metabolism of docetaxel by human cytochromes p450: … · vitro docetaxel metabolism by human liver...

ICANCER RESEARCH56. 58-65. January 1. 19961

ABSTRACT

The metabolism of docetaxel by human liver microsomes was investigated in vitro and compared with that of paditaxel. A main docetaxelmetabolite was generated by human liver microsomes in the presence ofNADPH: retention time in high pressure liquid chromatography and itsion fragmentation in mass spectrometry were identical to those of theauthentic derivative hydroxylated at the butyl group ofthe C53side chain.Kinetic measurements and chemical and Immunological inhibitions demonstrated that CYP3A was implicated in the hydroxylation of docetaxel:Km (2 ELM)and Vm values of docetaxel for human liver microsomes werecomparable to those calculated for the formation of metabolitep-hydroxyphenyl C3' paditaxel (M4). Docetaxel hydroxylation correlated only withthe CYP3A content of microsomes and with CYP3A-dependent 6(1-hy.droxylation of testosterone and 16-hydroxylation of dehydroepiandros

terone. The formation of hydroxydocetaxel was strongly reduced byCYP3A inhibitors such as ketoconazole, midaZOlam,erythromycin, testosterone, orphenadrine, and troleandomycin, whereas quinidine(CYP2D6), hexobarbital, tolbutamide, and mephenytoin (CYP2C) had noorlittleeffect.

The hydroxylation of docetaxel exhibited a highly positive correlationwith the formation of metabolite M4 of paditaxel (r = 0.929, P < 0.0001,n 12), but not with its 6-hydroxylation (r 0.48, P > 0.15). Docetaxel

abolished the hydroxylation of paclitaxel metabolite M4, but was totally

inactive on its 6a-hydroxylation. Conversely, pacitaxel reduced significantly the hydroxylation of docetaxel. We examined in vitro the possibleinteraction among docetaxel, paclitaxel, and drugs which could be amadated during chemotherapy. Cisplatin, verapamil, doxorubicin, vinblas.tine, and vincristine at concentrations usually recommended did notmarkedly modify taxoid metabolism. Ranitidine and diphenylhydraminebad no effect, but 100 @LMcimetidine partially inhibited the formation of6a.hydroxypaclitaxel.

Pretreatment of patients with barbiturates strikingly stimulated docetaxel hydroxylation, whereas no acceleration of docetaxel hydroxylationwas noticed in a patient receiving steroids.

INTRODUCTION

Different laboratory approaches including antisense RNA and antibodies have been recently developed for the treatment of humancarcinomas. However, for economical and practical reasons, the use ofclassical chemotherapy will remain the most useful treatment duringthe next decades. In this respect, new microtubule poisons exhibit apotent and highly promising antiproliferative efficiency. In contrastwith Vinca alkaloids, which prevent the assembly of microtubules, themechanism of action of taxoids primarily involves the stabilization ofmicrotubule assembly, thereby disrupting the cytoskeleton architec

hire and disturbing the orderly progression through mitosis (1, 2).Taxoids demonstrate a high antimitotic activity on experimental tumors and in animal models (3â€”7).In clinical trials, taxoids have beenshown to be active antineoplastic agents in patients with solid tumors,

particularly cisplatin-refractory ovarian cancer and also in breast,lung, head, and neck cancers (8â€”12).

Two taxoids (Fig. 1) are under current clinical evaluations: paclitaxel (Taxol), mainly extracted from the bark of the Pacific yew

(Taxus brevifolia; Ref. 13), and docetaxel (Taxotere), a semisyntheticmolecule prepared from lO-deacetylbaccatin Ill isolated from theleaves of the European yew (Taxus baccata; Refs. 14 and 15). In vivo,paclitaxel was mostly biotransformed at the hepatic level and generated hydroxylated derivatives recovered unconjugated in rat (16) andhuman bile (17, 18). In humans, metabolites result from the hydroxylation of paclitaxel at position 6 of the taxane ring (M5), at the paraposition of the phenyl ring at the C3' on the C13 side chain (M4), andat both positions leading to a dihydroxy derivative (M3; Fig. 1). Invitro studies on the biotransformation of paclitaxel by human livermicrosomes confirmed these observations: Cresteil et a!. (19) firstconcluded that CYP2C accounted for the formation of 6a-hydroxypaclitaxel, which is the major metabolite formed in vivo and in vitro,while CYP3A supported the second hydroxylation reaction. In contradiction to these conclusions, Kumar et al. (20) claimed that CYP3Awas responsible for the formation of 6a-hydroxypaclitaxel. Recentinvestigations conducted by Harris et a!. (21) and Rahman et a!. (22)confirmed the former conclusions and extended the identification ofthe major isoform involved in the 6a-hydroxylation of paclitaxel tothe CYP2C8.

In contrast to paclitaxel, the biotransformation pathway of docetaxel is similar in animals and humans. The initial metabolite resultsfrom the hydroxylation of the methyl of the tert-butyl group at the@side chain of docetaxel (Fig. 1), suggesting the participation of cytochrome P450 isoform(s). This metabolite is further oxidized andcyclized (23) in humans as in several animal species studied (24).

In this study, we intended to compare the metabolic pathwaysinvolved in paclitaxel and docetaxel biotransformation in the humanliver. Preliminary studies tentatively identified the CYP3A as the siteof hydroxylation for docetaxel (25). Herein, we investigated the invitro docetaxel metabolism by human liver microsomes, and wecompared docetaxel and paclitaxel metabolic pathways to estimate therelative participation of individual P450 isoforms. The CYP3A subfamily has already been implicated in the biotransformation of severalmolecules such as verapamil (26), vinblastine (27), and tamoxifen(28). Since clinical protocols have been designed to combine manyprescriptions during chemotherapy, in the second part of this study,we were concerned by the impact of drug association on the in vitrometabolism of paclitaxel and docetaxel to predict possible drug interactions modifying the pharmacokinetics of the active compounds andtheir cytotoxic effects.

