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

¹ Department of Pharmaceutical Sciences-UCL, Pharmacokinetics and Metabolism Unit, Laboratory of Mass Spectrometry, 7246, Av. E. Mounier, B-1200 Brussels, Belgium.

² Experimental Immunology Unit-UCL, Clos Chapelle aux Champs, 3056, B-1200 Brussels, Belgium.
^a Author for correspondence. Fax 32 2 2624150;

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
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.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Rapamycin (sirolimus, AY-22,989, C₅₁H₇₉NO₁₃), isolated from a strain of Streptomyces hygroscopus (1), is a 31-membered macrolide lactone with a molecular mass of 913.6 Da. Although rapamycin was originally developed as an antifungal and antitumor drug, the focus of current interest has shifted towards its immunosuppressive properties (2). Rapamycin binds to the same intracellular binding protein in lymphocytes (FKBP or FK-506 binding protein) as its structural homolog, the immunosuppressive drug FK-506 (3), and inhibits the S6p70-kinase (4), an immunosuppressive mechanism of drug biological action quite different from that of FK-506 (tacrolimus), which inhibits the phosphatase activity of calcineurin in vitro (5).¹

The cytochrome P-450 3A-dependent mixed-function oxygenase system is responsible for the metabolism of rapamycin (6). Some hydroxylated and (or) demethylated metabolites of rapamycin were isolated and identified from human liver microsomes and rat small intestinal microsomes, among them the 41-O-demethyl rapamycin metabolite (7), also called 39-O-demethyl rapamycin, following the numbering of atoms of the Cambridge Structure Data Bank (8).

We describe in this study the isolation of the 39-O-demethyl rapamycin metabolite from pig liver microsomes and the structural identification of this compound by fast atom bombardment (FAB) and electrospray tandem mass spectrometry (MS-MS). We also discuss the results of in vitro immunosuppressive activity of this metabolite in terms of structure–activity relation using the mixed lymphocyte reaction (MLR).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Chemicals and reagents.
Rapamycin (white powder, batch AY-22989–21) was graciously supplied by Wyeth-Ayerst Research. "Spectrograde" solvents (acetonitrile, dichloromethane, and isopropanol) used in extraction or analytical procedures were purchased from Labscan Unit T26 and hexane from Alltech Associates, Applied Science Labs. NADP, glucose-6 phosphate, and glucose-6-phosphate dehydrogenase grade II (5 g/L) were purchased from Boehringer Mannheim. The matrix used in FAB/MS, 3-nitrobenzyl alcohol, was manufactured by Aldrich-Chemie. Demineralized and filtered (MilliQ water purification system; Millipore-Waters) water was used. All cell culture reagents were obtained from Gibco Labs.

Animals.
Female Landrace Belgium pigs (~16 kg) were maintained in individual cages and were given free access to commercial food pellets (Versele-Laga) and water.

Preparation of pig liver microsomes.
After intracardiac injection of 20 mmol of a KCl solution (1 mol/L), the liver was removed, weighed, cut into seven pieces of ~100 g, and stored in a refrigerator at -80 °C. The pieces of liver were removed, weighed, washed with ice-cold 3 mmol/L imidazole homogenizing medium containing 0.5 mol/L sucrose, minced with scissors, and fractionated according to a described method (9) to produce a microsomal fraction containing 4.62 g/L protein and 0.43 nmol of cytochrome P-450/mg protein determined according to published standard procedures (10)(11).

Rapamycin microsomal incubation medium and extraction of the metabolites.
To the NADPH-generating medium (1.6 mL) containing 5.08 mg of NADP, 0.4 mL of MgCl₂ (0.5 mol/L), 10 mg of glucose-6 phosphate, and 1.2 mL of Tris (pH 7.4) were added 5 mL of pig liver microsomes, 1.2 µL of glucose-6-phosphate dehydrogenase (specific activity 350 kU/g), and 5 µg of rapamycin/mg protein dissolved in acetonitrile (1 g/L), in a total volume of 6.6 mL. This mixture was incubated for 1 h at 37 °C in 50-mL Erlenmeyer flasks and transferred to a centrifuge tube. Fourteen milliliters of dichloromethane were added, vortex-mixed for 2 min, and centrifuged for 20 min at 3800g. The aqueous phase was discarded and the residue remaining after evaporation of the organic phase under reduced pressure was dissolved in 800 µL of isopropanol. The resulting solution was then submitted to HPLC analysis.

