Nicotine metabolism and urinary elimination in mouse: in vitro and in vivo

This study aimed at elucidating the in vivo metabolism of nicotine both with and without inhibitors of nicotine metabolism. Second, the role of mouse CYP2A5 in nicotine oxidation in vitro was studied as such information is needed to assess whether the mouse is a suitable model for studying chemical inhibitors of the human CYP2A6. The oxidation of nicotine to cotinine was measured and the ability of various inhibitors to modify this reaction was determined. Nicotine and various inhibitors were co-administered to CD2F1 mice, and nicotine and urinary levels of nicotine and four metabolites were determined. In mouse liver microsomes anti-CYP2A5 antibody and known chemical inhibitors of the CYP2A5 enzyme blocked cotinine formation by 85-100%, depending on the pre-treatment of the mice. The amount of trans-3-hydroxycotine was five times higher than cotinine N-oxide, and ten times higher than nicotine N-1-oxide and cotinine. Methoxsalen, an irreversible inhibitor of CYP2A5, significantly reduced the metabolic elimination of nicotine in vivo, but the reversible inhibitors had no effect. It is concluded that the metabolism of nicotine in mouse is very similar to that in man and, therefore, that the mouse is a suitable model for testing novel chemical inhibitors of human CYP2A6.     

Nicotine metabolism and urinary elimination in mouse: in vitro and in vivo

This study aimed at elucidating the in vivo metabolism of nicotine both with and without inhibitors of nicotine metabolism. Second, the role of mouse CYP2A5 in nicotine oxidation in vitro was studied as such information is needed to assess whether the mouse is a suitable model for studying chemical inhibitors of the human CYP2A6. The oxidation of nicotine to cotinine was measured and the ability of various inhibitors to modify this reaction was determined. Nicotine and various inhibitors were co-administered to CD2F1 mice, and nicotine and urinary levels of nicotine and four metabolites were determined. In mouse liver microsomes anti-CYP2A5 antibody and known chemical inhibitors of the CYP2A5 enzyme blocked cotinine formation by 85–100%, depending on the pre-treatment of the mice. The amount of trans-3-hydroxycotine was five times higher than cotinine N-oxide, and ten times higher than nicotine N-1-oxide and cotinine. Methoxsalen, an irreversible inhibitor of CYP2A5, significantly reduced the metabolic elimination of nicotine in vivo, but the reversible inhibitors had no effect. It is concluded that the metabolism of nicotine in mouse is very similar to that in man and, therefore, that the mouse is a suitable model for testing novel chemical inhibitors of human CYP2A6.     

Alkylation of RNA by vinyl bromide metabolites in vitro and in vivo

[1,2-¹⁴C]Vinyl bromide was incubated with rat liver microsomes, NADPH, and polyadenylic acid, polycytidylic acid, or RNA, respectively. Part of the adenosine moieties in RNA or in polyadenylic acid were alkylated and labelled 1,N⁶-ethenoadenosine structures were formed. Part of the cytidine moieties were converted into 3,N⁴-ethenocytidine. In addition, a further unidentified cytidine alkylation product was observed which was not seen in experiments using [1,2-¹⁴C]vinyl chloride. When rats were exposed to [1,2-¹⁴C]vinyl bromide, radioactive ethenoadenosine and ethenocytidine were present in hydrolysates of liver RNA. A further alkylation product was observed in the RNA hydrolysates which did not occur in experiments using [¹⁴C]vinyl chloride. The data show that vinyl bromide metabolites alkylate nucleic acids; although in general in this respect vinyl bromide and vinyl chloride behave similarly, some differences are observed in the alkylation behaviour of both compounds.     

