A Phase I and pharmacological study was performed to evaluate the feasibility, maximum tolerated dose (MTD), dose-limiting toxicities (DLTs), and pharmacokinetics of the anthrapyrazole losoxantrone in combination with paclitaxel in adult patients with advanced solid malignancies. Losoxantrone was administered as a 10-min infusion in combination with paclitaxel on either a 24- or 3-h schedule. The starting dose level was 40 mg/m2 losoxantrone and 135 mg/m2 paclitaxel (as a 24- or 3-h i.v. infusion) without granulocyte colony-stimulating factor (G-CSF). Administration of these agents at the starting dose level and dose escalation was feasible only with G-CSF support. The following dose levels (losoxantrone/paclitaxel, in mg/m2) of losoxantrone and paclitaxel as a 3-h infusion were also evaluated: 50/135, 50/175, 50/200, 50/225, and 60/225. The sequence-dependent toxicological and pharmacological effects of losoxantrone and paclitaxel on the 24- and 3-h schedules of paclitaxel were also assessed. The MTD was defined as the dose at which >50% of the patients experienced DLT during the first two courses of therapy. DLTs, mainly myelosuppression, occurring during the first course of therapy were noted in four of six and five of eight patients treated with 40 mg/m2 losoxantrone and 135 mg/m2 paclitaxel over 24 and 3 h, respectively, without G-CSF. DLTs during the first two courses of therapy were observed in one of six patients at the 50/175 (losoxantrone/paclitaxel) mg/m2 dose level, two of four patients at the 50/200 mg/m2 dose level, one of four patients at the 50/225 mg/m2 dose level, and two of five patients at the 60/225 mg/m2 dose level. The degree of thrombocytopenia was worse, albeit not statistically significant, when 24-h paclitaxel preceded losoxantrone, with a mean percentage decrement in platelet count during course 1 of 80.7%, compared to 43.8% with the reverse sequence (P = 0.19). Losoxantrone clearance was not significantly altered by the sequence or schedule of paclitaxel. Cardiac toxicity was observed; however, it was not related to total cumulative dose of losoxantrone. An unacceptably high rate of DLTs at the first dose level of 40 mg/m2 losoxantrone and 135 mg/m2 paclitaxel administered as either a 24- or 3-h i.v. infusion precluded dose escalation without G-CSF support. The addition of G-CSF to the regimen permitted further dose escalation without reaching the MTD. Losoxantrone at 50 mg/m2 followed by paclitaxel (3-h i.v. infusion) at 175 mg/m2 with G-CSF support is recommended for further clinical trials.

The anthrapyrazole losoxantrone (C1–941; DuPont Pharmaceuticals Company, Wilmington, DE) was synthesized with the rationale of developing cytotoxic agents with a similar chemical structure to anthracyclines but with the distinctive feature of dissociation between antitumor activity and cardiotoxicity (1). Mechanistically, the cardiotoxicity of anthracyclines is related, at least in part, to their ability to undergo one electron reduction at the quinone oxygen to form a semiquinone. The enzymatic interaction between the semiquinone and molecular oxygen yields a superoxide anion, which gives rise to a highly reactive oxygen species (2, 3). Losoxantrone lacks the sugar moiety of doxorubicin, retains the aromatic ring structure that is responsible for its intercalation into DNA (4), and differs from the anthracendione mitoxantrone in the central quinone moiety by having an imino group instead of a keto group (Fig. 1), which precludes the formation of free radicals (5). Indeed, in the fetal mouse heart model, losoxantrone caused substantially less free radical formation in rat liver microsomal preparations and fewer cardiotoxic effects than did doxorubicin (5, 6). Losoxantrone, like doxorubicin and mitoxantrone, intercalates into DNA (4), induces single- and double-stranded DNA breaks (7), and inhibits topoisomerase II (8).

Losoxantrone demonstrated remarkable and broad antitumor activity in preclinical studies. In the National Cancer Institute drug evaluation screen, losoxantrone was active against a variety of cell lines derived from central nervous system, leukemias, and lung tumors, among others (8). Losoxantrone was more active than several other anthrapyrazoles and mitoxantrone and was as effective as doxorubicin against various murine tumors, including mammary adenocarcinoma 16C, colon adenocarcinoma 11a, the Ridgway osteogenic sarcoma (9), and mammary carcinomas arising in ras transgenic mice (10).

The activity of losoxantrone in preclinical models and its potential to induce less cardiotoxicity than most anthracyclines, anthracendiones, and other anthrapyrazoles provided the impetus for the clinical development of this agent. A Phase I study of losoxantrone demonstrated that the DLT5 was leukopenia and that its MTD on a short-infusion, every-3-weeks schedule was 55 mg/m2(11). Two Phase II studies of losoxantrone in patients with advanced breast cancer demonstrated significant antitumor activity, with an overall response rate of 43–63% (12, 13). In the larger of the two studies (13), 1% of the patients developed congestive heart failure, and 4% experienced >20% asymptomatic decline in LVEF.

In light of the increasing recognition that paclitaxel, particularly on a 3-h infusion schedule, enhances the potential for anthracycline-related cardiotoxicity (14) and that paclitaxel-anthracycline regimens have notable antitumor activity in women with metastatic breast cancer, with CR rates as high as 40% (14), a combination of paclitaxel and losoxantrone combination is a seemingly rational developmental alternative with a potentially greater therapeutic index than doxorubicin and paclitaxel. The enhancement of doxorubicin’s cardiotoxicity by paclitaxel, which is likely due to reduced hepatic clearance and/or increased cellular retention of doxorubicin and doxorubicinol by paclitaxel itself and/or its polyoxyethylated castor oil formulation vehicle (Cremophor EL; Ref. 15) may not be operative with paclitaxel-losoxantrone regimens. This reason and the remarkable single-agent activities of losoxantrone (12, 13) and paclitaxel (16, 17) in patients with breast cancer and other neoplasms, the vastly different mechanisms of cytotoxic actions of these agents, and the lack of overlapping nonhematological toxicities were the impetus to evaluate the feasibility of administering losoxantrone and paclitaxel in combination. The principal objectives of this study were to: (a) determine the MTDs of the combination of paclitaxel and losoxantrone; (b) characterize the principal DLTs of the combination of paclitaxel and losoxantrone; (c) describe the pharmacokinetics of both losoxantrone and paclitaxel in combination; (d) determine whether the sequence of drug administration results in significantly different toxicological and pharmacological profiles; and (e) seek preliminary evidence of antitumor activity in patients with advanced solid malignancies.

