Introduction

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Several strategies developed to elucidate the molecular basis of information transfer in biological systems are beginning to emerge and are sometimes discovered by a careful analysis of the biological effects of natural products such as CyA and FK-506 produced by soil microorganisms. In order to mediate their effects, CyA, FK-506 and rapamycin must each bind with high affinity to a cytosolic target protein that belongs to the immunophilin group. The first immunophilin to be identified was cyclophilin, which complexes with CyA. The macrolide immunosuppressant drug FK-506 (tacrolimus), inhibits cytokine gene transcription in a manner identical to that of CyA, whereas rapamycin exhibits a completely different mode of action (Shieh et al., 1989; Kuo et al., 1992). The 12-kDa cytosolic protein FKBP-12 (Standaert et al., 1990; Harding et al., 1989; Lane et al., 1991; Siekirka et al., 1989) is the recognized binding protein. The fact that calcineurin forms a complex not only with CyA-cyclophilin but also with FK-506-FKBP may explain the identical mode of action (Friedman and Weissman, 1991; Liu et al., 1991). Tacrolimus, a neutral 21-member macrolide, C₄₄H₆₉NO₁₂ (803 Da), contains a hemiketal function, an --diketonamide, a lactone function and the pipecolinyl ring, which are functional groups described as making part of the binding region to the FK-506 binding protein (Braun et al., 1995). Comparing, via MLR, the in vitro immunosuppressive activity of the FK-506 metabolites with those of known immunosuppressive agents (FK-506, CyA and IMM-125) is of pharmacological and clinical importance because some metabolites may retain the immunosuppressive potency of the parent compounds and/or exhibit cytotoxic effects. The two-way MLR is currently used to test the allogenic stimulation mimicking the rejection that occurs in human transplantation (Zarling et al., 1976).

Tacrolimus (fig. 1) is metabolized by the liver and intestinal cytochrome P-450 3A-dependent mixed-function oxygenase enzymic system (Sattler et al., 1992) to several metabolites, including O-demethylated, hydroxylated, O-demethylated hydroxylated and dihydrodiol metabolites (Lhoëst et al., 1995; Christians et al., 1991; Lhoëst et al., 1994).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Tautomers of tacrolimus.

The isolation of the FK-506 C₁₉-C₂₀ epoxide metabolite from pig liver microsomes (often thought to provide metabolic profiles closely related to human liver microsomes), and its identification by electospray ms-ms and NMR spectroscopy, will be described. The in vitro immunosuppressive activity of this metabolite was found comparable to that of known immunosuppressive drugs such as CyA and SDZ-IMM-125, a hydroxyethyl derivative of D-serine CyA. The stability of the metabolite under the conditions used for cell culture, as well as cell viability data, are discussed.

	Materials and Methods

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Chemicals and reagents. The following drugs and chemicals were kindly provided by or obtained from the sources indicated: FK-506 (Fujisawa Pharmaceuticals Co., Japan), spectrograde solvents such as methanol, acetonitrile and dichloromethane used in extraction or analytical procedures (Labscan Limited Unit T26, Dublin, Ireland), hexane (Alltech Associates, Inc., Applied Science Labs, Deerfield, IL.), NAD, NADP, glucose-6-phosphate and glucose-6-phosphate dehydrogenase (5 g/l) (Boehringer, Mannheim, FRG), analytical grade Tris(hydroxymethyl)-aminomethane-hydrochloride (0.2 M) and NaOH (1 N) used to prepare buffered solutions (Merck, Darmstadt, FRG) and the matrix used in FAB/MS, 3-nitrobenzyl alcohol and benzo(a)pyrene-7(8H)one (Aldrich-Chemie, Steimheim, FRG). Demineralized and filtered (Milli-Q water purification system) water was used.

Animals. Female Landrace Belgium pigs (±25 kg) were maintained in individual cages and were given free access to commercial food pellets (Versele-Laga sa, Deinze, Belgium) and water.

