Andreas Gescher

Pharmacokinetics and Tissue Disposition of Indole-3-carbinol and Its Acid Condensation Products after Oral Administration to Mice

Indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) are promising cancer chemopreventive agents in rodent models, but there is a paucity of data on their pharmacokinetics and tissue disposition. The disposition of I3C and its acid condensation products, DIM, [2-(indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane (LTr1), indolo[3,2b]carbazole (ICZ) and 1-(3-hydroxymethyl)-indolyl-3-indolylmethane (HI-IM) was studied, after oral administration of I3C (250 mg/kg) to female CD-1 mice. Blood, liver, kidney, lung, heart, and brain were collected between 0.25 and 24 h after administration and the plasma and tissue concentrations of I3C and its derivatives determined by high-performance liquid chromotography. I3C was rapidly absorbed, distributed, and eliminated from plasma and tissues, falling below the limit of detection by 1 h. Highest concentrations of I3C were detected in the liver where levels were approximately 6-fold higher than those in the plasma. Levels of DIM, LTr1, and HI-IM were much lower, although they persisted in plasma and tissues for considerably longer. DIM and HI-IM were still present in the liver 24 h after I3C administration. Tissue levels of DIM and LTr1 were found to be in equilibrium with plasma at almost every time point measured. In addition to acid condensation products of I3C, a major oxidative metabolite (indole-3-carboxylic acid) and a minor oxidative metabolite (indole-3-carboxaldehyde) were detected in plasma of mice after oral administration of I3C. ICZ was also tentatively identified in the liver of these mice. This study shows for the first time that, after oral administration to mice, I3C, in addition to its acid condensation products, is absorbed from the gut and distributed systemically into a number of well-perfused tissues, thus allowing the possibility for some pharmacological activity of the parent compound in vivo.

Comparison of the cytotoxicity in vitro of temozolomide and dacarbazine, prodrugs of 3-methyl-(triazen-1-yl)imidazole-4-carboxamide

SummaryThe present study tested the hypothesis that the experimental antineoplastic imidazotetrazinone temozolomide degrades in the biophase to 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) and exerts its cytotoxicity via this species. MTIC is a metabolite of the antimelanoma agent dacarbazine and is thought to be responsible for the antineoplastic activity of the latter. Cytotoxicity in vitro was investigated in TLX5 murine lymphoma cells. MTIC and temozolomide were cytotoxic in the absence of mouse-liver microsomes, whereas dacarbazine required metabolic activation. The generation of MTIC from either dacarbazine, its primary metabolite 5-[3-(hydroxymethyl)-3-methyl-triazen-1-yl]-imidazole-4-carboxamide (HMMTIC) or temozolomide was studied by reversedphase high-performance liquid chromatography in incubation mixtures under the conditions of the cytotoxicity assay. MTIC was found in incubations of temozolomide with or without microsomes. Dacarbazine yielded MTIC (and HMMTIC) only when microsomes were included in the incubation mixture. Although the mode of action of temozolomide seems to be similar to that of dacarbazine, the results obtained in this study show that these agents differ markedly in their ability to generate the active species MTIC.

Characterisation of urinary metabolites of temozolomide in humans and mice and evaluation of their cytotoxicity

SummaryThe experimental antineoplastic agent temozolomide was not metabolised in vitro at a measurable rate by mouse liver fractions. In contrast, the temozolomide analogue 3-methylbenzotriazinone was metabolicallyN-demethylated by hepatic microsomes to yield benzotriazinone. The major route of excretion of [14C]-labelled temozolomide in mice was via the kidneys. An acidic metabolite of temozolomide, probably a conjugate, was found in the urine of mice, but its identity could not be established unambiguously. Spectroscopic analysis and chemical tests revealed that it possesses an intact NNN-linkage. Another metabolite was found in the urine of patients but not of mice. This metabolite was identified as the 8-carboxylic acid derivative of temozolomide. Unlike the unknown species, this metabolite was cytotoxic against TLX5 lymphoma cells in vitro.