MATERIALS AND METHODS

Chemicals. Paclitaxel (Taxol, NSC 125973) was kindly provided by

Bristol-Myers Squibb (Wallingford, CT) and docetaxel (Taxotere, RP 56976,NSC 628503) by RhÃ´nePoulenc Rorer (Antony, France). All chemicals werefrom the highest available grade and purchased from Sigma, except pentoxyresorufin (Molecular Probes, Eugene, OR). Quinidine was a gift from Dr.P. Dayer, erythromycin was provided by Abbott (Rungis, France), racemicmephenytoin was generously supplied by Sandoz (Basel, Switzerland), mida

58

Received 8/1/95; accepted 10/31/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

@ This work was supported by a grant from l'Association pour la Recherche sur IcCancer (B. M., M. W.), la Ligue Nationale Contre Ic Cancer (T. C.), RhÃ´nePoulenc Sante(B. M., T. C., M. W.), and the Conseil Regional Midi-PyrÃ©nÃ©es(B. M.).

2 To whom requests for reprints should be addressed.

Metabolism of Docetaxel by Human Cytochromes P450: Interactions with Paclitaxeland Other Antineoplastic Drugs'

Isabelle Royer, Bernard Monsarrat, Michelle Sonnier, Michel Wright, and Thierry Cresteil2

Laboratoire de Pharmacologie et Toxicologie Fondamentales, UPR 8221, 205 route de Narbonne, 31077 Toulouse (1. R., B. M., M. WI, and institut National de Ia Sante et de IaRecherche MÃ©dicaleU75, UniversitÃ©RenÃ©Descartes, 156 rue de Vaugirard. 75730 Paris, Cedex 15 (M. S.. 7'. C.], France

on July 6, 2021. © 1996 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

METABOLISM OF DOCETAXEL BY HUMAN CYTOCHROMES P450

Reilly et a!. (35), and the biotransformation of paclitaxel was performed as

previously reported (19).Metabolism of Docetaxel. The conditions of incubation of docetaxel with

liver microsomes were derived from those set for paclitaxel. Microsomalproteins were incubated in I ml 100 mMsodium phosphate buffer (pH 7.4), 10mM MgCl2, 20% glycerol containing 0.1 mM NADP and 1 mM glucose6-phosphate as an NADPH-generating system. Docetaxel (10 mMin methanol)was added at a final concentration of 25 @.tM,except when otherwise indicated.Incubations carried out for 30 mm at 37Â°Cafter the addition of glucose6-phosphate dehydrogenase were stopped by the addition of 2.5 ml ethylacetate. The parent drug and its derivative were extracted twice by 2.5 ml ethyl

acetate. Then, the extracts were pooled, evaporated to dryness, and analyzed

using HPLC.For kinetic measurements, docetaxel concentrations ranged from I to 50

pM. For immunoinhibitions, liver microsomes were incubated at 23Â°C for 15

mm with increasing amounts of preimmune or anti-CYP3A4 IgG prior to theaddition of other reagents (19). Inhibition studies were performed in the

presence of 10 jAMquinidine, 20 @Morphenadrine, 50 @MDHEA, 160 ,.@Mtestosterone, and 400 @.LMerythromycin or 500 MMhexobarbital, tolbutamide,racemic mephenytoin, and midazolam. Cisplatin and diphenylhydramine

(1 @.LM-lmM)and verapamil, cimetidine, and ranitidine (10 ,.@M-l0mM)weredissolved in buffer. Doxorubicin, vinblastine, vincristine, and ketoconazolewere dissolved in methanol and added to the incubation mixture at concentrations ranging from 0.1 to 100 @Min the presence of a fixed amount ofmethanol. Troleandomycin (10 giM)was preincubated for 15rain at 37Â°Cin thepresence of NADPH to generate a complex with CYP3A prior to the additionof reagentsfor docetaxel incubation.

In experiments designed to estimate the inhibitory effect of docetaxel onpaclitaxel metabolism, docetaxel (from 1 to 100 @M)was incubated with 50.LMpaclitaxel. Conversely, paclitaxel concentrations between 10 and 100 pM

were added to incubation mixtures containing 25 pM docetaxel. After twoextractions with 2.5 ml ethyl acetate, residues were divided in two aliquots and

analyzed for the respective content in metabolites according to the separationprocedures described below.

Characterization and Quantitatlon of Docetaxel Metabolites. Ethyl acetate extracts evaporated to dryness were dissolved in 200 p1 acetonitrile:water

CsV

0

Fig. 2. Typical HPLC elution profile of docetaxel and its metabolite (F6) extractedafter in vitro incubation of docetaxel with human liver microsomes. A prominent peak ofunmodified docetaxel (@40 mm) and one peak corresponding to hydroxydocetaxel(metabolite F6) are visible. All other minor peaks (5) were present when NADPH wasomitted, whereas peak F6 was absent.

PACLITAXEL: Ri = H, R2 = HMetabolite M4: Ri â€”OH, R2 = HMetabolite M5: Ri â€”H, R2 = OH

@@YPÃ§1@D

@ Ri 0

x,@@

0 0@0CH30

aDOCETAXEL: Ri - CH3Metabolite F6: Ri = CH2OH

Fig. I . Chemical structure of paclitaxel and docetaxel and their metabolites. Arrows,major respective sites of hydroxylation of CYP3A and CYP2C8.

zolam by Roche (Basel, Switzerland), and ketoconazole by Janssen (Beerse,Belgium).

