[CANCER RESEARCH 49, 833-837, February 15, 1989]

Excretion and Metabolism of Mitoxantrone in Rabbits1

Bruno Richard, GérardFahre,2 Isabelle Fahre, and Jean-Paul Cano2'3

INSERM U-278, Laboratoire Hospitalo-Universitaire de Toxicocinétiqueet de Pharmacocinétique,Facultéde Pharmacie, 27 Boulevard Jean Moulin, I3385 Marseille,Cedex S, France; and Institut I. Paoli-J. Calmettes, 232 Boulevard de S" Marguerite, 13273 Marseille, Cedex 9, France

ABSTRACT

The hepatic clearance of mitoxantrone was evaluated in rabbits usingboth bile-duct cannulated animals and freshly isolated hepatocytes insuspension or in primary culture. Mitoxantrone metabolic behavior wasassessed by high-performance liquid chromatography using a methodwhich specifically resolved mitoxantrone from its mono- and dicarboxylicacid derivatives. Excretion of mitoxantrone in bile and urine was studiedover a 6-h period of observation following i.v. bolus injection of 0.04,0.20, and 1.0 mg [MC]mitoxantrone/kg. Bile route represented the mainexcretion pathway for mitoxantrone and its metabolites—mainly themonocarboxylic acid derivative. Biliary excretion was very rapid (maximum biliary concentration achieved 9 to 18 min following drug administration) and amounted to 29.5 ±9.3%, 27.6 ±7.9%, and 28.3 ±3.8% ofadministered drug, respectively. Urinary excretion amounted to 7.3 ±0.2%, 7.1 ±4.6%, and 6.0 ±1.5%, respectively. Both biliary and urinaryexcretions of mitoxantrone and its metabolites remained linear over therange of concentrations routinely used in clinic. Metabolism of mitoxantrone was first studied using rabbit hepatocytes in suspension. Sincemetabolic rate was slow under these incubation conditions (observationperiod, 1 h), mitoxantrone metabolism was investigated in primary cultures of rabbit hepatocytes. Mitoxantrone was rapidly accumulated withinthe cells and metabolized to its various metabolites which rapidly effluxedin the extracellular medium. After a 48-h exposure of hepatocytes to abroad range of mitoxantrone concentrations (1 to 20 UM),it could be seenthat (a) drug accumulation and metabolism did not exhibit saturationprocesses, (h) mitoxantrone was the main intracellular form, while (c)metabolites rapidly effluxed in the extracellular compartment and (d)the monocarboxylic acid derivative represented the main extracellularmetabolite. This data demonstrates the important role played by the liverin the pharmacokinetic behavior of mitoxantrone and suggests a carefuldrug monitoring in patients with severe liver dysfunction.

INTRODUCTION

Mitoxantrone (Novantrone*; l,4-dihydroxy-5,8-bis((2-[(2-hydroxyethyl)amino]ethyl)amino)-9,10-anthracenedione dihy-drochloride; NSC 301739) is a new anthracenedione derivativecurrently used for the treatment of breast cancer and of patientswith acute nonlymphocytic leukemia (1-3). This anticancerdrug has shown antitumor activity equal or superior to that ofAdriamycin in a number of animal tumor systems and in thetumor stem cell assay (4, 5).

Different laboratories have investigated the pharmacokineticbehavior of mitoxantrone following various schedules of administration (6) with a view to optimizing the clinical use of thiscompound. However, few attempts have been made to elucidateits metabolism and the enzyme systems responsible for itsdisposition. Different authors have investigated the eliminationpathways of mitoxantrone in different species (7-10) includinghumans (11, 12). They found that mitoxantrone and drug-

Received10/13/87;revised6/14/88,9/20/88;accepted10/13/88.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This research was supported by Lederle-INSERM «Grant86-009 and bygrants from "La FédérationNationale des Centres de Lutte Contre le Cancer"and "La FédérationDépartementaledes Bouches du Rhônedes Centres de LutteContre le Cancer."