In vitro metabolism of teratogens by differentiating rat embryo cells

Rapid and accurate prediction of teratogenic hazard had been achieved using cultures of differentiating limb mesenchyme (LB) and midbrain (CNS) cells from 13-day-old rat embryos. In this study we have used these cultures to examine the role of metabolism in the in vitro teratogenic activity of diphenylhydantoin (DPH) and cyclophosphamide (CPA). Two approaches were used. The first involved modulation of cytochrome P-450 activity by co-incubation in vitro with a variety of inhibitors at concentrations that were non-cytotoxic to the cells. This enhanced the toxicity of DPH by 13-82% in LB and by 3-52% in CNS cells. Benzimidazole and ellipticine caused the greatest enhancement and SKF 525A the least. DPH appears to be the proximate teratogen and there appear to be embryo-tissue cytochrome P-450s that assist in its detoxification. Following prior transplacental induction, CPA was toxic in vitro to LB cells from beta-naphthoflavone-pretreated mothers. CPA was non-toxic in cells of control, phenobarbitone- or 3-methylcholanthrene-treated embryos. Thus there appear to be inducible levels of cytochrome P-448 in embryo cells. In the second approach, positive immunocytochemical staining of the cells with both monoclonal and polyclonal P-450 antibodies identified phenobarbitone, beta-naphthoflavone- and 3-methylcholanthrene-inducible cytochrome P-450s at a constitutive level. Cytochromes P-448 (beta-naphthoflavone type) and P-450 (phenobarbitone type, PB3 fraction) were inducible, confirming that cytochrome P-450s are in fact present in the embryo cells.     

Biotransformation of lovastatin. I. Structure elucidation of in vitro and in vivo metabolites in the rat and mouse

Structures of in vitro microsomal and in vivo metabolites of lovastatin, a new cholesterol-lowering drug, were elucidated with the combined application of HPLC, UV, fast atom bombardment-MS, and NMR spectroscopy. Liver microsomes from rats and mice catalyzed the biotransformation of lovastatin, primarily at the 6'-position of the molecule, to form 6'-hydroxy-lovastatin and a novel 6'-exomethylene derivative. Hydroxylation at the 6'-position occurred stereoselectively, giving 6'-beta-hydroxy-lovastatin. Stereoselective hydroxylation at the 3"-position of the methylbutyryl side chain and hydrolysis of the lactone group to the corresponding hydroxy acid were the other two pathways of microsomal metabolism. 3'-Hydroxy-iso-delta 4',5'-lovastatin was isolated, but is not believed to be a direct metabolite since 6'-beta-hydroxy-lovastatin rearranges to this compound under mildly acidic conditions. The major metabolites excreted in bile of rats treated with the hydroxy acid form of the drug were identified as the 3'-hydroxy analog and a taurine conjugate of a beta-oxidation product of lovastatin. The pentanoic acid derivative of lovastatin, formed by beta-oxidation of the heptanoic acid moiety, was a major metabolite in livers of mice dosed with the hydroxy acid form of lovastatin. The microsomal metabolites, in their hydroxy acid forms, were active inhibitors of HMG-CoA reductase. The relative enzyme inhibitory activities of hydroxy acid forms of lovastatin, 6'-beta-hydroxy-, 6'-exomethylene-, and 3"-hydroxy-lovastatin were 1, 0.6, 0.5, and 0.15, respectively.     

Irreversible in vivo and in vitro binding of thiabendazole to tissue protein of pregnant mice

In vivo experiments showed that [¹⁴C]thiabendazole is irreversibly bound to protein of the liver and embryo in pregnant mice, but not to DNA and RNA in these tissues. In experiments carried out in vitro, the binding of [¹⁴C]thiabendazole to microsomal protein of the liver was proportional to time and protein concentration, was dependent upon the presence of oxygen and NADPH and was inhibited by carbon monoxide or SKF-525A, indicating that it was mediated by the cytochrome P-450-dependent monooxygenase system. The binding capacity of microsomes in various tissues was examined, and that of the liver microsomes was found to be more pronounced than that of other tissues.     

In vivo suppression of delayed hypersensitivity by thiabendazole and diethylcarbamazine

Several anthelminthic agents, such as niridazole and metronidazole, have been demonstrated to have striking effects on the immune system, apparently independent of their antiparasitic activities. In the present study, we have examined the effect of thiabendazole and diethylcarbamazine on two parameters of delayed hypersensitivity, lung granuloma formation around Schistosoma mansoni eggs and delayed footpad edema in response to schistosome egg antigens. Both drugs caused significant reduction of lung granuloma size in unsensitized animals when given as daily doses for 8 days. Thiabendazole was the more potent suppressant, affecting granuloma size in unsensitized animals when given as single dose and in sensitized animals using a multiple dose regimen. Diethylcarbamazine was without effect on granuloma size in sensitized animals, but inhibited delayed foodpad swelling when given daily for 6 days. Neither drug affected granulomas induced by non-antigenic plastic beads. These data support the hypothesis that some of the clinical activities of these drugs may be mediated by interference with host response to antigenic stimuli.     