Tissue Collection. All protocols were conducted according to the recommendations of the Ethical Committee of the Institut National de la Sante et deIa Recherche MÃ©dicale.Adult liver samples (n â€”12) were obtained fromdonors for kidney transplantation. Occasionally, one kidney was obtained

under the same conditions. Donors had no severe chronic pathology andgenerally died from a traffic accident. They had no repeated drug consumption.No information was available regarding their smoking and drinking habits.Alternatively, liver samples were collected from three fetuses (22, 26, and 27weeks, respectively) and eight children ages 12 h to 6 months as previouslyreported, as well as from five children given barbiturates, benzodiazepines, orsteroids for therapeutic purposes (19). Samples were frozen in liquid nitrogenimmediately after removal and kept at â€”80Â°C.

Microsome Preparation and Determination of Individual P450 IsoformContent. Frozen tissues were thawed in ice-cold saline, homogenized,andmicrosomes were prepared as previously reported (29). Cytochrome P450 was

measured according to the procedure of Greim (30), and the protein contentwas determined according to Lowry et a!. (31). The individual P450 isoformcontent of liver microsomes was quantified as detailed elsewhere (19).

Monooxygenase Activities. Except when otherwise specified, reactionswere performed with 0.3 nmol total P450. Pentoxyresorufin dealkylation wasfollowed by spectrofluorometry according to the method of Burke et a!. (32).The 6@3-hydroxylationof testosterone and the 16-hydroxylation of DHEA3were measured with â€˜4Csubstrates, and the metabolites were separated byTLC: the spot corresponding to authentic 63-testosterone and 16-hydroxyDHEA were visualized, scraped, and counted (33). Tolbutamide hydroxylationwas carried out with 0.1 nmol P450 essentially following the procedure ofKnodell et a!. (34). Diazepam demethylation was assayed as described by

3 The abbreviations used are: DHEA, dehydroepiandrosterone; HPLC, high pressure

liquid chromatography; IC5@,concentration giving half maximum inhibition.

rq)

0

Retention time (tnln)

59



6-OH PM4 PP450CYP2CCYP3ACYP2EICYP2D6CYP1A2CYP4AIPRODTest6@3ToIbDMDiazF6

Doe0.480.930.310.330.87â€”0.240.790.090.100.340.860.780.646-OHP0.630.410.720.330.21â€”0.340.130.170.820.400.830.89M4P0.730.420.87â€”0.280.260.190.060.380.920.860.68P4500.480.190.030.600.320.190.110.580.620.43CYP2C0.390.340.310.050.450.770.230.880.87CYP3Aâ€”0.410.810.470.200.200.810.630.61CYP2EI0.28â€”0.490.630.31â€”0.38â€”0.160.01CYP2D60.310.62â€”0.13â€”0.430.850.14CYPIA20.190.220.240.100.33CYP4AI0.30â€”0.270.040.51PROD0.240.610.81Test

6f30.630.57Tolb0.91


Table I Correlations between total cvtochrome P450 content, individual P450 isoform content, and related monooxygenase activities in adult human liver microsomesÂ°

a Total P450 content was spectrally determined, and the content of each individual isoform was estimated immunochemically. Monooxygenase activities supported by CYP2C

(DMDiaz, diazepam demethylation; ToIb. tolbutamide hydroxylation), CYP3A (Test 6@3,testosterone 6@3hydroxylase), and pentoxyresorufin dealkylase (PROD) were assayed withhuman liver microsomes (n 12), and activities were correlated with monooxygenase activities toward docetaxel (F6 Doe) and paclitaxel (6 OH P. 6a-hydroxylation of paclitaxel;M4P: metabolite M4 of paclitaxel), P450 isoform content (expressed as arbitrary units@ mg â€p̃rotein) and total P450 content (nmol . mg â€p̃rotein).

reaction mixture or when the incubation was performed with microsomes isolated from human kidney. The main absorption peak elutingat 40 mm corresponded to docetaxel, whereas the peak of the majormetabolite eluted at 22 mm and exhibited the characteristic UVspectra of docetaxel derivatives. The retention time of this metabolitecorresponded exactly to those of hydroxydocetaxel, which has beenchemically characterized by mass spectrometry and nuclear magneticresonance spectroscopy after isolation from both rat bile (24, 37) andhuman feces after treatment with docetaxel (23). Moreover, usingHPLC combined with mass spectrometry, it became possible to directly identify taxoids metabolites after separation by their massspectrum (36). We have applied this new approach to analyze humanliver microsomes extracts. The atmospheric pressure chemical ionization mass spectrum of the hydroxydocetaxel metabolite exhibitedthe corresponding intense molecular ion series: m/z 824 (MHI andm/z 806 (824-H2O); taxane ring series: m/z 527 and m/z 509, and thecharacteristic side chain at the C13 ion series at m/z 298 and m/z 182.

Fig. 3. In vitro inhibition by drugs of the docetaxel metabolism by human livermicrosomes. Inhibitions were performed as detailed in â€œMaterialsand Methods.â€•Resultsare the means Â±SDs of individual experiments performed in duplicate with a single adultpreparation, and expressed as the percentage of activity measured in the absence ofchemicals. 0, orphenadrine; Er, erythromycin; T, testosterone; TAO, troleandomycin;Mid, midazolam; H, hexobarbital; To, tolbutamide; MP, mephenytoin.

wox

(20:80, v/v) and analyzed using HPLC. A linear gradient of 33â€”57%acetonitrile in water was used as eluent at a flow rate of 1 mllmin. Quantification ofmetabolites and parent compound was performed using a linear calibrationcurve made with the corresponding pure compounds between 1 and 2.5 nmoland 0.02 and 0.2 nmol for docetaxel and hydroxydocetaxel, respectively. TheHPLC system included a Waters model chromatograph equipped with twomodel 510 pumps and an U6K injector and were linked to a Waters M99lphotodiode array for UV detection at 235 nm. Injections of 20â€”100pAweremade onto the analytical column (0.46 X 25 cm, Beckman ODS 5 @mC18column). To characterize the presence of docetaxel metabolites in microsomepreparation extracts, we used liquid chromatography combined with a newatmospheric pressure chemical ionization mass spectrometry interface as recently reported (36). Separation column and chromatographic conditions werethe same as those described above: 200 pAof a mixture of acetonitrile:watercorresponding to the microsome extract were introduced into the HPLC-mass

spectrometry system (FinniganMat TSQ 700; San Diego, CA) in the full scanmode (m/z 100â€”1200)at 2 ms/step.

