Study of Dose Escalation and Sequence Switching of Administration of the Combination of Docetaxel and Doxorubicin in Advanced Breast Cancer

Docetaxel has shown impressive antitumor activity in untreated and pretreated patients with breast cancer in multiple Phase II clinical trials and also in a variety of tumors, such as lung cancer, ovarian cancer, head and neck cancer, and others (1). In Europe and the United States, the recommended dose as a single agent was 100 mg/m² every 3 weeks, and the recommended dose as a single agent in Japan was 60 mg/m² every 3–4 weeks. The response rate for patients with breast cancer who received docetaxel as a first-line chemotherapy was 59%, and the response rate for patients with breast cancer who received docetaxel as a second-line chemotherapy was 41% (2). Anthracyclines are considered the most effective agents for management of breast cancer, and the inclusion of doxorubicin in a combination regimen increases the response rate and duration of survival (3). Therefore, it is logical and promising to choose doxorubicin as the agent to be used in combination with docetaxel.

The sequence of drug administration has been worthy of study in cancer chemotherapy. Rowinsky et al. (4) reported that the administration of cisplatin before paclitaxel induced significantly more severe neutropenia than the opposite sequence in part because of the lower paclitaxel CL.² Similarly,drug-drug interaction was observed for cisplatin-topotecan (5) and paclitaxel-cyclophosphamide (6, 7, 8). In contrast, no sequence-dependent toxicity or sequence-dependent pharmacokinetic interaction was reported in combinations of paclitaxel-carboplatin (9) and paclitaxel-topotecan (10). However, there has been no report of an evaluation of the sequence of administration of the combination of doxorubicin and docetaxel. It was suggested that docetaxel is metabolized mainly by liver cytochrome P450 isoenzymes of CYP3A and that doxorubicin inhibits docetaxel metabolism in human hepatocytes (11). Therefore,it is important to evaluate whether schedule-dependent differences in toxicity and pharmacokinetics exist between two sequences of docetaxel and doxorubicin administration.

The present Phase I study was conducted to determine the MTD of two combinations of docetaxel and doxorubicin and to evaluate the schedule-dependent pharmacokinetic and pharmacodynamic interactions of these drugs in breast cancer patients with tumors that were estrogen receptor negative or refractory to prior hormone therapy.

Patient eligibility criteria were as follows: (a) metastatic or recurrent advanced breast cancer with histological or cytological confirmation; (b) no prior chemotherapy except adjuvant chemotherapy; (c) estrogen receptor negative or refractory to prior hormone therapy; (d) age, 20–75 years; and(e) good performance status (performance status of 0–2 by Eastern Cooperative Oncology Group criteria). Other eligibility criteria included a WBC count of ≥4,000/μl, a neutrophil count of≥2,000/μl, a platelet count of ≥100,000/μl, hemoglobin ≥9.5 grams/dl, serum albumin ≥ 3.0 grams/dl, total bilirubin level ≤ 1.5 mg/dl, AST and alanine aminotransferase levels ≤ 2.0 times the upper limit of the normal range, blood urea nitrogen level within the normal limit,PaO₂ level ≥ 60 torr, normal electrocardiography, and normal cardiac LVEF (≥50%). For patients with liver metastasis of breast cancer, AST and alanine aminotransferase values were required to be less than 3 times the upper limit of the normal range. Written informed consent was obtained from all patients. The institutional review board of the National Cancer Center of Japan approved the protocol. This study was conducted according to the guidelines of the Ministry of Health and Welfare of Japan.

