Preclinical Evaluation of the Cardiotoxicity of Taxane–Anthracycline Combinations Using the Model of Isolated Perfused Rat Heart

Toxicology and Applied Pharmacology 163, 135–140 (2000) doi:10.1006/taap.1999.8847, available online at http://www.idealibrary.com on

Preclinical Evaluation of the Cardiotoxicity of Taxane–Anthracycline Combinations Using the Model of Isolated Perfused Rat Heart Denis Platel,* ,† Paul Pouna,* ,† Simone Bonoron-Ade`le,‡ and Jacques Robert* ,† ,1 *Institut Bergonie´, Bordeaux, France; †University Victor Segalen, Bordeaux, France; and ‡INSERM U441, Pessac, France Received March 24, 1999; accepted November 8, 1999

Preclinical Evaluation of the Cardiotoxicity of Taxane–Anthracycline Combinations Using the Model of Isolated Perfused Rat Heart. Platel, D., Pouna, P., Bonoron-Ade`le, S., and Robert, J. (2000). Toxicol. Appl. Pharmacol. 163, 135–140. Paclitaxel strongly potentiates the cardiotoxicity of doxorubicin in the clinical setting. In this study, we aimed (1) to determine whether this potentiation could be reproduced in an ex vivo model and, if so, (2) to select drugs and protocols that did not cause this potentiation. The effect of paclitaxel and docetaxel on the cardiotoxicity induced by doxorubicin and epirubicin was studied using the model of isolated perfused rat heart. Cardiac performances were evaluated after several combination protocols administered every 2 days over a period of 12 days, and anthracycline concentrations in the heart and liver were determined on Day 12. When administered simultaneously, paclitaxel strongly potentiated the cardiotoxicity of doxorubicin ex vivo, and this effect was not due to Cremophor EL, the solvent used in the formulation of paclitaxel. The potentiation of anthracycline cardiotoxicity could be avoided by the replacement of doxorubicin by epirubicin, and/or of paclitaxel by docetaxel. Cardiotoxic potentiation was also avoided by the introduction of a 24-h lag time between the repetitive injections of doxorubicin and docetaxel. The concentration of doxorubicin and its cardiotoxic metabolite, doxorubicinol, in the heart and liver was not significantly altered by the taxanes, but that of epirubicin was increased twofold both in the heart and the liver. These results show that the potentiation of doxorubicin-induced cardiotoxicity by paclitaxel can be reproduced with an ex vivo model, and that it is not related to an increase in tissue concentration of the drug or active metabolite. Our model, therefore, may be useful for the selection of anthracycline-containing protocols with no increased risk of cardiotoxicity for the patients. © 2000 Academic Press

Key Words: cardiotoxicity; anthracyclines; taxanes; cancer; chemotherapy.

Anthracyclines have been established for more than two decades as among the most effective antineoplastic agents for the treatment of hematological malignancies and solid tumors. 1 To whom correspondence should be addressed at Institut Bergonie´, 180 rue de Saint-Gene`s, 33076 Bordeaux-cedex, France. Fax: (33) 556 33 33 89; E-mail: [email protected].

They have allowed a significant improvement in the treatment of breast cancer, both in the adjuvant and in the palliative settings. The combination of doxorubicin (often replaced by epirubicin in Europe) with cyclophosphamide and 5-fluorouracil now represents the standard chemotherapy of advanced and metastatic breast cancer. More recently, the demonstration of the high efficiency of taxanes (paclitaxel and docetaxel) in breast cancer has encouraged the study of taxane–anthracycline combinations for the treatment of this disease. Such combinations have achieved high response rates in the clinic. One study, using a combination of doxorubicin (50 mg/m 2) and paclitaxel (175 mg/m 2 over 3 h) achieved an objective response rate of 94% in patients with metastatic breast cancer (Gianni et al., 1995). This high activity was confirmed later by other authors (Gehl et al., 1996; Hortobagyi et al., 1997). Similar response rates have been obtained with the combination of doxorubicin with docetaxel (Misset et al.,1998) and it is expected that such combinations will rapidly become the standard first-line treatment of metastatic breast cancer. The impressive results published by Gianni et al. (1995) and others (Gehl et al., 1996; Hortobagyi et al., 1997), however, were obtained at the expense of an increase in the cardiotoxicity of doxorubicin. The cardiotoxicity of anthracyclines is the major factor limiting their use. Chronic anthracycline-induced cardiomyopathy usually occurs within 1 year after completion of the treatment; its frequency remains low below a cumulative dose of 550 mg/m 2, but rapidly increases above this dose. Cardiomyopathy is fatal in more than 20% of patients, and is rarely reversible once it has occurred (Shan et al., 1996). Therefore, an increase in the risk of congestive heart failure is not tolerable in combination protocols. In the experience of Gianni et al. (1995), the risk of congestive heart failure associated with a doxorubicin–paclitaxel combination was 18%, at a median dose of doxorubicin of 480 mg/m 2, which makes this highly efficient combination the most cardiotoxic to date. One of the pitfalls in anthracycline cardiotoxicity is the lack of predictive tools that allow evaluation of the potential cardiotoxic risk of a new anthracycline or of a new combination. In the clinic, the best predictive parameters of cardiac injury are the decrease of both the left ventricular ejection fraction (LVEF) by isotopic ventriculography, and the shortening frac-

