Peter B. Farmer

Some studies of the active intermediates formed in the microsomal metabolism of cyclophosphamide and isophosphamide

Abstract Evidence is presented in support of the following metabolic pathways, in the liver, of the antitumour agent cyclophosphamide 2-[bis(2-chloroethyl)amino]-tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide. The drug is first converted, presumably by the mixed-function oxidases, into 4-hydroxycyclophosphamide which may then break down by elimination of acrolein from its tautomeric form, aldophosphamide, to yield phosphoramide mustard [N,N-bis(2-chloroethyl)phosphorodiamidic acid], a known cytotoxic agent. In competition with this process is the enzymic conversion of 4-hydroxycyclophosphamide (by dehydrogenation) and aldophosphamide (by oxidation) into the known in vivo metabolites of cyclophosphamide, 4-ketocyclophosphamide and carboxyphosphamide, respectively, each of which has low cytotoxicity. 4-Hydroxycyclophosphamide, which was too unstable to allow identification directly by conventional procedures, was trapped by reaction with ethanol. The resulting two, apparently isomeric, ethyl derivatives, (1) were amenable to mass spectrometry, (2) yielded acrolein 2,4-dinitrophenylhydrazone on treatment with acidic 2,4-dinitrophenylhydrazine, (3) were hydrolysed in water (pH 4.3), each isomer apparently regenerating 4-hydroxycyclophosphamide, (4) were highly toxic to Walker tumour cells in culture. Phosphoramide mustard was also isolated after in vitro metabolism of cyclophosphamide. On the basis of a bioassay involving Walker tumour cells in whole animals it appeared that, of the known metabolites of cyclophosphamide, only phosphoramide mustard possesses the cytoxicity and biological half-life appropriate to the active antitumour metabolite. Four other metabolites of low cytotoxicity were isolated and identified, namely, 4-ketocyclophosphamide, carboxyphosphamide, 2-(2-chloroethylamino)tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide, and 3-hydroxypropyl-N,N-bis(2-chloroethyl)phosphorodiamidate. The significance of metabolic detoxification processes in relation to the selective cytotoxicity of cyclophosphamide towards tumour cells in vivo is discussed. The metabolic activation of isophosphamide appears to follow a pathway similar to that of cyclophosphamide.

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.

MONITORING EXPOSURE TO 4,4'-METHYLENEDIANILINE BY THE GAS CHROMATOGRAPHY-MASS SPECTROMETRY DETERMINATION OF ADDUCTS TO HEMOGLOBIN

The determination of the covalently bound reaction products of 4,4-methylenedianiline (MDA) to hemoglobin was investigated as a possible method for biological dosimetry in humans. The extent of binding to rat hemoglobin of MDA was determined by dosing animals with the 14C-ring-labeled compound. Two adducts were released from the hemoglobin on hydrolysis under mildly basic conditions which were identified as MDA and N-acetyl-MDA and accounted for between 36 and 45% of the total radioactivity bound to the protein. A quantitative assay procedure was subsequently developed for measuring both of the base released adducts in rat hemoglobin. The method utilized solvent extraction followed by derivatization with pentafluoropropionic anhydride and subsequent separation and quantitation by capillary gas chromatography with selective ion monitoring mass spectrometry using deuterium-labeled analogues of MDA and N-acetyl-MDA as internal standards. A dose-response relationship was established in orally dosed rats between production of each of the hemoglobin released adducts and dose of MDA (1-12 mg/kg). The possible use of such adduct determinations as dosimeters for industrial workers exposed to MDA is discussed.

Arsenic and Ayurveda

Manufacture of an Ayurvedic arsenic-containing compound is described, which is currently in use in India to control blood counts of patients with haematological malignancies. The efficacy and side effects of this compound are evaluated in the light of the fact that arsenic was recognised to be of use in the control of blood counts from patients with chronic myeloid leukaemia as long as 100 years ago, in the West.