All patients were treated while hospitalized until the second cycle was completed. The study was designed as a prospective randomized crossover trial. Study design is shown in Fig. 1. Consecutive patients were randomized to either the doxorubicin after docetaxel sequence (docetaxel→doxorubicin sequence) or the docetaxel after doxorubicin sequence (doxorubicin→docetaxel sequence). There was no time interval between the administration of doxorubicin and docetaxel as described below. The sequence of drug administration was switched for the second course. Namely, in the first and second cycle,each patient received both sequences. If the patient responded to the treatment or the disease was stable, treatment was repeated every 3 or 4 weeks after the WBC count recovered to 4000/μl until the total dose of either docetaxel or doxorubicin reached 500 mg/m². The sequence of drug administration in the third and subsequent cycles was chosen according to the toxicity and the patient’s preference. Blood counts were monitored at least three times per week until the second cycle was completed. Docetaxel was administered by i.v. drip infusion over 1 h. Premedication by corticosteroid hormone was not permitted in this trial because corticosteroids contribute to leukocytosis and neutrophilia on the day of chemotherapy and might affect the metabolism of docetaxel by liver cytochrome P450. In addition, premedication of steroid hormones is not regarded as standard treatment of docetaxel at the dose of 60 mg/m² in Japan. Doxorubicin was administered by a 5-min bolus i.v. injection just after or before the administration of docetaxel. An initial dose of 50 mg/m² docetaxel was adopted because the dose was considered to be the minimal effective dose based on a Japanese Phase I trial for breast cancer (12). The initial dose of doxorubicin was determined by the minimal dose in the cyclophosphamide,doxorubicin, and 5-fluorouracil combination.

Toxicity was evaluated for the first two cycles according to the National Cancer Institute common toxicity criteria (13). DLT was determined by evaluation after the first cycle of therapy and was defined as grade 4 neutropenia for 7 days or more, grade 4 neutropenia associated with infection for 3 days accompanied by fever(>38°C), or any grade 3 or higher toxicity except anemia, alopecia,anorexia, vomiting, and general malaise. If DLT occurred in one or two of the first three patients at a specific dose level and sequence in the first cycle, three additional patients were treated at that dose level and sequence. The MTD was defined as the dose level and sequence one level below that at which at least three of six patients developed DLT in the first cycle. Once patients had a temperature of >38°C,they were treated with broad spectrum antibiotics after blood culture for a bacteriological survey. When neutropenia occurred as a DLT, the use of G-CSF was permitted until the neutrophil count recovered to 2000/μl. Patients who vomited once after drug administration received a serotonin antagonist, granisetron, and, in these cases, antiemetic prophylaxis with granisetron was used for subsequent cycles. Patients who experienced a DLT but were suspected of achieving a response were allowed to receive retreatment at a reduction of one dose level. To determine cardiotoxicity by combination of the two drugs, the LVEF was determined at baseline and serially monitored before the second,fourth, and sixth cycles and after the completion of treatment. Relative dose intensity of docetaxel and doxorubicin per week was calculated by dividing the administered dose at each level by the actual dose of level one for each drug. If the relative dose intensity did not increase, the dose level of the highest intensity was considered to be the recommended dose for the combination.

Patients with measurable lesions were evaluated for antitumor response to the combination. Complete response was defined as the disappearance of all measurable or assessable disease and all signs and symptoms of disease for at least 4 weeks. Partial response was defined as a reduction of 50% or greater in the sum of the products of the perpendicular diameters of all measurable lesions and the appearance of no new lesions for at least 4 weeks. Stable disease was defined as a less than 50% reduction or a less than 25% increase in the sum of the products of two perpendicular diameters of all measurable lesions and the appearance of no new lesions. Progressive disease was defined as an increase of 25% in the size of any lesion or the development of any new lesions. A breast surgeon and physician not related to this study reviewed the evaluation of responses.

To study pharmacokinetics, blood samples in all patients were obtained from venous catheters placed in the arm contralateral to the drug infusion. In the doxorubicin→docetaxel sequence, blood samples were collected before the infusion; immediately after completion of the infusion of doxorubicin; at 15 and 30 min during the infusion of docetaxel; immediately after the end of the infusion of docetaxel; 5,20, and 35 min after the end of the infusion of docetaxel; and 1, 3, 4,7, 24, 48, and 72 h after the end of the infusion of docetaxel. In the opposite sequence, that of docetaxel→doxorubicin, blood samples were collected before treatment; 30 min after the start of the docetaxel infusion; just after the end of docetaxel infusion; just after the infusion of doxorubicin; at 15, 30, and 55 min after the infusion of doxorubicin; and at 2 h and 55 min, 3 h and 55 min, 6 h and 55 min, 23 h and 55 min, 47 h and 55 min,and 71 h and 55 min after the end of the infusion of doxorubicin. Heparinized plasma obtained from patients was stored at −20°C until analyzed.