2 Present address: Sanofi Recherche, 32, Rue du Pr. J. Blayac, 34082 Mont

pellier Cedex, France.1To whom requests for reprints should be addressed.

related material were principally excreted via the bile route andthat urine was a minor excretory pathway. However, somediscrepancies occurred concerning mitoxantrone metabolites.In rats, mitoxantrone would appear to be metabolized to highlypolar derivatives, which have recently been identified as glucu-rono- and glutathione-conjugates (13). As opposed to this,major metabolites of mitoxantrone recovered in urine of humans (11, 12) or in primary cultures of human hepatocytes (14)were the mono- and dicarboxylic acids resulting from oxidationof the terminal hydroxyl groups of the side chain(s) (12).

This report investigates (a) the elimination pathways of mitoxantrone and its metabolites in bile-duct cannulated rabbitsand (h) the kinetics of mitoxantrone metabolism in primarycultures of rabbit hepatocytes.

MATERIALS AND METHODS

Drugs and Reagents. Mitoxantrone (M, 517.4), its mono- and dicarboxylic acid derivatives as well as 14C-labeled mitoxantrone were gen

erously supplied by Lederle Laboratories (Pearl River, NY). Radioactive[2-A>'i/ro.ryefA>'/-l4C]mitoxantrone (specific activity, 11.2 mCi/mmol)was 95% radiochemically pure as assessed by HPLC4 and was used

without further purification. Type IV collagenase was purchased fromSigma Chemical Co.

Chromatographie solvents were of analytical grade. Other chemicalsand reagents were obtained from regular commercial sources.

In Vivo Experiments. Experiments were performed in male NewZealand rabbits weighing 1.8-2.5 kg. Animals were anesthetized withurethane (2.0 g/kg) and kept at constant body temperature with warming lamps throughout the experiment. Preloading was achieved bycontinuous infusion into the left ear vein of a solution containing 0.3%NaCl and 1.3% glucose at 1.0 ml/min (15). A laparotomy was performed and the bile duct was cannulated with Vygon tubing (0.5-mminternal diameter) and tightly ligated. Bile samples were drawn atselected times after the i.v. injection and up to the sixth hour. Urinewas collected by way of a vesicourethral catheter.

[14C]Mitoxantrone was injected as a bolus (never exceeding 2 min)

in the left ear vein. Injected doses were 0.04, 0.20, and 1.00 mg/kg.In Vitro Experiments. Hepatocytes prepared from male New Zealand

rabbits weighing 0.6-1.0 kg, were obtained by a modification (16, 17)of the collagenase perfusion technique of Berry and Friend (18). Priorto experimentation, animals were anesthetized with urethane (2.0 g/kg). In brief, the liver was successively perfused with EGTA-supple-mented Kreb's Henseleit buffer and EGTA-free Kreb's Henseleit buffer,

followed by single-pass perfusion with 500 ml of the same mediumcontaining Sigma Type IV collagenase (0.5 mg/ml). The softened liverwas excised and the hepatocytes were resuspended in Leibovitz L-15medium containing 0.25% gelatine. The hepatocytes were separatedfrom nonparenchymal cells by repeated centrifugations (50 x g for 3min) and washing with the same medium. A single-cell suspension wasobtained by filtration through 150- and 60-^m nylon mesh. The viabilityof the cell suspension, as assessed by the exclusion of trypan blue was90% or higher.

Hepatocytes in suspension (5 x IO6cells/ml) were incubated at 37°C

in Leibovitz LI5 medium. The pH was maintained at 7.4 by passingwarm and humidified 95% Oz-5% CO? over the cell suspension. Thehepatocyte suspension was stirred throughout the incubation by aTeflon paddle as previously described (17).

The cell culture technique was similar to that described by other

4The abbreviation used is: HPLC, high-performance liquid chromatography.

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EXCRETION AND METABOLISM OF MITOXANTRONE IN RABBITS

authors (19, 20). Hepatocytes, resuspended in Ham's F-12 medium(0.85-1.0 x 10* cells/1.5 ml of medium) supplemented by 10% fetalcalf serum, were innoculated in 6-well plastic dishes (35 mm diameter).At the end of a 12-h exposure period, during which cells attached tothe plastic dishes, the medium and the dead cells were removed. Thehepatocytes were then incubated in serum-free Ham's F-12 medium in

the presence of various mitoxantrone concentrations. Extracellularmedia were recovered at the time points indicated. The monolayerswere rinsed twice with phosphate buffered saline (pH 7.4) and removedfrom the dishes by scraping into the same buffer. Both intra- andextracellular compartments were analyzed for total radioactivity byliquid scintillation counting and then by HPLC for quantification ofthe drug and its metabolites (14).