Preparation of pig liver microsomes. The liver was removed, weighed, and cut into seven pieces of about 100 g, which were frozen at 80°C. Then a known mass of liver was washed with 3 mmol/l ice-cold imidazole homogenizing buffer containing 0.5 mol/l sucrose, was blotted with filter paper and was minced with scissors. The minced liver was treated and fractionated according to recognized methods (Amar-Costesec et al., 1974). Protein and cytochrome P-450 concentrations were determined (Lowry et al., 1951; Omura and Sato, 1964) according to published standard procedures.

FK-506 microsomal incubation medium and extraction of metabolites. The NADPH-generating medium (1 ml), containing 0.88 mg NADPH, 2.54 mg NADP⁺, 0.2 ml MgCl₂ (0.5 mol/l), 15 mg glucose-6-phosphate and 0.6 ml Tris (pH 7.4), was preincubated in a Gallenkamp (Grant Instruments, Cambridge, UK) shaking incubator for 15 min at 37°C in small Erlenmeyer flasks. To this solution we added 2.5 ml of pig liver microsomes, 6 µl (5 g/l) of glucose-6-phosphate dehydrogenase (specific activity 350 kU/g) and 20 to 50 µg of FK-506 dissolved in ethanol (20 µl). This mixture was incubated for 2 hr at 37°C. The incubation medium was subsequently transferred to a centrifuge tube, and 7 ml of dichloromethane was added. After 2 min of mixing (vortex mixer), the tube was centrifuged for 10 min at 3200 × g. The aqueous phase was discarded, and the residue from the evaporation of the dichloromethane phase was dissolved in 1 ml of acetonitrile/water (3:7). The resulting solution was subsequently washed with 1.5 ml of hexane (2 min of vortex mixing), which was discarded after centrifugation for 5 min at 800 × g. The acetonitrile-water phase was extracted again with 2 ml of dichloromethane. After 2 min of mixing (vortex mixer) and centrifugation for 10 min at 3200 × g, the water phase was discarded and the dichloromethane layer was evaporated to dryness under a stream of nitrogen. The residue was dissolved in 50 µl of isopropanol, and the resulting solution was then subjected to HPLC analysis. In order to accumulate the required amounts of metabolites for electrospray ms-ms, we repeated the described experiment 10 times and combined the residues.

HPLC. The HPLC system consisted of an isocratic pump (Waters model 6000 A), a Waters U6K injector, a variable-wavelength Pye Unicam (Cambridge, UK) LC-UV detector connected to an HP 3392 A integrator (Hewlett-Packard, Palo Alto, CA). FK-506 metabolites were first separated on an Alltech (Gent, Belgium) Rsil CN column (10-µm, length 250 mm, I.D. 10 mm) using hexane/isopropanol 730:270 as the mobile phase. The flow rate was adjusted to 3 ml/min, and the UV detector was set at 210 nm. Under those conditions, two groups of peaks were observed: at retention times of 18 to 20 min (group 1) and 20 to 30 min (group 2). Eluate corresponding to group 2 was collected and subsequently rechromatographed on a Macherey-Nagel (Oensingen, Switzerland) C₈ Nucleosil column (5-µm, length 250 mm, I.D. 10 mm) at 60°C. The mobile phase was a mixture of acetonitrile/water (52.5:47.5), and the flow rate and UV detector settings were 2.75 ml/min and 210 nm, respectively; the oven temperature was 60°C. Under those conditions, a peak was detected in the HPLC chromatogram at the retention time of 14.7 min. The eluate corresponding to this peak was 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, Zürich, Switzerland) and evaporated to dryness under a stream of nitrogen. A quantity of 450 µg was accumulated to obtain sufficient amounts for evaluation of their in vitro immunosuppressive activity, for electrospray mass spectrometry and for NMR spectroscopy.