Differences between rodents and humans in the metabolic toxification of N,N-dimethylformamide☆

The widely used industrial solvent N,N-dimethylformamide (DMF) causes liver damage in occupationally exposed persons and is suspected of involvement in the generation of certain occupational malignancies. Here the extent of the biotransformation of DMF to three urinary metabolites has been compared in humans and rodents. The metabolites, which were quantified by gas chromatography (GC) are N-(hydroxymethyl)-N-methylformamide (HMMF), which yielded N-methylformamide on GC analysis, a species which decomposed to formamide on GC analysis, and N-acetyl-S-(N-methylcarbamoyl) cysteine (AMCC), measured after derivatization with ethanol to give ethyl N-methylcarbamate. Ten volunteers who absorbed between 28 and 60 mumol/kg DMF during an 8-hr exposure to DMF in the air at 60 mg/m3 excreted in the urine within 72 hr between 16.1 and 48.7% of the dose as HMMF, between 8.3 and 23.9% as formamide, and between 9.7 and 22.8% as AMCC. AMCC, together with HMMF, was also detected in the urine of workers after occupational exposure to DMF. The portion of the dose (0.1, 0.7, or 7.0 mmol/kg given ip) which was metabolized in mice, rats, or hamsters to HMMF varied between 8.4 and 47.3% of the dose; between 7.9 and 37.5% were excreted as formamide and only between 1.1 and 5.2%, as AMCC. The results suggest that there is a quantitative difference between the metabolic pathway of DMF to AMCC in humans and rodents. It is argued that the hepatotoxic potential of DMF may be linked to the extent of its metabolic conversion to AMCC.

N-methylformamide: Antitumour activity and metabolism in mice

The antitumour activities of N-methylformamide, N-ethylformamide and formamide against a number of murine tumours in vivo (Sarcoma 180, M5076 ovarian sarcoma and TLX5 lymphoma) have been estimated. In all cases N-methyl-formamide had significant activity, formamide had marginal or no activity and N-ethylformamide had no significant activity. N-methylformamide and N-ethylformamide were equitoxic to the TLX5 lymphoma in vitro. Formamide was found as a metabolite in the plasma and urine of animals given N-methylformamide and N-ethylformamide, but excretion profiles do not support the hypothesis that formamide is an active antitumour species formed from N-alkylformamides. No appreciable metabolism of N-methylformamide occurred under a variety of conditions with liver preparations in vitro. N-methylformamide, but not N-ethylformamide or formamide, reduced liver soluble non-protein thiols by 59.8% 1 h after administration of an effective antitumour dose.

N-hydroxymethylpentamethylmelamine, a major in vitro metabolite of hexamethylmelamine

Abstract N-Hydroxymethylpentamethylmelamine (HMPMM) was identified by HPLC and by GLC-MS after derivatization, as a metabolite of the anticancer drug hexamethylmelamine (HMM) in incubation mixtures with fortified mouse liver 9000 × g and microsomal preparations. HMPMM formation was dependent on the presence of NADPH and oxygen. N-demethylated metabolites were also found. HMPMM displays appreciable chemical stability and 29% was recovered after 60 min incubation in buffer. HMPMM constituted more than 50% of total HMM metabolites in 30 min incubations. The known chemical reactivity of carbinolamines means that HMPMM could be involved in the pharmacological or toxic effects of HMM.

Identification by proton NMR of N-(hydroxymethyl)-N-methylformamide as the major urinary metabolite of N, N-dimethylformamide in mice☆

Urine samples from mice which had received N,N-dimethylformamide were investigated by high field 1H-NMR spectroscopy. The most prominent signals in the N-CH3 region had chemical shifts identical with those of N,N-dimethylformamide (delta 2.85, 3.01) and N-(hydroxymethyl)-N-methylformamide (delta 2.91, 3.05). Resonances downfield of delta 7.5 (from formyl protons) also coincided with those of the reference formamides. When [14C]methyl-labelled N,N-dimethylformamide was injected and urine samples investigated by radio thin layer chromatography, the major area of radioactivity corresponded to the Rf of N-(hydroxymethyl)-N-methylformamide. Dimethylamine and methylamine were found to be minor metabolites of N,N-dimethylformamide.

