Identification and quantitative determination of glutathione-related urinary metabolites of fotemustine, a new anti-cancer agent.

1. Potential sulphur-containing metabolites of the anticancer agent, fotemustine, were synthesized, namely thiodiacetic acid (TDA), S-2-hydroxyethyl N-acetyl-L-cysteine (2-HE-NAC), N-acetyl-L-cysteine (NAC), S-methyl N-acetyl-L-cysteine (M-NAC), S-carboxymethyl-L-cysteine (CM-Cys), S-carboxymethyl N-acetyl-L-cysteine (CM-NAC), their corresponding sulphoxides and sulphones. Their chemical structures and stabilities were confirmed and derivatization methods were developed for their analysis by sulphur-selective g.l.c. (g.l.c.-FPD) and g.l.c.-mass spectrometry. 2. Four methods for isolation of potential metabolites of fotemustine were developed. Quantification of metabolites, derived in various ways was carried out by g.l.c.-atomic emission detection (AED) or g.l.c.-mass spectrometry. 3. Male Wistar rats (n = 4) were given a single i.p. dose of 40 mg/kg fotemustine. Urine excretion of TDA (18.4 +/- 1.9% in 24 h) and TDA sulphoxide (12.0 +/- 1.6% in 24 h) was significant; 32.7 +/- 4.6% of the fotemustine dose was excreted as TDA, and TDA sulphoxide in 48 h. NAC was excreted in rat urine at 1% of the dose. No other potential glutathione-derived metabolites of fotemustine were excreted. 4. Male Wistar rats (n = 4) were also treated i.p. with fotemustine at 5, 20 and 40 mg/kg, to investigate dose dependency and the time course of excretion of TDA. Excretion of TDA in 48 h urine decreased from 32 +/- 2 to 17 +/- 2% dose (mean +/- SD) with increasing dose of fotemustine.

Main urinary metabolites of two cysteamine-containing 2-(chloroethyl) nitrosoureas in rats.

Urine is the major route of excretion of N'-(2-chloroethyl)-N-[2-(methylsulfinyl)ethyl]-N'-nitrosourea (CMSOEN2), N'-(2-chloroethyl)-N-[2-(methylsulfonyl)ethyl]-N'-nitrosourea (CMSO2EN2), and their metabolites in the rat. Labeling the two compounds with 14C in three different positions facilitated their metabolic study in animals. The 14C-ethyl species were chosen in order to investigate the presence of unchanged compounds and that of the denitrosated forms. With the same 14C label position, we showed that isolated metabolites derived from this part of the molecule were degradation products of alkylated glutathione and/or cysteine. They are common to both CMSOEN2 and CMSO2EN2, namely thiodiacetic acid and its sulfoxide, the sum of which represents about half of urinary radioactivity. N-acetyl carboxymethylcysteine and N-acetyl hydroxyethylcysteine, accounting for approximately 6% to 7% of the eliminated 14C radioactivity, were also characterized. However, four minor metabolites corresponding to less than 10% of the excreted radioactivity remained unidentified. With the [14C]cysteamine and [14C]carbonyl labels related to the isocyanate moiety behavior, we indirectly showed that more than 60% to 70% of the excreted metabolites were carbamoylation products of endogenous substrates. A small amount of free amines, 2-methylsulfinylethylamine and/or 2-methylsulfonylethylamine, representing 15%-16% of the eliminated radioactivity, was also detected. The total data confirm the predominant function of glutathione and/or cysteine in the detoxifying system of the chloroethyl moieties and reveal the unexpected but important role played by carbamoylation reactions in the metabolic fate of the drug isocyanate moieties.

Determination of tauromustine and its demethylated metabolites in plasma and urine.

A sensitive, selective and precise high-performance liquid chromatographic method for simultaneous determination of tauromustine and its demethylated metabolites in plasma and urine has been developed. It is based on solid-phase extraction on C18 sorbent and separation on a semipolar column. The analytical procedure is described in detail. The method has been validated with respect to linearity, recovery, selectivity, precision and detection limit. The stability of the determined substances in various media has also been studied.

Pharmacokinetic studies on 1-(2-chloroethyl)-3-isobutyl-3-(beta-maltosyl)-1-nitrosourea (TA-077). III. Pharmacokinetics of a new nitrosourea antitumor agent TA-077 in humans (a phase I study).

A new nitrosourea antitumor agent TA-077, 1-(2-chloroethyl)-3-isobutyl-3-(beta-maltosyl)-1-nitrosourea, was intravenously administered to 15 cancer patients at doses ranging from 7 to 100 N (1 N = 30 mg/m2) in a phase I clinical trial. Time courses of blood concentrations of TA-077 and its active metabolite TA-G, 3-beta-D-glucopyranosyl analog of TA-077, were followed. The TA-G concentration reached a maximum at 7.0 +/- 2.3 min, and decreased thereafter with a half-life of 12.9 +/- 2.8 min. The time-course patterns and various pharmacokinetic parameters of TA-077 and TA-G were similar to those in the guinea pig, which, like humans, lacks plasma maltase activity. The 2 h-urinary excretion rate of TA-G in the above patients ranged from 0.15 to 7.7% of the dose. The areas under the concentration-time curve and maximal concentration values were both linearly correlated to the dose with correlation coefficients of 0.78 and 0.82, respectively. Repeated administration of TA-077 (29 to 40 N) for 5 or 6 consecutive days did not affect the pharmacokinetic parameters of TA-077 and TA-G in 7 cancer patients except for slight increases in the half-life and area under the curve of blood TA-G.

