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Styrene monooxygenase and epoxide hydratase activities were studied in the hepatic and extrahepatic microsomal fractions of male and female rats, mice, guinea pigs and rabbits. The highest specific acitivity of both enzymes was present in the liver where levels of epoxide hydratase were higher than monooxygenase. The two enzyme systems are present in all the tissues of both sexes of the species considered. A particularly high ratio of monoxygenase to hydratase activities was found in mouse and rabbit lungs.

The relationship between indenolol (an investigational agent) plasma levels and the drug's effect on blood pressure and heart rate was investigated after single and repeated once daily administration at two dosage levels (60 mg and 120 mg) in two different groups of patients with first or second stage hypertension, according to the World Health Organization classification. The pharmacokinetic data were indicative of a first order absorption-elimination curve; time of maximum plasma levels was 1.5 to two hours, and elimination half-life was four hours. The drug did not accumulate in the central compartment after repeated administrations. A long-lasting decrease of both resting and isometric exercise systolic pressure values was recorded after acute indenolol administration. Diastolic pressure was affected only by repeated administrations. The lower dose (60 mg daily) of indenolol did not affect heart rate, whereas the higher dose (120 mg daily) decreased this parameter. A steady state of pressure values and heart rate was reached after 14 days of once daily treatment.

5H-Dibenzol[b,f]azepine-10,11-epoxide and 10,11-dihydro-10,11-dihydroxy-5H-dibenzo[b,f]azepine were identified by gas chromatography-mass spectrometry in rat urine as the main biotransformation products in the metabolism of 5H-dibenzo[b,f]azepine (iminostilbene). The presence of these metabolites was confirmed in vitro by incubating iminostilbene with rat liver microsomal enzymes.

The 10,11-epoxide, 10-hydroxy, and 10,11-dihydrodiol metabolites of protriptyline were identified in rat urine collected after the administration of 40 mg/kg ip of protriptyline. Mass spectrometric characterization confirmed the structure of these metabolites.

A specific and sensitive method is described for the simultaneous determination of carbamazepineand carbamazepine-10,11-epoxide in biological specimens by combined gas chromatography-mass spectrometry. Cytenamide is used as the internal standard for quantitation. Kinetics of the distribution of the drug and its metabolite in plasma and in different areas of rat brain are reported. Determinations are possible at the nanogram level.

Studies of the cell killing effectiveness of ftorafur were carried out against 5-fluorouracil (5-FU). It appeared that in divided doses ftorafur is both relatively less toxic in terms of LD50 and bone marrow stem cell killing and less effective against L1210 and a mouse osteosarcoma than 5-FU. Pharmacokinetic analysis revealed that upon repeated administration of 5-FU there is an increase in late drug serum levels and a decreased clearance of 5-FU from the serum. Repeated administration of ftorafur is not associated with significant differences in serum levels of 5-FU or ftorafur.

The metabolism of styrene was studied in the rat after intraperitoneal administration of the cold and the 14C-labeled compound. In addition to phenylethylene glycol, mandelic acid, benzoic acid and hippuric acid, phenolic metabolites, namely, 4-vinylphenol, p-hydroxymandelic acid, p-hydroxybenzoic acid, and p-hydroxyhippuric acid, were identified in the urine of the treated animals. These biotransformation products were characterized by mass spectrometry and by comparative thin layer chromatography with standard compounds. Results of covalent binding studies of 14C-phenylethylene glycol to rat liver microsomal proteins suggest that these phenolic compounds may be formed as a result of chemical rearrangements of unstable arene oxides, reactive intermediates possibly implicated in styrene toxicity.

The interaction between cyclophosphamide (CPA) and phenobarbital (PB) was investigated in B6D2F, mice, checking both the antileukemic and immunosuppressive activity together with the serum levels of CPA and its metabolites. A reduced cytotoxic activity of CPA has been observed when PB is given for 2 days before CPA and an interval of at least 6 hours elapses between the last treatment of PB and the administration of CPA. On the contrary, when PB is given simultaneously with CPA for 2 or 4 consecutive days, an increased antileukemic activity of CPA occurs. In the experimental condition where PB decreases the activity of CPA, serum levels of CPA, assayed by means of a new specific gas-chromatographic method, and of its NBP-alkylating metabolites, indicate that this effect may be explained on a pure pharmacokinetic basis. However, for the situation where an increased effect of CPA was observed under the influence of PB, pharmacokinetic data did not provide a clear explanation.