Inhibition of thiabendazole metabolism in the rat.

1. A single oral dose of desmethylimipramine (80 mg/kg) administered to rats inhibited the hepatic microsomal hydroxylation of thiabendazole (45%), aniline (30%), biphenyl (30%) and ethylmorphine (15%) in vitro at 5 h after dosage; there was no decrease in cytochrome P-450 or b5. 2. A single oral dose of ethoxyquin (200 mg/kg) to rats inhibited the hepatic microsomal hydroxylation of thiabendazole (65%), aniline (40%) and biphenyl (40%) in vitro at 1 h after dosage; inhibition was less at 5 h. There were no changes in the contents of cytochromes P-450 and b5. 3. The max. plasma concn. of thiabendazole occurred 2--4 h after oral dosing (50--200 mg/kg) to rats. Thiabendazole (100 mg/kg) administered orally 30 min after oral ethoxyquin (400 mg/kg) or thiabendazole (200 mg/kg) administered orally 30 min after oral desmethylimipramine (80 mg/kg) delayed absorption of the thiabendazole and resulted in markedly markedly decreased plasma concentration of the anthelmintic. 4. Simultaneous administration of ethoxyquin (300 mg/kg) potentiated the anthelmintic effect of thiabendazole (750 mg/kg) on the helminth parasite, Nematospiroides dubius, in the mouse. Desmethylimipramine showed no similar potentiation.     

Identification of novel phencyclidine metabolites formed in vitro by rabbit microsomal metabolism

1. Phencyclidine (PCP) was incubated with rabbit liver and brain microsomal fractions, and the structures of metabolites formed by oxidation determined by g.l.c.-mass spectrometry.

2. The formation of several known mono- and di-hydroxylated metabolites, as well as two new metabolites, was seen in the liver preparations.

3. Hydroxylated PCP metabolites were also formed after incubation of PCP with brain microsomes, indicating that PCP biotransformation may occur in the brain itself.     

Disposition of 8-methoxypsoralen in the rat. Induction of metabolism in vivo and in vitro and identification of urinary metabolites by thermospray mass spectrometry

The pharmacokinetics and metabolism of 8-methoxypsoralen (8-MOP) were measured in the catheterized rat after pretreatment for 3 days with phenobarbital (PB), beta-naphthoflavone (BNF), 8-MOP, or vehicle. After an iv injection of 10 mg/kg of [14C]8-MOP, timed blood samples were collected and analyzed using a sensitive and specific assay for [14C]8-MOP. Total body clearance of 8-MOP increased from 0.55 +/- 0.06 liter/kg/hr in control rats to 5.6 +/- 0.4, 2.7 +/- 0.4, and 1.2 +/- 0.0 liters/kg/hr in rats pretreated with BNF, PB, and 8-MOP, respectively, indicating that all three compounds are inducers of 8-MOP metabolism. The pattern of urinary metabolites was altered by the enzyme inducers. The urinary excretion of the sulfate conjugate of 5-hydroxy-8-methoxypsoralen was increased from 10 to 40% of the dose after pretreatment with PB. This intact conjugate was identified using thermospray and fast atom bombardment mass spectrometry. Pretreatment with 8-MOP and BNF increased 2- and 3-fold, respectively, the urinary excretion of a labile sulfate conjugate of 5,8-dihydroxypsoralen. Metabolism of 8-MOP was demonstrated in the 9000 g supernatant and microsomes of rat liver and shown to be inducible by pretreatment of rats with BNF, PB, and 8-MOP. 8-MOP was metabolized in incubations with liver microsomes at rates of 0.22 +/- 0.06, 0.38 +/- 0.06, 0.78 +/- 0.07, and 0.91 +/- 0.03 nmol/min/mg of protein for vehicle, 8-MOP-, PB-, and BNF-pretreated rats, respectively. Results of our investigation indicate that the success of therapy with 8-MOP may be influenced by pharmacokinetic interactions with other drugs.     