RE@U' â€œ Fig. 4. Immunoinhibition of docetaxel biotransformation by anti-CYP3A4 immuno@, J.@I@ globulins in adult human liver microsomes. Microsomal proteins (0.3 nmol P450) were

preincubated for 10 mm at 23Â°Cwith IgG prior to the addition of other reagents. AfterIn Vitro Metabolism of Docetaxel by Human Microsomes. The incubation,metaboliteswereextractedandquantified.Resultsarethe meansÂ±SDsof

metabolic HPLC profile of docetaxel incubated with human liver individualexperimentsperformedinduplicateontwoadultpreparations,andexpressedas. . . the percentage of activity measured in the absence of IgG. Basal activities in the absence

microsomes tn the presence of NADPH demonstrated a major new of IgGwere1.2and0.92pmol. min â€˜@ mg â€p̃rotein.0, incubationswithpreimmunepeak (Fig. 2) undetectable when NADPH was omitted from the IgG;â€¢,incubationswithanti-CYP3A4IgG.

60

0 Er T TAOMId H To MP

anti CYP3A IgG(.g/nmol P450)



200

100

0


ketoconazole

-4@--@:Fig. 5. Effect of increasing concentrations of

cisplatin, verapamil, or ketoconazole on docetaxeland paclitaxel metabolism by human liver microsomes. Inhibitions were performed with increasingconcentrations of chemicals as detailed in â€œMaterials and Methods.â€•Results are given as the meanvalues of experiments performed in duplicate andexpressed as the percentage of activity measured inthe absence of chemicals. A, hydroxydocetaxel(F6); 0, 6-hydroxypaclitaxel (metabolite M5): â€¢,metabolite M4 of paclitaxel.

100

50

liit4 lOi*M100pM 1mM

Combined evidence from the retention time and the characteristicfragment ions suggest that this compound corresponds to a hydroxylated derivative of docetaxel. In agreement with the data obtained withthe pure reference compound, this docetaxel metabolite resulted froma monohydroxylation reaction at the tert-butyl group of the@@ sidechain (Fig. 1).

Pharmacokinetic Determinations. The formation of the hydroxydocetaxel metabolite by human liver microsomes was relatively low,with a mean value of 2.6 Â±0.6 pmol . mm@ . mg I, while extremevalues ranged from 0 to 7.5 pmol . min â€˜@ mg I@ This rate wascomparable to those observed for the formation of metabolite M4 ofpaclitaxel (5.3 Â±1.4 pmol â€m̃in@@ mg I) when incubations wereperformed with the same microsomal fractions, and was far below therate of formation of 6a-hydroxypaclitaxel (120 Â±30 pmol â€m̃in Img I@ The time dependence of the reaction was observed for up to 30mm with human liver microsomes. Moreover, the formation of metabolites increased with doses of docetaxel (1â€”50,.LM)following atypical monophasic Michaelis-Menten kinetics, with an apparent Kmof 2.1 Â±0.3 @LM(calculated from three separate experiments) and anextrapolated Vmof 10 pmol . min I . mg @.Thus, a concentration of25 pM docetaxel was routinely used in additional 30-mn incubations.

Identification of P450 Isoform Involved in Docetaxel Biotransformation. Several approaches have been used to unequivocallyidentify the P450 isoform responsible for the biotransformation ofdocetaxel. First, we compared the rates of hydroxylation of docetaxel

Fig. 6. Effect of increasing concentrations ofdoxorubicin, vinblastine, and vincristine on docetaxel and paclitaxel metabolism by human livermicrosomes. Inhibitions were performed in thepresence of increasing concentrations of chemicalsas detailed in â€œMaterialsand Methods.â€•Results aregiven as the mean values of experiments performedin duplicate, and expressed as the percentage ofactivity measured in the absence of chemicals. A,hydroxydocetaxel (F6); 0, 6-hydroxypaclitaxel(metabolite M5): â€¢,metabolite M4 of paclitaxel.

O.litI4 liii4 lOi*14lOOitt4

and paclitaxel by human liver microsomes (Table 1). A highly positive correlation occurred between the hydroxylation of the lateralchain of docetaxel at C13 and the hydroxylation of the correspondinglateral chain of paclitaxel (metabolite M4) in 12 samples(P < 0.0001), whereas no correlation was found with the production

of 6a-hydroxypaclitaxel (r = 0.48; P > 0.15).The formation of metabolite M4 of paclitaxel has been assigned to

CYP3A (19). Thus, we further compared the rate of hydroxylation ofdocetaxel with other CYP3A-dependent monooxygenase activitiesand with the CYP3A content of the microsomal preparations used toassay the hydroxylation of docetaxel. As expected, the docetaxelhydroxylation was correlated with the 6(3-hydroxylation of testosterone (r = 0.864; P < 0.001) and with the 16-hydroxylation of DHEA(r 0.837; P < 0.001), as well as with the CYP3A content (Table 1).Conversely, no correlation was observed between docetaxel hydroxylation and two CYP2C-dependent monooxygenases, or with theCYP2C content of microsomes. Similarly, no positive relation couldbe noticed with the microsomal total P450 content or the content inother individual P450 isoforms.

