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.

Different tacrolimus epoxides and dihydrodiol epoxides arising from the chemical oxidation of the parent drug are described. Open-chain tautomeric forms involving the lactone function were identified for the tacrolimus epoxides. Moreover, the identification by electrospray and electrospray linked scan mass spectrometry of an SDZ-RAD C16-C27 O-demethyl 17, 18-19, 20-21, 22 tris-epoxide new metabolite isolated from pig liver microsomes is reported. The in vitro immunosuppressive activity, using mixed lymphocyte reactions of the two macrolide reported oxidation compounds are discussed.

Isolation from rat urine and human liver microsomes and identification by electrospray and nanospray tandem mass spectrometry of new malagashanine metabolites.

Malagashanine has been isolated from indigenous madagascan Strychnos myrtoides alkaloids used traditionally to treat malaria. This alkaloid was found to enhance the action of chloroquine against chloroquine-resistant strains of Plasmodium falciparum when combined with classical antimalarial drugs (chloroquine, quinine). The present study was carried out in order to investigate by electrospray mass and tandem mass spectrometry and NMR spectroscopy the structure of two new metabolites isolated from rat urine and human liver microsomes. We were able to demonstrate the presence of two new metabolites of malagashanine corresponding to a malagashanine N-demethylated metabolite and to the oxidation of malagashanine in the alpha-position of the N-methyl group to produce a carbinolamine function. The latter metabolite may be subject to ring and open-chain tautomerism effects and dimeric species were detected in the electrospray mass spectrum.

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 from pig liver microsomes, identification by electrospray tandem mass spectrometry and in vitro immunosuppressive activity of a rapamycin tris-epoxide metabolite.

It was demonstrated that rapamycin is metabolized in vitro by pig liver microsomes under the influence of the cytochrome P450-dependent mixed function oxygenase system to a rapamycin tris-epoxide metabolite, as demonstrated by electrospray tandem mass spectrometry. The in vitro immunosuppressive activity of this metabolite was found to be lower than that of rapamycin, probably because the rapamycin effector sector was structurally modified. The effector region of rapamycin was recognized to include the conjugated double bonds of this compound and metabolic reactions affecting this region may change the binding affinity of the rapamycin-FKBP binary complex towards another pharmacological receptor bound to the binary complex. Moreover, metabolic modifications in the effector region are probably able to induce a change in the binding affinities of the rapamycin-FKBP binary complex, including the pipecolic acid moiety and the lactone function of 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.

In vitro immunosuppressive activity, isolation from pig liver microsomes and identification by electrospray ms-ms of a new FK-506 C19-C20 epoxide metabolite.

In order to mediate their effects, cyclosporin A and FK-506 must each bind with high affinity to a cytosolic target protein that belongs to the immunophilin group. FK-506 forms complexes with the FK-506 binding protein FKBP, mainly FKBP-12, and these complexes possess immunosuppressive activity through their ability to interact with another target, the abundant serine threonine phosphatase calcineurin. Evaluating the immunosuppressive activities of the FK-506 metabolites by comparing them with known immunosuppressive agents via mixed lymphocyte reaction is of clinical importance because some metabolites may retain the pharmacological activity of the parent drug or exhibit cytotoxic effects. FK-506 is metabolized by the cytochrome P-450-dependent mixed-function oxygenase system in different animal species, and we are reporting the isolation from pig liver microsomes, and the identification by electrospray ms-ms, of the FK-506 C19-C20 epoxide metabolite. We found that this new metabolite exhibits reduced in vitro immunosuppressive activity compared with FK-506 and has approximately the same immunosuppressive potency as other known immunosuppressive drugs, such as cyclosporin A and IMM-125, a hydroxyethyl derivative of D-serine cyclosporin A. We were able to demonstrate that after incubation of the FK-506 metabolite in human mixed lymphocyte reaction cultures for 6 days, the compound was stable under the conditions used for cell culture as evidenced by electrospray-ms data. A weak direct cytotoxic effect (< 30% cell death) was observed only at the highest concentrations (2500 and 5000 ng/ml), which shows that the mixed lymphocyte reaction inhibition cannot be due to a toxic effect.

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.

The in vitro immunosuppressive activity of the C15-demethylated metabolite of FK-506 is governed by ring- and open-chain tautomerism effects.

The FK-506 C15-demethylated metabolite, isolated from human liver microsomes and identified by fast atom bombardment mass spectrometry and NMR spectroscopy, is under the influence of ring- and open-chain tautomerism effects as demonstrated by fast atom bombardment mass spectrometry. Ring- and open-chain tautomerism effects are strongly influencing the in vitro immunosuppressive potency expressed by the measure of the inhibition of the mixed lymphocyte reaction, as demonstrated by the clear differences observed in immunosuppressive activity between the FK-506 C15-demethylated ring- and open-chain tautomeric conformations and depending on the tautomer-receptor specific interactions modulating the immunosuppressive activity of the FK-506 C15-demethylated metabolite.

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.

Isolation and identification of a novel isomerized epoxide metabolite of FK-506 from erythromycin-induced rabbit liver microsomes.

A novel metabolite of FK-506-a C13, C15, C31 O-demethyl C19 hydroxymethyl C36 isomerized epoxide of FK-506 obtained from erythromycin-induced rabbit liver microsomes--was isolated by HPLC and identified by FAB/MS and NMR. The existence of ring-chain tautomers for this metabolite is demonstrated by NMR spectroscopy. FAB/MS, and HPLC.

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.

Isolation and mass spectrometric identification of two metabolites of FK 506 from rat liver microsomal incubation media.

A transient epoxide in equilibrium with its possible isomeric forms, most probably induced easily by neighboring-group assistance of a hydroxy group, and a dihydrodiol formed under the influence of the hydrase enzymic activity and/or by simple hydrolysis were isolated by HPLC and identified by FAB/MS from rat liver microsomal incubation media.