Introduction

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Losoxantrone (7-hydroxy-2-[2-[(2-hydroxyethyl)amino]ethyl]- 5-[[2-[(2-hydroxyethyl)amino]ethyl]amino]anthra[1,9-cd]pyrazol-6(2H)-one, dihydrochloride) (also known as CI-941, DuP 941, or biantrazole, Fig. 1) is a member of the anthrapyrazole class of DNA intercalating agents in Phase III development in the U.S. Although not completely elucidated, the mechanism of action of losoxantrone is related to its ability to intercalate into DNA and block cell cycle division (Showalter et al., 1986). The clinical pharmacokinetics of losoxantrone have been well characterized during Phase I studies in cancer patients (Foster et al., 1992; Diab et al., 1999); however, the nature and extent of losoxantrone's elimination pathway have not been elucidated. In humans, renal excretion accounts for less than 10% of the dose (Graham et al., 1992). Two identified urinary metabolites (Blanz et al., 1993; Proksch et al., 1994; Richards and Sun, 1995) represent less than 0.6% of the administered i.v. dose. There is no evidence to suggest the presence of glucuronide conjugates in human urine. Therefore, in humans, the primary elimination pathway is presumably through the feces.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Structures for losoxantrone and three metabolites along with chromatograms showing separation by HPLC. Structures for losoxantrone (1), its monocarboxylic acid metabolites (2 and 3), and its dicarboxylic acid metabolite (4) are shown in the top panel. A, chromatogram showing separation of 1, 2, 3, and 4 by HPLC using UV/Vis detection. Radiochromatographic profiles are shown for unextracted [14C]losoxantrone (B), extracted quality control feces sample (C), and extracted patient feces sample (D).

Attempts to elucidate losoxantrone metabolism using in vitro metabolic systems have not been successful; therefore, this study was designed to determine the time course, as well as the metabolic profile, for the elimination of [¹⁴C]losoxantrone when given as a single i.v. dose to patients with refractory solid tumors during their first course of chemotherapy.

	Materials and Methods

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Study Medication. Individual vials of radiolabeled losoxantrone contained 20 µCi/ml of [¹⁴C]losoxantrone and 10 mg/ml of losoxantrone in sterile normal saline. The [¹⁴C]losoxantrone (98.4% radiochemically pure, lot CFQ7074-1) was synthesized at Amersham Pharmacia Biotech (Buckinghamshire, England) under Good Manufacturing Practices conditions and was uniformly labeled on the phenolic ring. Nonradiolabeled losoxantrone was supplied in vials containing 25 mg of lyophilized losoxantrone per vial and was reconstituted with normal saline.

Study Design. This was an open label, multiple dose study in which four patients with advanced solid tumors received 10 min losoxantrone infusions every 3 weeks. One hundred microcuries of radiolabeled drug was mixed with sufficient unlabeled drug to make up the 50 mg/m² infusion dose for the first course of therapy. The protocol and informed consent form were reviewed and approved by the Institutional Review Board, and each patient gave written informed consent before receiving the study medication. Radiation dosimetry calculations (Snyder et al., 1975) estimated that the radiolabeled dose used in this study would result in exposures (~3000 milliroentgen-equivalent-man) well below allowable limits (Massé and Miller, 1985).

Sample Collection. In the first patient, blood (plasma), urine, and feces were collected for up to 2 weeks after [¹⁴C]losoxantrone administration for the determination of total radioactivity, parent drug, and metabolites (feces only). Based on data from the first patient, blood, urine, and fecal samples were collected for 1 day, 1 week, and 2 weeks, respectively, for the remaining patients.

Analyses of Total Radioactivity in Plasma, Urine, and Feces. Total radioactivity was determined in plasma and urine by direct liquid scintillation counting (LSC¹) (model 1900CA or 2250CA, Packard Instruments, Downers Grove, IL) of 0.2- to 1.0-ml aliquots to which 15 ml of Ultima Gold Scintillation fluid (Packard) was added. Fecal samples were freeze-dried, reweighed, and pulverized to a fine homogeneous powder. Duplicate aliquots of ~0.1 g were added to Combusto-Cones (Packard), combusted in a sample oxidizer (Tri-Carb model B306, Packard), and radioactivity was determined by LSC. A specific activity individualized for each patient (mg/m² dose/100 µCi; range: 0.83-1.10 mg/µCi) was used to convert dpm concentrations to losoxantrone equivalents (ng/ml). The validated lower limit of quantification was 7 dpm (after background subtraction) based on precision and accuracy estimates of 15%.

