Georges Lhoëst

Identification and quantitation of 1,2-epoxybutene-3 as the primary metabolite of 1,3-butadiene

Abstract 1,3-Butadiene was incubated in the presence of rat liver microsomes supplemented with an NADPH-generating system. One of the major metabolites of butadiene was found to be 1,2-epoxybutene-3, which was analysed by electron-capture gas—liquid chromatography after its derivatization with pentafluorophenylhydrazine. The effects of variation of several incubation parameters and of different pretreatments of the animals on its formation kinetics were evaluated.

Subnanogram estimation of the proximate carcinogen N-hydryxy-2-fluorenylacetamide by gas-liquid chromatography.

A very sensitive method, using electron-capture gas chromatography, has been developed for the quantitative estimation of N-hydroxy-2-fluorenylacetamide, the proximal carcinogenic metabolite of N-2-fluorenylacetamide. After incubation of the carcinogenic parent arylamide with rat liver microsomes, the N-hydroxy derivative produced is converted into N-chloro-2-fluorenylamine by treatment with hydrochloric acid; the amine is extracted with cyclohexane and transformed into N-chloro-2-fluorenyltrifluoroacetamide with trifluoroacetic anhydride. As little as 0.06 ng of the latter compound can be readily detected by gas-liquid chromatography using an electron-capture detector.

Isolation and identification of new rapamycin dihydrodiol metabolites from dexamethasone-induced rat liver microsomes.

1. Rapamycin is metabolically transformed in rat liver microsomes to 3,4- and 5,6-dihydrodiol metabolites under the influence of the cytochrome P-450 mixed function oxygenase system. These metabolites were produced from dexamethasone-induced as well as from non-induced rat liver microsomes. The comparison of the ion spray mass spectra of the 5,6-dihydrodiol with the 3,4-dihydrodiol of rapamycin shows clearly that dihydrodiols were formed in two distinct positions of rapamycin. 2. FAB mass spectra as well as electrospray mass spectra of two additional peaks isolated from the same chromatographic run confirm the presence of a 3,4-dihydrodiol metabolite of rapamycin as also strongly suggested by UV spectra. Hplc reinjection of each individual peak always resulted in chromatograms showing a combination of the same three peaks and therefore are to be considered as tautomers of the 3,4-dihydrodiol of rapamycin. 3. These tautomeric conformations were found to have no immunosuppressive potency, most probably due to important structural and stereochemical modifications of the rapamycin binding domain to the binding protein (FKBP-12) and/or to important metabolic structural modifications of rapamycin effector domain.

Isolation from pig liver microsomes, identification by tandem mass spectrometry and in vitro immunosuppressive activity of an SDZ-RAD 17,18,19,20,21,22-tris-epoxide.

Macrolide immunosuppressive drugs such as tacrolimus (FK506) and sirolimus (rapamycin) are compounds largely used in modern immunosuppressive therapy and considered as powerful immunosuppressive agents. Some of these molecules are still under clinical development as, for example, SDZ-RAD (40-O-(2-hydroxyethyl)rapamycin), an immunosuppressive drug closely related to rapamycin. SDZ-RAD has a molecular mass of 957.57 Da (C53H83NO14) and shares the same common intracellular receptor as tacrolimus, the FK-506 binding protein (FKBP-12). SDZ-RAD exerts its pharmacological effect by binding to a different effector protein, inhibits the S6p 70-kinase and interrupts a different signal transduction pathway than tacrolimus. Both SDZ-RAD and rapamycin are metabolized mainly by the cytochrome P-450 3A4-dependent mixed function oxygenase enzyme system to hydroxylated and demethylated metabolites. We describe here the isolation from pig liver microsomes of a novel SDZ-RAD metabolite identified by electrospray tandam mass spectrometry as a new SDZ-RAD 17,18,19,20,21,22-tris-epoxide metabolite. The in vitro immunosuppressive activity as measured by the mixed lymphocyte reaction is more or less comparable to that of SDZ-RAD, although its pharmacological mode of action may be different from that classically described for rapamycin.

Gas chromatographic and mass fragmentographic assays of carcinogenic polycyclic hydrocarbon epoxide hydratase activity.