Eligibility.

Patients with solid tumors that were refractory to conventional therapy or for whom no standard therapy existed were candidates for this study. Eligibility requirements included: (a) age of ≥18 years; (b) WHO performance status of ≤2; (c) a life expectancy of at least 8 weeks; (d) no chemotherapy in the preceding 3 weeks; (e) adequate hematopoietic (ANC of ≥ 1500/μl, platelet count of ≥100,000/μl), hepatic (serum bilirubin of ≤1.5 mg/dl), and renal (serum creatinine of ≤2.0 mg/dl) functions; and (f) no coexisting medical problems that might compromise compliance with the study. Due to concern about the potential for cardiotoxicity of the study drug combination, patients were ineligible for treatment if they had: (a) a pretreatment LVEF of <45%, as determined by radionuclide scanning or ≥10% absolute reduction in LVEF within 6 months prior to this study; (b) prior treatment with a cumulative dose of doxorubicin of >400 mg/m2 or dose of mitoxantrone of >125 mg/m2; or (c) a history of congestive heart failure, myocardial infarction within 1 year of study entry, unstable angina, active cardiomyopathy, or ventricular arrhythmia requiring therapy.

Dosage, Dose Escalation, and Sequencing.

The study was designed to escalate the doses of both paclitaxel and losoxantrone until the MTD was reached. Initially, the sequence of administration of paclitaxel and losoxantrone was alternated to evaluate any sequence-dependent toxicity. In addition, paclitaxel was initially administered i.v. over 24 h; however, when a 3-h i.v. infusion schedule was demonstrated to be safe and possibly less toxic (18), the paclitaxel infusion was shortened to 3 h. Losoxantrone was administered i.v. over 10 min at all dose levels. Patients were retreated every 3 weeks, provided they had recovered to grade ≤1 from drug-related toxicities, except alopecia. At least three patients were treated at each dose level. The first three patients were treated with 40 mg/m2 losoxantrone followed by 135 mg/m2 paclitaxel administered over 24 h. The next three patients were treated at the same dose level and schedule, but with paclitaxel administered prior to losoxantrone. The following eight patients were treated with losoxantrone 40 mg/m2 and paclitaxel 135 mg/m2 administered over 3 h instead of 24 h, with four receiving losoxantrone prior to paclitaxel and the other four receiving the reverse sequence. When myelosuppression precluded dose escalation, the study was amended to require prophylactic administration of G-CSF, and four patients were treated with losoxantrone 40 mg/m2 followed by paclitaxel 135 mg/m2 administered over 3 h with G-CSF. With the addition of G-CSF, dose escalation continued with the following dose levels of losoxantrone/paclitaxel (mg/m2) evaluated: 50/135, 50/175, 50/200, 50/225, and 60/225. All patients received losoxantrone followed by paclitaxel administered over 3 h and prophylactic G-CSF. Dose reduction of 10% for losoxantrone and/or paclitaxel in subsequent courses was allowed for patients experiencing DLT. Intrasubject dose escalation was not permitted.

Toxicities were graded according to the NCI Common Toxicity Criteria. DLT was defined as at least one of the following: (a) ANC of <500/μl for >5 days or associated with fever exceeding 38°C; (b) platelet count of <25,000/μl; or (c) grade 3 or 4 nonhematological toxicity (excluding alopecia, nausea, or vomiting).

The MTD was defined as the dose at which >50% of the patients (or two of three patients, if only three patients were treated at a dose level) experienced DLT during courses 1 and 2 of therapy. The recommended dose for Phase II–III studies was one dose level below the MTD.

Drug Administration.

Losoxantrone was supplied by DuPont Pharmaceuticals Company (Wilmington, DE) in vials containing 25 mg of lyophilized powder for injection. Each vial was reconstituted with 2.5 ml of sterile water to achieve a concentration of 10 mg/ml. The calculated total dose was further diluted in 50 ml of 0.9% sodium chloride and administered i.v. over 10 min. All patients received prophylactic antiemetics with serotonin antagonists and dexamethasone.

Paclitaxel (Taxol®; Bristol Myers Squibb Company, Princeton, NJ) was supplied as a concentrated sterile solution of 6 mg/ml in 50% polyoxyethylated castor oil (Cremophor EL) and 50% dehydrated alcohol, USP. The total dose of paclitaxel was diluted in 500 ml of 5% dextrose or 0.9% sodium chloride. Paclitaxel was administered as a continuous i.v. infusion over 24 h in the first six patients and as a 3-h i.v. infusion in all subsequent cohorts. Prior to paclitaxel administration, all patients were premedicated with 20 mg of dexamethasone p.o. 12 and 6 h prior to paclitaxel and both 50 mg of diphenhydramine i.v. and 300 mg of cimetidine i.v. 30 min prior to paclitaxel.

Recombinant methionyl human G-CSF (Neupogen; Amgen, Thousand Oaks, CA) was supplied in ampules that contained 0.6 mg of cytokine in 2 ml of sterile water. When the feasibility of further dose escalation of the combination with G-CSF was explored, 5 μg/kg G-CSF was administered s.c., starting at 24 h after chemotherapy and continuing until day 12 or until the ANC increased to >10,000/μl.

Pretreatment and Follow-up Studies.

Medical histories, physical examinations, electrocardiograms, and routine hematology and chemistry studies were performed prior to each treatment. Complete blood counts and differential white blood counts were obtained weekly in patients who were not assigned to G-CSF, biweekly in patients assigned to G-CSF, and every other day in patients with grade 3–4 myelosuppression. The chemistry tests were repeated prior to each course of therapy. MUGA scans were obtained after every two courses and at study termination in patients who received more than eight courses. Tumors were measured after every two courses, and therapy was continued in the absence of evidence of disease progression. A CR was defined as the absence of any symptoms related to the cancer and a complete disappearance of all disease on two separate measurements performed at a minimum interval of 4 weeks. A PR required at least 50% decrease from baseline in the sum of the bidimensional product of all measurable tumors on two separate measurements performed at a minimum interval of 4 weeks.