Sample Collection. Immediately before, and at various times after i.v.injection of the radiolabeled drug (every 1-2 min during the first hour,and every 20 min up to the sixth hour), bile samples were directlycollected in disposable tubes at 4°C.These samples were carefullyprotected from light and kept frozen at -20°C until analysis, which

was performed not later than 24 h after collection.Urine was continuously collected and total diuresis was recorded at

regular intervals: samples were kept frozen at —¿�20°Cin tubes protected

from light until HPLC analysis. Bile and urine samples were analyzedby HPLC for metabolite quantification without further processing.

Analytical Method. Analyses of both biological fluids and cellularmedia were performed with a Hewlett-Packard 1084B gradient liquidChromatograph equipped with a 10 (mi particle size (",* ¿tBondapak

column (30 cm x 3.9 mm; Waters Millipore S.A.). Elution was carriedout at 1.0 ml/min and absorbance was recorded at 254 nm. The mobilephase consisted of formiate buffer (pH 4.0; 1.6 HIM;solvent A) andacetonitrile/water (48/52; v/v; solvant B) (14, 21). The solvent programmer was set to deliver 45% to 60% of solvant B along a 15-minlinear gradient. Eluent from the column was analyzed by a radioactiveflow detector (Radiomatic Instruments). Under these HPLC conditions, mitoxantrone was baseline-separated from its various metabolites. Metabolites were identified according to their retention timesrelative to their standards.

RESULTS

Biliary and Urinary Excretions of Mitoxantrone and Its Metabolites. The biliary excretion of mitoxantrone and of itscarboxylic acid derivatives was studied over 6 h following i.v.injection of increasing doses of mitoxantrone, respectively 0.04,0.20, and l.OOmg/kg.

Fig. 1 illustrates the kinetics of total radiolabel in bile as afunction of time for each dosage regimen. Excretion of radio-label was very rapid: the time required to reach the peakconcentration ranged from 9 to 18 min. The maximal biliaryconcentrations of total radiolabel were 2.54 ±0.91, 13.16 ±2.91, and 74.19 ±24.41 Mi/ml for the different doses administered.

The quantification of unchanged drug and of its differentmetabolites was investigated after HPLC analysis of each bilesample. A typical chromatogram of mitoxantrone and metabolites in bile of rabbit is shown in Fig. 2A. Biliary excretiondata is shown in Table 1. In the first 6 h of collection, followingi.v. injection of 0.04, 0.20, and 1.00 mg/kg ['"Cjmitoxantrone,

the total of unchanged drug and metabolites excreted in thebile, amounted to 29.5 ±9.3% (n = 3), 27.6 ±7.9%, and 28.3±3.8% of total administered drug, respectively. Mitoxantronemetabolites, and in particular the monocarboxylic acid derivative, were present in appreciable amounts (Table 1). Anotherpolar derivative was observed despite the mono- and the dicar-boxylic acid derivatives which were identified by coelution withstandard metabolites under our Chromatographie conditions.Its structure remained unidentified since only very smallamounts of this latter compound were accumulated in the bile.

<cr

LUooo

mu

80

60

40

20

O

12

q„=i mg/kg

' •¿�, ,r i i Vvv'0.2

0.04

•¿�TV »V.,'*

HOURSFig. 1. Typical biliary1 profiles of total radiolabel in bile-duct cannulated

rabbits. Increasing doses (qa = 0.04, 0.20, and 1.00 mg/kg) of [14C]mitoxantronewere administered by short i.v. injections in the left ear vein of bile-duct cannulatedrabbits; radiolabel in bile fractions was determined by liquid scintillation counting.•¿�,individual quantifications obtained in different animals (three for each dose);O, mean of three results obtained in different animals.

The kinetics of biliary elimination for mitoxantrone and eachof its metabolites were further investigated after HPLC analysisof each bile sample. Fig. 3 illustrates the patterns of mitoxantrone and its mono- and dicarboxylic acid derivatives obtainedafter the i.v. injection of 0.20 mg [l4C]mitoxantrone/kg in bile-duct cannulated rabbits. Mitoxantrone and drug-related materials (mono- and dicarboxylic acid derivatives) achieved a maximum biliary concentration of 9.2, 1.7, and 0.2 Mg/ml, respectively, during the first 30 min of collection. Their levels thendecreased very rapidly to reach undetectable values after the6th hour. In Table 2 are reported the terminal half-life valuesat the elimination phase for mitoxantrone and its mono- anddicarboxylic acid derivatives for each dosage regimen.