135

0041-008X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

136

PLATEL ET AL.

tion by echocardiography. However, the onset of these alterations can be sudden, and the subsequent decision to stop the treatment may come too late to prevent congestive heart failure. Therefore, clinical trials of taxane–anthracycline combinations must be conducted with caution and may not be able to avoid fatal side effect. We have recently developed and validated an ex vivo model for the preclinical evaluation of anthracycline cardiotoxicity (Pouna et al., 1996). This model was able to correctly predict the relative levels of cardiotoxicity of several anthracyclines, and also the protective effect of dexrazoxane. The model was also used to test the paclitaxel– doxorubicin combination and several variants of this protocol. We show in this report that paclitaxel potentiates the cardiotoxicity of doxorubicin, and that this effect is not related to Cremophor EL, the solvent used for the formulation of paclitaxel. We also show that the introduction of a time interval between the administrations of doxorubicin and paclitaxel can avoid the potentiation of the cardiotoxicity of doxorubicin. Alternative combinations, such as replacing doxorubicin by epirubicin or paclitaxel by docetaxel, were also investigated and did not potentiate anthracycline cardiotoxicity. MATERIALS AND METHODS Drugs and Chemicals Doxorubicin and epirubicin were kindly provided as clinical formulations by Pharmacia and Upjohn (Saint-Quentin-en-Yvelines, France). They were diluted with sterile water to a final concentration of 5 mg/ml, divided into aliquots, and stored at 4°C until use for a maximum of 2 weeks. Paclitaxel and docetaxel were kindly provided as clinical formulations by Bristol-MyersSquibb (Paris La De´fense, France) and Rhoˆne-Poulenc Rorer (Antony, France), respectively. They were extemporaneously diluted with 0.9% NaCl (1:3.8). Cremophor EL was purchased from Sigma Chimie (Saint-QuentinFallavier, France) and dissolved in the required amount of ethyl alcohol to reach the same concentration as in the clinical formulation of paclitaxel. All solvents and chemicals were of analytical grade. Special care was taken with the water used for the perfusion medium; sterile apyrogenic water was used, which was prepared for parenteral injections in humans. Experimental Animals All animal experiments described in this report were done in accordance with the guidelines of the Institut National de la Sante´ et de la Recherche Me´dicale. Several experimental treatments were administered ip to male Sprague–Dawley rats (age 10 –12 weeks) every other day for 11 days. The rats were weighed every 2 days and assessed for possible abnormalities such as ascites, diarrhea, or epistaxis. Rats were killed on the 12th day after the first injection, hearts were removed and perfused, cardiac functional parameters were monitored as described below, and the hearts were weighed after the end of the experiment. Independent series of animals were treated similarly but, after removal of the heart, approximately 120-mg portions of both the left ventricular myocardium and the liver were sampled and kept frozen for the evaluation of the anthracycline cardiac concentration as described below. The following treatments were performed: (1) 0.9% NaCl solution, for control; (2) doxorubicin, 3 mg/kg/day; (3) paclitaxel, 2.5 mg/kg/day; (4) Cremophor EL in ethyl alcohol, 220 mg/kg/day; (5) docetaxel, 2.5 mg/kg/day; (6) doxorubicin, 3 mg/kg/day, plus paclitaxel, 2.5 mg/kg/day; (7) doxorubicin, 3 mg/kg/day, plus docetaxel, 1.5 mg/kg/day; (8) doxorubicin, 3 mg/kg/day, plus docetaxel, 2.5 mg/kg/day; (9) doxorubicin, 3 mg/kg/day, plus Cremophor