Pharmacokinetic Sampling and Assay Methodologies.

Blood samples for pharmacokinetic analyses of losoxantrone and paclitaxel were obtained from indwelling venous catheters placed in the arm contralateral to the drug infusion. Losoxantrone pharmacokinetics were determined in 13 patients during the first course of therapy following administration of losoxantrone 40 mg/m2 and paclitaxel 135 mg/m2 as a 3- or 24-h infusion either before or immediately after losoxantrone administration. None of these patients received G-CSF. Blood samples were obtained pretreatment and at the following times from the start of losoxantrone infusion: 5, 10, 20, 30, 40, 65, and 90 min and 2, 3, 4, 6, 8, 12, 14, 24, 30, 48, 72, and 96 h. Blood samples for losoxantrone pharmacokinetics were collected without anticoagulant, immediately transferred to plastic centrifuge tubes containing a citrate buffer, and centrifuged. Next, plasma was separated and stored at −20°C until analysis. Losoxantrone plasma levels were determined using a high-performance liquid chromatographic method with a visible detection at 492 nm and a lower limit of sensitivity of 1.3 ng/ml, as described previously (19). The precision and accuracy of the method was within 15%, with a mean extraction efficiency of 99.7 ± 12.7%. The standard curves were linear over a concentration range of 1.3–17 ng/ml in plasma.

Paclitaxel pharmacokinetics were determined in 17 patients during their first course of therapy. The 17 patients received doses ranging from 175 to 225 mg/m2. Losoxantrone pharmacokinetics were not simultaneously characterized in these patients. All patients received their respective paclitaxel dose as a 3-h infusion immediately after a 10-min losoxantrone infusion, followed by G-CSF support. Blood samples for paclitaxel pharmacokinetics were obtained pretreatment and at the following times from the start of paclitaxel infusion: 1.5, 3, 3.3, 3.6, 4, 4.5, 5, 7, 9,12, 16, 24, 36, 48, and 72 h. Blood samples for paclitaxel pharmacokinetics were collected with anticoagulant and immediately centrifuged at 3000 rpm for 12 min. The separated plasma was then transferred to 5-ml polypropylene cryovials using a plastic pipette and stored at −20° C. Paclitaxel plasma concentrations were determined using high-performance liquid chromatography with UV detection and a sensitivity limit of 10 ng/ml. The precision and accuracy of the method was within 15%, with a mean extraction efficiency of 90 ± 9%. The standard curves were linear over a concentration range of 10.0–2500 ng/ml.

Pharmacokinetic and Pharmacodynamic Analysis.

Individual losoxantrone and paclitaxel pharmacokinetic parameters were calculated using standard model-independent methods (20). AUC values were calculated using the linear trapezoidal method as implemented in PK-IMS Version 2.0 (DuPont Pharmaceuticals Company), and were extrapolated to infinity. Other calculated pharmacokinetic parameters included the half-lives (t1/2), mean residence time (MRT), the apparent volume of distribution (Varea), the volume of distribution at steady-state (Vss), and systemic clearance (Cl). Values for maximum plasma concentration (Cmax) were the observed peak plasma concentrations.

Losoxantrone and paclitaxel pharmacokinetic parameters were also estimated using model-dependent methods. Individual losoxantrone and paclitaxel concentrations were fitted with a linear three-compartment model using the method of weighted least squares regression, as implemented in PCNONLIN (SCI Software; Statistical Consultants, Inc., Lexington, KY). Estimated pharmacokinetic parameters included the disposition of half-lives (t1/2α, t1/2β, and t1/2γ) and the central volume of distribution (Vc).

The relationships between losoxantrone and paclitaxel systemic exposure (Cmax and AUC) and toxicity during course 1 were explored. Parameters reflecting toxicity included NCI Common Toxicity Criteria grade neutropenia and thrombocytopenia, grade 4 neutropenia for >5 days or with fever, the occurrence of DLT, and percentage decrement in ANC or platelets during course 1. The relationships between the absolute decrement in LVEF from baseline to lowest value documented during all courses of treatment and losoxantrone drug exposure during course 1 and the total cumulative dose of losoxantrone were also evaluated.

Statistical Analyses.

The Wilcoxon rank sums test was used to evaluate the effects of study drugs sequence on hematological toxicity and the influence of losoxantrone and paclitaxel sequence on losoxantrone’s pharmacokinetics and to compare values of systemic exposure between different categories of toxicity. The JMP statistical software program (Version 3.1; SAS institute, Cary, NC) was used in the statistical analyses.

Clinical Study

The characteristics of the 41 patients entered on the study are listed in Table 1. Six patients had received prior doxorubicin, with a median dose of 110 mg/m2 (range, 90–380 mg/m2). A total of 162 courses of losoxantrone and paclitaxel were administered at five dose levels (Table 2). The median number of doses was four (range, one to nine). Thirty-seven patients received at least two courses of study drugs. The median total losoxantrone dose was 160 mg/m2 (range, 40–400 mg/m2).

Toxicity

Hematological Toxicity

Myelosuppression was the main toxicity of losoxantrone and paclitaxel. Tables 3 and 4 depict the incidences of hematological toxicities per patient and course, respectively. The combination of 40 mg/m2 losoxantrone and 135 mg/m2 paclitaxel over 24- or 3-h infusion, without G-CSF, produced significant toxicity with DLTs (grade 4 neutropenia of >5 days, grade 4 thrombocytopenia, or febrile neutropenia) occurring in 5 of 6 patients (83%) and 14 of 24 courses (58%) when paclitaxel was administered as a 24-h infusion and in 5 of 8 patients (62%) and 15 of 32 courses (47%) when paclitaxel was administered as a 3-h infusion. DLT occurring during the first course of therapy was noted in four of six and five of eight patients treated with 40 mg/m2 losoxantrone and 135 mg/m2 paclitaxel over 24 or 3 h, without G-CSF, respectively. Prior to G-CSF prophylaxis, dose reduction of paclitaxel to 100 mg/m2 after the first course was common and occurred in 8 of 13 patients who received more than one course of therapy. The ANC nadir during course 1 of therapy tended to be lower with paclitaxel administered on a 24-h schedule (mean, 182/μl) compared to a 3-h infusion (mean, 301/μl); however, the difference did not reach statistical significance (P = 0.2).