The urinary excretion of mitoxantrone and its metaboliteswas also studied over the same period of time. Urinary excretiondata is given in Table 1. The total amount of unchangedmitoxantrone and its different metabolites excreted in the urinein the first six hours after i.v. injection of 0.04, 0.20, and 1.00mg/kg [l4C]mitoxantrone accounted for 7.3 ±0.2% (n = 3), 7.1

±4.6%, and 6.0 ±1.5% of the injected dose, respectively. Thetotal amount of metabolites represented only a very low percentage of excreted drug. A typical chromatogram of mitoxantrone and its metabolites in rabbit urine is shown in Fig. IB.

Metabolism of Mitoxantrone by Rabbit Hepatocytes. Theaccumulation and metabolism of mitoxantrone were first studied in freshly isolated rabbit hepatocytes in suspension. Hepatocytes were incubated over l h with increasing [14C]mitoxan-

trone concentrations and, respectively, 1.0, 10.0, and 100.0/iM.Under these experimental conditions, radiolabeled drug wasrapidly and intensively accumulated within the hepatocytes witha transmembrane chemical gradient (intra- over extracellularconcentration) of approximately 200 for each drug concentration studied, demonstrating that transport was not saturated

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EXCRETION AND METABOLISM OF MITOXANTRONE IN RABBITS

a.u

MITOXANTRONE

B. urine

C hepatocytes

10 15

MINUTES

Fig. 2. HPLC chromatograms of mitoxantrone and its different metabolitesobtained under various conditions. A, bile sample after i.v. injection of 1 mg/kg[MC|mitoxantrone; B, urine sample after i.v. injection of 1 mg/kg |'4C]mitoxan-trone; C, extracellular medium analyzed after a 24-h incubation of primary cultureof rabbit hepatocytes with ['4C]mitoxantrone.

Table 1 Biliar)- and urinary excretions of miloxantrone and ils variousmetabolites following i.v. injection off'C/mitoxantrone (n = 3)

Injected dose (mg/kg)

BileMitoxantroneMono-COOHDi-COOHPolarsUrineMitoxantroneMono-COOHDi-COOHPolars0.047.1±1.6°3.4

±1.50.6±0.60.4±0.52.5

±0.9°0.2+0.10.1

±0.050.1±0.050.2040.5

±13.512.3±4.01.1±0.60.2

±0.511.3±

10.51.5±2.01.3

±2.20.1±0.31.00204.2

±4755.4±11.312.4

±0.610.7±3.444.8

±14.56.0±1.84.5±0.94.7±1.9

" Results are expressed in ¿ig/kgof body weight.

over this concentration range. However under these experimental conditions only 10% of the initial drug was metabolized,mainly to the monocarboxylic acid derivative. So as to enablemitoxantrone metabolism to be more fully investigated, studieswere undertaken using primary cultures of rabbit hepatocytes.

The metabolic pattern of mitoxantrone was evaluated in bothintra- and extracellular compartments following incubation ofthe hepatocytes over 48 h with 5 /¿M['4C]mitoxantrone. Un

changed mitoxantrone disappeared rapidly to attain an extracellular concentration of 0.70 ±0.18 pM after 48 h (Fig. 4).The rapid decrease of extracellular mitoxantrone is attributableto its large-scale accumulation within the cells and its subsequent metabolism. Three metabolites appeared in the extracellular compartment. Two of them were identified (Fig. 1C) asthe mono- and dicarboxylic acid derivatives of mitoxantroneon the basis of their retention times relative to the standard

Fig. 3. Kinetics of mitoxantrone and its metabolite biliary excretion. Kineticsof biliary excretion for mitoxantrone and its mono- and dicarboxylic acid derivatives obtained after i.v. injection of 0.20 mg [uC]mitoxantrone/kg in bile duct-

cannulated rabbits.

Table 2 Half-life values for mitoxantrone and its metabolites following i.v.injection of { "CJmitoxantrone

Injected dose (mg/kg)

CompoundanalyzedMitoxantrone

Mono-COOHDi-COOH0.041.3

±0.8°

1.1 ±0.20.8 ±0.10.201.7

+ 0.11.4 ±0.22.0 ±0.31.001.3

±1.11.5 ±0.11.4 + 0.2

" Half-life values are expressed in hours.