Frozen plasma samples were thawed at ambient temperature and then vortexed and centrifuged for 5 min at 3000 rpm to remove fibrous materials that can clog extraction columns. The assay was carried out at the Ibaraki Laboratory of Rhône-Poulenc Rorer Japan.

Docetaxel concentrations in plasma were determined by HPLC (reverse phase) using the Inertsil ODS 2 column (5 μm; 4.6 × 250 mm; GL Sciences, Tokyo, Japan) with UV detection (14). The method involves a solid-phase extraction (Bond Elut C2; Varian, Harbor, CA). Docetaxel and the internal standard were determined by an UV detector adjusted at 225 nm, and peak height was used for quantification. The lower limit of the assay was 7.5 ng/ml, and the linearity was confirmed up to 4000 ng/ml in plasma.

Doxorubicin and doxorubicinol, which is the major metabolite of doxorubicin in plasma, were determined by HPLC (reverse phase) using the Bondapak Phenyl column (10 μm; 3.9 × 300 mm; Waters,Milford, MA) with fluorescence detection (15). The method involves a solid-phase extraction (Bond Elut C2; Varian). Doxorubicin,doxorubicinol, and the internal standard (daunorubicin) were determined by a fluorescence detector adjusted at Ex 470 nm and Em 565 nm, and the peak height was used for quantification. The lower limit of the assay was 5 ng/ml for doxorubicin and 4.4 ng/ml for doxorubicinol, and linearity was confirmed up to 2000 ng/ml for doxorubicin and 262.5 ng/ml for doxorubicinol in plasma.

Pharmacokinetic parameters were calculated using the MULTI program described by Yamaoka et al. (16). C_max was taken from the actual value. The terminal rate constant (k) was determined by log-linear regression analysis of the terminal phase of the plasma concentration-time courses. The t_1/2values were calculated by the equation t_1/2 = 0.693/k. The AUC_0→∞ was calculated by the linear trapezoidal rule up to the last measurable data point with extrapolation to infinity. CL was calculated by dividing the dose of docetaxel or doxorubicin received by the AUC. V_ss was calculated by the equation CL × (mean residual time) − [infusion time (h)/2].

The neutrophil counts and the time under the curve were drawn, and the area under the grade 2 (<1500/μl), grade 3 (<1000/μl), and grade 4 (<500/μl) time curve was calculated by the trapezoidal rule,respectively. Regression curves for the relationship between AUC or CL and the area under the grade 2, 3, and 4 neutropenia time curves or the nadir of the neutrophil count for each sequence were made and compared,respectively.

Between November 13, 1995 and May 14, 1998, 25 patients were enrolled into the study. All patients were assessable for toxicity. The median number of cycles administered per patient was five (range, one to nine). Two of four patients treated with the doxorubicin→docetaxel sequence in the first cycle preferred the same sequence in the third and subsequent cycles. However, four of six patients who were given the docetaxel→doxorubicin sequence in the first cycle were treated with the opposite sequence in the third and subsequent cycles because of patient preference in one patient and toxicities in three patients. Patient characteristics are shown in Table 1. The median time from the last adjuvant chemotherapy was 16.1 months, with a range of 1–197 months. The median number of metastatic disease sites for all patients was two sites, with a range of one to five sites.