1. The metabolism in vitro of cyclobenzaprine by rat liver microsomal preparations was investigated.2. Four metabolic products, cyclobenzaprine N-oxide, 10,11-epoxycyclo-benzaprine N-oxide, desmethylcyclobenzaprine and cyclobenzaprine-10,11-epoxide were isolated and identified by comparing physical properties with those of authentic compounds.

Epichlorohydrin (ECH), a direct mutagen in vitro, did not induce chromosomal aberrations in bone-marrow cells of CD1 mice given single oral doses of 50 and 200 mg/kg in water. The ECH diol derivative (3-chloro-1,2-propanediol) was tested in vitro by a forward-mutation assay on the yeast Schizosaccharomyces pombe and showed a weak but significant mutagenic effect. The failure of ECH to induce mutagenic effects appears to be due to the rapid metabolic clearance of the compound in vivo. ECH blood kinetics at both doses, and at the same time the concentration of the diol, were determined. ECH rapidly disappeared from mouse blood, being no longer detectable 20 min after treatment. In contrast, 3-chloro-1,2-propanediol was measurable up to 5 h after dosage. No difference was observed in the kinetic and metabolic behavior of ECH after single and repeated doses (50 and 200 mg/kg/day for 7 days). When 3-chloro-1,2-propanediol was tested, neither glutathione depletion nor epoxide hydrolase inhibition (evaluated with both styrene-7,8-oxide and ECH as substrates) could be detected in mouse liver. Finally, no difference in ECH blood kinetics or metabolism were observed in experiments in which the compound was administered (200 mg/kg) intraperitoneally in water or orally as a solution in dimethyl sulfoxide.

5,5-Diphenylhydantoin irreversibly binds to rat liver microsomes and the process requires NADPH and O 2 . Protein binding was significantly enhanced when experiments were carried out with liver microsomal preparations from β-naphthoflavone and 3-methylcholanthrene pretreated animals whereas pretreatment with phenobarbital significantly reduced it. Carbon monoxide, β-diethylaminoethyl-diphenylpropylacetate and glutathione inhibited drug covalent binding to microsomal proteins. In contrast, enhanced drug binding was observed when trichloropropene oxide and cyclohexene oxide, two epoxide hydrolase inhibitors, were added to the incubation mixture. 5,5-Diphenylhydantoin in vitro metabolism was quantitatively determined by gas liquid chromatography with selected ion monitoring. A good correlation seems to exist between drug covalent binding and the microsomal process of 5,5-diphenylhydantoin hydroxylation to 5-(4-hydroxyphenyl)-5-phenylhydantoin. The results presented support a previous hypothesis on the intermediacy of arene oxides in the biotransformation of this drug.

A sensitive and specific method for the simultaneous determination of imipramine and desipramine in the blood plasma of depressed patients under treatment that involves separation by gas chromatography and detection by mass fragmentography is described. Concentrations were determined by focusing the mass spectrometer upon the ions at m/e 280 and 235 for imipramine and 308, 236 and 114 for desipramine (N-acetyl derivative), while promazine was used as the internal standard (ions at m/e 284 and 238). Determinations are possible at levels as low as 10 ng/ml in plasma.

1. The uptake of unlabelled and [(14)C]-desipramine was studied in rat isolated atria incubated in a medium containing concentrations of desipramine ranging from 200 pg/ml to 2 mug/ml. The uptake was found to be dose- and time-dependent. Equilibrium was not reached after 2-3 h of incubation unless concentrations of desipramine higher than 1 mug/ml were used.2. The washout curves of atria previously loaded with desipramine showed that the drug is slowly released and that this release is not influenced by its initial tissue concentration.3. The binding appeared to be at non-specific sites and not at sites where noradrenaline is stored since atria taken from rats treated with 6-hydroxydopamine accumulated the drug at the same rate as control atria.4. The inhibition of (-)-noradrenaline uptake and the potentiation of the chronotropic response to (-)-noradrenaline is correlated with the concentration of desipramine in atria for tissue levels of the drug ranging from 0.01 to 1 mug/g. Higher tissue levels show less potentiation of the effect of (-)-noradrenaline or even inhibition of the maximal response to (-)-noradrenaline. These concentrations of desipramine (> 7 mug/g) markedly depressed the atrial rate.5. The results show that despite the accumulation of desipramine by unspecific sites, concentrations of desipramine in the tissue are correlated with the pharmacological response. Furthermore a gradual shift from potentiation to inhibition of noradrenaline response can be obtained with the same bath concentrations of desipramine by increasing the time of incubation.