Triazines and related products. Part 19. 4-Amino-2-[2-(piperidin-1-ylazo)phenyl]quinazoline and its analogues

The reactions of anthranilonitrile and 5-bromoanthranilonitrile with sodium hydride in dimethyl sulphoxide yield 4-amino-2-(2-aminophenyl)quinazoline (7) and its dibromo-analogue (8), respectively. Nitrosation of the diamines affords unstable diazonium salts which cyclise to either quinazolino[3,2-c]- or quinazolino[1,2-c]-[1,2,3]benzotriazines. These tetracyclic triazines readily undergo ring opening in the presence of secondary-amines to form the title compounds. 1,3-Bis-(2-cyano-4-bromophenyl)triazene (12) is smoothly transformed into dibromoquinazolines (18) in boiling secondary amines.4-Amino-2-[2-(piperidin-1-ylazo)phenyl]quinazoline (17a) behaves as a masked diazonium compound and decomposes in mineral acids, in acetic acid containing copper-bronze, in hot ethylene glycol, on photolysis in methanol or ethanol, or on reduction. The triazene linkage of (17a) is resistant to alcoholic potassium hydroxide but the 4-aminoquinazoline nucleus is hydrolysed to the corresponding quinazolin-4(3H)-one system. Methylation of (17a) with methyl iodide in tetrahydrofuran affords an N(1) methiodide (29) which is hydrolysed to the corresponding 1-methylquinazolin-4(1H)-one (30) in aqueous alkali. The unusual properties of this and other 1-methylquinazolin-4(1H)-ones can be attributed to their dipolar character. This renders the 1-methyl group liable to removal in acidic conditions.

Studies of the metabolism of dimethylformamide in mice

It has been suggested that the industrial solvent N,N-dimethylformamide (DMF, OHCN(CH3)2) is metabolized extensively to its N~lesmethyl derivative N-methylformamide (NMF, OHCNHCH3) and to a minor extent to formamide (OHCNH2) in animals and man exposed to DMF vapours [1,2]. NMF is an antineoplastic agent with activity against a remarkably wide range of rodent tumours [3,4]. Therefore, NMF is currently undergoing clinical evaluation to test its therapeutic usefulness in patients with malignancies, even though in a clinical trial in 1956 seven patients who were treated with NMF showed symptoms of hepatotoxic i ty [5]. In view of the good ant i tumor activity of NMF in rodents and the finding that DMF is metabolized to NMF [1,2], one could expect DMF to exhibit significant anti tumor activity as it should be a prodrug of NMF. Lundberg et al. have recently associated the hepatotoxic potential of DMF with the metabolism of NMF [6]. Indeed NMF appears to decrease hepatic levels of glutathione in vivo [4] and also to induce lipid peroxidation in isolated hepatocytes (Lundberg et al., unpublished results). We report here the results of a study of the metabolism of DMF in vivo which was designed to clarify the mechanism by which DMF causes hepatotoxic i ty and further, to test the hypothesis that the plasma levels of NMF, obtained after injection of DMF

Assessment of viability of hepatocytes in suspension using the MTT assay.

The viability and state of proliferation of cells in culture is conveniently assessed using MTT, which is metabolically reduced to stain functionally intact cells. The hypothesis was tested that this assay can also be used in the quantitation of effects of toxicants on hepatocytes in suspension. Hepatocytes isolated from BALB/c mice were incubated without or with menadione, rotenone, N-methylformamide or paracetamol. Cellular damage was measured by either MTT assay or release into the medium of lactate dehydrogenase (LDH). Results obtained with the two tests were compatible in the case of toxicity inflicted by menadione or N-methylformamide. Rotenone decreased cell viability as indicated by the MTT assay immediately after addition of the agent, whereas measurement of LDH release did not detect this rapid toxic effect. The MTT assay detected paracetamol-induced damage within the first hour of exposure, but this was not detected by the LDH assay until 3 hr had elapsed.