We examined the inhibitory effect of well-defined substrates ofmonooxygenases. Substrates of CYP3A including erythromycin, testosterone, midazolam, orphenadrine, and troleandomycin elicited amarked reduction of docetaxel hydroxylation, whereas three substrates of CYP2C have no or little inhibitory effect (Fig. 3). In anotherset of experiments, DHEA, a substrate of CYP3A, was incubated

doxorubicin

61

cisplatin verapamil

10pM lOOiaM1mM10mM 0.luM lisM lOuM lOOiiM

vinbiastirte vincristine

0.111K 1pM lOuMlOOpM O.liaM 1pM 10@*MlOOuM




Fig. 7. Effect of increasing concentrations ofranitidine, cimetidine, and diphenylhydramine ondocetaxel and paclitaxel metabolism by humanliver microsomes. Inhibitions were perfonned inthe presence of increasing concentrations of chemicals as detailed in â€œMaterialsand Methods.â€•Resuits are given as the mean values of experimentsperformed in duplicate, and expressed as the percentage of activity measured in the absence ofchemicals. A, hydroxydocetaxel (F6); 0, 6-hydroxypaclitaxel (metabolite M5); â€¢, metaboliteM4 of paclitaxel.

with liver microsomes in the presence of docetaxel or paclitaxel:50 p.MDHEA substantially reduced the formation of the docetaxelmetabolite (by 86%) and the metabolite M4 of paclitaxel (by 78%),but moderately affected the 6a-hydroxylation of paclitaxel(

Table 2 Docetaxel hydroxylation in patients with drug history or under pathologicalconditionsPatientAgeDocetaxel

metabolite F6 CYP3A(pmol . n'a'@ mg â€˜ (arbitrary units@ mg â€˜

Disease Treatment and duration microsomal protein) protein)CYP2C(arbitrary units .

protein).mg50

86

77

79

834

days6 days

3 mo

9 yr

9 yrMaternofetal

infection None Not measurable 0.66Epilepsia Phenobarbital, 5 mg/kg/day 7.60 1.3Â°

for 5 days; pentobarbital, 2mg/kg/h for 3 days

Epilepsia Phenobarbital, 5 mg/kg/day I 3.3Â° 1.4Â°for 5 days

Medullar aplasia Prednisone, I mg/kg/day for 2.2 0.451 yr

Epilepsia Phenobarbital, 5 mg/kg/day; 26.8Â° 1.40pentobarbital, 2 mg/kg/h;pregnenolone, 2 mg/kg/dayfor 7 days; diazepam, 13mg, 1 day2.110

6.640

1.26

1.08

4.10a

Different from value in control patients within the same group of 9-year-old children are compared to control adults (0.57 for CYP3A and 1.13 for CYP2C).

METABOLISM OF DOCETAXEL BY HUMAN CY@OCHR@MESP450

(Fig. 8a). Conversely, the two pathways involved in the metabolismof paclitaxel were diversely affected by the addition of docetaxel tothe incubation mixture: the 6a-hydroxylation of paclitaxel remainedunchanged, whereas the formation of metabolite M4 was completelyabolished by docetaxel (Fig. 8b).

Ontogenesis and Induction of Docetaxel Metabolism. The lowlevel of docetaxel hydroxylation made this determination difficult. Noactivity (e.g., an activity 0.25). Microsomes obtained from apatient receiving prednisone for 1 year for the treatment of medullaraplasia did not show an increased docetaxel metabolism.

DISCUSSION

Clinical effectiveness of antineoplastic drugs depends on the balance between the cytotoxicity toward tumor cells and general toxicity.Usually, the therapeutic window is narrow and requires a carefuldosage. Thus, it is crucial to accurately estimate the biotransformationof antineoplastic drugs which occurs essentially at the hepatic leveland is catalyzed by cytochrome P450-dependent monooxygenases.Paclitaxel has been shown to undergo two pathways: the major leadsto its 6-hydroxylation and is catalyzed by CYP2C (19, 22), whereas aminor human metabolite results from the hydroxylation of the lateralchain by CYP3A isoform(s). These in vitro data have been supportedby the recovery of monohydroxylated and dihydroxylated metabolitesin bile of patients given paclitaxel.

In this report, we characterize the main metabolite of docetaxelformed by human liver microsomes and identify the P450 isoforminvolved in the hydroxylation reaction. Additionally, we assess druginteractions comparatively to the P450 isoforms involved in the hydroxylation of paclitaxel. For that purpose we used the human micro

some samples which have already allowed the characterization ofpaclitaxel metabolic pathways. Correlation studies, immunologicalinhibitions, and chemicals inhibitions support the conclusion that, invitro, the first docetaxel metabolite is formed by CYP3A isoforms.Kinetic measurements are consistent with this statement: the hydroxylation rate on the lateral chain of paclitaxel is similar to that ofdocetaxel and never exceeds 5â€”10%of the reaction occurring on thetaxane ring of paclitaxel catalyzed by CYP2C8. The lack of hydroxy

lation on the taxane ring of docetaxel is somewhat surprising and iscurrenfly under investigation.

The CYP3A content of microsomes obtained from the liver ofpatients treated with barbiturates exceeds the mean value measured incontrols of the same age. These samples present a markedly higheractivity toward docetaxel and confirm the implication of CYP3A inthe metabolic process. In the CYP3A subfamily, three major proteinshave been characterized. Their expression is age related: CYP3A7 isdetected in fetal livers (38, 39), whereas CYP3A4 (HLp or P450 NW)was the major P450 isoform present in adult livers. Recently, allfetuses and a fraction of the adult population were shown to containCYP3A5 RNA (40), although the existence of the protein was notdemonstrated. Data collected herein from fetal samples demonstratethat CYP3A (CYP3A7 and potentially CYP3A5) has no activitytoward docetaxel. Thus, the hydroxylation of docetaxel seems torestrict the catalytic efficiency to CYP3A4. This point will be furtherexamined with recombinant 3A4 and 3A7 proteins expressed in Coscells, as already done for testosterone and DHEA biotransformations.4

