Introduction

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Losoxantrone, 7-hydroxy-2-[2-[(2-hydroxyethyl)amino]ethyl]-5-[[2-[(2-hydroxyethyl)ami-no]ethyl]amino]-anthra[1,9-cd]pyrazol-6(2H)-one dihydrochloride (CI-941; DuP-941¹; biantrazole), is a member of the novel class of anthrapyrazole anticancer agents. This cytostatic compound is derived from anthracyclines (e.g., doxorubicin and daunorubicin) and mitoxantrone by chromophore modification of the anthracene-9,10-dione unit. In clinical trials (phase I and II), losoxantrone has been shown to be the most powerful anthrapyrazole. It was developed with the intention of diminishing the cardiotoxicity associated with anthracene-9,10-dione drugs. The cardiotoxicity represents the dosis-limiting side effect and is induced by free radicals created during the biotransformation of anthraquinonoid antitumor agents (Murdock et al., 1979; Smith, 1983; Zee-Cheng and Cheng, 1983) (Fig. 1).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Chemical structures of anthrapyrazoles CI-937, CI-941 1, PD111707, and the anthracenedione derivative mitoxantrone.

Both the synthetic preparation and the exceptional in vivo anticancer activity of losoxantrone are well known (Lown, 1983; Showalter et al., 1987; Reszka et al., 1988; Beylin et al., 1989; Fry, 1991; Calvert et al., 1994; Zhang et al., 1994). Several clinical studies of combination regimens with losoxantrone and paclitaxel in patients with advanced breast cancer were recently completed (Kaufman et al., 1998, 1999; Diab et al., 1999). Furthermore, the results of a phase II clinical trail in hormone-refractory metastatic prostate cancer were published (Huan et al., 2000).

The antitumor activity of losoxantrone, among other biochemical effects against tumor cells, is based on inhibiting the enzyme topoisomerase II (Fry, 1991; Leteurtre et al., 1994). Although numerous studies analyzing the pharmacokinetic behavior of the drug under various conditions were published, the metabolism of anthrapyrazole CI-941 is almost unknown until now. Only a small part of the CI-941 biotransformation, proved by metabolites in human urine (Blanz et al., 1993; Richards and Sun, 1995) and feces (Joshi et al., 2001), was elucidated. Further details about the metabolic pathway do not exist, such as the facility of participation of the CI-941 chromophore in oxidative or reductive biotransformation. The redox behavior of anthrapyrazole CI-941 was analyzed only under nonphysiological conditions [e.g., in studies using electrochemical methods (Anne and Moiroux, 1989) or pulse radiolysis (Graham et al., 1987)]. For a one-electron reduction step, the redox potential (E, 538 ± 10 mV) is very similar to that of mitoxantrone (E, 527 mV). However, radical anions of CI-941 caused by pulse radiolysis or cytochrome P450 reductase are unstable to air. If electron spin resonance spectroscopy is used for detection, these radicals are not proven in in vitro experiments (Anne and Moiroux, 1989). Consequently, losoxantrone is not a substrate for a one-electron reduction. Furthermore, for a two-electron reduction step, the electrochemical potential of CI-941 was shown to be much higher than the potential of mitoxantrone.

Using primary cultures of rat hepatocytes as in vitro model, our study describes an extensive metabolism of losoxantrone and elucidates the biotransformation of its chromophore. Additionally, the behavior of the CI-941 metabolism in human hepatoma (HepG2) cells was investigated.

The structures of the metabolites were analyzed by mass spectrometry using online coupled LC-MS, and two-dimensional NMR spectroscopy. Furthermore, the progress of the concentration ratios of the metabolites depending on the incubated starting concentration of CI-941 was analyzed.