HPLC.
The HPLC system consisted of a HP 1090 HPLC system (Hewlett-Packard) connected to a Diode-Array detector HP 1050. Rapamycin metabolites were first separated in normal phase on an Alltech Rsil column (10 mm, length 250 mm, i.d. 10 mm) fitted with a Lichrosorb CN precolumn (Merck) with a gradient of hexane (from 100 mL/L to 600 mL/L):isopropanol (from 900 mL/L to 400 mL/L) during 35 min, followed by an isocratic hexane:isopropanol (60:40) phase as the mobile phase. Flow rate and UV detector settings were 2 mL/min and 276 nm, respectively. Groups of peaks with retention times (RT) 27 to 55 min were then collected and after evaporation of the mobile phase the residue was dissolved in acetonitrile. The resulting solution was then rechromatographed on a reversed-phase HPLC system consisting of three columns in tandem (Nova-Pak-C₁₈ 4 µm, Waters, 3.9 x 150 mm Nucleosil-C₁₈,, 5 µm, 4 x 250 mm, Macherey-Nagel, and Supelco, LC-18 DB, 5 µm, 4.6 x 250 mm, Sercolab) at 40 °C by using a gradient of acetonitrile (from 480 mL/L to 1000 mL/L):water (from 520 mL/L to 0 mL/L) as the mobile phase. Flow rate and UV detector settings were 1 mL/min and 276 nm, respectively. Under these conditions, the major peaks were detected and collected. After evaporation of the mobile phase under reduced pressure, the residues were dissolved in acetonitrile, transferred to individual tubes, preweighed on a semimicro balance (Precisia), and evaporated to dryness under a stream of nitrogen.

FAB/MS.
FAB mass spectra were obtained with a Kratos (Kratos Analytical) MS80 RFA instrument. Metabolites (100 µg) were dissolved in dichloromethane. Small aliquots of the resulting solution were slowly transferred and evaporated on a copper probe tip (standard Kratos FAB probe) before adding a fixed amount (6 mg) of a 3-nitrobenzyl alcohol matrix. Xenon gas was used in the Kratos FAB source with a primary energy of 7.8 kV.

Electrospray MS-MS.
Electrospray MS-MS spectra were obtained with a Finnigan Mat LCQ and MSⁿ instrument. The source voltage was 5.05 kV, the capillary voltage 25.76 V, and the capillary temperature 220.10 °C. The compounds (100 µg) were dissolved in a mixture of acetonitrile:5 mmol/L aqueous solution of ammonium acetate (50:50) and the solution was infused with the aid of a syringe pump at a flow rate of 5 µL/min.