EL in ethyl alcohol, 220 mg/kg/day. In addition, similar experiments were performed after treatment with: (10) epirubicin, 3 mg/kg/day; (11) epirubicin, 3 mg/kg/day, plus paclitaxel, 2.5 mg/kg/day; and (12) epirubicin, 3 mg/kg/day, plus docetaxel, 2.5 mg/kg/day. In all combinations, anthracyclines and taxanes were administrated simultaneously. Finally, to study the influence of the schedule of doxorubicin–paclitaxel combinations, the drugs were administered sequentially, (13) doxorubicin (3 mg/kg/day) being injected on days 1, 3, 5, 7, 9, and 11, and paclitaxel (2.5 mg/kg/day) on days 2, 4, 6, 8, 10, and 12. Perfusion of Isolated Rat Hearts Rats were heparinized ip (500 IU/100 g body weight) and anesthetized with diethylether. The heart was quickly excised and soaked in Krebs–Henseleit solution at 4°C. Coronary perfusion was initiated through a short cannula in the aortic root and maintained at a constant pressure of 90 mm Hg in a nonrecirculating way by the Langendorff technique as described by Lorell et al. (1986). Perfusion pressure was measured by a P23Db transducer (Bentley Trantec) connected to the aortic infusion cannula. The heart was electrically paced at a rate of 300 beats/min (5 Hz) through stimulator-activated stainless steel electrodes placed on the heart. A latex balloon attached to one end of a polyethylene catheter was placed in the left ventricle through the mitral valve. The catheter was filled with water and the other end was linked to an electronic amplifier (Thomson Medical) by a second P23Db transducer. The coronary perfusion pressure and the left ventricular pressure were recorded by a computer that allowed continuous monitoring of heart rate, left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP ⫽ LVSP ⫺ LVEDP), and the maximal and minimal first derivatives of LVDP as a function of time (LV(dP/dt) max and LV(dP/dt) min), respectively. The perfusate consisted of a modified Krebs–Henseleit buffer, pH 7.4, containing NaCl (118 mM), KCl (4.7 mM), MgSO 4 (1.2 mM), KH 2PO 4 (1.2 mM), NaHCO 3 (25 mM), glucose (11 mM), CaCl 2 (0.95 mM), and insulin (10 IU/L). It was continuously bubbled with a mixture of 95% O 2/5% CO 2 and maintained at 37°C. The latex balloon inserted in the left ventricle was periodically dilated with distilled water in order to produce a LVEDP of 5 to 6 mm Hg. After 30 to 45 min stabilization, necessary to reach the maximal functional cardiac values, the preceding parameters were measured and recorded. The coronary flow progressively increased during stabilization and then remained constant for at least 30 min. Anthracycline Concentrations The samples obtained from the hearts and livers of rats treated with anthracyclines, alone or in combination with taxanes, were homogenized in physiological saline (2 ml for 100 mg tissue) with a tissue homogenizer (UltraTurrax). After addition of an adequate amount of internal standard (daunorubicin) and of 0.5 ml borate buffer (50 mM, pH 9.8) to 0.5 ml of the homogenate, anthracyclines and metabolites were extracted with 9 ml of chloroform/methanol 4/1 (v/v), according to Baurain et al. (1979). After mixing and centrifugation (10 min at 3000g), the solvent layer was recovered, evaporated to dryness, and reconstituted with 200 ␮l methanol. Calibration curves were obtained after incubating heart homogenates with doxorubicin or epirubicin in vitro for 15 min at room temperature. For both anthracyclines, a good linearity was obtained from 0.015 to 1.5 nmol/g tissue. Chromatography was performed on a Radial-Pack C18 column (Waters Associates, SaintQuentin-en-Yvelines, France) inserted in a compression device. The solvent was a mixture of ammonium formate buffer (60 mM, pH 4.0) and acetonitrile (68/32) delivered at 2 ml/min. Detection was by a laser-induced fluorescence recorder (Zeta Technology, Toulouse, France) with excitation and emission wavelengths of 488 and 550 nm, respectively. Retention times and peak areas were recorded by a microcomputer, using the PC1000 software (Thermo Quest, Les Ulis, France). Statistical Analysis of the Data The weight changes occurring in each group during treatment were compared using the Student’s t test after ANOVA assumption of the validity of the