Experimental Procedures

Reagents and Materials. N,N-Dimethylformamide (99.9+%, HPLC grade), ammonium trifluoroacetate, trifluoroacetic acid, and ammonium acetate were purchased from Sigma-Aldrich (Steinheim, Germany). Acetonitrile, methanol (LiChrosolv, gradient grade), ascorbic acid, and hydrogen peroxide were obtained from Merck (Darmstadt, Germany). The horseradish peroxidase (E.C. 1.11.1.7, type VI) was obtained from Boehringer Mannheim (Mannheim, Germany) and glutathione from Sigma (Deisenhofen, Germany). Losoxantrone was supplied by DuPont (Bad Homburg, Germany). PRP-1 resin (12-20 µm) was obtained from Hamilton (Darmstadt, Germany) and Sephadex LH20 from Amersham Biosciences AB (Uppsala, Sweden). HPLC-grade deionized water was generated using a Milli-Q-Plus PF water purification system (Millipore, Eschborn, Germany) for preparations of HPLC mobile phase and sample solutions. A centrifuge model 5415C (Eppendorf, Hamburg, Germany) was used for sample pretreatment.

HPLC Instrumentation. Chromatography was performed using Spectra-Physics equipment (Thermo Quest, Egelsbach, Germany). The system consisted of a SCM 400 vacuum membrane degasser, a SP8800 HPLC pump, and a Spectra-Focus fast-scanning LC spectrometer. Multispectral detection was performed in high-speed scanning mode, with a step width of 5 nm and a spectral range of 370 to 790 nm. For sample injection, a Gilson sample injector with a 2-ml sample loop and a Gilson dilutor model 401 equipped with a 5-ml syringe were used (Abimed, Langenfeld, Germany). Acquired data were processed by the Spectra-Focus software (Spectra-Physics). Separations were performed on Superspher 60 RP-select B (4 µm) HPLC cartridges (250 × 4 mm, LiChroCart system, Merck) protected with a LiChrospher 60 RP-select B (5 µm, 4 × 4 mm; Merck) guard column. Preparative amounts were separated on a glass column (300 × 10 mm; Superformance system, E.; Merck) filled with PRP-1 resin using the Spectra Physics system equipped with an EC6W switching valve (VICI, Schenkon, Switzerland) instead of a Gilson sample injector. Compounds were eluted with a gradient system described later.

Chromatographic Conditions. Analyses were performed using different gradient systems; the chromatographic conditions were optimized according to analytical and preparative demands. The solvent system consisted of 10 mM aqueous ammonium trifluoroacetate (adjusted to pH 2.2 by the addition of 500 µl/l of trifluoroacetic acid) (solvent A), acetonitrile (solvent B), and N,N-dimethylformamide (solvent C). Since the analytes were variously concentrated in different mediums (e.g., urine and cell extract), the gradient of this eluent system was adapted to the conditions for optimizing the separations. The next run was started after 15 min of equilibration. The flow rate was maintained at 1 and 2 ml/min in case of semipreparative separations. The following gradients were used: gradient I: start conditions (85% A/5% B/10% C, v/v/v), 0 to 10.0 min (75:15:10, v/v/v), 10.0 to 15.0 min (75:15:10, v/v/v), 15.0 to 21.0 min (30:60:10, v/v/v), 21.0 to 24.0 min (85:5:10, v/v/v); gradient II: 0 to 20.0 min (76:14:10, v/v/v), 20.0 to 25.0 min (30:60:10, v/v/v), 25.0 to 30.0 min (30:60:10, v/v/v), 30.0 to 35.0 min (76:14:10, v/v/v); gradient III: 0 to 5.0 min (85:5:10, v/v/v), 5.0 to 15.0 min (75:15:10, v/v/v), 15.0 to 21.0 min (30:60:10, v/v/v), 21.0 to 35.0 min (30:60:10, v/v/v), 35.0 to 45.0 min (85:5:10, v/v/v).

