, inhibits the cyclization of diaryl o -( α -hydroxy)tolyl phosphates.