These conclusions were further confirmed by drug interactions. Wefirst used several compounds known for their capacity to specificallyinhibit CYP3A monooxygenases such as endogenous testosterone andDHEA and exogenous drugs. All reduce the hydroxylation of docetaxel from 50 to 100%. Next, several drugs have been incubated invitro with docetaxel. Ketoconazole is the more potent inhibitor ofdocetaxel hydroxylation and paclitaxel minor metabolite M4 hydroxylation exhibiting@ below 1 @.LM.This observation is consistent withthe high affinity of ketoconazole for CYP3A-dependent activitiesexemplified by a K1toward vinblastine hydroxylation of 0.2 @LM(27).Verapamil, a calcium channel blocker, down-regulates the expressionof the mdr gene at the transcriptional level and thus could reverse theresistance of proliferative cells to anticancer drugs (41 , 42). Doses ofverapamil between 15 and 50 @Mare required to decrease in vitro themdr gene expression in leukemic cell lines. This range of concentration is compatible with that which efficiently reduces paclitaxel anddocetaxel metabolism, and is in agreement with the Km of verapamil

4 Unpublished data.

63




biotransformation by human microsomes (26). Cisplatin is widelyused in chemotherapy in association with taxoids (43); however, incontrast to other anticancer drugs, it was not a substrate for microsomal monooxygenases nor impaired docetaxel and paclitaxel metabolism, even at the highest dose studied. A major side effect of treat

ments with paclitaxel was associated with the use of Cremophor asvehicle. This compound induces the release of histamine leading toallergic reactions. This effect can be prevented by the coadministration of@ and H2 receptor blockers such as ranitidine, cimetidine, anddiphenylhydramine. Apparent Km of cimetidine and ranitidine forhuman liver microsomes are 0.6 and 5 mrvi, respectively (44, 45),explaining the higher inhibitory effect of cimetidine on paclitaxelmetabolism observed in vitro. However, due to the dose of cimetidineused in therapeutics, drug interaction should remained limited, asshown in the rat model (46).

The biotransformation of Vinca alkaloids and anthracyclines byliver microsomes was mainly supported by CYP3A (27, 47). Since thereported Km of these molecules for CYP3A were in the 10 p.M rangeas well as the Km of docetaxel and paclitaxel, it is not surprising that

the extent of inhibition caused by Vinca alkaloids on taxoid metabolism is modest at doses below 10 p.M.

These observations are of interest because the different classes ofanticancer drugs combined for chemotherapy act on different targetsat the cellular level, either on the cell structure through a modificationof the microtubule assembly or by intercalation into the DNA molecule; therefore, their biological effect could be additive or synergistic.The only limitation is relative to the drug disposition and depends onthe elimination capacity of active compounds by the organism. According to the P450 isoform involved in the elimination process andto the relative affinity of associated drugs toward that isoform, anaccumulation could overcome the beneficial effect of chemotherapy.Thus, the involvement of distinct P450 isoforms in the hepatic metabolism of paclitaxel and docetaxel might lead to different disposition of these two drugs in human patients.

ACKNOWLEDGMENTS

We thank Dr. J. L. Fabre, Dr. F. Lavelle, and Dr. 0. J. Sanderink fromRhÃ´nePoulenc Rorer for their assistance in obtaining docetaxel and forstimulating collaboration.

REFERENCES

1. Schiff, P. B., and Horwitz, S. B. Taxol stabilizes microtubuies in mouse fibroblastscells. Proc. Natl. Acad. Sci. USA, 77: 1561â€”1565,1980.

2. Ringel, I., and Horwitz, S. B. Studies with RP 56976 (Taxotere) a semisyntheticanalogue of Taxol. J. NatI. Cancer Inst., 83: 288â€”291, 1991.

3. Bissery, M. C., GuÃ©nard,D., GuÃ©riue-Voegelein, F., and Lavelle, F. Experimentalantitumor activity of Taxotere (RP 56976. NSC 628503): a Taxol analogue. CancerRes., 51: 4845â€”4852, 1991.

4. Silvestrini, R., Zaffaroni, N., Orlandi, L., and Oriana, S. in vitro activity of Taxoland Taxotere on primary cultures and established cell lines of human ovarian cancer.Stem Cells, II: 528â€”535, 1993.

5. Braakhuis, B. J. M., Hill, B. T., Dietel, M., Kelland, L. R., Aapro, M. S., Zoli, W., andLelieveld, P. In vitro antiproliferative activity of docetaxel (Taxotere@), paclitaxel(Taxol) and cisplatin against human tumour and normal bone marrow cells. Anticancer Res., 14: 205â€”208,1994.

6. Woo, D. D. L., Miao, S. Y. P., Pelayo, J. C., and Woolf, A. S. Taxol inhibitsprogression of congenital polycystic kidney disease. Nature (Lond.), 368: 750â€”753,1994.

7. Hill, B. T., Whelan, R. D. H., Shellard, S. A., McClean, S., and Hosking, L. K.Differential cytotoxic effects of docetaxel in a range of mammalian tumor cell linesand certain drug resistant sublines in vitro. Invest. New Drugs, 12: 169â€”182,1994.

8. Trimble, E. L., Adams, J. D., Vena, D., Hawkins, M. J., Friedman, M. A., Fisherman,J. S., Christian, M. C., Canetta, R., Onetto, N., Hayn, R., and Arbuck, S. G. Paclitaxelfor platinum-refractory ovarian cancer: results from the first 1,000 patients registeredto National Cancer Institute treatment referral center 9103. J. Clin. Oncol., 11:2405â€”2410, 1993.

9. Chevalier, B., Fumoleau, P., Kerbrat, P., Dieras, V., Roche, H., Krakowski, I., Azli,N., Bayssas, M., Lentz, M. A., and Van Glabbeke, M. Docetaxel is a major cytotoxicdrug for the treatment of advanced breast cancer: a phase II trial of the clinical

64

screening cooperative group of the European organization for research and treatmentof cancer. J. Clin. Oncol., 13: 314â€”322, 1995.