A sensitive and specific method for the quantitative determination of imipramine in human biological fluids is described. After extraction with n -hexane, imipramine is detected by gas-liquid chromatography-mass fragmentography. The procedure permits routine analysis on relatively small amounts of plasma (1–2 ml) containing as little as 10 ng/ml of imipramine without any interference from endogenous substrates.

Gas chromatography—chemical ionization mass fragmentography has been applied in the quantitative determination of purine and pyrimidine bases and nucleoside analogues that are widely used in cancer chemotherapy. A permethylation reaction makes them suitable for gas chromatographic analysis. Single ion detection by focusing the mass spectrometer on the abundant “quasimolecular ions” proved to be a highly specific and sensitive procedure for the identification and evaluation of these drugs and their metabolic products present in biological fluids in amounts as low as 100 ng/ml without any interference due to endogenous substrates.

Adriamycin (NSC-123, 127) is an anticancer drug of the anthracycline type widely used in the clinical practice for the treatment of different malignancies (1). Intercalation with DNA has been suggested as having a role in the mechanism of action of this drug (2, 3, 4). Recently, reports by Sinha (5) and Lucacchini et al. (6) suggested a covalent interaction of Adriamycin (AM) with DNA and proteins. These studies, however, were not conclusive since they were carried out by spectrophotometric and Chromatographie procedures. In the present study a covalent interaction between AM and microsomal proteins was demonstrated in a more “classical” covalent binding study with the 14 C-labelled compound.

On July 10, 1976, as a consequence of an accident at the ICMESA Chemical plant in Meda, not far from Milan, Italy, a large densely populated area (town of Seveso and its southern outskirts) was contamined with 2, 3, 7, 8-tetrachlorodibenzoparadioxin (TCDD) (1, 2).

Cyclobenzaprine (40 mg/kg ip) was administered to rats, and six urinary metabolites of this drug were identified. They were the 10,11-epoxide, the N -oxide, the desmethyl derivative, the hydroxylated and desmethylhydroxylated compounds, and the N -oxide hydroxylated at the 10- or 11-position. Mass spectrometric analysis confirmed their structures.

A sensitive and specific combined gas chromatographic-mass fragmentographic method for the determination of trazodone in rat plasma is described. After extraction with diethyl ether and washing of the extracts in order to prevent interference from endogenous materials, trazodone and the internal standad benperidol were separated on an OV-1 column. The minimum amount of trazodone detected was 20 ng by gas chromatography with the use of a flame ionization detector and 200 pg when using the mass fagmentographic technique. Plasma levels in rats treated with a single intravenous does (10 mg/kg) of trazodone are also reported.

A specific and sensitive method is described for the determination of saccharin in biological fluids. The compound is extracted as its methyl derivative following a salt-solvent pair procedure and assayed by GLC with either flame-ionization or mass fragmentographic detection using ethylated or trideuteromethylated saccharin, respectively, as the internal standard for quantitation. Detector response was linear over concentrations of 50 mg/ml--10 micrograms/ml with multiple-ion detection mass fragmentography and from 2 micrograms/ml up to milligram levels with flame-ionization detection. Interference from endogenous substrates was never observed. Plasma kinetics and urinary elimination of saccharin in healthy human volunteers given the sweetener orally, acutely (50 mg/60 kg of body weight) or for 5 days (130 mg/60 kg of body weight/day divided over the three main meals), also are reported.

Male rat liver intact nuclear preparations are able to metabolize adriamycin to reactive species that irreversibly interact with nuclear DNA and proteins in the presence of reduced NADPH. This process was not inhibited by 1 mM SKF-525A, suggesting that a nuclear monooxygenase enzymatic system was not involved.