Electrospray ms-ms. Electrospray ms-ms spectra were obtained with a Bruker Quistor ESQLC and MSⁿ instrument (Bruker-Franzen Analytik GmbH, Bremen, FRG). The source voltage was 4.0 kV; the capillary exit, skimmer 1 and skimmer 2 were set at 80.0, 42.0 and 5.5 V, respectively. The octapole offsets 1 and 2 were set at 5.5 and 3.0 V, and the flow of dry gas (N₂) was 8 l/min. The compounds (100 µg) were dissolved in a mixture of acetonitrile/5 mM 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.

NMR. ¹H NMR spectra were recorded at 300 K on a Bruker AMX-500 spectrometer (Bruker, Rheinstetten, FRG) equipped with an Aspect X-32 computer. Then 1.3 mg of FK-506 and 237 µg of metabolite 1 were dissolved in 0.5 ml of CD₃CN. Spectra were referenced relative to the solvent peak (CD₂HCN, d = 2.00 ppm). In order to obtain the spectra, 4096 transients were acquired with a relaxation delay of 10 s to yield 16 k data points, apodized using a 0.3-Hz exponential windows function and zero-filled to 64 k before Fourier transform.

Functional assays to determine the immunosuppressive effect. PBMNC were isolated from heparinized blood by density gradient centrifugation on LSM (Pharmacia). Isolated PBMNC were resuspended in enriched medium: RPMI 1640 medium (Gibco, Merelbeke, Belgium) supplemented with 100 U/ml penicillin, 100 mg/l streptomycin, 10 mM/l glutamine and 20% heat-inactivated fetal calf serum (Biosys, Compiègne, France). Bidirectional mixed lymphocyte reactions were performed with 1 × 10⁶ PBMNC of two MHC incompatible donors per well in 96-U-well microplates (Falcon, Lincoln, NE) containing 200 µl/well (final volume) and incubated for 6 days at 37°C, 5% CO₂ with 50 µl of the test compounds solution at different concentrations (from 5000 ng/ml to 1 ng/ml). After that time, 10 µl of a ³H thymidine solution (0.2 mCi/ml, Amersham International, Bucks, UK) was added to each well. Eight hours later, the ³H incorporation was measured by liquid scintillation counting in a Beta Counter (Beckman LSD 6000 SE; Beckman Instruments, Fullerton, CA). All results are the mean of three experiments. In each experiment, immunosuppressive activity measured for each concentration was expressed as the mean of three wells. Standard deviations were always less than 15% of the mean. The immunosuppressive effect of the solvent (acetonitrile/water 50:50) was also checked and was found negative.

Cell viability of MLR cultures. Cell viability was evaluated by MTT colorimetric assay (Mosmann, 1983; Niks and Otto, 1990). Briefly, 10 µl of a MTT solution in PBS was added to 100 µl of a 10⁶/ml cell suspension in two wells in microplates. The plates were incubated for 90 min at 37°C 5% CO₂. A third well with cells in PBS was used as control. In three other wells with the same cell suspension, 20 µl of a 10% Triton X100 solution was added to get 100% cell lysis. Then 100 µl of an isopropanol solution containing 0.04% HCl was added to each well and mixed before incubation, and 60 min later, ODs were measured in a Titertek Twin Reader Plus (Flow ICN, Asse, Belgium) at 540 nm wavelength and 690 nm as reference. Percent viability was calculated as follows:

(1)

The assay was performed by adding the same concentrations of products as those tested in the mixed lymphocyte cultures.

Extraction of the FK-506 metabolite after incubation with MLR cultures. We added 50 µg of FK-506 metabolite or diluent (ACN/H₂O) to a 1-ml suspension of the bidirectional MLR (cells at 10⁶/ml) in 6-well microplates. After incubation for 6 days at 37°C 5% CO₂, the plates were centrifuged and the supernatants harvested for extraction. To the supernatants was added 10 ml of water, and this aqueous solution was extracted twice with 20 ml of dichloromethane in a separating funnel. The dicloromethane layers were evaporated to dryness under vacuum, and 250 µl of the residues dissolved in 500 µl of acetonitrile was injected on a same reverse-phase HPLC column and in the same conditions as previously defined. A small peak was collected between 13.5 and 14.5 min that was not present in the blank.