10. Francis, P. A., Rigas, J. R., Kris, M. 0., Pisters, K. M. W., Orazem, J. P., Woolley,K. J., and Heelan, R. T. Phase II trial of docetaxel in patients with stage LII and IVnon-small-cell lung cancer. J. Clin. Oncol., 12: 1232â€”1237,1994.

11. Vanoosterom, A. T., and Schriivers, D. Docetaxel (Taxotere@), a review of preclinicaland clinical experience. 2. Clinical experience. Anticancer Drugs, 6: 356â€”368, 1995.

12. Rowinsky, E. K., Ross, C., and Donehower, M. D. Paclitaxel. N. EngI. J. Med., 332:1004â€”1014, 1995.

13. Wani, M. C., Taylor, H. L., Wall, M. E., Coggon, P., and McPhail, A. T. Plantantitumor agents. VI: The isolation and structure of taxol, a novel antileukemic andantitumor agent from Torus brev(folia. J. Am. Chem. Soc., 93: 2325â€”2327, 1971.

14. Cohn, M., GuÃ©nard,D., GuÃ©ritte-Voegelin, F., and Potier, P. Process for preparingderivatives of baccatin III and lO-deacetyl baccatin III. Eur. Patent Application. EP253,738 01/20/88; U. S. patent 492401 2; granted May 8, 1990.

15. GuÃ©ritte-Voegelin, F., GuÃ©nard,D., Lavelle, F., Le Goff, M. T., Mangatal, L., andPotier, P. Relationships between the structure of Taxol analogues and their antimitoticactivity. J. Med. Chem., 34: 992â€”998,1991.

16. Monsarrat, B., Maid, E., Cros, S., GarÃ¨s, M., GuÃ©nard,D., GuÃ©ritte-Voegelin, F.,and Wright, M. Taxol metabolism: isolation and identification of three major metabolites of taxol in rat bile. Drug Metab. Dispos., 18: 895â€”901,1990.

17. Monsarrat, B., Alvinerie, P., Wright, M., Dubois, J., GuÃ©riue-Voegelein, F., GuÃ©nard,D., Donehower, R., and Rowinsky, E. Hepatic metabolism and biliary clearance ofTaxol in rats and human. J. Natl. Cancer Inst. Monogr., 15: 39â€”46, 1993.

18. Harris, J. W., Katki, A., Anderson, L. W., Churny, G. N., Paukstelis, J. V., andCollins, J. M. Isolation, structural determination and biological activity of 6a-hydroxytaxol. The principal human metabolite of Taxol. J. Med. Chem., 37:706â€”709,1994.

19. Cresteil, T., Monsarrat, B., Alvinerie, P., TrÃ©luyer,J. M., Vieira, I., and Wright, M.Taxol metabolism by human liver microsomes: identification of cytochrome P450isozymes involved in its biotransformation. Cancer Res., 54: 386â€”92, 1994.

20. Kumar, G. N., Wale, U. K., and Walle, T. Cytochrome P450 3A-mediated humanliver microsomal taxol 6a-hydroxylation. J. Pharmacol. Exp. Ther., 268: 1160â€”1165,1994.

21. Harris, J. W., Rahman, A., Kim, B. R., Guengerich, F. P., and Collins, J. M.Metabolism of taxol by human hepatic microsomes and liver slices: participation ofcytochrome P450 3A4 and an unknown P450 enzyme. Cancer Res., 54: 4026â€”4035,1994.

22. Rahman, A., Korzekwa, K. R., Grogan, J., Gonzalez, F. J., and Harris, J. W. Selectivebiotransformation of Taxol to 6a-hydroxytaxol by human cytochrome P450 2C8.Cancer Res., 54: 5543â€”5546, 1994.

23. Monegier, B., Gaillard, C., Sable, S., and Vuilhorgne, M. Structures of the majorhuman metabolites of docetaxel (RP 56976, Taxotere@). Tetrahedron Lett., 35:3715â€”3718,1994.

24. Gaillard, C., Monsarrat, B., Vuilhorgne, M., Royer, I., Monegier, B., SabÃ©,S.,GuÃ©nard,D., Gires, P., Archimbaud, Y., Wright, M., and Sanderink, G. J. Docetaxel(TaxotÃ¨re). Metabolism in the rat. Proc. Am. Assoc. Cancer Res., 35: 428, 1994.

25. Bruno, R., and Sanderink, 0. J. Pharmacokinetics and metabolism of TaxotereÂ®(docetaxel). Cancer Surv., 17: 305â€”313,1993.

26. Kroemer, H. K., Echizen, H., Heidemann, H., and Eichelbaum, M. Predictability ofthe in vivo metabolism of verapamil from in vitro data: contribution of individualmetabolic pathways and stereoselective aspects. J. Pharmacol. Exp. Ther., 260:1052â€”1057,1992.

27. Zhou-Pan, X. R., SÃ©rÃ©e,E., Zhou, X. J., Placidi, M., Maurel, P., Barra, Y., andRharnani, R. Involvement of human liver cytochrome P450 3A in vinblastine metabolism: drug interactions. Cancer Res., 53: 5121â€”5126, 1993.

28. Jacolot, F., Simon, I., Dreano, Y., Beaune, P., Riche, C., and Berthou, F. Identification of the cytochrome P450 3A family as the enzymes involved in the N-demethylation of tamoxifen in human liver microsomes. Biochem. Pharmacol., 41:1911â€”1919,1991.

29. Cresteil, T., Flinois, J. P., POster, A., and Leroux, J. P. Effect of microsomalpreparations and induction on cytochrome P450-dependent monooxygenases in fetaland neonatal rat liver. Biochem. Pharmacol., 28: 2057â€”2063, 1979.

30. Greim, H. Synthesesteigerung und abbauhemmung bei der vermchrung der mikrosomalen cytochrome P450 und b5 durch phenobarbital. Arch. Pharmacol., 266:261â€”275,1970.

31. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurementwith the Folin reagent. J. Biol. Chem., 193: 265â€”275,1951.