Styrene's capacity to induce chromosomal aberrations was studied in bone marrow cells of CD1 male mice. No mutagenic effect could be detected after either a 4-day treatment course with daily oral doses of 500 mg/kg or a 70-day course with daily oral doses of 200 mg/kg. Urinary elimination of styrene metabolites related to styrene-7,8-oxide formation (i.e. phenylethylene glycol, mandelic acid, benzoic acid, phenylglyoxylic acid and total mercapturic acids) was quantitatively evaluated in the group of mice given the 200 mg/kg dose. In parallel, kinetic studies were made on styrene and styrene-7,8-oxide blood concentrations in the same group of animals. These determinations were carried out on days 1 and 70 of treatment by spectrophotometric, gas chromatographic and mass fragmentographic procedures. Not even nanograms of styrene-7,8-oxide were found in the blood of styrene-treated mice. This suggests that the metabolite does not migrate from the cellular compartment where it is formed being immediately metabolized or irreversibly bound to cellular structures. This observation could well explain the lack of mutagenic effects observed.

A series of analytical techniques are described which may give a deeper insight into the mechanisms of styrene toxicity. These techniques include: ultraviolet (UV) photometry, thin layer chromatography (TLC), liquid scintillation counting (LSC), gas liquid chromatography (GLC), mass spectrometry (MS), mass fragmentography (MF) and mass chromatography (MC). Styrene, styrene-7,8-oxide, reduced glutathione and the activities of toxifying (styrene-7,8 -oxide forming monooxygenase) and detoxifying (styrene-7,8-oxide hydrase and glutathione-S-styrene-7,8-oxide transferase) enzymes were assayed in the liver, brain, lungs, heart, perirenal fat, spleen and kidneys of male mice with a view to building an experimental model able to predict which body districts are most likely to be targets for styrene toxicity. The results described show a good correlation between our in vitro and in vivo data and reports in the literature concerning the in vivo toxicity of this vinyl compound towards discrete tissues in man and laboratory animals. New perspectives on styrene toxicity are opened by the identification in rat urine of 4-vinylphenol, p-hydroxymandelic acid, p-hydroxybenzoic acid and p-hydroxyhippuric acid. This ancillary metabolic pathway leading to the formation of phenolic type metabolites has been seen to result from chemical rearrangements of unstable arene oxides, reactive species which may be implicated in styrene toxicity.

Styrene was quantitatively determined by head space gas chromatography-selected ion monitoring using ethylbenzene as the internal standard. The lower limit of sensitivity of this procedure is 500 pg per milliliter of blood or gram of wet tissue and detector response (flame ionization or selected ion monitoring) was linear up to microgram concentrations without any interference from endogenous compounds. The kinetics of styrene distribution in blood and tissues (brain, heart, lungs, liver, kidneys, spleen, and perirenal fat) of CD2F1 mice receiving a single intraperitoneal administration of styrene (200 mg/kg) were also investigated.

Caffeine was found not to bind covalently to liver microsomal proteins from mice, rats and rabbits. Microsomes metabolized caffeine only to a limited extent, the highest rate (about 2% of the substrate concentration) being obtained with rabbit microsomal preparations. The rat liver perfusion technique represents a good model for in vitro caffeine biotransformation studies and therefore for covalent binding experiments. After 2 h perfusion caffeine was extensively metabolized mainly to dimethyl and monomethyl xanthines, a minor pathway to 1,3,7-trimethyluric acid was also seen. However, covalent binding studies using the liver perfusion technique did not reveal any appreciable amount of caffeine metabolites irreversibly bound to either microsomal and total proteins and to DNA.

Brain monoamines may be involved not only in the electroconvulsive threshold but also in the anticonvulsant activity of antiepileptic drugs in laboratory animals. Increased susceptibility to electroconvulsions has been reported in mice and rats treated with drugs which deplete brain monoamines, such as reserpine, a-methylparatyrosine and parachlorophenylalanine (1–4). These findings, together with reports on the anticonvulsant activity of two amine precursors, 1-dopa and 5-hydroxytryptophan (4, 5), and d-amphetamine (3), have led to the suggestion that brain monoamines may play a role in seizure susceptibility

The covalent binding of hexamethylmelamine (HMM) and its metabolites was studied in liver, tumor, blood, kidney, spleen, lung, brain, heart, and small intestine after a single IP injection of 2,4,6-14C-hexamethylmelamine (50 mg/kg) to C57Bl/6J female mice bearing 20-day-old M5076/73A ovarian cancer. Covalent binding to tissue macromolecules was measured 2, 10, and 40 h after injection of the drug. At 2 h liver and small intestine showed the highest levels of irreversibly bound metabolites, the lowest being found in brain and heart. Except in the small intestine, where a decrease was observed between 2 and 10 h, the level of covalent binding was constant up to 40 h. HMM metabolism was also studied. Tissue distribution of pentamethylmelamine (PMM), 2,2,4,6-tetramethylmelamine (TMM), and 2,4,6-trimethylmelamine (TriMM) was determined at the three times considered. At 2 h the drug was already extensively metabolized, TriMM being the major metabolite among those determined.