137

TAXANE–ANTHRACYCLINE CARDIOTOXICITY

TABLE 1 General Toxicity Induced by Various Treatments

Control Cremophor EL (220 mg/kg/day) Paclitaxel (2.5 mg/kg/day) Docetaxel (2.5 mg/kg/day) Doxorubicin (3 mg/kg/day) Epirubicin (3 mg/kg/day) Doxorubicin (3 mg/kg/day) ⫹ Cremophor EL (220 mg/kg/day) Doxorubicin (3 mg/kg/day) ⫹ Paclitaxel (2.5 mg/kg/day), simultaneously every other day Doxorubicin (3 mg/kg/day) ⫹ Paclitaxel (2.5 mg/kg/day), alternately Doxorubicin (3 mg/kg/day) ⫹ Docetaxel (1.5 mg/kg/day) Doxorubicin (3 mg/kg/day) ⫹ Docetaxel (2.5 mg/kg/day) Epirubicin (3 mg/kg/day) ⫹ Paclitaxel (2.5 mg/kg/day) Epirubicin (3 mg/kg/day) ⫹ Docetaxel (2.5 mg/kg/day)

Early deaths

Body weight variation (%)

Diarrhea

Epistaxis

0/11 0/9 0/7 0/8 1/12 0/9

⫹30 ⫾ 1 ⫹29 ⫾ 2 ⫹8 ⫾ 2* ⫺8 ⫾ 4* ⫺15 ⫾ 2* ⫺7 ⫾ 1*

0/11 0/9 0/7 0/8 4/11 0/9

0/11 0/9 0/7 0/8 8/11 3/9

1/8

⫺24 ⫾ 2†

7/7

6/7

4/11

⫺18 ⫾ 7

6/7

6/7

7/18 1/11 1/9 4/11 7/17

⫺22 ⫾ 1 ⫺29 ⫾ 2†† ⫺25 ⫾ 3† ⫺17 ⫾ 2†† ⫺25 ⫾ 2††

6/11 7/10 5/8 5/7 9/10

9/11 6/10 6/8 4/7 7/10

Note. Values are presented as means ⫾ SEM. Significant differences are as follows: drug alone vs control: *p ⬍ 0.001; drug combination vs anthracycline alone: †p ⬍ 0.01; ††p ⬍ 0.001.

test. However, since the variances of the data obtained in the different groups for the parameters of cardiac function were markedly different, we used the Mann–Whitney nonparametric test to analyze the results. Comparisons were made between untreated animals and those treated with a single drug, and between animals treated with an anthracycline and those treated with a combination of this anthracycline with another drug. All data are expressed as means ⫾ SEM. Since more than 20 comparisons were made for each parameter, statistical significance was determined as p ⬍ 0.01.

RESULTS

General Toxicity of Treatments (Table 1) Doxorubicin treatment induced mortality, significant weight loss, as well as side effects such as epistaxis and diarrhea. Epirubicin promoted similar effects, although to a lesser extent. Paclitaxel alone, at the dose of 2.5 mg/kg/day, limited the gain of body weight, and docetaxel, at the same dose, led to a 7% weight loss. Cremophor EL, the paclitaxel vehicle, was devoid of any side effect. The combination of doxorubicin with paclitaxel (simultaneously or alternately) strongly increased the extent of these side effects and the number of early deaths. Cremophor EL and docetaxel at 1.5 and 2.5 mg/kg/day, respectively, both potentiated the doxorubicin-induced general toxicity. Paclitaxel and docetaxel also increased the general toxicity of epirubicin. Effect of Various Treatments on Cardiac Function (Table 2) Doxorubicin at a dose of 3 mg/kg/day induced significant alterations of the cardiac functional parameters in rats. There was a 20% reduction in heart contractility (LV[dP/dt] max) and a 33% reduction in heart relaxation (LV[dP/dt] min), which reflect