The major toxicity of the combination was grade 4 neutropenia. One patient receiving the doxorubicin→docetaxel sequence at dose level 1 experienced febrile neutropenia for 1 day. All three patients receiving the docetaxel→doxorubicin sequence at dose level 2 experienced grade 4 neutropenia for more than 7 days, but the neutropenia in all patients resolved without G-CSF, and the MTD in this sequence was 50 mg/m² docetaxel and 40 mg/m² doxorubicin (level 1). Therefore, the doxorubicin→docetaxel sequence was administered subsequently through level 4, reaching dosages of 50 mg/m² doxorubicin and 70 mg/m² docetaxel. Duration of grade 4 neutropenia of all patients is shown in Table 2. Duration of grade 4 neutropenia at dose level 2 was significantly longer with the docetaxel→doxorubicin sequence than with the opposite sequence (P = 0.0062).

Although both error of intrapatient variability (P =0.0224) and the cycle effect (P = 0.0226) were significant, the crossover design in this study was considered to be valid for evaluation (Table 3). G-CSF was used for 3 days in one patient at level 3 who experienced grade 4 neutropenia for 9 days. Two patients at level 4 experienced DLT; one patient experienced grade 4 neutropenia for more than 7 days, and G-CSF was administered for 1 day, and one patient experienced febrile neutropenia for more than 3 days, and G-CSF was administered for 2 days. The other three patients at level 4 experienced febrile neutropenia for 1 day. Although the incidence of DLT at level 4 (DLT was seen in two of six patients at this level) did not fulfil the definition of MTD in the original protocol, further dose escalation was stopped because the relative dose intensity was not increased despite the dose escalation as described below. We concluded that level 4 was the MTD in the doxorubicin→docetaxel sequence. There was no documented sepsis. G-CSF was used in eight patients during 14 different cycles of administration of the doxorubicin→docetaxel sequence. Delay of subsequent cycles was observed in two patients at level 2 and three patients at level 4.

One patient experienced grade 3 anemia and another 20 patients experienced grade 1 or 2 anemia in the first cycle. Thrombocytopenia was not observed in the first cycle. Grade 1 and grade 2 thrombocytopenia were observed in three patients during the subsequent cycles.

Nonhematological toxicities in the first cycle are shown in Table 4. One patient at dose level 1 experienced an allergic reaction. Although the patient was able to complete the first cycle of the combination after the i.v. administration of 20 mg of dexamethasone, 5 mg of chlorpheniramine maleate, and 20 mg of famotidine, she was not assessable for the second cycle of chemotherapy because she was removed from the study due to an allergic reaction before completion of the second cycle, despite premedication. One patient at level 4 experienced severe systemic skin eruption due to allergy just after the beginning of docetaxel administration and was removed from the study. One patient receiving the doxorubicin→docetaxel sequence at dose level 3 experienced reversible grade 3 diarrhea without abdominal pain, and no recurrences were observed on retreatment at a one-dose level reduction. Fifteen patients experienced grade 1 or 2 diarrhea during the subsequent cycles. Nausea and vomiting were mild and generally well controlled after a prophylactic serotonin antagonist was used. Grade 1 and 2 oral mucositis was observed in 13 patients at all cycles. One patient experienced grade 3 elevated AST, and another 10 patients experienced grade 1 or 2 liver damage at all cycles. Fluid retention was not noted in the absence of corticosteroid premedication. All patients developed alopecia, which was generally complete by the end of the second cycle. With regard to cardiotoxicity, despite the median cumulative doxorubicin dose being 220 mg/m² (range, 40–360 mg/m²), no congestive heart failure was observed. No patient had a LVEF decrease of more than 20%, with the exception of one patient whose LVEF decreased from 82% to 65% after six cycles of chemotherapy.

Relative dose intensity of each level in the doxorubicin→docetaxel sequence is shown in Table 5. Although two of six patients at dose level 4 experienced DLT, this dose level was considered to be the MTD of the doxorubicin→docetaxel sequence because the relative dose intensity of level 4 was not increased in contrast with that of level 3 (Table 5). Therefore, the determination of the MTD in the doxorubicin→docetaxel sequence was not based on observation of the DLT but on relative dose intensity.