<_1D-¡_jLUÜ

erh-

X

4 .

3 .

2 .

0 12 24

HOURS

Fig. 4. Time course of mitoxantrone metabolism. Freshly isolated rabbithepatocytes in primary culture were incubated over 48 h with 5 >iM[14C]mitox-

antrone and at previously selected times, aliquot parts of the extracellular compartment were analyzed by HPLC.

metabolites. The third metabolite corresponds to a very polarderivative since its retention time under these HPLC conditionswas identical to that of void volume. Although its identityremains unknown, it was not a degradative product generatedduring the incubation period. Indeed, when [14C]mitoxantronewas incubated for 48 h in hepatocyte-free medium, and themedium was analyzed by HPLC, neither metabolites nor degradative compounds were generated.

The monocarboxylic acid derivative first appeared in theextracellular compartment, i.e., 30 min after mitoxantroneincubation. Its level increased over the entire period of observation up to 1.28 ±0.50 fiM. The dicarboxylic acid derivativeappeared later. Its level remained lower but similar to that ofpolar derivatives (0.49 ±0.18 /IM and 0.39 ±0.14 ¿tM,respectively). After a 48-h incubation period, 75.0 ±5.0% of extracellular radiolabel was accounted for by metabolites amongwhich the monocarboxylic acid was the main derivative.

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EXCRETION AND METABOLISM OF MITOXANTRONE IN RABBITS

The intracellular compartment was also analyzed for itsmetabolite content. Results (expressed in nmol/106 cells) are

shown in Table 3.The intracellular content was analyzed following the incuba

tion of hepatocytes in primary culture with increasing [I4C]

mitoxantrone concentrations and for various exposure times.Unchanged mitoxantrone represented the main intracellularform at each time point studied, while under the same incubation conditions, the monocarboxylic acid derivative representedthe main extracellular form (Table 3).

Metabolism of mitoxantrone was also investigated after a 48-h exposure of hepatocytes in primary culture to increasing [I4C]

mitoxantrone concentrations ranging between 1 and 20 MM.Fig. 5 illustrates the 24-h extracellular concentrations of unchanged mitoxantrone and of its various metabolites, as afunction of the initial extracellular mitoxantrone concentration.Over this range of concentration, accumulation of both themono- and dicarboxylic acid derivatives remained linear, whilethe extracellular concentration of the polar derivative(s)achieved plateau values for initial mitoxantrone concentrationsranging between 10 and 20 MM.

The extracellular concentration of unchanged mitoxantroneincreased as a linear function of initial extracellular mitoxantrone concentrations of up to 15 MM.

Table 3 Analysis of intracellular radiolabel after exposure of primary cultures ofrabbit hepatocytes to increasing drug concentrations

Exposuretime(h)1

JIM2612485

nM26124820

MM261248Drug

concentration"Mitoxantrone0.22

±0.030.24±0.010.19

±0.030.16±0.021.19

+0.081.10±0.101.02

+0.040.96+0.124.72

±0.285.34±0.374.52+0.275.27±0.68mono-COOH0.04

±0.040.06±0.010.06±0.020.06+0.010.14

±0.040.17±0.030.21+0.040.33

+0.080.51

±0.160.73±0.090.93±0.171.87±0.56di-COOH000.01

±0.010.02±0.020.04

±0.030.07±0.060.08±0.020.11

±0.020.07

±0.120.17±0.160.31±0.100.31

±0.27Polar00000.05

±0.050.12±0.050.09

±0.040.11±0.050.36

±0.140.19±0.170.21

±0.200.15+ 0.27

" Resultsare expressedin nmol/10*cells.

i 3

LUO 1

oo o

mono COOH

di COOH

10 15

MITOXANTRONE

20

uM

Fig. 5. Metabolism of mitoxantrone as a function of extracellular mitoxantrone concentration. Freshly isolated rabbit hepatocytes in primary culture wereincubated with increasing [14C]mitoxantrone concentrations. After a 48-h expo

sure, extracellular compartment was recovered and analyzed by HPLC. Data arethe average of two or the mean of three different experiments.

DISCUSSION

This report demonstrates that the liver plays an importantrole in the pharmacokinetic behavior of mitoxantrone.