Mass Spectrometry. Mass spectrometric analyses were conducted using ionspray ionization on an API III triple quadrupole mass spectrometer (Sciex, Thornhill, ON, Canada) online coupled to the HPLC system. The LC-MS investigations were carried out using an isocratic solvent system that consisted of 10 mM aqueous ammonium trifluoroacetate, pH 2.2, acetonitrile, and N,N-dimethylformamide (80:10:10, v/v/v). The HPLC solvent flow (1 ml/min) was split up by use of a Milli-Mite needle valve (Series 1300; Hoke, Frankfurt, Germany) into a capillary flow of ~5 µl/min. To avoid a large dead volume, the valve was used to control the flow through one outlet of a stainless steel tee union placed directly after the outlet of the spectrometer. The third outlet was coupled via a fused-silica capillary (100-µm inner diameter, 30-cm length) to the mass spectrometer. This arrangement allowed the detection of a compound with a time delay of only 1 to 2 s between the registration of the light absorption of the compound and the detection of the quasi-molecular ions of the same compound in the mass spectrometer. The operating parameters for the measurements were as follows: ionspray voltage, 4800 V; orifice voltage, 70 V. Mass spectral data were acquired and processed with a Sciex MacSpec data system.

Nuclear Magnetic Resonance Spectroscopy. High-resolution NMR spectra were obtained on a Bruker AMX-400 spectrometer (Bruker Physics, Karlsruhe, Germany) interfaced to a X32 computer and equipped with an inverse triple-resonance probe. The two-dimensional data set consisted of homonuclear total correlation (TOCSY) spectra, inverse-detected heteronuclear multiple-quantum correlation (HMQC) spectra, and heteronuclear multiple-bond correlation (HMBC) spectra (Bax et al., 1983) optimized for long-range coupling (ⁿJ(CH) = 7 Hz). The sweep width in the F2-dimension was 4000 Hz; chemical shifts were referenced to the solvent peak. The inverse-detected HMQC incorporated a bilinear rotation decoupling pulse sequence to suppress the signals of protons bound to ¹²C. During acquisition, proton decoupling was achieved with a globally optimized alternating-phase rectangular pulse composite pulse sequence. The HMBC experiment was acquired without decoupling and without suppression of direct ¹J(CH) correlations. Data processing consisted of zero filling up to 2 kilobytes (F2) and 1 kilobyte in the F1-dimension. A squared sine-bell-shaped weighting function shifted by /3 and a baseline correction was applied in both dimensions.

Rat Hepatocytes. Microscopic observation of the cells during incubations with CI-941 1 showed a rapid uptake and subsequent compartmentalization of CI-941 into vesicles. Analogous behavior was reported for moxantrazole CI-937 (Renner et al., 1995).

Incubations with Rat Hepatocytes.

Analytical scale Male Sprague-Dawley rats (220-280 g) were purchased from Interfauna (Tuttlingen, Germany). All animals received a standard diet (Alma H 1003; Botzenhardt, Kempten, Germany) and water ad libitum. Isolated hepatocytes were prepared by collagenase perfusion according to Seglen (1976). A modification of the isolation procedure reported by Gebhard et al. (1990) was applied. The viability of the cells assessed by trypan blue exclusion was greater than 85%. Isolated hepatocytes were resuspended in Williams' medium E supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 µg/ml), dexamethasone (0.1 µM), and newborn calf serum (Sebio, Walchsing, Germany) at a density of 1.25 × 10⁶ cells/ml. The cells were plated in collagen-coated six-well plastic dishes with a 35-mm diameter (Greiner, Nürtingen, Germany). Each well was inoculated with 1.25 × 10⁶ cells. At the end of a 2-h exposure period of incubation in an atmosphere of 5% CO₂/95% air at 37°C during which cells were attached to the plastic dishes, the medium and the dead cells were removed. The hepatocytes were covered with 1 ml of incubation medium containing CI-941 1. The incubation medium consisted of an aqueous solution of 137 mM NaCl, 5.5 mM KCl, 1.2 mM MgSO₄ · 7 H₂O, 0.79 mM Na₂HPO₄ · 2 H₂O, 0.1 mM KH₂PO₄, 10 mM Hepes, and 5 mM glucose, pH 7.4. All experiments were performed in triplicate. After the incubation period (20 h; 37°C in air), the cells were scraped off and transferred into a 2-ml plastic cup (Eppendorf). Additionally, the wells were washed with 100 µl of 1 M ascorbic acid to ensure complete transfer of the cells and to prevent further oxidation. This suspension was sonicated (Sonifier B-12; Branson Sonic, Danbury, CT) for lysis of the cells and then centrifuged at 14,000g for 6 min to remove cell debris.