32. Burke, M. D., Thompson, S., Elcombe, C. R., Halpert, J., Haaparanta, T., and Mayer,R. T. Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series ofsubstrates to distinguish between different induced cytochromes P450. Biochem.Pharmacol., 34: 3337â€”3345, 1985.

33. Chung, L. W. K., and Chao, H. Neonatal imprinting and hepatic cytochrome P450.Comparison of testosterone hydroxylation in a reconstituted system between neonatally imprinted and nonimprinted rats. Mol. Pharmacol., 18: 543â€”549,1980.

34. Knodell, R. G., Hall, S. D., Wilkinson, G. R., and Guengerich, F. P. Hepaticmetabolism of tolbutamide: characterization of the form of cytochrome P-450 involved in methyl hydroxylation and relationship to in vivo disposition. J. Pharmacol.Exp.Ther.,241:1112â€”1119,1987.

35. Reilly, P. E. B., Thompson, D. A., Mason, S. R., and Hooper, W. CytochromeP450111A enzymes in rat liver microsomes. Involvement in C3-hydroxylation ofdiazepam and nordazepam but not N-dealkylation of diazepam and temazepam. Mol.Pharmacol., 37: 767â€”774,1990.

36. Royer, I., Alvinerie, P., Ho, L. K., Wright, M., and Monsarrat, B. Paclitaxel metabolites in human plasma and urine: identification of 6a-hydroxytaxol. 7-epitaxol andtaxol hydrolysis products using liquid chromatography/atmospheric-pressure chemi




cal ionization mass spectrometry. Rapid Commun. Mass Spectrom, 9: 495â€”502,1995.37. Wright, M., Monsarrat, B., Royer, I., Rowinsky, E. K., Donehower, R. C., Cresteil,

T., and GuÃ©nard,D. Metabolism and pharmacology of taxoids. In: V. Farina (ed),Chemistry and Pharmacology of Taxol and Its Derivatives, pp. 131â€”164.New York:Elsevier, 1995.

38. Cresteil, T., Beaune, P., Kremers, P., Celier, C., Guengerich, F. P., and Leroux,J. P. Immunoquantification of epoxide hydrolase and cytochrome P-450 isoenzymes in fetal and adult human liver microsomes. Eur. J. Biochem., 151:345â€”350, 1985.

39. Komori, M., Nishio, K., Kitada, M., Shiramatsu, K., Muroya, K., Soma, M.,Nagashima, K., and Kamataki, T. Fetus-specific expression of a form of cytochromeP450 in human livers. Biochemistry, 29: 4430â€”4433, 1990.

40. Schuetz, J. D., Beach, D. L., and Guzelian, P. S. Selective expression of cytochromeP450 CYP3A mRNAs in embryonic and adult human liver. Pharmacogenetics, 4:11â€”20,1994.

41. Muller, C., Goubin, F., Ferrandis, E., Cornil-Scharwtz, I., Bailly, J. D., Bordier, C.,BÃ©nard,J., Sikic, B. I., and Laurent, G. Evidence for transcriptional control of humanmdrl gene expression by verapamil in multidrug-resistant leukemic cells. Mol.Pharmacol., 47: 51â€”56,1995.

42. Muller, C., Bailly, J. D., Goubin, F., Laredo, J., Jaffrezou, J. P., Bordier, C., and

Laurent, G. Verapamil decreases P-glycoprotein expression in multidrug resistanthuman leukemic cell lines. mt. J. Cancer, 56: 749â€”754, 1994.

43. Rowinski, E. K., Gilbert, M. R., McGuire, W. P., Noe, D. A., Grochow, L. B.,Forastiere, A. A., Ettinger, D. S., Lubejko, B. G., Satorius, S. E., Cornblath, C. B.,Hendricks, C. B., and Donehower, R. C. Sequences of taxol and cisplatin: a phase Iandpharmacologicstudy.J. Clin.Oncol.,9: 1692â€”1703,1991.

44. Klotz, U., Arvela, P., Pasanen, M., Kroemer, H., and Pelkonen, 0. Comparativeeffects of H2-receptor antagonists on drug metabolism in vitro and in vivo. Pharmacol. Ther., 3: 157â€”161,1987.

45. Knodell, R. 0., Browne, D. G., Gwozdz, G. P., Brian, W. R., and Guengerich, F. P.Differential inhibition of individual human liver cytochromes P450 by cimetidine.Gastroenterology, 101: 1680â€”1691, 1991.

46. Klecker, R. W., Jamis-Dow, C. A., Egorin, M. J., Erkmen, K., Parker, R. J., Stevens,R., and Collins, J. M. Effect of cimetidine, probenecid and ketoconazole on thedistribution. Biliary secretion and metabolism of (3H] Taxol in the Sprague-Dawleyrat. Drug Metab. Dispos., 22: 254â€”258, 1994.

47. Lewis, A. D., Lau, D. H. M., Duran, G. E., Wolf, C. R., and Sikic, B. I. Role ofcytochrome P-450 from the human CYP3A gene family in the potentiation ofmorpholino doxorubicin by human liver microsomes. Cancer Res., 52: 4379â€”4384,1992.

65



1996;56:58-65. Cancer Res Isabelle Royer, Bernard Monsarrat, Michelle Sonnier, et al. Interactions with Paclitaxel and Other Antineoplastic DrugsMetabolism of Docetaxel by Human Cytochromes P450:

Updated version

http://cancerres.aacrjournals.org/content/56/1/58

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/56/1/58To request permission to re-use all or part of this article, use this link


http://cancerres.aacrjournals.org/content/56/1/58http://cancerres.aacrjournals.org/cgi/alertsmailto:[email protected]://cancerres.aacrjournals.org/content/56/1/58http://cancerres.aacrjournals.org/

metabolism of docetaxel by human cytochromes p450: … · vitro docetaxel metabolism by human liver...

Documents