the cardiotoxic effects already shown with this model. In contrast, paclitaxel alone at a dose of 2.5 mg/kg/day induced no significant alteration of cardiac function. When doxorubicin and paclitaxel were administered in combination using the same protocol and the same doses, there was a dramatic increase in the alterations of the cardiac functional parameters compared with doxorubicin alone: the decrease in heart contractility and relaxation was 39 and 46% of the control values, respectively. To determine whether this potentiation of doxorubicin cardiotoxicity was the result of paclitaxel itself or to the solvent used for its clinical formulation, the effect of Cremophor EL on these parameters was investigated. Cremophor EL alone appeared to have a slight, nonsignificant inotropic effect with an increase of LV(dP/dt) max and LV(dP/dt) min of 21 and 14%, respectively, compared to control animals. Also, there was no significant difference in any cardiac parameter between doxorubicin alone and the combination doxorubicin–Cremophor EL. This excludes the participation of Cremophor EL in the potentiation of doxorubicin cardiotoxicity by paclitaxel, despite the significant increase in the general toxicity symptoms induced by doxorubicin. The doxorubicin–paclitaxel combination was compared to combinations replacing doxorubicin by epirubicin, and paclitaxel by docetaxel. When used at the same dose, epirubicin alone had a slightly lower cardiotoxicity than doxorubicin (p ⫽ 0.02 for both contractility and relaxation), as already shown with this model (Pouna et al., 1996). When epirubicin was combined with paclitaxel, as was doxorubicin, there was no potentiation of cardiotoxicity. When used alone at 2.5 mg/kg/

138

PLATEL ET AL.

TABLE 2 Effect of Various Treatments on Cardiac Functional Parameters Number evaluable Control Cremophor EL (220 mg/kg/day) Paclitaxel (2.5 mg/kg/day) Docetaxel (2.5 mg/kg/day) Doxorubicin (3 mg/kg/day) Epirubicin (3 mg/kg/day) Doxorubicin (3 mg/kg/day) ⫹ Cremophor EL (220 mg/kg/day) Doxorubicin (3 mg/kg/day) ⫹ Paclitaxel (2.5 mg/kg/day), simultaneously every other day Doxorubicin (3 mg/kg/day) ⫹ Paclitaxel (2.5 mg/kg/day), alternately Doxorubicin (3 mg/kg/day) ⫹ Docetaxel (1.5 mg/kg/day) Doxorubicin (3 mg/kg/day) ⫹ Docetaxel (2.5 mg/kg/day) Epirubicin (3 mg/kg/day) ⫹ Paclitaxel (2.5 mg/kg/day) Epirubicin (3 mg/kg/day) ⫹ Docetaxel (2.5 mg/kg/day)

LVDP (mm Hg) 118.4 ⫾ 126.4 ⫾ 97.2 ⫾ 111.4 ⫾ 87.5 ⫾ 109.2 ⫾

9 9 7 8 11 9

5.4 6.6 5.4 6.9 4.4** 7.6

LV(dP/dt) max (mm Hg)

LV(dP/dt) min (mm Hg)

2812 ⫾ 178 3427 ⫾ 194 2676 ⫾ 118 2698 ⫾ 225 2212 ⫾ 90* 2741 ⫾ 183

⫺2169 ⫾ 128 ⫺2466 ⫾ 143 ⫺1856 ⫾ 142 ⫺2088 ⫾ 197 ⫺1441 ⫾ 68** ⫺1856 ⫾ 148 ⫺1458 ⫾ 165

7

88.8 ⫾ 7.7

2174 ⫾ 206

7

54.3 ⫾ 5.6†

1350 ⫾ 140†

11 10 8 7 10

97.5 ⫾ 6.7 92.4 ⫾ 10.2 84.3 ⫾ 8.7 97.4 ⫾ 2.8 98.5 ⫾ 8.6

2228 ⫾ 233 2006 ⫾ 284 1833 ⫾ 235 2175 ⫾ 70 2427 ⫾ 280

⫺765 ⫾ 115† ⫺1572 ⫾ 138 ⫺1457 ⫾ 193 ⫺1329 ⫾ 170 ⫺1612 ⫾ 48 ⫺1588 ⫾ 148

Note. Values are presented as means ⫾ SEM. Significant differences are as follows: drug alone versus control: *p ⬍ 0.01; **p ⬍ 0.001; drug combination versus anthracycline alone: †p ⬍ 0.001.

