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 and cotinine effects on development of two-cell mouse embryos in vitro

The independent and interactive effects of nicotine and cotinine on the development of cultured two-cell embryos were investigated. Cultures were maintained for 120 h and developmental stages of embryos were scored after 72 h and at the termination of culture. Concentrations of nicotine at or below 0.5 mM, and concentrations of cotinine at or below 0.008 mM, did not adversely affect development. In addition, neither nicotine nor cotinine produced synergistic effects at higher concentrations at which both independently impaired development. These data show, therefore, that nicotine and its major metabolite, cotinine, significantly interfere with preimplantation development of mouse embryos only at concentrations far in excess of those anticipated to be present in the blood of an “average” smoker. Thus, we conclude that the well documented adverse effects of smoking during pregnancy are unlikely to be attributable to a direct effect of nicotine or cotinine on the preimplantation embryo.     

Nicotine and cotinine effects on development of two-cell mouse embryos in vitro

The independent and interactive effects of nicotine and cotinine on the development of cultured two-cell embryos were investigated. Cultures were maintained for 120 h and developmental stages of embryos were scored after 72 h and at the termination of culture. Concentrations of nicotine at or below 0.5 mM, and concentrations of cotinine at or below 0.008 mM, did not adversely affect development. In addition, neither nicotine nor cotinine produced synergistic effects at higher concentrations at which both independently impaired development. These data show, therefore, that nicotine and its major metabolite, cotinine, significantly interfere with preimplantation development of mouse embryos only at concentrations far in excess of those anticipated to be present in the blood of an "average" smoker. Thus, we conclude that the well documented adverse effects of smoking during pregnancy are unlikely to be attributable to a direct effect of nicotine or cotinine on the preimplantation embryo.     

Nicotine elimination and tolerance in non-dependent cigarette smokers

Although most smokers are nicotine-dependent, recent studies suggest that some very light smokers (chippers, who smoke fewer than five cigarettes per day) may smoke for decades without developing dependence. It was considered that slowed nicotine elimination and/or reduced nicotine tolerance might underlie chippers' ability to maintain smoking at such low levels. To evaluate this hypothesis, we studied the elimination kinetics and pharmacodynamics of nicotine in chippers and matched regular smokers. Plasma nicotine levels and cardiovascular responses were observed for several hours after subjects were administered uniform doses of tobacco smoke. Chippers did show less chronic nicotine tolerance, but only on some response measures. Their rates of nicotine elimination equaled those of regular smokers. This finding, when coupled with other data about chippers' smoking patterns and nicotine absorption, establish that chippers cannot maintain substantial plasma nicotine levels between cigarettes, and thus suggest that attempts to maintain minimal trough levels of nicotine do not underlie chippers' smoking.     

Nicotine metabolism in mammals

Over the past decade studies on nicotine metabolism have been advanced by use of new methods involving enzyme purification, preparation of specific antibodies and HPLC. In in vivo experiments of nicotine metabolism, a number of new metabolites have been identified, and most major and minor metabolites have been quantitatively determined, which make it now possible to investigate the contribution of each pathway to overall nicotine metabolism. In in vitro experiments, C-oxidation and N-oxidation have been precisely characterized. Despite extensive investigations, metabolic regulation of nicotine metabolism still remains unclear.     

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.     

New metabolites of thiabendazole and the metabolism of thiabendazole by mouse embryo in vivo and in vitro

Thiabendazole [2-(4'-thiazolyl)benzimidazole; TBZ], a teratogen in ICR mice, is known to be mainly metabolized to 5-hydroxy-TBZ (5-OH-TBZ) and its conjugates in domestic and laboratory animals. Besides the known metabolites of TBZ, 4-hydroxy-TBZ and 2-acetylbenzimidazole (ABI) were identified as new metabolites of TBZ in the urine of F344 rats and ICR mice. 5-OH-TBZ and ABI, as well as TBZ, were found in the embryos of ICR mice given TBZ orally on day 10 of gestation. In the whole-embryo culture system, 5-OH-TBZ and ABI in the medium, and TBZ, 5-OH-TBZ and ABI in the embryo were detected after 24 hr of culture in 25 or 50 micrograms TBZ ml. However, the amount of metabolites in the embryo in vitro was very small compared with that detected in vivo, whereas the amount of TBZ was comparable. Furthermore, the mouse embryo homogenate, at organogenesis, metabolized TBZ to 5-OH-TBZ or ABI. The specific activity required by this homogenate to form 5-OH-TBZ or ABI was less than 1/1000 of that of the liver microsomal fraction. The results suggested that mouse embryos at organogenesis could metabolize TBZ, although most of the metabolites in the embryo in vivo came from the dam.     