Liver production of N-alkylated porphyrins caused in mice by treatment with substituted dihydropyridines: Evidence that the alkyl group on the pyrrole nitrogen atom originates from the drug

The porphyrogenic drug, 3,Sdiethoxycarbonyl1,4-dihydro-2,4,6_trimethylpyridine causes a marked inhibition of the enzyme protohaem ferro-lyase (EC 4.99.1 .l) in the liver of rats, mice and chick embryos [l-4] an effect which is thought to be responsible for the very pronounced accumulation of protoporphyrin seen in this type of experimental porphyria. We have isolated a potent inhibitor of protohaem ferro-lyase from the liver of mice made porphyric by treatment with this drug [5,6] and have identified the inhibitor as N-methyl protoporphyrin [7]. Inhibition of protohaem ferro-lyase has also been obtained both in vivo and in vitro with synthetic N-alkylated porphyrins [7-91, and the size of the alkyl group present on the pyrrole nitrogen atom has been shown to be important for the inhibitory effect, N-ethylmesoporphyrin being less active than N-methylmesoporphyrin [8]. Isotopic experiments have suggested that the N-methylated protoporphyrin produced by treatment with 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine originates from liver haem [5,6,10], but the source of the methyl group bound onto the pyrrole nitrogen atom has not yet been determined. The following findings have raised the possibility that the methyl group may originate from the 4-methyl substituent of the drug: under relatively mild chemical conditions certain dihydropyridines lose their 4-alkyl substituent on oxidation and that this alkyl group can be donated to suitable nucleophiles [ 111. A series of dihydropyridine analogues have been compared for their ability to inhibit liver protohaem ferro-lyase

Monitoring exposure to acrylamide by the determination of S-(2-carboxyethyl)cysteine in hydrolyzed hemoglobin by gas chromatography-mass spectrometry

Acrylamide is a potent cumulative neurotoxin in animals and man. In vivo exposure to this electrophile results in the formation of a covalently bound reaction product with cysteine residues in hemoglobin. This adduct yields on acid hydrolysis S-(2-carboxyethyl)cysteine which has been analyzed by capillary gas chromatography with mass spectrometry. Globin isolated from the blood of rats exposed to acrylamide was spiked with an internal standard (globin treated in vitro with d3-acrylamide) and was then hydrolyzed with 6 N HCl. The protein hydrolysate was fractionated on a Dowex 50W H+ ion exchange column and the amino acids in the partially purified extract were determined as N-heptafluorobutyryl methyl esters using an OV-1701 fused silica capillary column. Quantitation was made by chemical ionization (isobutane) selective ion monitoring in which the ions m/z 386 (M-OCH3)+ derived from derivatized S-(2-carboxyethyl)cysteine in the sample and the corresponding ion m/z 389 from the added deuterium-labeled internal standard were monitored. The dose-response relationship between production of hemoglobin adduct and dose of acrylamide (0.1 mg/kg-5 mg/kg) is curved, showing an increasing slope with increasing doses of acrylamide.

Metabolism of aniline mustard [N,N-di-(2-Chloroethyl)-aniline]

The possible metabolic activation of the antineoplastic agent N,N-di-(2-chloroethyl) aniline (aniline mustard) is discussed. Conversion of aniline mustard into the glucuronide (p-di-2-chloroethylaminophenyl-β-d-glucopyranosid)uronic acid was mediated by a rat liver homogenate containing the appropriate cofactors. The glucuronide was a major metabolite in the serum and bile after administration of aniline mustard to rats and after isolation and purification it was identified as its methyl ester by mass spectrometry. The use of Amberlite XAD-2 resin facilitated the isolation from serum of the glucuronide and another metabolite, N-(2-chloroethyl)-4-hydroxyaniline. The implication of these findings for the clinical application of aniline mustard is discussed.