The sequence-dependent toxicity of losoxantrone and paclitaxel was explored prior to G-CSF support at the first dose level. Although the degree of neutropenia was not related to the sequence of study drugs administration on either the 24- or 3-h schedule of paclitaxel, with the mean percentage decrement in ANC during course 1 of 96.6% when paclitaxel over 24-h infusion preceded losoxantrone, 100% with the reverse sequence (P = 0.31), 95% when paclitaxel over 3-h infusion preceded losoxantrone, and 92.5% with the reverse sequence (P = 0.56), the degree of thrombocytopenia was worse, albeit not statistically significant, when 24-h paclitaxel preceded losoxantrone with a mean percentage decrement in platelet count during course 1 of 80.7% compared to 43.8% with the reverse sequence (P = 0.19). This trend of more severe thrombocytopenia was not noted with the 3-h schedule of paclitaxel with the mean percentage decrement in platelet counts during course 1 of 37.7% when paclitaxel preceded losoxantrone compared to 40.5% with the reverse sequence (P = 0.9).

These toxicity data provided the rationale for prophylactic G-CSF support, for administering losoxantrone before paclitaxel, and for administering paclitaxel as a 3-h infusion in all subsequent cohorts. The combination of losoxantrone 40 mg/m2 followed by paclitaxel 135 mg/m2 and G-CSF prophylaxis was, indeed, much better tolerated with only 1 of 18 courses associated with a DLT (grade 4 neutropenia for >5 days).

With the first dose level well tolerated, dose escalation proceeded with ascending losoxantrone dose to 50 mg/m2 and a fixed dose of paclitaxel at 135 mg/m2. With these doses not producing DLT in any course, the paclitaxel dose was escalated in three subsequent cohorts to 175, 200, and 225 mg/m2, respectively, and a fixed dose of losoxantrone at 50 mg/m2. These dose levels were associated with DLTs in ≤25% of all courses and dose escalation continued with the final dose level exploring 60 mg/m2 losoxantrone and 225 mg/m2 paclitaxel. This dose level was associated with DLTs in 2 of 5 patients and 2 of 14 courses. DLTs during the first two courses of therapy with paclitaxel/losoxantrone were observed in one of six patients at the 50/175 mg/m2 dose level, two of four patients at the 50/200 mg/m2 dose level, one of four patients at the 50/225 mg/m2 dose level, and two of five patients at the 60/225 mg/m2 dose level.

Grade 4 thrombocytopenia developed in 1 patient at each of the following dose levels of losoxantrone/paclitaxel (in mg/m2): 40/135 (24 h), 50/175, and 50/225. Grade 3 thrombocytopenia was the highest grade of thrombocytopenia, developing in one patient at each of the following dose levels of losoxantrone and paclitaxel (in mg/m2): 40/135 (3 h, no G-CSF), 40/135 (with G-CSF), 50/200, and 50/225. No grade 3 or 4 thrombocytopenia occurred in any of the five patients treated at the highest dose of losoxantrone/paclitaxel (60/225 mg/m2).

Red cell toxicity was minimal. Grade 4 anemia (hemoglobin, <6.5 mg/dl) occurred in 1 patient treated with 40 mg/m2 losoxantrone followed by 135 mg/m2 paclitaxel (3-h infusion) without G-CSF. Grade 3 anemia (hemoglobin, 6.5–7.9 mg/dl) developed in three patients at 40 mg/m2 losoxantrone and 135 mg/m2 paclitaxel, one patient at 50 mg/m2 losoxantrone and 200 mg/m2 paclitaxel, and one patient at 50 mg/m2 losoxantrone and 225 mg/m2 paclitaxel. No grade 3 or 4 anemia was seen at the highest dose level of losoxantrone and paclitaxel.

Nonhematological Toxicity

Cardiac Toxicity.

Two patients developed congestive heart failure. The first patient with congestive heart failure, which was not likely to be related to the study drugs, was a 61-year-old woman with metastatic lung cancer and no prior anthracycline therapy, who was treated at 40 mg/m2 losoxantrone followed by 135 mg/m2 paclitaxel (24 h), with a dose reduction of paclitaxel to 100 mg/m2 after course 1 secondary to myelosuppression. Her total cumulative dose of losoxantrone over five courses of therapy was 200 mg/m2. At baseline, LVEF was 61%, and after course 4, it was 67%. Following course 5, she developed cellulitis of the left arm while she was neutropenic and was hospitalized. Twenty-four h later, she developed hypotension and received i.v. fluids and packed RBCs. She then developed increasing dyspnea and an echocardiogram revealed significant mitral regurgitation with an estimated LVEF of 61%. Her dyspnea was probably related to fluid overload in the setting of significant mitral insufficiency.

The second patient with congestive heart failure was a 47-year-old woman with pleural mesothelioma. She had previously received doxorubicin at a total cumulative dose of 100 mg/m2 and received seven courses of losoxantrone 50 mg/m2 (total cumulative dose, 350 mg/m2, and 225 mg/m2 paclitaxel). At baseline and after course 6, LVEF was 51 and 52%, respectively. After course 7, she developed congestive heart failure manifested by dyspnea with a decrease in LVEF to between 39 and 43%. She was treated with furosemide and digoxin with significant clinical improvement and received additional therapy with paclitaxel alone and, subsequently, after progression, with BCNU and temozolomide.

In addition, one patient had a decline from pretreatment LVEF exceeding 20%, but without symptoms of congestive heart failure. He had colon carcinoma, no prior anthracycline therapy, and received six courses of losoxantrone 50 mg/m2 (total cumulative dose, 300 mg/m2) and 175 mg/m2 paclitaxel. The LVEF was 58% at baseline, 53% after cycle 4, declined to 33% about 14 days after course 6 (grade 2 toxicity), and the patient was taken off study due to this decline in LVEF. A repeat MUGA 3 months later revealed an increase in LVEF to 47%. Finally, eight patients had asymptomatic declines in LVEF of between 10 and 20%.

No relationship was found between the cumulative dose of losoxantrone and absolute change in LVEF (Fig. 2).

Other Significant Nonhematological Toxicity.