Identification of major urinary metabolites of ACNU, 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitroso urea hydrochloride in rats.

Metabolites of 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1- nitrosourea hydrochloride (ACNU) in rat urine were investigated. After intravenous administration of 14C-ACNU into rats, four major radioactive metabolites and two minor ones were detected in the urine by two-dimensional thin-layer chromatographic analysis. The main metabolite was identified to be an imidazolidinone compound, 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-5-hydroxy-2-imidazolidinone (M-D). One of the other major metabolites was identified to be a nitrosated compound of the main metabolite i.e., 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]- 5-hydroxy-3-nitroso-2-imidazolidinone (M-C). These were new types of metabolites which have not been reported in the metabolic study of other chloroethylnitrosourea derivatives. Compared with authentic compounds, two metabolites were identified to be a denitrosated derivative of ACNU i.e., 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)urea (M-B), and a cyclized pyrimidopyrimidine compound which lacks the ethylene moiety of ACNU, i.e., 3,4-dihydro-7-methylpyrimido[4,5-d]pyrimidin-2-(1H)-one (M-A). The two minor metabolites were supposed to be compounds derived from M-A. Discussions were made on mechanism of formation of these metabolites in vivo.

Pharmacological disposition of 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea in mice.

1-(2-Chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea (PCNU; NSC 95466) is a lipid-soluble nitrosourea that is presently in clinical trial. We have studied the pharmacological disposition of [ethyl-14C]PCNU in mice using an i.v. drug dose of 20 mg/kg/animal. Disappearance of total radioactivity from plasma was biphasic with mean half-lives of the two exponential phases of 21.7 min and 27.4 hr, respectively. The plasma half-life of intact drug was 29 min, and levels of intact drug, as measured by thin-layer chromatography, fell below detectable levels by 4 hr. The area under the plasma concentration-time curve for intact drug was 32.72 nmol X hr/ml. Computer analysis of the data for total radioactivity (PCNU equivalents), based upon an open two-compartment model, yielded values of the pharmacokinetic parameters K12, K21, and K10 of 1.49 hr-1, 0.25 hr-1, and 0.19 hr-1, respectively. The highest peak organ level of drug was 168.9 nmol of PCNU equivalents per g tissue in the liver 1 hr after drug administration. Maximum levels in kidney, lungs, heart, and spleen were observed at 5 min, with values of 119.5, 115.4, 80.3, and 66.7 nmol of PCNU equivalents per g of tissue, respectively. A high peak drug level in brain (50.6 nmol/g) agreed with the prediction that PCNU can cross the blood-brain barrier. The levels of intact drug relative to total radioactivity at 30 min were 60% in brain, 55% in heart, and 48% in spleen. The concurrent value in liver was 7% of the total radioactivity, suggesting that metabolism or decomposition of PCNU occurs in this organ. The principal excretory route of [ethyl-14C]PCNU was urinary, with a cumulative excretion of 62% in the first 24 hr.

Main metabolites of 1-(2-chloroethyl)-3-[1'-(5'-p-nitrobenzoyl-2',3'-isopropylidene)-alpha, beta-D-ribofuranosyl]-1-nitrosourea and 1-(2-chloroethyl)-3-(2',3', 4'-tri-O-acetyl-alpha, beta-D-ribopyranosyl)-1-nitrosourea in rats.

The metabolism of two glycosylnitrosoureas, 1-(2-chloroethyl)-3-[1'-(5'-p-nitrobenzoyl-2',3'-isopropylidene)-alpha, beta-D-ribofuranosyl]-1-nitrosourea (RFCNU) and 1-(2-chloroethyl)-3-(2',3',4'-tri-O-acetyl-alpha, beta-D-ribopyranosyl)-1-nitrosourea (RPCNU), has been investigated in the rat. With the label on the carboxyl moiety of RFCNU, we have shown that hydrolysis of the 4-nitrobenzoyl ester occurred to a large extent in vivo; 4-nitrobenzoic acid and its glucuronide were the major urinary metabolites. Two other minor metabolites and their glucuronides were identified as 4-aminobenzoic acid and 4-acetamidobenzoic acid. With the label on the chloroethyl moieties of RFCNU and RPCNU, we have shown that chloroethanol was a major degradation product of this alkylating part of the molecule. The concentration of chloroethanol in plasma vs. time has been determined. In urine, four metabolites derived from alkylated glutathione, namely thiodiacetic acid and its sulfoxide, N-acetylcarboxymethylcysteine, and N-acetylhydroxyethylcysteine, have been identified.

Disposition and metabolism in 1-(2-chloroethyl)-3-(2',3',4'-tri-o-acetyl, ribopyranosyl)-1-nitrosourea in rats.