day, paclitaxel presented no significant cardiotoxicity. The combination of docetaxel (at 1.5 or 2.5 mg/kg/day) with doxorubicin (3 mg/kg/day), did not significantly increase the alteration of the cardiac functional parameters induced by doxorubicin, despite a marked increase in general toxicity. Similarly, the combination of epirubicin and docetaxel did not increase the alterations of the cardiac performances induced by epirubicin. Finally, the possibility that the potentiation of doxorubicin cardiotoxicity by paclitaxel could be reduced by the introduction of a lag time between administrations was explored. To this end, rats were treated alternately with each drug, instead of simultaneously every other day; in these conditions, there was

no significant increase in cardiac toxicity over that of doxorubicin alone. Concentrations of Anthracyclines in Heart and Liver (Table 3) Paclitaxel and docetaxel did not significantly modify the accumulation of doxorubicin in both the heart and the liver, despite a trend toward an increased doxorubicin accumulation in the heart when associated with paclitaxel. Similarly, Cremophor EL tended to enhance the cardiac accumulation of doxorubicin but also did not potentiate the alterations of the cardiac performances induced by the anthracycline. Doxorubicinol concentrations were low (⬍10% of the doxorubicin

TABLE 3 Tissue Accumulation of Doxorubicin and Epirubicin

Number evaluable Doxorubicin (3 mg/kg/day) Doxorubicin (3 mg/kg/day) ⫹Cremophor EL (220 mg/kg/day) Doxorubicin (3 mg/kg/day) ⫹ Paclitaxel (2.5 mg/kg/day), simultaneously every other day Doxorubicin ( 3 mg/kg/day) ⫹ Paclitaxel (2.5 mg/kg/day), alternately Doxorubicin (3 mg/kg/day) ⫹ Docetaxel (1.5 mg/kg/day) Epirubicin (3 mg/kg/day) Epirubicin (3 mg/kg/day) ⫹ Paclitaxel (2.5 mg/kg/day) Epirubicin (3 mg/kg/day) ⫹ Docetaxel (2.5 mg/kg/day)

Heart (nmol/g tissue)

Liver (nmol/g tissue)

17

5.0 ⫾ 0.9

15.4 ⫾ 3.8

11

6.4 ⫾ 1.5

19.3 ⫾ 4.6

12

7.2 ⫾ 1.7

11.7 ⫾ 4.0

5 10 13 10 11

4.1 ⫾ 1.2 3.7 ⫾ 0.7 2.9 ⫾ 0.4 6.0 ⫾ 0.6** 6.2 ⫾ 1.5*

not done 6.0 ⫾ 1.3 12.6 ⫾ 3.1 29.6 ⫾ 4.0* 20.2 ⫾ 2.0*

Note. Values are presented as mean ⫾ SEM. Significant differences are as follows: drug combination versus anthracycline alone: *p ⬍ 0.05; **p ⬍ 0.01.

139

TAXANE–ANTHRACYCLINE CARDIOTOXICITY

concentrations) and similar, whatever the treatment used (data not shown). However, both paclitaxel and docetaxel significantly enhanced the concentration of epirubicin in the heart and liver. DISCUSSION

The mechanism of anthracycline cardiotoxicity is generally considered to be a result of a one-electron reduction of the molecule, followed by the generation of oxygen free radicals, which damage the mitochondrial membrane and impair energy production (Olson and Mushlin, 1990). Functional alterations in cardiac contractility and relaxation, therefore, appear as the primary events leading to heart failure in humans. Thus, an ex vivo model that can evaluate these alterations represents an important experimental tool. The model we have implemented appears to be rapidly predictive of the clinical cardiotoxicity of anthracyclines. In the present study, we show that the potentiation of doxorubicin cardiotoxicity by paclitaxel in the clinical setting could have been predicted if this combination had been evaluated with our model. In addition, we show that the replacement of either drug by one of its most common analogs, i.e., epirubicin for doxorubicin and docetaxel for paclitaxel, does not lead to potentiation of cardiotoxicity, and that the resulting combinations, therefore, can be tested more safely in patients for the evaluation of anticancer efficacy. Similarly, as we have shown in the model, the introduction of a delay between the administration of doxorubicin and paclitaxel decreases the cardiotoxicity of the combination in the clinic (Amadori et al., 1996; Hudis et al., 1998). Recently, clinical trials aimed at the evaluation of combinations including epirubicin or docetaxel instead of doxorubicin and paclitaxel have been undertaken, and the preliminary results now available have not shown any dramatic increase in the cardiotoxicity of the anthracycline (Conte et al., 1997; Misset et al., 1998). However, the evaluation of the cardiotoxicity of any drug or drug combination is relatively difficult and requires the registration of a sufficient number of events before comparisons can reach statistical significance. We believe that our results can encourage and accelerate the development of such clinical trials. The mechanism involved in the potentiation of doxorubicin cardiotoxicity by paclitaxel remains unknown. It has been postulated that Cremophor EL, the solvent used for the formulation of paclitaxel in clinics, was involved in this potentiation. Indeed, Cremophor EL is responsible for the nonlinearity of paclitaxel pharmacokinetics (Sparreboom et al., 1996); it also alters the pharmacokinetics of doxorubicin (Webster et al., 1996). However, in our experimental conditions, Cremophor EL plays no direct role in the potentiation of the cardiotoxicity of doxorubicin by paclitaxel, despite a strong increase in the general toxicity of doxorubicin. Cremophor EL displays some positive inotropic effect of its own, at the limit of significance, but this effect disappears when combined with paclitaxel,