	Results

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The residue resulting from peak 2 collected by normal-phase HPLC after incubation of FK-506 with pig liver microsomes and extraction of the microsomal medium was rechromatographed on a reverse-phase HPLC column. A metabolic peak with retention time 14.75 min was collected (fig. 2). The electrospray mass spectrum (fig. 3) reveals the presence of quasi-molecular ions of mass m/z = 837 (M + NH₄)⁺, 842 (M + Na)⁺ and 858 (M + K)⁺, which indicates that an oxidation process has taken place under the influence of the cytochrome P-450-dependent mixed-function oxygenase system. The fact that a fragment ion of mass m/z = 821 (M  O + NH₄)⁺ is observed in the electrospray mass spectrum indicates that a FK-506 epoxide was formed at one of the isolated FK-506 double bonds. This is confirmed by the electrospray ms-ms spectrum of the sodium adduct m/z = 842 (fig. 4), which shows the presence of daughter ions of mass m/z = 824 (842  H₂O)⁺, 814 (842  CH₂CH₂)⁺, 806 (842  2H₂O)⁺, 798 (842  ethylene  O)⁺, 786 (842  CH₂CH-CHO)⁺, 768 (842  CH₂CH-CHO  H₂O)⁺, 731 (842  111)⁺, 713 (842  111  H₂O)⁺, 687 (842  ethylene  O  111)⁺, 669 (842  ethylene  O  111  H₂O)⁺)⁺, 632 (842  210)⁺, 616 (842  210  O)⁺, 604 (842  238)⁺, 588 (842  210  O  ethylene)⁺, 576 (842  210  CH₂CH-CHO)⁺, as illustrated in the fragmentation pathways of figures 5, 6 and 7, in agreement with a metabolic biotransformation leading to a FK-506 C₁₉-C₂₀ epoxide.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Reverse-phase HPLC chromatogram showing the presence of the new FK-506 metabolite of retention time 14.75 min (rt14.75).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Electrospray mass spectrum of the FK-506 metabolite rt14.75.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Electrospray ms-ms spectrum of the sodium adduct m/z = 842.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Fragmentation pathways leading to the fragments 731 and 669.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Fragmentation pathways leading to the fragments 798, 786 and 768.

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Fragmentation pathways leading to the fragments 632, 616 and 604.

The NMR spectrum of this metabolite (500 MHz, CD₃-CN) was not of sufficient quality to assign all the signals because of the insufficient amount of material collected for this minor metabolite, but it nevertheless enabled us to confirm unambiguously the presence of the vinylic proton at the C₃₇ position (5.79 ppm) already assigned for FK-506 (Hane et al., 1992) and proved that the C₃₆-C₃₇ double bond of the metabolite remained unchanged. Because metabolism of the C₂₇-C₂₈ double bond was not observed, as clearly evidenced by electrospray ms-ms (fig. 7), epoxidation of the remaining double bond occurred at the FK-506 C₁₉-C₂₀ position. Moreover, the electrospray ms-ms mass spectrum (fig. 8) of the ion m/z = 824 selected as the parent ion reveals the presence of daughter ions of mass m/z = 806 (824  H₂O)⁺, 780 (824  CH₂CH₂  O)⁺, 713 (824  111)⁺, 695 (824  111  H₂O)⁺, 669 (824  111  CH₂CH₂  O)⁺, 651 (824  111  H₂O  CH₂CH₂  O)⁺, which confirms that ethylene and oxygen may be lost, giving rise to enhanced conjugation in this moiety of the molecule. The loss of oxygen and of oxygen and ethylene from the FK-506 sodium adduct was not observed, and the peculiar fragmentation pathways observed for the new FK-506 metabolite are to be attributed to the close proximity of the FK-506 C₁₉-C₂₀ epoxide and the C₃₆-C₃₇ double bond.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Electrospray ms-ms spectrum of m/z = 824.