The combination of losoxantrone and paclitaxel was mildly emetogenic, with only one patient developing severe (grade 3) nausea and vomiting at losoxantrone and paclitaxel doses of 50 mg/m2 and 200 mg/m2, respectively. The incidence of mucositis was 9.8%, with four patients developing grade 1 mucositis. Neurological toxicities, principally paresthesia and hyperesthesia, were mild and occurred in eight (19.5%) and seven (17.1%) patients, respectively. Pronounced or total alopecia (grade 2) developed in 18 patients (29%) and mild alopecia (grade 1) occurred in five patients (12%). Two patients developed grade 3–4 hyperbilirubinemia. One patient with a mucoepidermoid tumor of the parotid gland, who received eight courses of 50 mg/m2 losoxantrone and 225 mg/m2 paclitaxel, developed grade 4 hyperbilirubinemia (peak bilirubin, 4.6 mg/dl) and elevation in LDH after a RBC transfusion and therefore the elevations were possibly due to transfusion-related hemolysis. The hyperbilirubinemia resolved prior to the next course of therapy, and the patient received five additional courses of study drugs with no dose reduction and no further hyperbilirubinemia. The second patient, with colon cancer and no liver metastases, developed grade 3 hyperbilirubinemia (peak bilirubin, 2.3 mg/dl) after the second course of 40 mg/m2 losoxantrone and 100 mg/m2 paclitaxel (dose reduction from 135 mg/m2 in course 1 secondary to fever and neutropenia), and the hyperbilirubinemia resolved prior to the next course of therapy. The patient received three additional courses of therapy, with a dose reduction in losoxantrone to 30 mg/m2 without further hyperbilirubinemia.

Antitumor Activity

One patient had a CR, and three patients had PRs. The patient with CR had an adenoid cystic carcinoma of the scalp with cervical lymph node metastases. He had prior radiation therapy to the cervical and supraclavicular nodes and no prior systemic chemotherapy and was treated with 40 mg/m2 losoxantrone followed by a 3-h infusion of 135 mg/m2 paclitaxel for four courses with complete clinical and radiological resolution of his metastases that lasted for 2 months prior to developing new pulmonary nodules. The three PRs included: a patient with non-small cell lung carcinoma with prior cisplatin and etoposide therapy who was treated with a 24-h infusion of 135 mg/m2 paclitaxel followed by 40 mg/m2 losoxantrone (4-month duration of response); a patient with transitional cell carcinoma of the bladder with prior therapy with methotrexate, vinblastine, doxorubicin, and cisplatin/carboplatin who was treated with 50 mg/m2 losoxantrone followed by 3-h infusion of 175 mg/m2 paclitaxel and G-CSF (unknown duration of response); and a patient with adenocarcinoma of the esophagus with prior radiation therapy to the esophagus concomitant with cisplatin and 5-FU who was treated with 50 mg/m2 losoxantrone followed by 3-h infusion of 225 mg/m2 paclitaxel and G-CSF (4-month duration of response).

Pharmacokinetic and Pharmacodynamic Studies

Plasma sampling for losoxantrone pharmacokinetics was performed in 13 patients following administration of 40 mg/m2 losoxantrone and 135 mg/m2 paclitaxel as a 3- or 24-h infusion either before or immediately after losoxantrone administration. Paclitaxel pharmacokinetics were determined in 17 patients who received paclitaxel doses ranging from 175 to 225 mg/m2 dose over 3 h immediately after losoxantrone. Losoxantrone pharmacokinetics were not simultaneously characterized in these patients. The pharmacokinetic parameters of losoxantrone and paclitaxel are listed in Tables 5 and 6, respectively, and representative disposition curves of losoxantrone are shown in Fig. 3. All data were optimally fitted to a three compartment model with i.v. infusion input for estimating the half-lives of t1/2α, t1/2β, and t1/2γ and the central volume of distribution (VC).

Losoxantrone pharmacokinetics were characterized by a rapid distribution phase (mean t1/2α = 0.13 h; range, 0.04–0.23 h), a mean t1/2β of 1.5 h (range, 0.38–3.2 h), and a long terminal elimination phase (mean t1/2γ = 35 h; range, 13–48 h). The Vss was large (mean = 456 liters/m2; range, 165-1257 liters/m2), and Cl was high (mean = 341 ml/min/m2; range, 217–526 ml/min/m2). Similarly, paclitaxel pharmacokinetics were characterized by rapid distribution into tissue (mean t1/2α = 0.36 h; range, 0.11–0.77 h), a mean t1/2β of 2.4 h (range, 1.1–6.1 h), and a long terminal elimination phase (mean t1/2γ = 27 h; range, 17–78 h). Mean paclitaxel Vss and Cl were 78 liters/m2 (range, 45–262 liters/m2) and 166 ml/min/m2 (range, 51–278 ml/min/m2), respectively.

The sequence-dependence pharmacokinetic effects of losoxantrone were evaluated for different paclitaxel schedules. No significant differences (P < 0.05) in losoxantrone AUC or Cl were noted based on the sequences of losoxantrone and paclitaxel on either the 24- or 3-h schedule of paclitaxel.

No linear relationship was found between losoxantrone or paclitaxel Cmax, AUC, or Cl and grade of neutropenia, grade of thrombocytopenia, grade 4 neutropenia for >5 days, and percentage decrement in ANC or platelets. Similarly, no relationship was found between course 1 losoxantrone exposure and the percentage decrement in LVEF.