which indicates an interaction between both drugs. Alterations of the pharmacokinetics of doxorubicin by paclitaxel have been found by Gianni et al. (1997) and Holmes et al. (1996). When paclitaxel (24-h infusion) was administered before doxorubicin (48-h infusion), a decrease in doxorubicin clearance was shown (Holmes et al., 1996). However, when administered as a 3-h infusion, the effect of paclitaxel on doxorubicin pharmacokinetics was much smaller and concerned mainly the linearity of the kinetics (Gianni et al., 1997). Prior to these detailed studies, it had been shown that doxorubicinol accumulates in plasma and tissues to a higher extent when paclitaxel was given in combination with doxorubicin (Berg et al., 1994). In contrast, paclitaxel tended to reduce epirubicinol concentrations in plasma in another study of an epirubicin–paclitaxel combination (Conte et al., 1997). Our results do not confirm these data, since we did not observe any significant difference in doxorubicinol tissue concentration when doxorubicin was administered alone or in combination with paclitaxel or docetaxel. In addition, the taxane-induced increase in epirubicin concentration observed in the present study was not associated with a potentiation of cardiotoxicity of this anthracycline, although general toxicity was greatly potentiated. Thus, the potentiation of doxorubicin cardiotoxicity by paclitaxel cannot be ascribed to an increase in the cardiac accumulation of the drug, and the mechanism by which such a potentiation occurs remains to be determined. ACKNOWLEDGMENTS This work was supported in part by a grant from Bristol-Myers-Squibb. We are grateful to Mrs. Liliane Tariosse, Mr. Ge´rard Gouverneur, and Mrs. Christine Garcia for technical assistance. Dr. Ricardo Bellott is acknowledged for his help and advice for HPLC. Dr. Simone Mathoulin-Pe´lissier and Mrs. Ve´ronique Picot are acknowledged for their assistance in statistical analysis of the data.

REFERENCES Amadori, D., Frassineti, G. L., Zoli, W., Tienghi, A., Ravaioli, A., Giunchi, D. C., Gentile, A., and Salzano, E. (1996). A phase I/II study of paclitaxel and doxorubicin in the treatment of advanced breast cancer. Semin. Oncol. 23(Suppl. 1), 19 –23. Baurain, R., Deprez-De Campaneere, D., and Trouet, A. (1979). Rapid determination of doxorubicin and its fluorescent metabolites by high pressure liquid chromatography. Anal. Biochem. 94, 112–116. Berg, S. L., Cowan, K. H., Balis, F. M., Fisherman, J. S., Denicoff, A. M., Hillig, M., Poplack, D. G., and Oshaughnessy, J. A. (1994). Pharmacokinetics of Taxol and doxorubicin administered alone and in combination by continuous 72-hour infusion. J. Natl. Cancer Inst. 86, 143–145. Conte, P. F., Baldini, E., Gennari, A., Michelotti, A., Salvadori, B., Tibaldi, C., Danesi, R., Innocenti, F., Gentile, A., Dellanna, R., Biadi, O., Mariani, M., and Del Tacca, M. (1997). Dose-finding study and pharmacokinetics of epirubicin and paclitaxel over 3 hours: A regimen with high activity and low cardiotoxicity in advanced breast cancer. J. Clin. Oncol. 15, 2510 –2517. Gehl, J., Boesgaard, M., Paaske, T., Jensen, B. V., and Dombernowsky, P. (1996). Combined doxorubicin and paclitaxel in advanced breast cancer: effective and cardiotoxic. Ann. Oncol. 7, 687– 693.