Functional assays to determine the immunosuppressive effect.
Mononuclear cells were isolated from human peripheral blood by density gradient centrifugation on Ficoll–Hypaque medium (International Medical, d = 1.077). After washing, the cells were suspended in enriched RPMI medium at a concentration of 2 x 10 cells/L (12). The enriched RPMI medium consisted of 770 mL/L RPMI medium 1640 (Gibco), 200 mL/L fetal calf serum (Biosys), 10 mL/L glutamine, 10 mL/L penistreptomycin (5000 kU/L), and 10 mL/L gentamicin (50 g/L). In microplates, 10 cells/well (50 µL) were incubated at 37 °C and 5% CO₂ for 5 days with 50 µL of the metabolite solution, 10 nonirradiated MHC-incompatible allogeneic cells (50 µL), and 50 µL of enriched medium. The concentration range of the metabolite and of the parent compound rapamycin was 0.125, 1.25, 12.5, 25, 100, 500, and 1000 µg/L. To each well was added 10 µL of a [¹ H]thymidine solution [7.4 GBq (0.2 Ci)/L, Isotopchim]. Cell cultures were harvested with an automated multiwell harvester (Skatron) that first aspirates cells, lyses them, and transfers their DNA onto filter paper, while allowing unincorporated [¹ H]thymidine to wash out. The incorporation was determined by liquid scintillation counting (beta counter cpm, Beckman LS 6000SE) after an additional 8-h incubation. The potential inhibitory response of each solution was calculated in cpm and expressed as the percentage of inhibition of a normal response (MLR performed in absence of any solution). Each culture, including positive and negative controls, was performed in triplicate, and then repeated five times. Pure rapamycin and solvent additions were the positive and negative controls, respectively.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The reversed-phase chromatogram resulting from the incubation of rapamycin in the presence of pig liver microsomes is illustrated in Fig. 1 and reveals the presence of metabolic peaks of RTs 8.5 min, 11.2 min, 18.6 min, and 23.2 min. The remaining chromatographic peaks observed at 25.9 min, 26.7 min, and 27.8 min are tautomers of unchanged rapamycin as confirmed by FAB/MS. The metabolite at RT 11.2 is a 5,6-dihydrodiol metabolite already identified from rat liver microsomes (13).

View larger version (18K): [in this window] [in a new window]   Figure 1. Reversed-phase HPLC chromatogram resulting from the incubation of rapamycin in the presence of pig liver microsomes.

The FAB mass spectrum of the metabolite at RT 23.2 (Fig. 2 ) is characterized by the presence of quasimolecular ions of mass m/z = 922 (MNa) and 938 (MK) and by fragmentation ions of mass m/z = 906 (M - CH₃OH)K, 890 (M - CH₃OH)Na, 785 (M - 115)H, 769 (M - CH₄ - 115)H, 755 (M - 3H₂O - pipecolic acid)K, and 737 (769 - CH₃OH)H, proving that a demethylation process occurred under the influence of the cytochrome P-450-dependent mixed-function oxygenase system as illustrated in the fragmentation pathway of Fig. 3 . Because ring–chain tautomerism effects exist between the lactone function and the corresponding carboxylic acid with the formation of a double bond at the C₂₅–C₂₆ positions, a fragmentation process in the position of the double bond is observed giving rise to a fragment ion of mass m/z = 785, demonstrating that demethylation occurred at the rapamycin C₃₉ position. Also, the fact that fragment ions are observed resulting in the elimination of pipecolic acid indicates that some intramolecular interactions between the C₃₉ hydroxy group and the C₁₆ carbonyl are possible, giving rise to a carbinolamine intermediate that is able to lose pipecolic acid, as already observed for tacrolimus (14)(15).

View larger version (21K): [in this window] [in a new window]   Figure 2. FAB mass spectrum of metabolite of RT 23.2 min.

View larger version (25K): [in this window] [in a new window]   Figure 3. Relevant fragmentation ions of metabolite of RT 23.2 min.

These observations were confirmed by electrospray MS (Fig. 4 ) and MS-MS measurements on this rapamycin metabolite characterized mainly by the presence of quasimolecular ions of mass m/z = 917 (M NH₄), 922 (M Na), and 938 (M K), confirming that rapamycin was submitted to a phase I demethylation reaction. When the sodium adduct (m/z = 922) was selected as the parent ion, daughter ions of mass m/z = 890 (M - CH₃OH)Na, 872 (M - CH₃OH - H₂O)Na, 614 (M - 308)Na^, 582 (M - 308 - CH₃OH)Na, and 564 (M - 308 - CH₃OH - H₂O)Na were observed, as shown in the Fig. 5 , confirming again that demethylation occurred at the rapamycin C₃₉ position. The daughters of the parent ion m/z = 890 were found to be 872 (890 - H₂O), 779 (890 - 111), 761(890 - pipecolic acid), 735(890 - 111 - CO₂), 717(890 - 111 - CO₂ - H₂O), 582(890 - 308), and 564 (890 - 308 - H₂O) (data not shown). The fact that pipecolic acid or 2-formyl piperidine 2-ene, as also observed for tacrolimus metabolites (14), may be lost from the parent ion m/z = 890 confirms that intramolecular interactions of free hydroxy groups with the C₁₆ rapamycin carbonyl function are possible to produce a carbinol-amine intermediate.

