Introduction

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Taxoids are natural compounds isolated from the bark or leaves of yew trees and screened for their antiproliferative properties. Taxol or paclitaxel was initially selected on the basis of its cytotoxic properties upon various cell lines and tumor-bearing mice; its biological action was attributed to the capacity to bind tubulin and to stabilize microtubules, thus modifying the cytoskeleton architecture and blocking the cell cycle in phase G2/M (Wani et al., 1971; Schiff et al., 1979). However, the limited availability of paclitaxel led to the development of analogs, which retained the biological activity on tubulin assembly but were readily synthesized in large quantities. This led to the discovery of docetaxel (Taxotere) a semisynthetic compound derived from 10-deacetylbaccatin III with an affinity for microtubules 2 times higher than paclitaxel (Ringel and Horwitz, 1991). Paclitaxel and docetaxel are widely used with success in patients with various tumors, including ovarian or breast carcinomas resistant to conventional chemotherapy (Rowinski et al., 1992).

The therapeutic efficiency of taxoids is the result of the balance between their biological action on tubulin and rate of degradation. The elimination of paclitaxel and docetaxel from the body occurs mainly through the biliary route. Five metabolites are isolated from the bile of a patient given paclitaxel and three are hydroxylated (Monsarrat et al., 1993). The complete chemical structure of these metabolites has been established by mass spectrometry and NMR spectrometry (Monsarrat et al., 1993, 1998; Harris et al., 1994a; Monegier et al., 1994; Royer et al., 1995); the major metabolite formed is a monohydroxylated derivative at C6 of the taxane ring not present in rat bile (Monsarrat et al., 1990). Hydroxylated product on the phenyl at C3' is also observed in human bile, as well as the dihydroxylated molecule at C3' of the lateral chain and C6 of the taxane ring. Two distinct monooxygenases are involved in the hydroxylation of paclitaxel in the human liver; the formation of the C3'-derivative is clearly assigned to CYP3A4, whereas the 6-hydroxylated derivative is the major metabolite produced by a CYP2C protein identified as CYP2C8 (Cresteil et al., 1994; Harris et al., 1994b; Rahman et al., 1994).

In contrast with paclitaxel, the biotransformation of docetaxel is similar in animals and humans (Vuilhorgne et al., 1995). The chemical structure of purified metabolites was elucidated by mass spectrometry and NMR spectrometry and demonstrates that the metabolism of docetaxel involves an initial oxidation on the tert-butyl of the lateral side chain before its cyclization, both reactions catalyzed by CYP3A4 (Monegier et al., 1994; Marre et al., 1996; Royer et al., 1996; Shou et al., 1998). These metabolites were also isolated from feces collected from patients given docetaxel (Sparreboom et al., 1996). No hydroxylation on the taxane ring of docetaxel or on the phenyl group of the lateral chain has been reported in in vitro and in vivo studies. Thus, relatively minor modifications on the taxoid molecule have major consequences on the site of oxidation and the identity of the P450¹ isoform implicated in the reaction. Furthermore, the oxidative metabolism of paclitaxel and docetaxel strikingly reduced their biological activity. Thus, the 6-hydroxypaclitaxel is more than 30-fold less potent than paclitaxel in the in vitro inhibition of cell proliferation (Harris et al., 1994a; Kumar et al., 1995; Sparreboom et al., 1995). Similarly, the hydroxylated metabolite of docetaxel shows poor in vitro cytotoxicity in P388 cells and in B16 tumor-bearing mice (Vuilhorgne et al., 1995). Consequently, the biotransformation of taxoids at the hepatic level leads to the inactivation of the active parent molecule and consequently to a reduction of its therapeutic effect.

