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.

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

SDZ-IMM-125 N-methyl leucine 9 hydroxylated in the gamma 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. The in vitro 50% inhibitory concentration, tested against bidirectional mixed lymphocyte reaction was 80 microg/l indicating that this metabolite does not retain in 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.

Isolation, identification and immunosuppressive activity of a new IMM-125 metabolite from human liver microsomes. Identification of its cyclophilin A-IMM-125 metabolite complex by nanospray tandem mass spectrometry.

The isolation from human liver microsomes and identification by electrospray mass spectrometry and tandem mass spectrometry of a new metabolite of IMM-125 resulting from the biotransformation of the amino acid 1 vinylic methyl group to a carboxylic acid, called the IMM-125-COOH metabolite, is described. It was found that the complex of this new metabolite with cyclophilin A is formed less easily than the corresponding cyclophilin A-IMM-125-CH2OH main metabolite and cyclophilin A-IMM-125 complexes. However, when formed, the IMM-125-COOH metabolite-cyclophilin A complex requires more collision-induced dissociation (CID) to dissociate the complex than the complexes formed with the two other ligands. The nanospray tandem mass spectrum of the IMM-125-COOH metabolite-cyclophilin A complex (m/z 1755) gives rise to cyclophilin A-ligand complexes of m/z 1751 by elimination of CO2 and of m/z 1749 by loss of CO2 and H2O or glycerol. Since immunosuppressive activity is known to be dependent on the formation of a binary complex between cyclophilin A and the drug and since the target for the binary complex was found to be the calcium- and calmodulin-dependent protein phosphatase, calcineurin, it could be interesting to measure for structurally related immunosuppressive drugs the CID energy necessary to dissociate the binary complexes in order to evaluate whether a correlation with the phosphatase activity could be derived.

Isolation and identification of a C39 demethylated metabolite of rapamycin from pig liver microsomes and evaluation of its immunosuppressive activity.

We studied in vitro metabolism of rapamycin using pig liver microsomes. After extraction of the metabolites from the incubation medium, the crude metabolite extract was submitted to normal and subsequently to reversed-phase HPLC chromatography. We describe in the current study a metabolite of retention time 23.2 min collected from reversed-phase HPLC and identified by fast atom bombardment mass spectrometry (MS) and electrospray MS-MS as a C39 demethylated rapamycin metabolite. In vitro immunosuppressive activity of this metabolite, determined by the mixed lymphocyte reaction, was negligible compared with that of the parent compound. The decrease of in vitro immunosuppressive activity compared with the parent compound is likely to be attributed to important structural modifications of the rapamycin binding region to the FK-506 binding protein.

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.

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.

15-Desmethyl FK-506 and 15,31-desmethyl FK-506 from human liver microsomes: isolation, identification (by fast atom bombardment mass spectrometry and NMR), and evaluation of in vitro immunosuppressive activity.

15-Desmethyl FK-506 and 15,31-desmethyl FK-506 metabolites extracted from incubated human liver microsomes were separated by normal and reversed-phase HPLC. The metabolites were identified by fast atom bombardment/mass spectrometry and nuclear magnetic resonance spectroscopy. The in vitro 50% inhibitory concentration, tested against bidirectional mixed lymphocyte reactions, was 325 mg/L for 15,31-desmethyl FK-506 and 4 mg/L for 15-desmethyl FK-506, indicating that these products of FK-506 demethylation retain in vitro immunosuppressive activity.