Semipreparative scale. For the synthesis of the metabolites in a semipreparative scale, isolated hepatocytes were resuspended in a manner identical to the analytical scale (1.25 × 10⁶ cells/ml; see above). To set down the cells, the suspension was incubated for 2 h in an atmosphere of 5% CO₂ and 95% air at 37°C. After removal of the medium, an aqueous solution containing the drug 1, 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄ · 7 H₂O, 1.2 mM KH₂PO₄, 25.0 mM NaHCO₃, 0.05 mM glucose, and 1.0 mM CaCl₂, pH 7.4, was added resulting in a hepatocyte density of 1.25 × 10⁶ cells/ml. Six milliliters of this cell suspension were added to collagen-coated plastic dishes with a 90-mm diameter (Greiner). Consequently, each dish was inoculated with 7.5 × 10⁶ cells. Each series consisted of 35 dishes and was performed five times.

Incubations with Human Hepatoblastoma Cells (HepG2).

Cultivation of HepG2 cells HepG2 hepatoblastoma cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Eggenstein, Germany) supplemented with 2 mM glutamine, 10% fetal calf serum, 40 U/ml streptomycin, and 50 U/ml penicillin, as described elsewhere (Fahrner et al., 1993). They were passaged every week at the time they reached confluence. Stocks were kept frozen in liquid nitrogen. Frozen cells were thawed, cultured for 1 week, and passaged before use. For the experiments described here, Dulbecco's modified Eagle's medium was replaced by Williams' medium E to provide conditions comparable to primary hepatocytes.

Incubation of losoxantrone. Losoxantrone was added in a concentration ranging from 10 to 50 µM. Each concentration was incubated in a humidified atmosphere of 95% air and 5% CO₂ at 37°C for 24 h in triplicate. The following work-up was analogous to the sample preparation used for rat hepatocytes. The series of experiments was repeated twice, independently from each other. During the sample preparation for a HPLC analysis (100-µl injection volume), the intra-and extracellular area of the hepatoma cells were not differentiated.

Isolation and Purification of Metabolites.

Analytical scale Freshly prepared primary cultures of rat hepatocytes were incubated with losoxantrone for 24 h at 37°C, with each drug-concentration in triplicate. In a range of concentrations between 10 and 100 µM, losoxantrone was dissolved in sterilized transport medium. 1.25 × 10⁶ cells each received 1 ml of a CI-941 solution and were incubated immediately. After the incubation, the cells were isolated together with the solution surrounding the hepatocytes.

Semipreparative scale. The first chromatographic purification step was performed on a glass column (400 × 35 mm) filled with Sephadex LH 20 using an isocratic eluent system, water/methanol (7:3, v/v). The fraction containing the reaction products was concentrated to approximately 5 ml. Further purification was achieved by semipreparative HPLC (see gradient I). After evaporation to a volume of about 3 ml, the buffer salts in the fractions were removed via chromatography on Sephadex LH 20 (see above). Finally, the solutions were lyophilized after vacuum evaporation of the organic modifier.

Quantitative analysis. After separation of the cell debris, each supernatant was isolated and weighed to calculate the individual loss of water evaporated during the 24-h incubation of the drug solution in six-well plastic dishes. To normalize to the CI-941 starting concentration, the supernatants were corrected by a factor resulting from the weight difference before and after incubation. By incubating 1 ml of different CI-941 concentrations in the same cell density throughout, we assumed a specific gravity of 1 g/ml for each solution at the beginning of incubation. Consequently, the concentrations of nonmetabolized losoxantrone and its metabolites determined by HPLC were corrected. Hereby, the metabolism of 1 could be compared with the conditions at the starting point of each experiment.