A method developed for measuring the antitumor agent ftorafur and its biotransformation product 5-fluorouracil was applied to biological specimens. After extraction with ethylacetate, ftorafur, 5-fluorouracil, and the internal standard 2-methyl-4-hydroxy-6-chloromethylpyrimidine are converted to their chloromethyldimethylsilyl derivatives and assayed by glc, using either an electron-capture or a flame ionization detector. The minimum detectable amount is 100 pg/injection for ftorafur and 50 pg/injection for 5-fluorouracil employing electron-capture detection. Linearity was found up to microgram amounts of both substances, without any interference from endogenous substrates. Preliminary data are reported on the comparative serum kinetics of ftorafur and 5-fluorouracil in mice.

A sensitive and specific method involving gas chromatographic separation with nitrogen—phosphorus selective detection has been developed and applied for the determination of hemamethylmelamine in mouse plasma. Following extraction with n-hexane, the drug is quantitatively evaluated using triethylenemelamine as external standard. The lower limit of sensitivity of the method is 10 ng/ml of mouse plasma and detector response was linear up to 2 μg/ml. The plasma disappearance curve after a single intraperitoneal dose of hexamethylmelamine to mice is reported. Pentamethylmelamine and N2,N2,N4,N6-tetramethylmelamine were identified in mouse plasma as hexamethylmelamine metabolites by combined gas chromatography—mass spectrometry.

1. The mono-oxygenase activity that forms epoxides has been studied in rat liver microsomes using as substrates carbamazepine and cyclobenzaprine, tricyclic drugs which form stable epoxides in vivo and in vitro.2. A simple gas chromatographic method has been used to determine the amount of epoxide formed and the linearity of the enzymic reaction with time and protein concentration has been demonstrated.3. Pre-treatment with carbamazepine increases the rate of formation of carbamazepine epoxide in rat liver microsomal preparations.4. The effect of SKF 525-A on the formation of these epoxides has also been studied.

On incubation with rat liver microsomes, N,N-dimethyl-5H-dibenzo[a,d]cycloheptene-Δ5,γ-propylamine is converted to the N-oxide. The identification of this metabolite has been achieved by the use of gas chromatography, mass spectrometry and thin-layer chromatography.

The plasma kinetics and urinary elimination of saccharin were studied in 3 groups each of 5 healthy male volunteers given the sweetener as three different single oral doses (50, 150 and 333 mg 60 kg body weight). Saccharin concentrations were determined by gas liquid chromatography-stable isotope dilution mass fragmentography. The compound was rapidly absorbed through the gastrointestinal tract, reaching plasma peak concentrations between 30 and 60 min after intake. Plasma saccharin elimination was also fast, with a monoexponential pattern of decay. At the 3 doses studied saccharin was excreted in urine within a few hours, about 60% of the dose being excreted unchanged at 6 h and 76% at 24 h.

Caffeine metabolism to 6-amino-5-[ N -methylformylamino]-1,3-dimethyluracil was studied in the isolated, perfused rat liver. The [2- 14 C]-labelled drug and metabolites were separated by thin-layer chromatography or high-pressure liquid chromatography. The chemical structure of 6-amino-5-[ N -methylformylamino]-1,3-dimethyluracil was confirmed by mass spectrometry and it was quantitatively determined by liquid scintillation counting. 6-Amino-5-[ N -methylformylamino]-1,3-dimethyluracil is one of the major metabolites of caffeine found in the perfusion medium. The kinetics of caffeine elimination and of the uracil metabolite formation were studied up to 2 h perfusion time using livers from control rats and rats pretreated with phenobarbital, β-naphthoflavone or 3-methylcholanthrene. Phenobarbital pretreatment did not modify the rate of caffeine elimination or the extent of 6-amino-5-[ N -methylformylamino]-1,3-dimethyluracil formation. In contrast, there was a highly significant inducing effect on both drug elimination and formation of the uracil metabolite in perfusions of livers from β-naphthoflavone- and 3-methylcholanthrene-pretreated animals.