Myelosuppression was the principal DLT of the combination of losoxantrone and paclitaxel and prevented dose escalation from the first dose level of 40 mg/m2 losoxantrone and 135 mg/m2 paclitaxel, without G-CSF support. DLTs were observed during 14 of 24 (58%) courses and 15 of 32 (47%) courses of therapy with 40 mg/m2 losoxantrone and 40 mg/m2 paclitaxel 40 mg/m2 administered over 24 and 3 h, respectively, without G-CSF support. In addition, the doses of paclitaxel after the first course were reduced in 8 of 13 (62%) patients, indicating that this dose level is intolerable without G-CSF. Because single-agent losoxantrone produced grade 4 neutropenia in 74% of patients in a Phase II study (13), the severe degree of myelosuppression observed when losoxantrone is combined with another myelosuppressive agent, paclitaxel, is not surprising. The addition of prophylactic G-CSF support allowed further dose escalation of both losoxantrone and paclitaxel. The MTD was defined as the dose at which >50% of the patients experienced DLT during course 1 and/or 2 of therapy. On the basis of this definition, the MTD was not reached with DLTs observed in one of six patients at the losoxantrone/paclitaxel 50/175 mg/m2 dose level, two of four patients at the 50/200 mg/m2 dose level, one of four patients at 50/225 mg/m2 dose level, and two of five patients at 60/225 mg/m2 dose level during the first two courses of therapy with losoxantrone and paclitaxel. On the basis of these results, the recommended dose level for further studies is 50 mg/m2 losoxantrone and 175 mg/m2 paclitaxel with G-CSF and was based on the recommended dose for each agent when used alone (11, 16). Using the same recommended doses for each agent in combination has the potential for significant antitumor activity. The preliminary results of a Phase III study comparing 50 mg/m2 losoxantrone and 175 mg/m2 paclitaxel with G-CSF, which was based on the results of this study, to 175 mg/m2 paclitaxel alone as a first-line cytotoxic therapy in patients with metastatic breast cancer, but in whom adjuvant anthracycline or mitoxantrone therapy was allowed, revealed that grade 3 and 4 neutropenia occurred in 69% of patients and grade 4 thrombocytopenia occurred in 11%, which is similar to the incidence of these toxicities in this study, and that there was an overall response rate of 53% (21). The response rate in the paclitaxel arm was only 15%. The response rate to losoxantrone and paclitaxel in the Phase III study is similar to the response rate (53%) observed in a recent Phase II trial of the combination of 60 mg/m2 doxorubicin and 200 mg/m2 paclitaxel as a 3 h infusion with G-CSF as first-line chemotherapy in patients with metastatic breast cancer and no prior adjuvant doxorubicin (22).

This study presented an opportunity to explore the toxicological and pharmacological effects produced by alternate sequences of losoxantrone and paclitaxel, which are particularly important because sequence-dependent toxicological and pharmacological effects between paclitaxel and doxorubicin have been observed (23). The administration of paclitaxel on a protracted (24-h) schedule followed by doxorubicin had been demonstrated to be associated with more severe toxicity, which is explained by a reduction in the clearance of doxorubicin by paclitaxel (15, 23). Although this interaction occurs on all schedules of paclitaxel, the sequence-dependent toxicological and pharmacological effects have been observed only with a prolonged (24-h infusion) paclitaxel schedule. Except for a slightly greater magnitude of myelosuppression, based on percentage decrement in the platelet counts during course 1 when paclitaxel is administered prior to losoxantrone over 24 but not 3 h, which did not reach statistical significance, there was no evidence of sequence dependent toxicological effects. This conclusion however, should be considered preliminary because the sequence-dependent toxicity was evaluated in a limited number of patients (six patients on the 24-h schedule of paclitaxel), and the patients did not serve as their own controls.

In this study, losoxantrone pharmacokinetics were characterized by a high total plasma clearance and a slow rate of elimination, suggesting extensive tissue distribution. Paclitaxel pharmacokinetic parameter values were consistent with data from previously published studies of paclitaxel given as a 3-h infusion. For example, in the study by Schiller et al.(24) of single-agent paclitaxel administered over 3 h, paclitaxel Cl over the dose range of 210–250 mg/m2 ranged between 162 and 198 ml/min/m2, which is similar to the Cl range of 152–180 ml/min/m2 observed in this study.

The principal reason for developing losoxantrone is its potential for less cardiotoxicity than doxorubicin. In this study, one patient developed CHF related to study drugs, and nine additional patients experienced >10% asymptomatic reduction in LVEF. However, no relationship could be established between the total cumulative dose of losoxantrone and the change in LVEF. Interestingly, cardiotoxicity was also noted in both arms of the Phase III trial comparing the combination of paclitaxel and losoxantrone to paclitaxel as a single agent in patients with metastatic breast cancer with two patients in the losoxantrone and paclitaxel arm and 1 patient in the paclitaxel arm developing CHF.(21) In addition, both Phase II studies of single agent losoxantrone in advanced breast cancer also reported cardiotoxicity with a decline in LVEF of >5% in 10 of 15 patients in one study (12) and 1 and 4% incidence of CHF and >20% asymptomatic decline in LVEF, respectively, in the second study (13). The frequency and severity of cardiotoxicity, its relationship to the cumulative dose of losoxantrone, and the risk benefit ratio of losoxantrone compared to doxorubicin await further studies.

In summary, the combination of 50 mg/m2 losoxantrone and 175 mg/m2 paclitaxel with G-CSF was well tolerated and is the recommended dose for further disease directed studies. This combination, at these doses, has demonstrated significant activity in a Phase III study in patients with metastatic breast cancer who had prior exposure to anthracyclines. Further evaluation of the efficacy and toxicity, including cardiotoxicity, of this combination is warranted. Whether losoxantrone has a superior therapeutic index compared to doxorubicin would require a direct comparison of the two agents in a Phase III setting.

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

        
1

Presented, in part, at the Annual Meeting of the American Society of Clinical Oncology, held May 14–17, 1994, in Dallas, TX.

                                
5

The abbreviations used are: DLT, dose-limiting toxicity; MTD, maximum tolerated dose; LVEF, left ventricular ejection fraction; CR, complete response; ANC, absolute neutrophil count; G-CSF, granulocycte colony-stimulating factor; PR, partial response; AUC, area under the concentration-time curve; CHF, congestive heart failure.

Fig. 1.

Chemical structures of doxorubicin, mitoxantrone, and losoxantrone.

Fig. 1.

Chemical structures of doxorubicin, mitoxantrone, and losoxantrone.

Close modal
Fig. 2.

The relationship between total losoxantrone dose and absolute decrease in LVEF.

Fig. 2.

The relationship between total losoxantrone dose and absolute decrease in LVEF.

Close modal
Fig. 3.

Plasma disposition curves for losoxantrone. ▵, losoxantrone followed by 24-h paclitaxel infusion; □, 24-h paclitaxel infusion followed by losoxantrone; ⋄, losoxantrone followed by 3-h paclitaxel infusion; ○, 3-h paclitaxel infusion followed by losoxantrone.

Fig. 3.