	Results

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For the chromatographic separation of losoxantrone and its metabolites, a new method was developed. With respect to the suitability of drug analyses for direct coupling of the HPLC system to a mass spectrometer, a volatile buffer salt was used. Because the separation of strong basic compounds like losoxantrone is most efficient at low pH values, an ammonium trifluoroacetate-trifluoroacetic acid buffer was chosen. The organic modifier used was acetonitrile. Good separation was achieved in the presence of buffer salt; however, simple acidification to pH 2.2 with 500 µl of trifluoroacetic acid/l of water did not effect any separation. After addition of ammonium trifluoroacetate ranging from 10 to 200 mM, the separation was excellent. The variation of the buffer concentration had no detectable influence on the separation quality. However, the addition of 10% N,N-dimethylformamide as a third component significantly improved the peak shape of late-eluting compounds. Tests with aminoalkylamino-substituted anthracene-9,10-diones and structurally related anthrapyrazoles revealed that this solvent system can be used universally for the separation of such compounds. Furthermore, it could be demonstrated that this solvent is compatible with thermospray ionization, continuous-flow fast atom bombardment, and ionspray ionization. These ionization methods are most frequently used for HPLC-MS coupling.

Biotransformation of Losoxantrone in Primary Cultures of Rat Hepatocytes. During the metabolism in rat hepatocytes, CI-941 derivatives are created in very different amounts. However, the amounts of metabolites produced by cell colonies in six-well plates were still sufficient for both HPLC and mass spectrometric analyses (direct injections and online LC-MS couplings). The estimated amount of losoxantrone metabolized within 24 h was nearly 20%. When analyzing cell lysates directly without any drug extraction, the detection limit of CI-941 1 was 150 pg/100 µl.

View larger version (42K): [in this window] [in a new window]   Fig. 2.   Top, UV/VIS spectra of 1, 2, and 3; bathochromic shift of the metabolites 2 and 3 after hydroxylation of the phenolic substructure of 1; bottom, UV/VIS data of CI-941 1 and its metabolites 2 to 13.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Top, HPLC chromatogram of CI-941 1, its metabolites (2 to 13), and phenol red contained in cell medium (max, 430 nm; Ind) (a 24-h incubation); middle, corresponding RIC for the quasi-molecule ion [M + H]+ (m/z 442); bottom, isolated metabolite 2 spiked with PD111707.

Biotransformation of the chromophore of CI-941 1. In rat hepatocytes, two metabolites (2 and 3), containing an oxidized anthraquinonoid chromophore, were identified by LC-MS analysis. From selected ion monitoring [reconstructed ion current (RIC); Fig. 3], metabolites 2 (retention time, 11.7 min) and 3 (retention time, 14.6 min) can be identified as hydroxylation products of losoxantrone.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   MS/MS mass spectrum of CI-941 1 (quasi-molecule ion [M + H]+; m/z 426) and the metabolite 2 ([M + H]+; m/z 442).

View larger version (42K): [in this window] [in a new window]   Fig. 5.   Fragmentation pattern of metabolites 2, 4, 5, and 8.

Oxidative activation of the metabolite 2. The biotransformation of 1 into the hydroquinonoid hydroxylation products 2 and 3 enables the formation of the corresponding quinonoid products by oxidative activation catalyzed by cytochrome P450. These intermediates are electrophilic species able to react with nucleophilic substrates (e.g., glutathione) according to a Michael addition. In fact, the elucidation of metabolite 10 (Fig. 6) confirmed this reaction behavior. The compound is represented by the chromatographic peak at 10.9 min. The molecular mass derived from the ionspray mass spectrum shows the formation of a monoglutathione conjugate of 2. However, mass spectrometry fails in defining the site of substitution in the molecule (see below). The predominant cleavage of the -glutamyl peptide bonding yielding [M + H  129]⁺ ions at m/z 618, which can be observed in the daughter ion mass spectrum, is typical for glutathione conjugates. The pronounced fragment ion at m/z 472 may be formed by fission of the sulfur-carbon bond in the glutathionyl substituent and transfer of a phenolic hydrogen to the eliminated neutral. The same CS-bond fission associated with transfer of a hydrogen atom from the eliminated neutral to sulfur results in the formation of the ion at m/z 474 ("reduced form"). By comparison with results derived from CI-937, the detection of the fragment ions, m/z 472 and 474 suggests a substitution pattern of the glutathionyl group at position C-9 in the ortho-hydroquinonoid ring subsystem of 2. Analogous fragment ions could be detected during tandem mass analyses of glutathione conjugates of CI-937 (Renner et al., 1995).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Glutathione conjugate 10 of metabolite 2 and CID fragments at m/z 474 and 472.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   RIC of CI-941 1, metabolites 2 to 8, and 10 to 13.