This important conclusion is based upon the following observations.

First in rabbits, mitoxantrone and its metabolites were rapidlyand intensively excreted via the bile route. Over a 6-h period ofobservation, 25 to 30% of the administered drug was eliminatedin bile. Drug-related material, and the monocarboxylic acidderivative in particular, represented approximately 30% of theexcreted dose. Urinary excretion of mitoxantrone and its metabolites was, to the contrary, low, amounting to less than 6 to8% of the dose during a 6-h observation period. Both the biliaryand urinary excretions of mitoxantrone and its metabolitesremained linear over a range of doses routinely used in clinic,e.g., 0.04 to 1.0 mg/kg. The intense biliary excretion of drugshown in rabbit was in agreement with previous reports onstudies with rats (8-10), dogs (7), and humans (11, 12). Thepresent study demonstrates that metabolites, and most importantly the monocarboxylic acid derivative, represent a highpercentage of the excreted material. Ehninger et al. (8), usingthe isolated perfused rat liver model, also reported the presenceof three mitoxantrone metabolites. The major metabolite, themost polar, accounted for 80% of the excreted material. On thebasis of the respective Chromatographie conditions, this metabolite would not correspond to the mono- and dicarboxylic acidderivatives we identified in rabbits, but rather to our polarmetabolite. This (these) latter metabolite(s) would appear tocorrespond to a conjugate derivative with glucuronic acid orglutathione (13). This suggests large interspecies differences inmetabolic patterns between rat and rabbit.

Second, using freshly isolated rabbit hepatocytes in primaryculture we demonstrated that mitoxantrone was intensivelymetabolized to mono- and dicarboxylic acid derivatives. Another metabolite was also recovered in the extracellular compartment but its level remained low over the entire period ofobservation. Although this latter remained unidentified it may,in accordance with its characteristics determined under ouranalytical conditions, correspond to a conjugate derivative.Metabolites synthesized in the intracellular medium rapidlyeffluxed in the extracellular compartment, representing approximately 70% of the extracellular material after a 48-h exposureto mitoxantrone. The metabolites identified in the extracellularmedium following exposure of rabbit hepatocytes in primaryculture to mitoxantrone, are qualitatively and quantitativelysimilar to those observed in bile. Previous reports from ourlaboratory (14) have shown that mitoxantrone is also intensivelymetabolized by both rat and human hepatocytes in primaryculture. While mono- and dicarboxylic acid derivatives were themain derivatives recovered after exposure of human hepatocytesto mitoxantrone, the main derivative observed in rats was apolar one perhaps corresponding to the conjugate derivative(s)observed by Ehninger (8) and Wolf (13). From our studies inrat (14), rabbits, and humans (14), it can be assumed that therabbit is more closely related to humans than the rat at least asfar as the metabolic pattern is concerned. The appearance andthe accumulation of the mono- and dicarboxylic acid derivativesincreased in a concentration-dependent manner, when hepatocytes in primary culture were exposed to mitoxantrone concentrations ranging between 1 and 20 MM.This concentration rangecorresponds to the medium and high plasma levels routinelyencountered in clinic (21). The approximate linearity observedfor intra- and extracellular accumulation and metabolism has

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EXCRETION AND METABOLISM OF MITOXANTRONE IN RABBITS

also been observed by others (22, 23). Burns et al. (22) showedin L-1210 leukemia cells that passive diffusion is the mechanismof mitoxantrone uptake, since the uptake remained linear overa broad concentration range, temperature-dependence was limited and transport was not impeded by a wide range of metabolicinhibitors.

This first detailed analysis of mitoxantrone excretion andmetabolism in rabbit, demonstrates the important role playedby the liver in the pharmacokinetic behavior of this new anti-tumor drug. Thus, liver dysfunction may result in decreasedclearance of mitoxantrone. Therefore, and in agreement withother authors (24-26), drug dosages should be reduced inpatients with severe liver dysfunction.

ACKNOWLEDGMENTS

We would like to thank Nicole Lénaand Georges De Sousa for theirkind cooperation and Sylvaine Just for her technical collaboration.

REFERENCES

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1989;49:833-837. Cancer Res   Bruno Richard, Gérard Fabre, Isabelle Fabre, et al.   Excretion and Metabolism of Mitoxantrone in Rabbits

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