The in vitro immunosuppressive activity (fig. 9) of this FK-506 new metabolite was similar to the immunosuppressive potency of CyA and of IMM-125 and weakened compared with FK-506. In order to determine whether the epoxide was stable under the conditions used for cell cultures, we incubated the FK-506 new metabolite (50 µg) for 6 days in MLR cultures. The supernatant from the incubation mixture was extracted with dicloromethane, and the organic phase was evaporated to dryness. An acetonitrile solution of the residue was injected on a reverse-phase HPLC column, and a small HPLC peak not found in the blank was collected between 13.5 and 14.5 min. The electrospray mass spectrum (ESI-ms) of this compound (fig. 10) revealed largely the same main fragmentation ion m/z = 812 as the one found in the ESI-ms of the FK-506 29-30 epoxide (fig. 3), which indicates that the compound extracted from the MLR cultures was not modified. In a second experiment that was performed with 50 µg of the 29-30 epoxide, the fragmentation ion of mass m/z = 812 was still present, but another fragment ion appeared at m/z = 826; it probably resulted from the direct loss of oxygen from the epoxide (M-O)Na⁺, and it confirms that the compound extracted from the MLR cultures supernatant was not modified. This metabolite induces a ±80% MLR inhibition in a range of concentrations between 5000 and 100 ng/ml. A weak direct cytotoxic effect (<30% cell death) is observed only at the highest concentration (2500 and 5000 ng/ml), which shows that the MLR inhibition cannot be due to a toxic effect (fig. 11).

View larger version (14K): [in this window] [in a new window]   Fig. 9.   In vitro immunosuppressive activity of the FK-506 C19-C20 epoxide metabolite.

View larger version (8K): [in this window] [in a new window]   Fig. 10.   Electrospray mass spectrum of the compound extracted from the MLR cultures.

View larger version (13K): [in this window] [in a new window]   Fig. 11.   Effect of FK-506, metabolite and diluent on cell viability.

	Discussion

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It is generally considered that FK-506 has a binding domain that interacts with FKBP-12 and an effector element that can interact with a second protein (Morris, 1994). In the currently accepted model, the complex of the immunosuppressive agent with its cognate protein inhibits cytoplasmic signal transduction. These complexes possess immunosuppressive activity through their ability to interact with another target, the abundant serine-threonine phosphatase calcineurin, which is inhibited by the immunophilin drug complexes at nanomolar concentrations.

Certain metabolites of FK-506 (Iwasaki et al., 1993; Iwasaki et al., 1995) were described as possessing in vitro immunosuppressive activity comparable to that of FK-506. In contrast, the C₃₁-O-desmethyl FK-506 metabolite and others such as the C₁₃, C₁₅ and C₁₃, C₁₅-O-desmethyl metabolites were reported to possess low or negligible immunosuppressive potency. The C₃₁-O-desmethyl FK-506 metabolite retained its immunosuppressive activity; the metabolic transformation did not occur either at the FK-506 binding domain or at the effector sector so that the structure of the FKBP-12-FK-506-calcineurin complex was not greatly modified. This was not the case for the FK-506-O-desmethyl metabolites, where ring-chain tautomerism effects were described (Iwasaki et al., 1995; Lhoëst et al., 1995) that probably affected the conformation not only of the FK-506 binding domain but also of the overall complexes, including the binding proteins.

We found that the FK-506 19-20 epoxide new metabolite exhibits reduced in vitro immunosuppressive activity compared with FK-506 and more or less the same immunosuppressive potency as CsA and IMM-125. Because metabolism occurred at the effector site of the FK-506 molecule, the structural changes at the FK-506 effector site may be important enough to decrease somewhat the immunosuppressive potency of this metabolite. We were able to demonstrate that this metabolite was stable under the conditions used for cell cultures. We also found that a direct cytotoxic effect (<30% cell death) was observed at concentrations of 2500 and 5000 ng/ml and that, consequently, the MLR inhibitions were not due to toxic effects.