Pharmacokinetic parameters of docetaxel, doxorubicin, and doxorubicinol in the first and second cycles at all dose levels are shown in Table 6. There were no significant differences in the pharmacokinetic parameters of docetaxel, doxorubicin, and doxorubicinol between the two sequences at dose levels 1 and 2. Pharmacokinetics of a representative case at dose level 2 are shown in Fig. 2.

The area under the grades 2, 3, and 4 neutropenia-time curve and nadir count of neutrophils at levels 1 and 2 are shown in Table 7. There was a significant difference between the two sequences in the correlation between the AUC of docetaxel and the area under the grade 4 neutropenia-time curve at levels 1 and 2, or when levels 1 and 2 were considered together(P = 0.0354). However, no significant differences were found between the two sequences in correlation between AUC, CL of docetaxel and nadir counts of neutrophils or area under the neutropenia-time curve of grade 2 and grade 3 at dose levels 1 and 2 or when dose levels 1 and 2 were considered together. Similarly, there were no significant differences between the two sequences with respect to correlations between the AUC, CL of doxorubicin and nadir counts of neutrophils or the area under the neutropenia-time curve of grades 2,3, and 4 at dose levels 1 and 2 or the combination of levels 1 and 2. Furthermore, there was no significant correlation between the AUC of doxorubicinol and those values.

Although the present study was carried out in a Phase I setting and two patients were unable to complete the treatment due to an allergic reaction, response was evaluated in all patients on the basis of intention to treat. Ten of 25 patients had a partial response. The response rate was 40% (95% confidence interval, 21–61%). Median progression-free survival from the initial day of this combination chemotherapy was 6 months, with a range of 1.3–12 months.

Docetaxel and doxorubicin are considered to be two of the most effective anticancer agents against breast cancer. Chemotherapy with a combination of both drugs has been considered to have promise. Therefore, this Phase I trial of docetaxel and doxorubicin in combination was conducted in breast cancer patients who were estrogen receptor negative or refractory to prior hormone therapy because these patients were considered to be the most suitable population for anticancer chemotherapy. In addition, our study was conducted to evaluate the effect of the sequence of administration of docetaxel and doxorubicin. The MTD in the docetaxel→doxorubicin sequence was 50 and 40 mg/m², respectively, and the MTD in the alternative doxorubicin→docetaxel sequence was 50 and 70 mg/m², respectively. The duration of grade 4 neutropenia was significantly longer in patients treated with the docetaxel→doxorubicin sequence than in patients treated with the opposite sequence (P = 0.0062). Therefore, it was suggested that the sequence of administration of these drugs affected myelosuppression.

Concerning interactions between drugs, there have been reports which have evaluated the effects of the order of administration of two drugs,especially with regard to paclitaxel and doxorubicin. In a study of the combination of a 24-h infusion of paclitaxel and bolus injection of doxorubicin either 4 h before or 4 h after the infusion of paclitaxel, Sledge et al. (17) reported that toxicity manifested by mucositis appeared more severe when paclitaxel preceded doxorubicin compared with the reverse sequence. In the simultaneous 72-h continuous infusion of both drugs, a greater concentration of doxorubicinol was shown with this combination in comparison with the administration of doxorubicin alone (18). The sequence of paclitaxel given over 24 h followed by doxorubicin given over 48 h was associated with greater toxicity and increased maximal plasma concentration and AUC of doxorubicin as reported by Holmes et al. (19). In these studies, the sequence of paclitaxel followed by doxorubicin was suggested to be more toxic, possibly because of pharmacokinetic interaction. In contrast, there was no sequence-dependent toxicity and pharmacokinetic difference between two sequences of 3-h paclitaxel infusion and bolus injection of doxorubicin (20). The present study is the first report of a prospective analysis of the pharmacodynamics and pharmacokinetics of drug interaction according to the order of administration of doxorubicin and docetaxel. Although there was no pharmacokinetic difference according to drug sequence in our study, and docetaxel may differ from paclitaxel in its pharmacokinetic interaction with doxorubicin, our results, which showed that the docetaxel→doxorubicin sequence was more toxic, might be reasonable from the viewpoint of findings of studies of paclitaxel and doxorubicin.