Plasma disposition curves for losoxantrone. ▵, losoxantrone followed by 24-h paclitaxel infusion; □, 24-h paclitaxel infusion followed by losoxantrone; ⋄, losoxantrone followed by 3-h paclitaxel infusion; ○, 3-h paclitaxel infusion followed by losoxantrone.

Close modal
Table 1

Dose escalation scheme

Losoxantrone dose, mg/m2Paclitaxel dose, mg/m2 (infusion time, h)SequenceaNo. of patientsNo. of courses
TotalWith DLTTotalWith DLT
Without G-CSF       
 40 135 (24) L→P 11 
  P→L 13 
 40 135 (3) L→P 18 
  P→L 14 
With G-CSF       
 40 135 (3) L→P 18 
 50 135 (3) L→P 12 
 50 175 (3) L→P 25 
 50 200 (3) L→P 
 50 225 (3) L→P 28 
 60 225 (3) L→P 14 
Total   41  162  
Losoxantrone dose, mg/m2Paclitaxel dose, mg/m2 (infusion time, h)SequenceaNo. of patientsNo. of courses
TotalWith DLTTotalWith DLT
Without G-CSF       
 40 135 (24) L→P 11 
  P→L 13 
 40 135 (3) L→P 18 
  P→L 14 
With G-CSF       
 40 135 (3) L→P 18 
 50 135 (3) L→P 12 
 50 175 (3) L→P 25 
 50 200 (3) L→P 
 50 225 (3) L→P 28 
 60 225 (3) L→P 14 
Total   41  162  
a

L, losoxantrone; P, paclitaxel.

Table 2

Patient characteristics

CharacteristicNo. of patients
Total no. of patients 41 
Median age, yr (range) 60 (25–79) 
WHO performance status  
 0 14 
 1 24 
 2 
Tumor type  
 Non-small cell lung 13 
 Head and neck 
 Melanoma 
 Colorectal 
 Esophagus 
 Sarcoma, unknown primary, bladder 2 each 
 Small cell lung, stomach, liver 1 each 
Previous treatment  
 Radiation and chemotherapy 14 
 Chemotherapy only 13 
 No treatment 
 Radiation only 
 Chemotherapy + immunotherapy 
 Chemotherapy + hormone 
 Chemotherapy + hormone + immunotherapy 
 Chemotherapy + radiation + hormone +  immunotherapy 
CharacteristicNo. of patients
Total no. of patients 41 
Median age, yr (range) 60 (25–79) 
WHO performance status  
 0 14 
 1 24 
 2 
Tumor type  
 Non-small cell lung 13 
 Head and neck 
 Melanoma 
 Colorectal 
 Esophagus 
 Sarcoma, unknown primary, bladder 2 each 
 Small cell lung, stomach, liver 1 each 
Previous treatment  
 Radiation and chemotherapy 14 
 Chemotherapy only 13 
 No treatment 
 Radiation only 
 Chemotherapy + immunotherapy 
 Chemotherapy + hormone 
 Chemotherapy + hormone + immunotherapy 
 Chemotherapy + radiation + hormone +  immunotherapy 
Table 3

Hematological toxicity (patients)

Losoxantrone dose, mg/m2Paclitaxel, mg/m2 (infusion time, h)SequenceaNo. of patientsNeutropenia gradeFebrile neutropeniabThrombocytopenia grade
TotalWith DLT344, >5 days34
Without G-CSF           
 40 135 (24) L→P 
  P→L 
 40 135 (3) L→P 
  P→L 
With G-CSF           
 40 135 (3) L→P 
 50 135 (3) L→P 
 50 175 (3) L→P 
 50 200 (3) L→P 
 50 225 (3) L→P 
 60 225 (3) L→P 
Losoxantrone dose, mg/m2Paclitaxel, mg/m2 (infusion time, h)SequenceaNo. of patientsNeutropenia gradeFebrile neutropeniabThrombocytopenia grade
TotalWith DLT344, >5 days34
Without G-CSF           
 40 135 (24) L→P 
  P→L 
 40 135 (3) L→P 
  P→L 
With G-CSF           
 40 135 (3) L→P 
 50 135 (3) L→P 
 50 175 (3) L→P 
 50 200 (3) L→P 
 50 225 (3) L→P 
 60 225 (3) L→P 
a

L, losoxantrone; P, paclitaxel.

b

Fever of >38°C and grade 4 neutropenia.

Table 4

Hematological toxicity (courses)

Losoxantrone dose, mg/m2Paclitaxel, mg/m2 (infusion time, h)SequenceaNo. of coursesNeutropenia gradeFebrile neutropeniabThrombocytopenia grade
TotalWith DLT344, >5 days34
Without G-CSF           
 40 135 (24) L→P 11 10 
  P→L 13 11 
 40 135 (3) L→P 18 
  P→L 14 
With G-CSF           
 40 135 (3) L→P 18 
 50 135 (3) L→P 12 
 50 175 (3) L→P 25 
 50 200 (3) L→P 
 50 225 (3) L→P 28 
 60 225 (3) L→P 14 
Losoxantrone dose, mg/m2Paclitaxel, mg/m2 (infusion time, h)SequenceaNo. of coursesNeutropenia gradeFebrile neutropeniabThrombocytopenia grade
TotalWith DLT344, >5 days34
Without G-CSF           
 40 135 (24) L→P 11 10 
  P→L 13 11 
 40 135 (3) L→P 18 
  P→L 14 
With G-CSF           
 40 135 (3) L→P 18 
 50 135 (3) L→P 12 
 50 175 (3) L→P 25 
 50 200 (3) L→P 
 50 225 (3) L→P 28 
 60 225 (3) L→P 14 
a

L, losoxantrone; P, paclitaxel.

b

Fever of >38°C and grade 4 neutropenia.