View larger version (16K): [in this window] [in a new window]   Figure 4. Electrospray MS spectrum of metabolite of RT 23.2 min.

View larger version (24K): [in this window] [in a new window]   Figure 5. Daughter ions of the parent MNa+ = 922 and relevant fragmentation pathways.

The in vitro immunosuppressive activity of MLR by the C₃₉ demethylated rapamycin metabolite is essentially zero until 25 µg/L (Fig. 6 ). The 50% immunosuppressive concentration (IC₅₀) of rapamycin is 2 x 10^-4 µg/L (data not shown) and of the C₃₉ demethylated metabolite 2 x 10 µg/L, which is 10^-6 times lower than that of rapamycin. In the higher concentration range, the immunosuppressive activity increases progressively from 25 to 1000 µg/L, where the last recorded value is similar to the one of rapamycin. These results are in agreement with the ones obtained recently for four rapamycin metabolites isolated from urine of renal transplant recipients, since the observed immunosuppressive activities in MLR (16) were small at a concentration of 40 µg. Probably the 39-O-demethyl metabolite was present among these four metabolites. The metabolite obtained from pig liver was estimated to be 5% of unchanged rapamycin (comparison between the surfaces under the curve in the HPLC chromatogram). The same metabolite was already isolated from human liver microsomes (7) and inhibited phytohemagglutinin-induced lymphocyte proliferation. The amount and the immunosuppressive activity of this metabolite are both low, leading to a negligible contamination in current therapeutic monitoring methods of rapamycin. Kessler et al. reported (17) that the binding region, including the C₁₁ to C₂₃ and C₃₇ to C₄₄ positions of rapamycin, is nearly identical to the one of tacrolimus. Intramolecular interactions of free hydroxy groups at the rapamycin C₁₆ position to produce a carbinolamine intermediate were observed in FAB as well as in electrospray MS. Also, intermolecular interactions with FKBP-12 may give rise to the formation of several tautomeric forms of the C₃₉ demethylated rapamycin metabolite, presenting different binding affinities for FKBP-12. The decrease of in vitro immunosuppressive activity is likely to be attributed to important structural modifications of the rapamycin binding region to FKBP-12, including intramolecular interactions of the C₃₉ hydroxy group with the lactone function, producing another ring tautomer very similar to the one described for iso-FK-506 (18)(19) and decreasing the binding affinity of this rapamycin metabolite for its pharmacological receptor FKBP-12.

View larger version (25K): [in this window] [in a new window]   Figure 6. In vitro immunosuppressive activity of the rapamycin C39 demethylated metabolite in the concentration range 0.125 to 1000 µg/L. , rapamycin; , 39-O-demethylated metabolite; , acetonitrile.

We conclude that rapamycin is metabolized in vitro under the influence of cytochrome P-450-dependent mixed-function oxygenase enzymic system to a C₃₉ demethylated rapamycin metabolite characterized by an in vitro immunosuppressive activity that is essentially zero.

	Footnotes

 
¹ Nonstandard abbreviations: FKBP, FK-506 binding protein; FAB, fast atom bombardment; MS, mass spectrometry; MLR, mixed lymphocyte reaction; and RT, retention time.

	References

Top Abstract Introduction Materials and Methods Results and Discussion References

 