140

PLATEL ET AL.

Gianni, L., Munzone, E., Capri, G., Fulfaro, F., Tarenzi, E., Villani, F., Spreafico, C., Laffranchi, A., Caraceni, A., Martini, C., Stefanelli, M., Valagussa, P., and Bonadonna, G. (1995). Paclitaxel by 3-hour infusion in combination with bolus doxorubicin in women with untreated metastatic breast cancer: High antitumor efficacy and cardiac effects in a dose-finding and sequence-finding study. J. Clin. Oncol. 13, 2688 –2699. Gianni, L., Vigano, L., Locatelli, A., Capri, G., Giani, A., Tarenzi, E., and Bonadonna, G. (1997). Human pharmacokinetic characterization and in vitro study of the interaction between doxorubicin and paclitaxel in patients with breast cancer. J. Clin. Oncol. 15, 1906 –1915. Holmes, F. A., Madden, T., Newman, R. A., Valero, V., Theriault, R. L., Fraschini, G., Walters, R. S., Booser, D. J., Buzdar, A. U., Willey, J., and Hortobagyi, G. N. (1996). Sequence-dependent alteration of doxorubicin pharmacokinetics by paclitaxel in a phase I study of paclitaxel and doxorubicin in patients with metastatic breast cancer. J. Clin. Oncol. 14, 2713– 2721. Hortobagyi, G. N., Holmes, F. A., Theriault, R. L., Rahman, Z., and Buzdar, A. U. (1997). Combination chemotherapy with paclitaxel and doxorubicin for metastatic breast cancer. Semin. Oncol. 24(Suppl. 11), 13–19. Hudis, C., Ricco, L., Seidman, A., Baselga, J., Currie, V., Fennely, D., Gilewski, T., Lebwohl, D., Moynahan, M., Raptis, G., Surbone, A., Sklarin, N., Yao, T. J., Keefe, D., and Norton, L. (1998). Lack of increased cardiac toxicity with sequential doxorubicin and paclitaxel. Cancer Invest. 16, 67–71.

Lorell, B. H., Wexler, L. F., Momomura, S., Weinberg, E., and Apstein, C. S. (1986). The influence of pressure overload left ventricular hypertrophy on diastolic properties during hypoxia in isovolumically contracting rat hearts. Circ. Res. 58, 653– 663. Misset, J. L., Dieras, V., Gruia, G., Bourgeois, H., Cvitkovic, E., Kalla, S., Bozec, L., Beuzeboc, P., Jasmin, C., Aussel, J. P., Riva, A., Azli, N., and Pouillart, P. (1999). Phase I dose-finding study of first-line docetaxel and doxorubicin in patients with metastatic breast cancer: High antitumor activity and absence of cardiac toxicity. J. Clin. Oncol., in press. Olson, R. D., and Mushlin, P. S. (1990). Doxorubicin cardiotoxicity: Analysis of prevailing hypothesis. FASEB J. 4, 3076 –3086. Pouna, P., Bonoron-Ade`le, S., Gouverneur, G., Tariosse, L., Besse, P., and Robert, J. (1996). Development of the model of rat isolated perfused heart for the evaluation of anthracycline cardiotoxicity and its circumvention. Br. J. Pharmacol. 117, 1593–1599. Shan, K., Lincoff, A. M., and Young, J. B. Anthracycline-induced cardiotoxicity. (1996). Ann. Intern. Med. 125, 47–58. Sparreboom, A., van Tellingen, O., Nooijen, W. J., and Beijnen, J. H. (1996). Nonlinear pharmacokinetics of paclitaxel in mice results from pharmaceutical vehicle Cremophor EL. Cancer Res. 56, 2112–2115. Webster, L. K., Cosson, E. J., Stokes, K. H., and Millward, M. J. (1996). Effect of the paclitaxel vehicle, Cremophor EL, on the pharmacokinetics of doxorubicin and doxorubicinol in mice. Br. J. Cancer 73, 522–524.