Ether and ester conjugates of metabolite 2 coupled with phenolic substituents. In addition to the detection of the 8-hydroxy-derivative 2, a series of metabolites, 4 to 8, derived from metabolite 2 were identified.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   MS/MS mass spectrum of metabolite 4 (quasi-molecular ion [M + H]+; m/z 618).

View larger version (21K): [in this window] [in a new window]   Fig. 9.   Ionspray tandem mass spectrum of 5 (quasi-molecular ion [M + H]+; m/z 522).

View larger version (10K): [in this window] [in a new window]   Fig. 10.   Proposed molecule structures of 6 and 7.

Enzymatic conversion of metabolite 4 and 5 catalyzed by -glucuronidase/arylsulfatase. The cleavage of the glycosidic bond catalyzed by -glucuronidase converted the metabolite 4 into the aglycon 2. The product of this in vitro reaction was verified chromatographically and compared with HPLC analyses of solutions containing this hydrolyzed product spiked with compound 2. Using arylsulfatase to hydrolyze an ester bond, compound 2 was also formed after an enzymatic conversion of metabolite 5.

Metabolites resulting from hydroxylation of the chromophore and side-chain oxidation. The MS/MS investigation of peaks 8 and 9 in the HPLC chromatogram (Fig. 3, top) revealed that these metabolites result from hydroxylation of the chromophore of 1 and oxidation of one of the two side chains. The fragmentation of the [M + H]⁺ ion of 8 (m/z 456) is illustrated in Fig. 5. The typical fragmentations of the quasi-molecular ions of 1 and 2 comprise the elimination of CH₂=CH-NH-CH₂-CH₂OH and H₂N-CH₂-CH₂OH. These dissociation are replaced by loss of CH₂=CH-NH-CH₂-COOH (m/z 355) and H₂N-CH₂COOH (m/z 381) in the daughter ion spectrum of 8. Furthermore, fragments at m/z 268, 294, and 308 originating from substantial side-chain cleavage are found in the MS/MS spectra of both compounds 2 and 8. The position of the oxidized side chain cannot be derived from these data; therefore, both, structures 8a and 8b could represent this metabolite (mass spectrum not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 11.   Daugther ion mass spectrum of metabolite 9.

Compounds derived by oxidation of the terminal carbinol group in one or both side chains of 1. The metabolites responsible for peaks 11, 12, and 13 of the chromatographic trace (Fig. 3, top) exhibit identical UV/VIS absorption (Fig. 2) when compared with losoxantrone. Thus, structural changes to the chromophoric unit are not associated with their formation. Online HPLC-MS showed that the three metabolites are formed by oxidation of the losoxantrone side chains. The electrospray mass spectrum of 11 (Fig. 12) exhibits a [M + H]⁺ ion at m/z 424 in accordance with the oxidation of one of the two terminal carbinol functionalities to an aldehyde group. Because the location of the aldehyde function cannot be established, both structural representatives (11a and 11b) are equally probable. Metabolite 12 results from further oxidation of 11 to a monocarboxylic acid ([M + H]⁺ at m/z 440). As shown in Fig. 12, the fragmentation pattern of metabolite 12 parallels the breakdown of the quasi-molecular ion of 11 in the formation of ions at m/z 252, 278, and 292. These ions are also found in the MS/MS spectrum of losoxantrone, again pointing to the fact that the anthrapyrazole ring system is not affected in the biotransformation of 1 into 11 and 12. These fragment ions also occur in the MS/MS spectrum of the biotransformation product 13, which is the dicarboxylic acid of losoxantrone showing a quasi-molecular ion at m/z 454. The retention times in the chromatographic separation of 1 and 11 to 13 are close together; however, the daughter ion spectra of each compound has been recorded by MS/MS separation of the individual quasi-molecular ions (Fig. 13).