1. Sehgal SN, Baker H, Vezina C. Rapamycin (AY-22,989), a new antifungal antibiotic. II. Fermentation, isolation and characterization. J Antibiot (Tokyo) 1975;28:727-730. [Medline] [Order article via Infotrieve]
2. Morris R. Rapamycins: antifungal, antitumor, antiproliferative, and immunosuppressive macrolides. Transplant Rev 1992;6:39-87.
3. Screiber SL. Chemistry and biology of the immunophilins and their immunosuppressive ligands. Science 1991;251:283-287. [Abstract/Free Full Text]
4. Dumont FJ, Su Q. Mechanism of action of the immunosuppressant rapamycin. Life Sci 1996;58:373-395. [ISI][Medline] [Order article via Infotrieve]
5. Liu J, Farmer JDJ, Lane WS, Friedman J, Weissman I, Screiber SL. Calcineurin is a common target of cyclophilin–cyclosporin A and FKBP–FK-506 complexes. Cell 1991;66:807-815. [ISI][Medline] [Order article via Infotrieve]
6. Sattler M, Guengerich FP, Yun CH, Christians U, Sewing KF. Cytochrome P-450 3A enzymes are responsible for biotransformation of FK-506 and rapamycin in man and rat. Drug Metab Dispos 1992;20:753-761. [Abstract]
7. Christians U, Sattler M, Schiebel HM, Kruse C, Radeke HH, Linck A, Sewing K-FR. Isolation of two immunosuppressive metabolites after in vitro metabolism of rapamycin. Drug Metab Dispos 1992;20:186-191. [Abstract]
8. Streit F, Christians U, Schiebel HM, Napoli KL, Ernst L, Linck A, et al. Sensitive and specific quantification of sirolimus (rapamycin) and its metabolites in blood of kidney graft recipients by HPLC/electrospray–mass spectrometry. Clin Chem 1996;42:1417-1425. [Abstract/Free Full Text]
9. Amar-Costesec A, Beaufay H, Wibo M, Thinès-Sempoux D, Feytimans E, Robbi M, et al. Analytical study of microsomes and isolated subcellular membranes from rat liver. J Biol Chem 1974;61:201-212.
10. Lowry OH, Rosebrough NJ, Farr AL, Randall RI. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. [Free Full Text]
11. Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomes. 1. Evidence for its hemoprotein nature. J Biol Chem 1964;239:2370-2378.
12. James SP. Immunologic studies in human. Coligan JE Kruisbeck AM Margulies DH Shevach EM Strober W eds. Current protocols in immunology 1992:71001-71010 National Institutes of Health New York. .
13. Nickmilder MJM, Latinne D, Verbeeck RK, Janssens W, Svoboda D, Lhoëst GJJ. Isolation and identification of new rapamycin dihydrodiol metabolites from dexamethasone induced rat liver microsomes. Xenobiotica 1997;27:869-883. [ISI][Medline] [Order article via Infotrieve]
14. Lhoëst GJJ, Maton N, Laurent A, Verbeeck RK, Latinne D. Characterization of a FK-506 C₃₆–C₃₇ dihydrodiol from erythromycin induced rabbit liver microsomes. Constantin E eds. Therapeutic aspects and analytical methods in cancer research 1994:164-182 Amudes Strasbourg. .
15. Lhoëst GJJ, Verbeeck RK, Maton N, Muthelet P, Latinne D. The in vitro immunosuppressive activity of the C₁₅-demethylated metabolite of FK-506 is governed by ring- and open-chain tautomerism effects. J Pharmacol Exp Ther 1995;274:622-626. [Abstract/Free Full Text]
16. Goodyear N, Murtly JN, Gallant HL, Yatscoff RW, Soldin JJ. Comparison of binding characteristics of four rapamycin metabolites to the 14 and 52 kDa immunophilins with their pharmacologic activity measured by the mixed-lymphocyte culture assay. Clin Biochem 1996;29:309-313. [ISI][Medline] [Order article via Infotrieve]
17. Kessler H, Haesner R, Schüler W. Structure of rapamycin: an NMR and molecular-dynamics investigation. Helv Chim Acta 1993;76:117-130.
18. Grassberger MA, Fehr T, Horvath A, Schultz G. Isolation of an isomer of FK-506 from fermentation of Streptomyces tsukubaensis and its chemical synthesis from FK-506. Tetrahedron 1992;48:413-430.
19. Lhoëst GJJ, Maton N, Verbeeck RK. Isolation and identification by FAB mass spectrometry and NMR spectroscopy of a demethylated metabolite of FK-506 from erythromycin-induced rabbit liver microsomes. Pharm Acta Helv 1993;68:35-41. [Medline] [Order article via Infotrieve]