The purpose of the present study is to investigate the metabolism of paclitaxel and docetaxel analogs to define the structural elements orientating the hydroxylation on the taxoid molecule and determining the kinetic parameters for individual analogs. Since the only structural differences between paclitaxel and docetaxel are the replacement of the phenyl ring of the side chain at C13 by a t-butyl-oxy group and the absence of the acetyl in position 10, we choose to explore separately the role of these modifications on the biotransformation of taxoids by P450 (for structure see Fig. 1). Incubations were conducted with human liver microsomes to assess the overall biotransformation and with recombinant human P450 to ascribe individual reaction to a single P450 isoform.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Chemical structure of taxoids.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. Paclitaxel was kindly provided by Bristol-Myers Squibb Co. (Wallington, CT) and docetaxel by Rhone Poulenc Rorer (Antony, France). 10-Deacetylpaclitaxel, 10-acetyldocetaxel, 10-deacetylbaccatin III, and baccatin III were isolated or synthesized as previously reported (Chauvière et al., 1981; Denis et al., 1988; Mangatal et al., 1989). All chemicals were from the highest commercially available grade.

Microsome Preparation. Human hepatic tissues were collected and microsomes prepared from frozen samples, as stated previously (Cresteil et al., 1979). The total cytochrome P450 content, the protein content, and the concentration of individual P450 isoforms were estimated as reported elsewhere (Cresteil et al., 1985, 1994).

Expression of Recombinant P450. COS-1 cells or Ad293 cells were maintained at 37°C under 5% CO₂ in Dulbecco's modified Eagle's medium supplemented with 4500 mg/l D-glucose, 10% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. The preparation of stable transfectants expressing CYP1A1, 1A2, 3A4, and 3A7 in Ad293 cells was detailed previously (Lacroix et al., 1997; Sonnier and Cresteil, 1998). A similar procedure was developed to generate stable transfectants of CYP2C8, 2C9, and 3A5. Full-length cDNA encoding CYP2C8, 2C9, and 3A5 were kindly provided by Dr. F. J. Gonzalez (NCI, Bethesda). Briefly, CYP3A5 cDNA was removed from pUC9 and 2C8 and 2C9 cDNAs from pUV-1 as EcoRI fragments and inserted in the correct orientation in the EcoRI site of pMT2 originated from the Genetics Institute (Cambridge, MA). Fifty micrograms of P450-pMT2 plasmid was transfected in COS-1 cells for transient expression by the calcium phosphate procedure, as reported by Lacroix et al. (1997). Stable transfectants were obtained by transfection of Ad293 cells with 60 µg of P450-pMT2 and 3 µg of pIBW-3 plasmid containing the neomycin resistance gene. Seventy-two hours after transfection, selection was initiated with geneticin sulfate (1 mg/ml culture medium) for 2 to 3 weeks. Resistant clones derived from single colonies were harvested and grown individually in culture medium containing 50 µg/ml geneticin for 2 to 3 additional weeks. Microsomes were prepared and their content in P450 protein was estimated by Western blot with antibodies purchased from Daiichi Chemicals (Tokyo, Japan) against the rat CYP2C6 and reacting with all human CYP2C proteins, from Daiichi Chemical for CYP3A4/5 or from Affiniti (Exeter, UK) for anti-CYP3A5. Clones selected showed the highest content in P450 protein and activity toward reference compounds; 6-hydroxylation of testosterone for CYP3A5 and dealkylation of methoxy trifluoromethylcoumarin for CYP2C. When tested with antibodies to CYP3A4/5 reacting with the two proteins, comparable intensities were obtained indicating that Ad293-3A4 and Ad293-3A5 expressed similar amount of CYP3A proteins. Immunochemically determined P450 level and activity expressed per milligram of microsomal protein were comparable in stable transfectants and liver microsomes.

Metabolism of Taxoids. Microsomal proteins corresponding to 0.3 nmol of P450 were incubated in a final volume of 1 ml of 50 mM sodium phosphate, pH 7.4, 5 mM MgCl₂, 10% glycerol with a NADPH-generating system consisting in 0.1 mM NADP, 1 mM glucose 6-phosphate. Taxoids (10 mM dissolved in methanol) were added to a final concentration of 50 µM except when otherwise indicated, and the reaction was initiated by the addition of glucose-6-phosphate dehydrogenase. After 30 min at 37°C, the reaction is stopped by the addition of 2.5 ml of ethyl acetate to extract the parent molecule and its metabolites. Extraction is repeated three times; organic phases were pooled and evaporated to dryness under a nitrogen stream. The residues, dissolved in 200 µl of acetonitrile/water (70:30, v/v), were analyzed by HPLC and HPLC/MS. For K_m determinations, taxoid concentrations ranged from 0 to 250 µM; double reciprocal plots allowed the calculation of apparent K_m and V_m. For structural determinations, incubations were carried out for 1 h and were scaled up by 10 to obtain a sufficient amount of metabolites.