A specific and very sensitive procedure for the determination of epoxide hydratase activity in hepatic microsomes is described. Any polycyclic hydrocarbon epoxide can be used as a substrate; in this study, benzo(a)anthracene-5,6-oxide, benzo(a)pyrene-4,5-oxide and 3-methylcholanthrene-11,12-oxide were utilized. The corresponding trans-diols formed during incubation are separated and evaluated using either an electron-capture gas chromatographic method for the determination of their chloromethyldimethylsilylated derivatives or gas chromatographic-mass fragmentographic measurement of their trimethylsilylated derivatives. Concentrations as low as 1 ng per millilitre of incubation mixture can be estimated.

Isolation and identification by FAB mass spectrometry and NMR spectroscopy of a demethylated metabolite of FK506 from erythromycin-induced rabbit liver microsomes.

A new demethylated metabolite, extracted from erythromycin-induced rabbit liver microsomes incubation media, isolated by HPLC and identified by FAB/MS and NMR, is described. Moreover, NMR spectra suggest the existence of tautomeric forms of this O-demethylated metabolite of FK506.

Identification and in vitro immunosuppressive activity of a SDZ-IMM-125 metabolite isolated from phenobarbital-induced rabbit liver microsomes

1. A metabolite of D-serine-cyclosporine A has been isolated from phenobarbital induced rabbit liver microsomes using hplc. 2. This metabolite was identified by FAB, electrospray mass spectrometry as well as nmr spectroscopy and is the result of metabolism of the vinylic methyl group of the 9-carbon amino acid unique to the cyclosporins, the first amino acid of this cyclic undecapeptide. This metabolite exhibits a significantly lower immunosuppressive activity than IMM-125 and CsA.

Cyclization reactions of IMM-125 and oxidation of cyclosporin A amino-acid 1 in the alpha position of the double bond lead to the loss of in vitro immunosuppressive activity

Cyclosporin A (CsA) and IMM-125, a hydroxyethyl derivative of D-serine CsA, are cyclic undecapeptides of molecular weight 1201.8 and 1261.8, respectively. The main metabolites still possessing the undecapeptide structure were found to be compounds resulting from the biotransformation of amino acids 4, 9 and 1. Under the influence of the hepatic cytochrome P-450-dependent monooxygenase system, CsA and IMM-125 amino acid 1 are metabolized to a mono-hydroxylated compound (metabolite M-17) and to a dihydrodiol. A metabolite M18 was found to be the result of a non-enzymic intramolecular formation of a tetrahydrofuran derivative from metabolite M17. Since the existence of a CsA dihydrodiol was reported and since epoxides are considered as the dihydrodiol precursors, the aim of the present work was to prove that the same non-enzymic intramolecular formation of a tetrahydrofuran ring could occur by nucleophilic attack of the amino-acid 1 beta -hydroxy group at the epsilon -position of the freshly formed epoxide by reaction of IMM-125 with m-chloroperbenzoic acid and cyclosporin A with selenium oxide. The immunosuppressive activity of the compounds, as measured by the mixed lymphocyte reaction and by the luciferase activity of a Jurkat-T-cell line stably transfected with the NF-AT/luc reporter plasmid, was found negligible for IMM-125 compared to the parent drug as well as for the cyclosporin A derivative. Structures of the IMM-125 and CsA derivatives were elucidated by electrospray mass-spectrometry and NMR spectroscopy.

Isolation, identification and immunosuppressive activity of SDZ-IMM-125 metabolites from human liver microsomes.

SummarySDZ-IMM-125 N-methyl leucine 9 hydroxylated in the γ position is a metabolite which was extracted from incubated human liver microsomes and subsequently separated by normal and reverse-phase HPLC. This metabolite was identified by fast atom bombardment mass spectrometry, electrospray-ms/ms mass spectrometry and nuclear magnetic resonance spectroscopy. Thein vitro 50% inhibitory concentration, tested against bidirectional mixed lymphocyte reaction was 80 μg/l indicating that this metabolite does not retainin vitro immunosuppressive activity most probably due to the structural modification of SDZ-IMM-125 in the recognized binding region to cyclophilin A reducing its binding affinity relative to the parent drug.