The antineoplastic activity in animals of 1-(2-chloroethyl)-3-(2',3',4'-tri-O-acetyl, ribopyranosyl)-1-nitrosourea (RPCNU) has been widely demonstrated. The present study deals with the disposition and the metabolism of three 14C-labeled species of RPCNU. The chemical plasma half-life of the drug was less than 5 min. Within the first min after injections, most of the radioactivity derived from ethyl-14C groups were recovered as volatile products. Among these, 2-chloroethanol was identified as a main component. Analysis of labeled species in urine after administration of [ethyl-14C]RPCNU showed that thiodiacetic acid and its sulfoxide were major metabolites of RPCNU (62% of the urinary radioactivity). Traces of N-acetylcarboxymethyl- and N-acetylhydroxyethylcysteine) were also detected. The only labeled species concentrating in particular tissues was that carrying the chloroethyl moiety. Uptake to high levels of [ethyl-14C]RPCNU did occur in liver, kidney, pancreas, thymus, and Harder's gland.

Pharmacokinetics of a new water-soluble nitrosourea derivative (ACNU) in human gliomas.

The pharmacokinetics of a newly developed water-soluble nitrosourea derivative (ACNU) following a single intravenous injection was investigated in 11 patients with gliomas. A major portion of ACNU was excreted within 2 hours. The distribution rate was very fast, and the elimination rate tended to be slow. More than 50% of ACNU moves into the tissue compartment. ACNU tended to move into glioma tissue well. The ACNU level in glioma tissue was above 1.0 microgram/gm 30 to 60 minutes ater injections. ACNU was detected at a higher concentration in malignant gliomas than in benign gliomas. These results suggest that ACNU is taken up by tumor tissue relatively rapidly and eliminated slowly, which leads to effective manifestation of its antitumor activity.

Urinary metabolites of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea.

Urinary metabolites of ring 14C-labeled 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (Methyl CCNU) from rats have been isolated and characterized by high-performance liquid chromatography and mass spectrometry. About 44% of the cyclohexyl moiety of CCNU was excreted in 24 hr and included approximately 10% of the excreted dose as free amines and 40% as conjugates that could be converted to amines by hydrolysis. Amine composition of free base plus hydrolyzable conjugates was 55% hydroxycyclohexylamines (3-trans, 3-cis, 4-cis, and 4-trans) and 30% cyclohexylamine. This strongly supports previous studies which indicated that CCNU is largely hydroxylated in vivo as well as in vitro. Rats pretreated with phenobarbital excreted high relative amounts of cis-4-hydroxy derivatives (41%), again showing a high degree of correlation between in vitro and in vivo results. Treatment of urine with beta-glucuronidase gave no apparent increase in free amines. However, sulfatase was about 25% as effective as alkaline hydrolysis for releasing free amines from whole urine. Major urinary metabolites were found to have m.w. of about 629, 413, 329, and 243 and represented 55%, 20%, 20%, and 5% of total excreted 14C, respectively. It was concluded that the higher m.w. metabolites may be conjugates of peptides possibly derived from active site-directed inactivation of specific enzymes. Previous work has shown that enzymes such as chymotrypsin and glutathione reductase are inhibited by isocyanates in this manner. Hydroxylated metabolites of Methyl CCNU had a pattern similar to that of CCNU. The major free (12%) and conjugated amine (54%) metabolites of Methyl CCNU in the urine in decreasing order of quantity present were cis-3-hydroxy-trans-4-methylcyclohexylamine, trans-4-methylcyclohexylamine, trans-4-hydroxymethylcyclohexylamine, and trans-3-hydroxy-trans-4-methyl-cyclohexylamine.

Disposition of the carcinostatic agent 1-(2-chloroethyl)-3-[1'-(5'-p-nitrobenzoyl-2',3'-isopropylidene)-alpha,beta-d-ribofuranosyl]-1-nitrosourea in animals.

The disposition of 1-(2-chloroethyl)-3-[1'(5'-p-nitrobenzoyl-2',3'-isopropylidene)-alpha,beta-D-ribofuranosyl]-1-nitrosourea (RFCNU) was investigated in animals following i.p. administration (35 micromol/kg) of the drug labeled with 14C in three different positions. The appearance and disappearance kinetics of the label in blood and plasma were strongly dependent upon the labeled species administered to animals. Protein binding of [carboxyl-14C]- and [carbonyl-14C]RFCNU greatly prolonged the blood and plasma half-life of these two labeled species. No intact RFCNU was found when rat plasma samples were analyzed using high-performance liquid chromatography, indicating the very short half-life of this compound. Most of the dose was recovered in the urine after administration of the carboxyl-14C and ethyl-14C species and in the expired air as 14CO2 after administration of the carbonyl-14C species. Except for the liver and kidney, no specific localization of the radioactivity was found when animals were given [carboxyl-14C]- or [carbonyl-14C]RFCNU. However, tissue distribution studies showed a marked specificity of the species carrying the ethyl-14C moiety for the liver, kidney, pancreas, and thymus, but not for the brain.