Investigation of exenatide elimination and its in vivo and in vitro degradation

Exenatide is a 39 amino acid incretin mimetic for the treatment of type 2 diabetes, with glucoregulatory activity similar to glucagon-like peptide-1 (GLP-1). Exenatide is a poor substrate for the major route of GLP-1 degradation by dipeptidyl peptidase-IV, and displays enhanced pharmacokinetics and in vivo potency in rats relative to GLP-1. The kidney appears to be the major route of exenatide elimination in the rat. We further investigated the putative sites of exenatide degradation and excretion, and identified primary degradants. Plasma exenatide concentrations were elevated and sustained in renal-ligated rats, when compared to sham-operated controls. By contrast, exenatide elimination and degradation was not affected in rat models of hepatic dysfunction. In vitro, four primary cleavage sites after amino acids (AA)-15, -21, -22 and -34 were identified when exenatide was degraded by mouse kidney membranes. The primary cleavage sites of exenatide degradation by rat kidney membranes were after AA-14, -15, -21, and -22. In rabbit, monkey, and human, the primary cleavage sites were after AA-21 and -22. Exenatide was almost completely degraded into peptide fragments <3 AA by the kidney membranes of the species tested. The rates of exenatide degradation by rabbit, monkey and human kidney membranes in vitro were at least 15-fold slower than mouse and rat membranes. Exenatide (1-14), (1-15), (1-22), and (23-39) were not active as either agonists or antagonists to exenatide in vitro. Exenatide (15-39) and (16-39) had moderate-to-weak antagonist activity compared with the known antagonist, exenatide (9-39). In conclusion, the kidney appears to be the primary route of elimination and degradation of exenatide.     

Drug metabolism in epileptics: in vivo and in vitro correlations.

1. The effect of inducing drug therapy on the relationship between in vitro (cytochrome P-450 content) and in vivo (antipyrine kinetics) was investigated by comparing eleven consecutively treated epileptics with two groups of controls, eleven subjects with normal liver histology and eleven disease matched non-epileptics, all underwent diagnostice liver biopsy. 2. The epileptics had significantly higher cytochrom P-450 level in biopsies and they also metabolized antipyrine faster than the controls. 3. Decrease in antipyrine half-life in epileptics was related with alterations in liver histology, whereas the level of cytochrome P-450 was not. 4. There was a linear relationship between these two indices of enzyme induction when regressed on logarithmic data.     

Drug metabolism in man: in vivo and in vitro correlations.

In man the liver drug metabolizing ability may be determined by assaying drug kinetics after its administration or by measuring activity of drug metabolizing enzymes from liver biopsies. Little is known about the relationship between these parameters. We investigated the problem by determining in vivo (antipyrine kinetics) and in vitro (cytochrome P-450) indices of drug metabolism in 150 consecutive patients with diagnostic liver biopsy. In patients with normal liver histology cytochrome P-450 content ranged 10.3 umole/g and the half-life 9.5 hrs. In general, alterations in liver histology were related to the changes in drug metabolism; the patients with severe changes had prolonged half-life and low cytochrome P-450, whereas the changes in those with slight parenchymal alterations were less pronounced. The relationship between in vivo and in vitro drug metabolism was non-linear in the whole material, and linear when comparing severe ill patients with controls or in subjects with closely equal liver histology. The results emphasize the importance of evaluating the liver histology when investigating in vivo and in vitro drug metabolism in man.     

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.     

Ethyl carbamate metabolism: in vivo inhibitors and in vitro enzymatic systems

The metabolism of ethyl carbamate and the localization of its metabolites have been shown to be almost completely inhibited by ethanol in the mouse [Waddell, Marlowe, Pierce: Food Chem. Toxicol.25, 527 (1987); Yamamoto, Pierce, Hurst, Chen, Waddell: Drug Metab. Dispos. 16, 355 (1988)]. The enzyme system catalyzing this metabolism which is inhibited by ethanol now has been further investigated in both in vivo and in vitro studies. There is a direct, highly significant relationship between the extent of metabolism of ethyl carbamate and covalent binding of metabolites to liver protein. Paraoxon, carbaryl, CCl4 ethanol, methimazole, 4-methylpyrazole, diethyl maleate, ethyl N-hydroxycarbamate, and t-butyl carbamate inhibit, to different extents, the metabolism of ethyl carbamate in vivo; SKF-525A, CoCl2, Cacyanamide, chloral hydrate, 2-oxo-4-thiazolidine carboxylic acid, allopurinol, and methyl carbamate do not. Porcine liver esterase, yeast aldehyde dehydrogenase and mouse liver catalase catalyzed the metabolism in vitro; dog or bovine catalase, acid phosphatase, alcohol dehydrogenase, or carbonic anhydrase did not under the conditions tested. Paraoxon, 4-methylpyrazole, carbaryl, and NaF significantly inhibited the hydrolytic activity of mouse liver homogenates toward p-nitrophenyl acetate; ethanol or ethyl carbamate did not. However, each of these, except 4-methylpyrazole, inhibited the metabolism of ethyl carbamate by mouse liver homogenate or porcine liver esterase to about the same extent. Ion exchange chromatography of mouse liver cytosol revealed that the fraction with ability to metabolize ethyl carbamate co-chromatographed almost exactly with the ability to hydrolyze p-nitrophenyl acetate. It is proposed that ethyl carbamate is metabolized in the mouse, at least partially, by esterases; however, metabolism by other enzyme systems cannot be excluded.     