Nitroimidazole bioreductive metabolism. Quantitation and characterisation of mouse tissue benznidazole nitroreductases in vivo and in vitro

We have investigated the nitroreduction of the 2-nitroimidazole benznidazole (BENZO) to its corresponding amine by murine normal tissues and tumours. In vivo concentrations of BENZO and its amine metabolite were measured by HPLC 3 hr after BENZO, 2.5 mmoles kg-1 i.p. This gave plasma and tissue BENZO concentrations of 96-160 micrograms ml-1 or g-1. Mouse plasma, KHT and RIF-1 tumour BENZO amine concentrations were very low (0.3-1.4 micrograms g-1); kidney and EMT6 tumours had intermediate levels; and liver contained very high amine levels (approximately 50 micrograms g-1). Three per cent of the BENZO dose was recovered as amine in the 24 hr urine, compared to 5% for the parent compound. Nitroreduction to the amine was demonstrated with liver and tumour preparations under N2 in vitro. The reaction was highly dependent on NADPH, and inhibited extensively in air. With liver microsomes and whole homogenates 2 and 3 moles respectively of BENZO were consumed per mole of amine formed. Inhibitor studies showed that NADPH: cytochrome P-450 (cytochrome c) reductase and cytochrome P-450 were both involved in BENZO reduction, predominantly at early and late reduction steps respectively. Aldehyde oxidase contributed to the cytosolic nitroreduction. Purified buttermilk xanthine oxidase also reduced BENZO to its amine under anaerobic conditions in vitro, but very inefficiently. The apparent Km and Vmax for BENZO amine production by whole liver homogenates were 0.148 mM and 1.45 nmole min-1 mg-1 protein respectively. Tumour homogenates were less active than liver; e.g. Vmax for the KHT tumour was 6-10-fold lower.     

Diclofenac metabolism in the mouse: novel in vivo metabolites identified by high performance liquid chromatography coupled to linear ion trap mass spectrometry

The metabolism of [(14)C]-diclofenac in mice was investigated following a single oral dose of 10?mg/kg. The majority of the drug-related material was excreted in the urine within 24?h of administration (49.7 %). Liquid chromatographic analyses of urine and faecal extracts revealed extensive metabolism to at least 37 components, with little unchanged diclofenac excreted. Metabolites were identified using a hybrid linear ion-trap mass spectrometer via exact mass determinations of molecular ions and subsequent multi-stage fragmentation. The major routes of metabolism identified included: 1) conjugation with taurine; and 2) hydroxylation (probably at the 4'-and 5-arene positions) followed by conjugation to taurine, glucuronic acid or glucose. Ether, rather than acyl glucuronidation, predominated. There was no evidence for p-benzoquinone-imine formation (i.e. no glutathione or mercapturic acid conjugates were detected). A myriad of novel minor drug-related metabolites were also detected, including ribose, glucose, sulfate and glucuronide ether-linked conjugates of hydroxylated diclofenac derivatives. Combinations of these hydroxylated derivatives with acyl conjugates (glucose, glucuronide and taurine) or N-linked sulfation or glucosidation were also observed. Acyl- or amide-linked-conjugates of benzoic acid metabolites and several indolinone derivatives with further hydroxylated and conjugated moieties were also evident. The mechanisms involved in the generation of benzoic acid and indolinone products indicate the formation reactive intermediates in vivo that may possibly contribute to hepatotoxicity.     

Structure elucidation of in vivo and in vitro metabolites of mangiferin

The in vivo and in vitro metabolism of mangiferin was systematically investigated. Urine, plasma, feces, contents of intestinal tract and various organs were collected after oral administration of mangiferin to healthy rats at a dose of 200mg/kg body weight. For comparison, mangiferin was also incubated in vitro with intestinal flora of rats. With the aid of a specific and sensitive liquid chromatography coupled with electrospray ionization tandem hybrid ion trap mass spectrometry (LC-ESI-IT-MS(n)), a total of thirty-three metabolites of mangiferin were detected and their structures were tentatively elucidated on the basis of the characteristics of their precursor ions, product ions and chromatographic retention times. The biotransformation pathways of mangiferin involved deglycosylation, dehydroxylation, methylation, glycosylation, glucuronidation and sulfation.     