Characterization and Quantification of Taxoids Metabolites. Metabolites and parent compounds were separated by reverse-phase HPLC, and peaks corresponding to the different products were quantified. The following HPLC conditions were used: 1) Ultrasphere ODS column, 4.6-mm × 25-cm, C₁₈ (Beckman Coulter, Inc., Fullerton, CA,); 2) isocratic eluent MeOH/H₂O 65:35 for paclitaxel and 10-deacetylpaclitaxel; linear gradient: 40 to 50% H₂O/MeOH, 0 to 50 min for baccatin III and 10-deacetylbaccatin III; linear gradient 30 to 60% H₂O/CH₃CN, 0 to 55 min for docetaxel and acetyldocetaxel; 3) UV detection at 235 nm and a flow rate of 0.8 ml/min. The HPLC system included a Waters (Milford, MA) model equipped with a 600 MS pump and an U6K injector and were linked to a Waters M991 photodiode array for UV detection at 235 nm. To characterize the structure of taxoid metabolites in microsome or cell extracts, we used on-line HPLC/MS with an atmospheric pressure chemical ionization-mass spectrometry interface mode, as previously reported (Royer et al., 1995, 1996). Chromatographic conditions were the same as those described above; microsome and cell extracts were dissolved in 100 µl of a mixture of acetonitrile/water and introduced into the HPLC/MS system (TSQ 700; Thermo Finnigan MAT, San Diego, CA). Mass spectrometry data were acquired continuously in the full-scan mode (m/z 150-1200) at 2 ms/step.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Metabolism of Paclitaxel. The biotransformation of paclitaxel by human liver microsomes has been extensively studied by us and others, and we checked in a first set of experiments the formation of known metabolites in our conditions (Fig. 2 and 3). As expected, two oxidation products were resolved by HPLC from incubation mixtures of liver microsomes with 50 µM paclitaxel and NADPH; the 6-hydroxy and the 3'-phenyl-hydroxy derivatives eluted at 9.5 and 16 min, respectively, in comparison with the parent molecule (20 min). These products were absent from incubations without NADPH. Their structures were explored by HPLC/MS and yielded fragments at m/z 286 (unchanged side chain at C13) and 569 (taxane ring) in the parent paclitaxel molecule shifting to either 302 or 585 after hydroxylation, which is in agreement with the structure already reported in the literature (Royer et al., 1995). The oxidation on the C2 benzoate ring could be excluded based on the shift of fragment from m/z 447 (lateral chain  C2 benzoate) to 463 observed only in material eluting at 9.5 min.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Structure of paclitaxel, 10-deacetylpaclitaxel, docetaxel, and 10-acetyldocetaxel metabolites verified by HPLC/MS.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Chromatogram of incubation of paclitaxel with human liver microsomes. Arrows refer to metabolites formed in the presence of NADPH eluting at 9.5 min (C3'-OH-paclitaxel) and 16 min (6-OH-paclitaxel).

Metabolism of 10-Deacetylpaclitaxel. The HPLC profile of incubation mixture of 10-deacetylpaclitaxel with liver microsomes in the presence of NADPH showed two peaks eluting at 11.5 and 12.5 min, respectively. In addition, the unchanged 10-deacetylpaclitaxel had a retention time of 16 min (Fig. 4). These two peaks were absent from incubations run in absence of NADPH and were identified as oxidation products. HPLC/MS analysis of 10-deacetylpaclitaxel generated a parent ion at m/z 812 and fragments at m/z 527 (intact taxane ring), 405 (taxane ring-C2 benzoate), and 286 (unchanged side chain at C13). The peak eluting at 11.5 min yielded a parent ion at m/z 828 and fragments compatible with an hydroxylation on the benzoate ring at C2, with fragments at m/z 543, 405, and 286, whereas the peak eluting at 12.5 min displayed a parent ion at m/z 828 and fragments at m/z 543, 421, and 286, indicating that oxidation occurred on the taxane ring, probably in position 6. 