Determination of N-7-[2H3]methyl guanine in rat urine by gas chromatography-mass spectrometry following administration of trideuteromethylating agents or precursors

A gas chromatographic-mass spectrometric method has been developed for the determination of N-7-[2H3]methyl guanine in urine in the presence of large natural levels of N-7-methyl guanine. Urine is fractionated on heptanesulfonic acid-treated C-18 Sep-pak cartridges, followed by derivatization to give a volatile N-heptafluorobutyryl-O6-2,3,4,5, 6-pentafluorobenzyl derivative which is separated on an SE52 fused silica capillary column. Using N-7-ethyl guanine as an internal standard, the total amount of N-7-methyl guanine is determined by gas chromatography-flame ionization detection. The percentage of N-7-[2H3]methyl guanine is then measured by gas chromatography-mass spectrometry, enabling the amount of deuterated base to be determined. Preliminary experiments with [2H3]methyl methanesulfonate in rats showed measurable excretion of N-7-[2H3]methyl guanine. 4-(Di[2H3]methylamino)antipyrine alone gave no detectable amount of alkylated base, but coadministration of nitrite resulted in excretion of deuterated N-7-methyl guanine.

The microsomal metabolism of some analogues of cyclophosphamide. 4-methylcyclophosphamide And 6-methylcyclophosphamide.

Abstract 4-Methylcyclophosphamide and 6-methylcyclophosphamide are, like cyclophosphamide, converted by rat liver microsomes into 4-hydroxy derivatives. 4-Hydroxy-4-methylcyclophosphamide was isolated directly, in admixture with the product [2-(2-chloroethylamino)tetrahydro-4-methyl-2 H -1,3,2-oxazaphosphorine 2-oxide] of dechloroethylation. P.m.r. data for the hydroxy derivative, which was also formed when 4-methylcyclophosphamide was treated with aqueous KMnO 4 , indicated that it exists in aqueous solution as the acyclic tautomer, 2-oxopropyl- N , N - bis -(2-chloroethyl)phosphorodiamidate. 4-Hydroxy-6-methylcyclophosphamide was trapped by reaction with ethanol, and afforded two isomeric ethoxy derivatives analogous to those previously reported from cyclophosphamide. Treatment of the products of metabolism of 4-methyl- and 6-methylcyclophosphamide with 2,4-dinitro-phenylhydrazine afforded, respectively, the 2,4-dinitrophenylhydrazones of methyl vinyl ketone and of crotonaldehyde. 4-Methylcyclophosphamide cannot form metabolites analogous to 4-ketocyclophosphamide and carboxyphosphamide, the relatively non-toxic metabolites of cyclophosphamide. The significance of this fact is discussed in relation to a mechanism which could account for the relatively selective cytotoxicity of cyclophosphamide in vivo towards neoplastic tissue. Conventional electron impact mass spectrometry has played an important role in the characterization of the products described in this study. 4-Hydroxy-4-methylcyclophosphamide was additionally characterized by the relatively novel technique of field desorption mass spectrometry.

The formation and metabolism of N-hydroxymethyl compounds—III: The metabolic conversion of N-methyl and N, N-dimethylbenzamides to N-hydroxymethyl compounds☆

The stability of metabolically-generated N-(hydroxymethyl) compounds was investigated using a series of N-methylbenzamides as model substrates. N-(Hydroxymethyl)-benzamide was characterized as a major metabolite of N-methylbenzamide in vitro, and was also identified as a urinary metabolite of N-methylbenzamide. N-(Hydroxymethyl) compounds were also found as metabolites of 4-chloro-N-methylbenzamide and 4-t-butyl-N-methylbenzamide in vitro. Thus substitution in the 4-position of the phenyl ring of derivatives of N-(hydroxymethyl)-benzamide did not affect their stability sufficiently to cause degradation to formaldehyde under the conditions used. N-(Hydroxymethyl)-N-methylbenzamide was identified as a metabolite of N,N-dimethylbenzamide in vitro. However, N-(hydroxymethyl)-N-methylbenzamide was less stable than N-(hydroxymethyl)-benzamide under alkaline conditions. Furthermore, N-(hydroxymethyl)-N-methylbenzamide, unlike N-(hydroxymethyl)-benzamide and its 4-substituted derivatives, was positive in the colorimetric assay for formaldehyde, presumably because of its degradation to produce formaldehyde. Thus substitution on the nitrogen atom which bears the methyl group in N-methylbenzamide markedly affected the stability of the N-methylol produced during oxidative metabolism. N-Formylbenzamide was identified as a metabolite of N-methylbenzamide in suspensions of mouse hepatocytes and also in vivo. The mechanism for its production probably involves the generation of N-(hydroxymethyl)-benzamide.