The effect of the sequence of administration on drug to drug interaction was evaluated for the combination of docetaxel with cisplatin or ifosfamide (21). Pronk et al.(22) found no significant differences in pharmacodynamics and pharmacokinetics between two schedules of docetaxel and cisplatin, but there appeared to be a trend toward a higher incidence of grade 3 and grade 4 leukocytopenia when the administration of cisplatin was followed by that of docetaxel. Similarly, toxicity was reduced when docetaxel preceded ifosfamide (21). Moreover,Baille et al. (23) indicated no pharmacokinetic interaction between docetaxel and several anticancer drugs from the review of results in Phase I trials. In our study, there was no significant difference in pharmacokinetic parameters of docetaxel,doxorubicin, and doxorubicinol between the two sequences. Similarly,Bellott et al. (24) reported that docetaxel did not change the pharmacokinetic profile of doxorubicin or its metabolite, doxorubicinol, in the pharmacokinetic study in which 75 mg/m² docetaxel was administered immediately after or 1 h after bolus infusion of 50 mg/m² doxorubicin. D’Incalci et al.(25) also indicated that a 1-h interval between the administration of docetaxel and doxorubicin did not change the AUC data compared with the immediate administration of the second agent. However, they described the important finding that docetaxel did not alter doxorubicin AUC, but doxorubicin increased the docetaxel AUC compared with the AUC of each drug administered singly. Moreover, it was reported that docetaxel increased doxorubicin levels in mice not only in many tissues but also in serum at 24 h (26). It was unknown whether these results were related to the pharmacodynamic differences found in our study. No active metabolites of docetaxel and doxorubicin were identified, and the observation that there were no significant differences in pharmacokinetics between the two sequences suggested that the difference might be related to factors such as cellular pharmacokinetics rather than the plasma concentration of the drugs or that the sequence of drug administration might affect the sensitivity of myeloid progenitor cells in bone marrow.

The French study, in which doxorubicin was given first as an i.v. 15-min bolus injection followed 1 h later by docetaxel as a 1-h infusion, indicated that the recommended dose for the combination of both drugs was either 75 mg/m² docetaxel plus 50 mg/m² doxorubicin or 60 mg/m² of both drugs (27, 28). Kennedy et al. (29) also reported that the MTD for doxorubicin and docetaxel was 40 and 60 mg/m², respectively, with the same schedule as the French study (27, 28). The MTD in our study, which seemed to be a little low, might have been affected by the avoidance of the standard use of corticosteroid premedication with docetaxel. However,similar doses in the doxorubicin→docetaxel sequence in our study suggested that there were no ethnic differences in the pharmacokinetics of docetaxel in agreement with population pharmacokinetic studies (30, 31). In fact, the difference in recommended dose for docetaxel between Japan and the United States and Europe was due to the criterion for DLT, that is, the duration of grade 4 neutropenia. Moreover, the French study (27, 28) recently reported that excellent response rates of 66% and 90% were achieved with the combination of docetaxel and doxorubicin in previously untreated metastatic breast cancer. The response rate in our study, which was similar to those with the single administration of docetaxel, might be due to bias in patient selection; however, the present study was performed in a Phase I setting, and the response rate was not the primary end point.

We concluded that a sequence-dependent toxicity of docetaxel and doxorubicin on neutropenia was suggested but that there was no pharmacokinetic sequence interaction between the two drugs. The sequence of 50 mg/m² doxorubicin followed by 60 mg/m² docetaxel is recommended for practical reasons for subsequent clinical trials, and the Phase II study is ongoing in Japan. It would be of interest to evaluate that sequence in comparison with the sequence of 50 mg/m²docetaxel followed by 40 mg/m² doxorubicin to evaluate whether the latter schedule, which has the same toxicity but at a lower dosage, would be associated with a higher or lower response rate.