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Chromatogram of incubation of 10-deacetylpaclitaxel with human liver microsomes. Arrows refer to metabolites formed in the presence of NADPH eluting at 11.5 min (C2-OH-10-deacetylpaclitaxel) and 12.5 min (C6-OH-10-deacetylpaclitaxel).

Metabolism of Docetaxel. The metabolism of docetaxel by human liver microsomes was consistent with those reported earlier; a single peak of product eluting at 23.5 min was observed and identified by mass spectrometry as the hydroxylated derivative on the tert-butyl of the lateral chain, shifting its mass from m/z 282 to 298, whereas a fragment at m/z 182 (lateral chain  tert-butyl  O-CO) was recovered in the product as in the native docetaxel molecule, indicating that oxidation occurred on the tert-butyl moiety.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Chromatogram of incubation of docetaxel with Ad293-3A4 cells. Arrows refer to metabolites formed by Ad293-3A4 cells and absent from incubation with Ad293 cells. The asterisk indicates the C3'-OH-docetaxel produced by human liver microsomes.

Metabolism of 10-Acetyldocetaxel. Metabolites generated during incubation of 10-acetyldocetaxel with liver microsomes in the presence of NADPH were resolved by HPLC and characterized by HPLC/MS (Fig. 6). The unchanged 10-acetyldocetaxel eluted at 49 min with a parent ion m/z 850 and yielded fragments at m/z 794 (tert-butyl), 734 (tert-butyl  acetyl at C10), 569 (side chain at C13), 509 (side chain at C13  acetyl at C10), and 282 (side chain at C13). It was accompanied by several peaks; the oxidation product eluted at 37 min was hydroxylated on the tert-butyl of the lateral chain (t-butyl-OH-10-acetyldocetaxel), with a parent ion at m/z 866 and showed fragments at m/z 794, 569, 509, and 298. The oxidation on the taxane ring (presumably in position 6) led to a compound eluted at 44 min (parent ion m/z 866), having fragments at m/z 810, 750, 585, 525, and 282. Two other peaks were monitored at 40 and 42 min. The material present in these peaks cannot be distinguished by mass fragmentography and was believed to result from oxidation in two different positions on the phenyl ring of the lateral chain at C3'-parent ion m/z 866, with fragments at m/z 810, 750, 569, and 298 (phenyl-OH-10-acetyldocetaxel).

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Chromatogram of incubation of 10-acetyldocetaxel with human liver microsomes. Arrows refer to metabolites formed in the presence of NADPH eluting at 37 min (t-butyl-OH-10-acetyldocetaxel), 40 and 42 min (phenyl-OH-10-acetyldocetaxel), and 44 min (6-OH-10-acetyldocetaxel).

Metabolism of Baccatin III. When the lateral chain at C13 is absent from taxoid molecules, the biotransformation rate is severely reduced and the low amount of metabolites of baccatin III and 10-deacetylbaccatin III formed during incubation with human liver microsomes or P450-expressing cell lines made illustrating their structure difficult. In our conditions, baccatin III was eluted at 36 min after two peaks containing baccatin derivatives with retention times of 26 and 31 min. Fragments of baccatin III (parent ion m/z 587) indicated ions at m/z 527 (acetyl), 405 (acetyl  benzoate at C2), and 345 (acetyl at C4  benzoate at C2  acetyl at C10). Fragments issued from the derivatives gave the same pattern with ions at m/z 543, 421, and 361 for a parent ion at m/z 603. This indicated that an oxygen atom was added to the taxane ring and was not compatible with an oxidation on the benzoate ring at C2, with expected fragments at m/z 603, 543, 405, and 345. These two hydroxylated metabolites on the taxane ring were produced only by Ad293-3A4 and Ad293-3A7 cells; all other P450-expressing cell lines were devoid of activity but retained the epimerization of baccatin. In addition to these two metabolites, a very low amount of hydroxylated metabolites of 7-epibaccatin can also be detected in HPLC but cannot be accurately analyzed in HPLC/MS.