Analyses of Parent Drug in Plasma and Urine. The extraction procedure was a previously published method for losoxantrone (Graham et al., 1989). Chromatographic separation of extracts was accomplished with a Waters (Milford, MA) Symmetry C₈ HPLC column (3.9 × 150 mm). The mobile phase consisted of acetonitrile/20 mM sodium heptane sulfonic acid/100 mM sodium acetate/40 mM tetrabutylammonium hydrogen sulfate (8:8:44:40, v/v/v/v) at a flow rate of 1 ml/min. A Waters fraction collector was used for collecting 2-ml fractions of the HPLC eluents. Fifteen milliliters of Ultima Gold was added to each vial, and radioactivity was measured by LSC as described for plasma total radioactivity.

HPLC Profiling of Radioactivity in Feces. Fecal samples containing the highest concentration of radioactivity from a patient (collected 80.7 h after dosing) were used for determining the parent drug radioactivity. Fecal samples (3 × 200 mg) or [¹⁴C]losoxantrone spiked control feces were repeatedly extracted with methanol and concentrated HCl (95:5 v/v, 5-10-ml volumes), and extracts were concentrated under a nitrogen stream to approximately 2 ml. A diluted aliquot was filtered using 0.45-µm Ultrafree centrifugal filters (Millipore Corp., Bedford, MA) and taken for analysis by LSC for tracking the recovery of radioactivity. A portion of the remaining filtrate (150 µl) was analyzed by HPLC equipped with a YMC (Wilmington, NC) phenyl column (100 × 4.6 mm) run in the isocratic mode using a mobile phase of ammonium phosphate buffer (pH 3, 54 nM)/isopropanol (93:7) at a flow rate of 1 ml/min. The eluent was mixed with Flo-Scint (Packard) in a 1:3 ratio and was monitored using a Radiomatic Flo-one/Beta model A500 radioactivity detector system (Packard). The remaining feces pellets were burned completely in a sample oxidizer.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

The urinary and fecal recoveries (mean ± S.D.) were 9.0 ± 2.3 and 60.9 ± 18.3%, respectively. Overall recovery (for up to 2 weeks) of total radioactivity was 69.9 ± 16.8%. A mean cumulative excretion plot is presented as an inset in Fig. 2. Radioactivity was excreted into urine within the first 4 h after dosing with [¹⁴C]losoxantrone and was essentially complete within the first 24 h. Fecal excretion was evident within the first 24 h and persisted for up to 9 days.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Plasma concentration-time curves of intact losoxantrone and losoxantrone equivalents of total radioactivity (mean ± S.D.). Inset, cumulative excretion plot of [14C] total radioactivity, expressed as a percentage of dose.

The concentration of [¹⁴C]losoxantrone closely paralleled the concentration of total radioactivity in plasma. Mean losoxantrone and total radioactivity concentrations in plasma are shown in Fig. 2. Intact losoxantrone plasma area under the curve represented 82% of the mean total radioactivity area under the curve (2198 ng·h/ml). Radioactivity in the urine accounted for 13% of the total radioactivity recovered. Intact losoxantrone accounted for the majority (88%) of the radioactivity recovered in urine (0-24 h).

Radioactivity in the feces accounted for the majority (87%) of the total radioactivity recovered, suggesting biliary or intestinal excretion. The major portion (77%) of this radioactivity was in the form of intact losoxantrone, based on radiochemical profiling and HPLC using UV/Vis retention time comparison to synthetic standards (Fig. 1). Extraction recoveries from control feces treated with [¹⁴C]losoxantrone as well as patient feces samples were more than 80% of the total radioactivity. Losoxantrone metabolites (2, 3, and 4), as previously identified in patient urine (Blanz et al., 1993; Proksch et al., 1994; Richards and Sun, 1995), were not evident in the feces sample from a patient given [¹⁴C]losoxantrone.

There are several potential mechanisms by which intravenously administered losoxantrone could be excreted intact in the feces. First, it could go directly into the bile from the liver, as has been described for the analogous anticancer agent mitoxantrone (Savaraj et al., 1982). Second, losoxantrone could be transported by intestinal P-glycoprotein and thereby secreted from the systemic circulation into the intestine. In vitro evidence that losoxantrone is a substrate for P-glycoprotein was demonstrated using a multidrug-resistant cell line (Chen et al., 1996). Resistance to losoxantrone by the human breast cancer cell line (MCF-7) was overcome by simultaneous exposure to thaliblastine, a known P-glycoprotein substrate. Separate studies have suggested that mitoxantrone and doxorubicin are substrates for intestinal P-glycoprotein in Caco-2 cells (Peters and Roelofs, 1992). Our study was not able to differentiate between these proposed mechanisms.