View larger version (15K): [in this window] [in a new window]   Fig. 12.   MS/MS spectrum of metabolites 11 to 13 (quasi-molecule ions [M + H]+; m/z 424, 440, and 454).

View larger version (18K): [in this window] [in a new window]   Fig. 13.   RIC of the metabolites 11 to 13 compared with nonmetabolized CI-941 1.

Structural elucidation of the glucuronic acid conjugate 4 by ¹H NMR spectroscopy. The determination of the substitution pattern of the conjugate 4 necessitates the reliable assignment of the resonances of all positions of the anthrapyrazole skeletons of CI-941 1 and compound PD111707 (identical with metabolite 3). Complete assignments of ¹H and ¹³C resonances of 1 and PD111707 were achieved by the combination of inverse-detected ¹H/¹³C heteronuclear NMR analyses (HMQC and HMBC; spectra not shown). The following ¹H/¹H TOCSY spectrum of PD111707 allowed a complete ¹H assignment (Fig. 14).

View larger version (30K): [in this window] [in a new window]   Fig. 14.   1H/1H TOCSY-NMR spectrum of PD111707 (solvent, CD3CN).

View larger version (21K): [in this window] [in a new window]   Fig. 15.   The range of the aryl protons of metabolite 4 (1H/1H COSY-NMR spectrum; solvent, D2O).

The Extent of the CI-941 Metabolism. The correlation of metabolism on losoxantrone-starting concentration was examined using freshly cultivated rat hepatocytes. For each incubation series (n = 5), 1 ml of a solution containing CI-941 in the range of 10 to 100 µM was added to 1.25 × 10⁶ cells each and incubated for 22 h. Within the incubation series each losoxantrone concentration was incubated in duplicate. The cells were immediately extracted and analyzed via HPLC.

Biotransformation in Human Hepatoma Cells (HepG2). When losoxantrone was incubated with HepG2 cells, the analysis by HPLC demonstrated an unambiguous behavior of the cells in metabolism (chromatogram not shown). Biotransformation of losoxantrone was not found. Only amounts of nonmetabolized CI-941 1 were detected in the lysate and the cell debris, respectively. Neither metabolites resulting from oxidation of the terminal carbinol group in a side chain of CI-941 1 nor metabolites derived by an ortho- or para-hydroxylation at aromatic carbon atom C-8 or C-10, respectively, was found in the HepG2 cells. Also, no follow-up compounds, which might be formed via an oxidative activation of the hydroxylated chromophore, were found.

Cytotoxicity in HepG2 Cells. The cytotoxicity of anthrapyrazole was determined using leakage of lactate dehydrogenase, according to the procedure described by Gebhardt and Fausel (2000). The determination of the cytotoxic effect on human hepatoma (HepG2) cells revealed that drug concentrations below 100 µM had no toxic influence in vitro, but a concentration of 400 µM resulted in necrosis of approximately 50% of the cells (data not shown).

Inhibition of the Cytochrome P450 in Rat Hepatocytes. The inhibition of the cytochrome P450 system by using 2-methyl-1,2-[3-dipyridyl]-propan-1-one (metyrapone) has often been described. The interaction of this propanone derivative with the reduced and oxidized form of the P450 enzyme system prevents its enzymatic activity.

	Discussion

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Until now, the biotransformation of losoxantrone (CI-941; 1) has widely been unknown. In spite of a generally low metabolic rate, this article describes a rather ramified metabolism of 1 found in rat hepatocytes.

CI-941 1 shows some structural similarities to mitoxantrone and the anthrapyrazole CI-937, the metabolic pathways of which have been studied by our group (Mewes, 1993; Renner et al., 1995). The main difference between 1 and mitoxantrone and CI-937 is the lack of the second hydroxy group in ring A (Fig. 1). The presence of two para-oriented hydroxy groups is a prerequisite of the immediate oxidative activation of mitoxantrone and CI-937, resulting in glutathionyl conjugates and, in the case of mitoxantrone, in an additional intramolecular cyclization product. Anthraquinonoid drugs presenting a para-hydroquinonoid substructure show an increase in cytotoxicity (Werbel et al., 1987), which could be caused by oxidative activation. Although the contribution of oxidative activation to the anticancer activity of the drugs remains to be determined, it was of interest to investigate the behavior of CI-941 lacking the second OH group.

