Isolation and identification of new rapamycin dihydrodiol metabolites from dexamethasoneinduced rat liver microsomes

Abstract

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.

Assessment and validation of the MS/MS fragmentation patterns of the macrolide immunosuppressant everolimus

“…Because of this, only incomplete HPLC separation of its metabolites could be achieved and the purity of the isolated metabolite fractions would have been insufficient for NMR spectroscopic analysis. Electrospray ionization (ESI)‐tandem mass spectrometry (MS/MS) has previously been used to identify the structures of several metabolites of the related sirolimus;6–9 however, no attempt was made to further validate the hypothetical fragment structures. It is obvious from these publications that alternative structures for several fragments were possible.…”

In vitro immunosuppressive activity of tacrolimus dihydrodiol precursors obtained by chemical oxidation and identification of a new metabolite of SDZ‐RAD by electrospray and electrospray‐linked scan mass spectrometry

“…1), with a molecular mass of 957.57 Da [C 53 H 83 NO 14 ], like CsA, FK506 and RAPA, is primarily metabolized in the liver by the cytochrome P-450 3A enzymes 17 the metabolites produced result in either a mass increase of 16 units, corresponding to single hydroxylation, or in a mass decrease of 14 units, resulting from single Odemethylation of SDZ-RAD. 18 Epoxides and dihydrodiols were recently isolated from pig and rat liver microsomes 19,20 and identified by electrospray mass spectrometry (MS).…”

“…22 In addition, we describe in this report the identification of an SDZ-RAD C 16 , C 27 O-demethyl 17, 18-19, 20-21, 22 trisepoxide new metabolite isolated from pig liver microsomes. The SDZ- RAD 17,[18][19][20][21]22 tris-epoxide has already been isolated from pig liver microsomes and was identified by electrospray tandem MS (MS/MS). 21 The new metabolite was identified by electrospray and by electrospray-linked scan mass spectrometry.…”

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

“…8 We have recently identified, from pig liver microsomes, metabolites of rapamycin corresponding to epoxides and dihydrodiols resulting from the epoxide hydrase enzyme activity. 9,10 We describe here the isolation, identification by electrospray tandem mass spectrometry (ESIMS/MS) and the in vitro immunosuppressive activity using mixed lymphocyte reactions (MLR) of an SDZ-RAD 17,18,19, 20,21,22-tris-epoxide.…”