Table 5

Losoxantrone pharmacokineticsa

L→PbP→L
40→135c (24 h)40→135 (3 h)135 (24 h)→40135 (3 h)→40
No. of patients 
Cmax (ng/ml) 4049 (1811–6286) 4409 (840–8920) 1856 (1226–2455) 3145 (2463–3960) 
AUC (ng · h/ml) 2367 (2249–2484) 1735 (1268–2821) 1851 (1419–2195) 2544 (1527–3075) 
Cl (ml/min/m2282 (268–296) 425 (236–526) 373 (304–470) 283 (217–437) 
Varea (liters/m2850 (749–951) 1196 (561–2057) 1333 (1104–1524) 1119 (720–1384) 
Vss (liters/m2304 (165–444) 480 (212–1257) 570 (339–865) 470 (251–710) 
MRT (h) 18 (10–25) 18 (8.1–42) 25 (19–31) 32 (14–55) 
t1/2 (h) 35 (32–37) 33 (15–48) 42 (37–52) 49 (34–74) 
t1/2α (h) 0.12 (0.08–0.15) 0.09 (0.04–0.14) 0.18 (0.12–0.23) 0.12 (0.06–0.16) 
t1/2β (h) 1.2 (1.2–1.3) 1.1 (0.38–1.8) 2.4 (1.7–3.2) 1.3 (0.84–1.6) 
t1/2γ (h) 37 (34–39) 29 (13–48) 36 (31–41) 39 (27–53) 
Vc (liters/m24.1 (2.9–5.2) 7.6 (1.4–14) 14 (8.3–24) 6.5 (2.9–9.4) 
L→PbP→L
40→135c (24 h)40→135 (3 h)135 (24 h)→40135 (3 h)→40
No. of patients 
Cmax (ng/ml) 4049 (1811–6286) 4409 (840–8920) 1856 (1226–2455) 3145 (2463–3960) 
AUC (ng · h/ml) 2367 (2249–2484) 1735 (1268–2821) 1851 (1419–2195) 2544 (1527–3075) 
Cl (ml/min/m2282 (268–296) 425 (236–526) 373 (304–470) 283 (217–437) 
Varea (liters/m2850 (749–951) 1196 (561–2057) 1333 (1104–1524) 1119 (720–1384) 
Vss (liters/m2304 (165–444) 480 (212–1257) 570 (339–865) 470 (251–710) 
MRT (h) 18 (10–25) 18 (8.1–42) 25 (19–31) 32 (14–55) 
t1/2 (h) 35 (32–37) 33 (15–48) 42 (37–52) 49 (34–74) 
t1/2α (h) 0.12 (0.08–0.15) 0.09 (0.04–0.14) 0.18 (0.12–0.23) 0.12 (0.06–0.16) 
t1/2β (h) 1.2 (1.2–1.3) 1.1 (0.38–1.8) 2.4 (1.7–3.2) 1.3 (0.84–1.6) 
t1/2γ (h) 37 (34–39) 29 (13–48) 36 (31–41) 39 (27–53) 
Vc (liters/m24.1 (2.9–5.2) 7.6 (1.4–14) 14 (8.3–24) 6.5 (2.9–9.4) 
a

Values are means (ranges).

b

L, losoxantrone; P, paclitaxel.

c

Doses are in mg/m2 (values in parentheses are infusion times).

Table 6

Paclitaxel pharmacokineticsa

L→Pb
50→175c (3 h)50→200 (3 h)50→225 (3 h)60→225 (3 h)
No. of patients 
Cmax (ng/ml) 4,772 (3,710–5,730) 4,745 (4,210–5,440) 7,328 (5,630–9,680) 5,930 (5,030–6,730) 
AUC (ng · h/ml) 21,143 (13,974–33,196) 20,938 (16,815–26,168) 33,162 (13,129–74,436) 20,823 (19,134–22,171) 
Cl (ml/min/m2152 (90–211) 163 (125–194) 169 (51–278) 180 (166–199) 
Varea (liters/m2205 (136–357) 277 (166–543) 177 (24–359) 207 (122–242) 
Vss (liters/m280 (45–135) 121 (66–262) 51 (4.0–84) 61 (55–68) 
MRT (h) 11 (6.6–16) 16 (7.2–37) 5.9 (2.9–8.2) 7.3 (6.7–7.7) 
t1/2 (h) 16 (10–23) 22 (9.9–50) 11 (5.1–20) 13 (8.2–16) 
t1/2α (h) 0.4 (0.2–0.57)d 0.26 (0.11–0.57) 0.31 (0.14–0.66)d 0.46 (0.25–0.77) 
t1/2β (h) 3.4 (2.5–4.3)d 1.9 (1.1–3.4) 2.7 (2.1–3.1)d 3.5 (2.4–6.1) 
t1/2γ (h) 21 (17–27)d 23 (10–51) 38 (17–78)d 24 (10–50) 
Vc (liters/m29.7 (6.8–14)d 8.8 (5.9–17) 6.6 (2.8–13)d 12.7 (9.2–20) 
L→Pb
50→175c (3 h)50→200 (3 h)50→225 (3 h)60→225 (3 h)
No. of patients 
Cmax (ng/ml) 4,772 (3,710–5,730) 4,745 (4,210–5,440) 7,328 (5,630–9,680) 5,930 (5,030–6,730) 
AUC (ng · h/ml) 21,143 (13,974–33,196) 20,938 (16,815–26,168) 33,162 (13,129–74,436) 20,823 (19,134–22,171) 
Cl (ml/min/m2152 (90–211) 163 (125–194) 169 (51–278) 180 (166–199) 
Varea (liters/m2205 (136–357) 277 (166–543) 177 (24–359) 207 (122–242) 
Vss (liters/m280 (45–135) 121 (66–262) 51 (4.0–84) 61 (55–68) 
MRT (h) 11 (6.6–16) 16 (7.2–37) 5.9 (2.9–8.2) 7.3 (6.7–7.7) 
t1/2 (h) 16 (10–23) 22 (9.9–50) 11 (5.1–20) 13 (8.2–16) 
t1/2α (h) 0.4 (0.2–0.57)d 0.26 (0.11–0.57) 0.31 (0.14–0.66)d 0.46 (0.25–0.77) 
t1/2β (h) 3.4 (2.5–4.3)d 1.9 (1.1–3.4) 2.7 (2.1–3.1)d 3.5 (2.4–6.1) 
t1/2γ (h) 21 (17–27)d 23 (10–51) 38 (17–78)d 24 (10–50) 
Vc (liters/m29.7 (6.8–14)d 8.8 (5.9–17) 6.6 (2.8–13)d 12.7 (9.2–20) 
a

Values are means (ranges).

b

L, losoxantrone; P, paclitaxel.

c

Doses are in mg/m2 (values in parentheses are infusion times).

d

Pharmacokinetic parameters were from only three patients.

We thank Meredith Sterling for editing the manuscript.

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