Metabolism of 10-Deacetylbaccatin III. Extracts prepared from incubations of NADPH with 10-deacetylbaccatin and liver microsomes contained a single peak considered a direct metabolite, with a retention time of 23 versus 24 min for 10-deacetylbaccatin. The structure of this metabolite was verified by HPLC/MS; the protonated molecular ion (m/z 545) and characteristic fragment ions were 16 mass units greater than 10-deacetylbaccatin, indicating that an oxygen atom was added to the taxane ring.

Quantitative Metabolism of Taxoids by Human Liver Microsomes. The overall metabolism of taxoids and the balance between the different oxidative routes were investigated in liver microsomes and reflected the participation of individual P450 isoforms to the overall metabolism of taxoids by microsomes. The rate of biotransformation was quite variable, ranging from 0.36 to 4% of the substrate concentration transformed in 30 min by 1 mg of microsomal protein when taxoids were incubated at a concentration of 50 µM. A general feature is related to the presence of an acetyl group in position 10 of the taxane ring, which strikingly activates the biotransformation rate by liver microsomes. The rate of reaction is 2- to 5-fold higher with baccatin III, 10-acetyldocetaxel, and paclitaxel than with 10-deacetylbaccatin III, docetaxel, and 10-deacetylpaclitaxel, respectively (Table 1).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The rationale of this study was based on the observation that two changes in the structure of the taxoid molecule could shift the major hydroxylation site from the C6 of the taxane ring of paclitaxel to the side chain at C13 in docetaxel. Therefore, we intend to evaluate the impact of individual modification of the taxoid molecule on the site of hydroxylation and extent of the reaction by P450-dependent monooxygenases.

Data are summarized in Table 2. CYP3A4 and to a minor extent CYP3A7 were the only P450 isoforms tested in this study capable to actively hydroxylate taxoid molecules lacking the side chain at C13, such as in baccatin III and 10-deacetylbaccatin III. The reaction occurred on the taxane ring probably in position 6, whereas CYP2C8 responsible for this reaction in paclitaxel had no activity. The presence of a tert-butyl group in the side chain at C3', such as in docetaxel and 10-acetyldocetaxel, increased the overall oxidation of taxoids by CYP3A isoforms and orientated the metabolism toward the formation of the oxidation product on the tert-butyl group. A hydroxylation on a tert-butyl group by CYP3A4 has already been described for terfenadine, the tertiary butyl group being liable to oxidation because of its high number of available primary carbons (Yun et al., 1993). The hydroxylated derivative was the unique primary metabolite formed from docetaxel and accounted for 74% of the total metabolite formation from 10-acetyldocetaxel by human liver microsomes, with a similar affinity for docetaxel and 10-acetyldocetaxel. CYP3A4 was the major contributor to this reaction, whereas CYP3A5 only moderately catalyzed this reaction, as already reported by Royer et al. (1996) and Shou et al. (1998). Actually the affinity of CYP3A4 for docetaxel has been reported to be 10-fold higher than those of CYP3A5 and could explain why the participation of CYP3A5 to the biotransformation of taxoids at a dose of 50 µM might be very low in relation with the low affinity of CYP3A5 for taxoids. Conversely, the presence of a benzamide group in the side chain favored the hydroxylation in position 6 of the taxane ring, with more than 60% of the metabolite formed from either paclitaxel or 10-deacetylpaclitaxel by CYP2C8.