The metabolism of N-hydroxyphenacetin in vitro and in vivo

N-Hydroxyphenacetin (100 mg/kg) injected i.p. into rats rapidly appeared in the blood and disappeared with a t1/2 of 14 min; phenacetin and 4-acetamidophenol were major metabolites in blood. Ferrihaemoglobin was formed, but 4-nitrosophenetole was not detected in blood. N-Hydroxyphenacetin injected i.p. into rats was excreted in the urine unchanged (partly conjugated 2.1% of the dose, 2% was excreted as phenacetin, 19% as 4-acetamidophenol) and 1.8% as 2-hydroxyphenacetin. In addition, small amounts of 3-hydroxyphenacetin (0.4%) and traces of N-[4-(2-hydroxyethoxy)phenyl]acetamide (beta-HAP) (0.05%) were found. Time-course kinetics have shown that N-hydroxyphenacetin is metabolized in vitro to phenacetin, 2- and 3-hydroxyphenacetin, and 4-acetamidophenol by microsomal and cytosolic preparations of rat and rabbit liver. However, after the initial reaction, the formation of phenacetin and 2- and 3-hydroxyphenacetin did not continue with time, indicating that these products were not formed enzymically. N-Hydroxyphenacetin incubated with rat erythrocytes formed ferrihaemoglobin; the relationship between ferrihaemoglobin, phenacetin and 4-nitrosophenetole concn indicated that N-hydroxyphenacetin was oxidized by oxyhaemoglobin to acetyl 4-ethoxyphenyl nitroxide, which yielded phenacetin and 4-nitrosophenetole spontaneously.     

The metabolism of N-hydroxyphenacetin in vitro and in vivo

1. N-Hydroxyphenacetin (100 mg/kg) injected i.p. into rats rapidly appeared in the blood and disappeared with a t_1/2 of 14 min; phenacetin and 4-acetamidophenol were major metabolites in blood. Ferrihaemoglobin was formed, but 4-nitrosophenetole was not detected in blood.

2. N-Hydroxyphenacetin injected i.p. into rats was excreted in the urine unchanged (partly conjugated 2·1% of the dose, 2% was excreted as phenacetin, 19% as 4-acetamidophenol) and 1·8% as 2-hydroxyphenacetin. In addition, small amounts of 3-hydroxyphenacetin (0·4%) and traces of N-[4-(2-hydroxyethoxy)phenyl]acetamide (β-HAP) (0·05%) were found.

3. Time-course kinetics have shown that N-hydroxyphenacetin is metabolized in vitro to phenacetin, 2- and 3-hydroxyphenacetin, and 4-acetamidophenol by microsomal and cytosolic preparations of rat and rabbit liver. However, after the initial reaction, the formation of phenacetin and 2- and 3-hydroxyphenacetin did not continue with time, indicating that these products were not formed enzymically.

4. N-Hydroxyphenacetin incubated with rat erythrocytes formed ferrihaemoglobin; the relationship between ferrihaemoglobin, phenacetin and 4-nitrosophenetole concn indicated that N-hydroxyphenacetin was oxidized by oxyhaemoglobin to acetyl 4-ethoxyphenyl nitroxide, which yielded phenacetin and 4-nitrosophenetole spontaneously.     

Identification of novel phencyclidine metabolites formed in vitro by rabbit microsomal metabolism.

1. Phencyclidine (PCP) was incubated with rabbit liver and brain microsomal fractions, and the structures of metabolites formed by oxidation determined by g.l.c.-mass spectrometry. 2. The formation of several known mono- and di-hydroxylated metabolites, as well as two new metabolites, was seen in the liver preparations. 3. Hydroxylated PCP metabolites were also formed after incubation of PCP with brain microsomes, indicating that PCP biotransformation may occur in the brain itself.