With primary cultures of rat hepatocytes as in vitro model, it could be shown that the oxidative metabolic pathway of losoxantrone is initiated by hydroxylation in the ortho- and para-position relative to the phenolic group in the CI-941 chromophore. This can be regarded as the key step for an extensive follow-up biotransformation. The ortho- and para-regioisomers 2 and 3 are detected in the ratio 10:1. This ratio does not consider secondary products such as 4 and 5 derived from 2.

The glucuronic acid derivative 4 could be enriched and purified to such an extent that a detailed structural analysis by ¹H NMR spectroscopy could be achieved. In particular, the ortho-substitution of ring A has been proven. Thus, the enzymatic cleavage of 4 into 2 indirectly furnishes evidence for the ortho-connectivity of the two OH groups in 2.

Similar to those observed in 2, bathochromic shifts for the UV/VIS absorption are found in metabolites obtained by hydroxylation of the chromophoric unit followed by oxidation of the side chains (8 and 9) or sulfation (5). This and the predominant formation of 2 in comparison with 3 can be taken as an argument to consider these metabolites as derivatives of 2. Furthermore, in the case of 5, enzymatic hydrolysis produced the primary metabolite 2.

Analogous to mitoxantrone and CI-937 (Mewes et al., 1993; Renner et al., 1995), the hydroxylation product 2 is metabolized to a glutathione conjugate 10. Its formation can be rationalized by oxidative activation yielding an intermediate ortho-quinone (Fig. 16). The strong electrophilic character of this intermediate enables the Michael addition of the nucleophilic glutathione. In principle, two regioisomeric structures are possible for the monoglutathione conjugate. Although 10a would be the result of a 1,6-addition, the isomeric 10b would be formed in a 1,4-addition.

View larger version (16K): [in this window] [in a new window]   Fig. 16.   Oxidative activation of 2 and a following Michael addition of glutathione.

The bathochromic shift of the _max value in the UV/VIS spectrum of 10 (_max, 501 ± 2 nm) is by far lower than the shifts found in the two monoglutathione conjugates of CI-937 (_max, 510 ± 2 and 512 ± 2 nm) (Fig. 16). The two hydroxy groups of CI-937 have the same para-orientation as in the losoxantrone metabolite 3. In fact, CI-937 and 3 differ only in the length of one side chain. This small structural change has no influence on the UV/VIS characteristics (_max, 507 ± 2 nm for CI-937 and 509 ± 2 nm for 3). The marked difference in the _max value of 10 and the _max values of the monoglutathione conjugates of CI-937 can be taken as evidence for 10 to be derived from the major hydroxylation product 2 rather than from 3. The corresponding glutathione conjugates of 3 could not be detected even after substantial enrichment of the cell extracts.

In addition to intercalation and inhibition of topoisomerase II, the observation of the monoglutathione conjugate 10, produced from an electrophilic intermediate after hydroxylation and oxidative activation by reaction with the nucleophilic glutathione, gives rise to speculations on a biochemical effect. In this case, losoxantrone would behave as a prodrug, which after structural modification would covalently bind to nucleophilic centers of biomolecules.

From the experimental results, it follows that losoxantrone undergoes oxidative biotransformations in rat hepatocytes. To confirm this to be the effect of the catalytic action of cytochrome P450, this system was inhibited by the addition of metyrapone. Under these conditions, the metabolism of the anthrapyrazole 1 in primary cultures of rat hepatocytes broke down completely. In contrast to the results of incubation experiments with rat hepatocytes, similar studies with human HepG2 cells did not show any biotransformation of CI-941 1. In the case of mitoxantrone, a thioether conjugate could be detected in small yields (Mewes, 1993; Mewes et al., 1993). Obviously, the P450 monooxygenase of human hepatoma cells is not able to initiate the hydroxylation of losoxantrone, which would be essential to produce an electrophilic intermediate required for a glutathione conjugation. Thus, in such tumor cells, any cytotoxic effect via oxidative activation of hydroxylated CI-941 is not likely.