Collectively these results suggested that the side chain plays a crucial role in determining the orientation of the taxoid molecule into the active site of P450 proteins. The presence of the side chain is required for a correct orientation of the substrate into the active site of CYP2C8 and to place the putative hydroxylation site (e.g., carbon 6 of the taxane ring) in the vicinity of the heme-oxygen complex. Its absence completely prevents any reaction by CYP2C8. The replacement of the tert-butyl group of the side chain by a phenyl ring facilitates the positioning of the substrate into the CYP2C8-active site and would probably suggest that hydrophobic interactions exist between the phenyl group of the side chain and hydrophobic residues of the CYP2C8 peptide chain via - bonding. Conversely, the presence of a benzamide group instead of a boc-amino group in the side chain reduces the hydroxylation by CYP3A4 through a decreased affinity for the protein. This effect may be related to different conformations of the side chain of paclitaxel and docetaxel, as proposed earlier (Dubois et al., 1993).

Another feature is the 2- to 5-fold increase of the overall hydroxylation rate of 10-deacetylbaccatin III, docetaxel, and 10-deacetylpaclitaxel by human liver microsomes when an acetyl group is added in position 10 of the taxane ring. This higher biotransformation results from either a stimulation of the catalytic velocity for baccatin and 10-acetyldocetaxel or an increased affinity for paclitaxel without modification of the catalytic efficiency. Both CYP3A4- and CYP2C8-dependent activities are stimulated, indicating that the presence of a hydrophobic group in position 10 stimulates the activity, but without striking modification of the balance between CYP3A4 and 2C8. However the addition of the acetyl group in position 10 of docetaxel allowed the 6-hydroxylation of 10-acetyldocetaxel by CYP2C8 but with a weaker affinity than the oxidation on the tert-butyl group by CYP3A4.

These observations attribute a major role in the substrate recognition by P450 to hydrophobic groups and to interaction between these hydrophobic groups and hydrophobic amino acid residues. Several key residues have been hypothesized to play an active role in the binding of substrates to CYP3A4 based on sequence alignment or after site-directed mutagenesis. Thus, aromatic residues Phe²¹⁵, Phe³⁰⁴, and Tyr³⁰⁷ (respectively Phe¹⁸¹, Phe²⁶³, and Tyr²⁶⁶ when aligned on BM3 or cyp102 from Bacillus megaterium) could interact with groups of 3A4 substrates but are also present in CYP3A5 and 3A7 (Lewis et al., 1996). Mutations in SRS4 within close proximity to the putative substrate-binding pocket have demonstrated altered oxidation products with progesterone but are similar in 3A4, 3A5, and 3A7 (Domanski et al., 1998). Similarly, the highly conserved residue Ser¹¹⁹ has been shown to play an important role in the active site topology of CYP3A4 but is also present in 3A5 and 3A7 (Roussel et al., 2000). To date no definitive clue has been provided regarding the identity of amino acids involved in substrate recognition and orientation, and the sequence homology between 3A4, 3A5, and 3A7 could not explain the difference observed in the transformation of taxoids. In this respect, the use of substituted taxoids allows exploring the orientation of substrates into the active site of CYP3A and constitutes a useful tool to probe the hydrophobic pocket of these enzymes.

The biological activity of taxoids tested upon microtubule assembly or in in vitro cytotoxic assays has demonstrated a partial relationship between the hydrophobicity of taxoids and their interaction with tubulin. Thus paclitaxel, 10-deacetylpaclitaxel, docetaxel, and 10-acetyldocetaxel showed a very similar potency to inhibit microtubule disassembly and modification of substituents at C10 has no or little effect on the activity except when very large groups are branched at C10 (Guénard et al., 2000). Furthermore, the addition of polar substituents has no effect in the tubulin assay. These data confirmed previous observations reporting that modifications at C10 (or C7) have generally no effect on the biological activity of taxoids (Parness et al., 1982). In contrast the potent biological activity of taxoids is generally reduced by modification on the lateral chain of paclitaxel and docetaxel (Lataste et al., 1984). Gueritte-Voegelein et al. (1991) and Guenard et al. (1993) reported that the configuration at position C2' and C3' of paclitaxel is important for the inhibition of microtubule assembly and postulated a positive correlation between the conformation of the side chain and the binding activity. These results differ from the striking difference reported here in the biotransformation of taxoids by P450 proteins and could be the basis for the design of molecule with high biological activity and low biotransformation.