In vivo metabolism of alpha-methyltryptamine in rats: identification of urinary metabolites

1. The in vivo metabolism of alpha-methyltryptamine (AMT), a psychoactive tryptamine analogue, was studied in rats. 2. Male Wistar rats were administered 10 mg kg(-1) AMT orally and 24-h urine fractions were collected. After enzymatic hydrolysis of the urine sample, the metabolites were extracted by liquid-liquid extraction and analysed by gas chromatography/mass spectrometry (GC/MS). 3. 2-Oxo-AMT, 6-hydroxy-AMT, 7-hydroxy-AMT and 1'-hydroxy-AMT were detected as metabolites of AMT.     

Marijuana use: Persistence and urinary elimination

Marijuana continues to be one of the most widely used illicit drugs in this country. Unlike most other drugs, it is persistent in the body and elimination occurs only gradually. Using the Syva EMIT Cannabinoid Assay test, a readily available semiquantitative immunochemical urine test for marijuana metabolites, a group of ten chronic marijuana users was monitored while hospitalized. The results of the monitoring are presented as are two representative case reports. Implications regarding the interpretation of results and interpretation of “significant” urinary marijuana levels are discussed. Specific implications for use in treatment programs and general primary care settings are also discussed.     

Toxicokinetics and metabolism of linuron in rabbit: in vivo and in vitro studies

1. Linuron (N′-(3,4-dichlorophenyl)-N-methoxy-N-methylurea) metabolism and kinetic behaviour were investigated after oral and i.v. administration to six New Zealand White female rabbits.

2. After i.v. dosage, linuron distributes quickly and widely to peripheral tissues and its is rapidly eliminated; rapid absorption was also observed after oral administration of the herbicide which undergoes extensive first pass metabolism in the liver.

3. The major metabolites obtained from both in vivo (serum samples) and in vitro (microsomal fractions incubated with linuron) experiments were identified by?h.p.l.c.-mass spectrometry as N′-(3,4-dichlorophenyl)-N-methoxyurea, N′-(3,4-dichlorophenyl) urea, and N′-(6-hydroxy-3,4-dichlorophenyl) urea.

4. Given the common metabolites reported in rat and rabbit, and the fact that linuron is a liver enzyme inducer in rat, it may be possible that linuron also induces the P450 system in rabbit. Hence, despite the low acute toxicity of linuron in rabbit, the intake of hay and feed contaminated by the herbicide could be a health risk for these breeding animals since it could modify the effectiveness of many drugs commonly used in veterinary practice and metabolized by the same liver enzymes.     

Mechanisms of cocaine hydrolysis and metabolism in vitro and in vivo: a clarification

There is confusion in the literature concerning the mechanisms by which the cocaine hydrolysis product, benzoylecgonine (BE), is formed in vitro and in vivo. Some authors assume that all BE is formed nonenzymatically. This review summarizes evidence that both enzymatic and nonenzymatic mechanisms exist. In vitro BE is produced exclusively by hydrolysis at alkaline pH, as esterases present in blood or serum do not catalyze formation of BE. In vivo BE is formed both nonenzymatically as well as through the action of esterases found in a number of tissues including hepatocytes. The enzymatic mechanism is the predominant one operating in vivo.     

In vitro and in vivo murine metabolism of spirogermanium.

We report the identification of spirogermanium (SG) metabolites derived from incubation of the drug with a mouse liver microsomal preparation as well as those obtained from the urine of mice injected with the drug. GC/MS data using electron impact and chemical ionization indicate that the major metabolic products appearing in the urine of mice are hydroxylated metabolites resulting from oxidation of the ethyl substituents on germanium. Thermospray (TSP) LC/MS data suggest that these hydroxy metabolites are further oxidized to an acid and a deethylated metabolite that has undergone hydroxylation of the germanium atom. In a separate experiment, human urine from a subject undergoing therapy with SG was subjected to TSP-LC/MS analysis. The SG metabolite pattern observed in the urine from human was similar to that observed in the mouse urine. These results suggest that the metabolic fate of SG in human is qualitatively similar to that found in the mouse.