On-line chemiluminescence determination of mitoxantrone by capillary electrophoresis.

A novel capillary electrophoresis (CE) with chemiluminescence (CL) detection method for the determination of mitoxantrone (MTX) has been developed, which based on the CL reaction of potassium ferricyanide with luminol in sodium hydroxide medium sensitized by MTX. Under optimum analytical conditions, MTX is determined over the range of 7.0×10(-8)-1.0×10(-6)M with a detection limit of 1.0×10(-8)M. The relative standard deviation (RSD) was 3.7%, 2.6% and 3.0% for 7.0×10(-8), 5.0×10(-7) and 1.0×10(-6)M MTX (n=11), respectively. In laboratory-built CE-CL apparatus, the proposed method has been applied to determination of MTX in commercial drug and spiked in human urine and plasma with satisfactory results.

Pharmacokinetic studies of mitoxantrone and one of its metabolites in serum and urine in patients with advanced breast cancer.

OBJECTIVE: Mitoxantrone (MTO) was administered to patients with advanced breast cancer either as free MTO (f-MTO) or liposomal MTO (1-MTO). The intra- and interindividual variations in serum pharmacokinetics of MTO were analysed. In addition, the excretion of MTO and its metabolite mitoxantrone dicarboxylic acid (MTOD) in urine was determined. METHODS: The concentration of MTO was measured by high-performance liquid chromatography in serum over a period of 24 h and the amount of MTO and the metabolite MTOD excreted in urine over 18 h was determined. Pharmacokinetic parameters of f-MTO and 1-MTO were calculated. RESULTS: 1-MTO had a significantly longer half-life of distribution in the deep (third) compartment and thus a larger area under the curve (AUC) than f-MTO. No difference was found with respect to distribution in the peripheral (second) compartment. The kinetics of MTO in serum did not significantly differ between patients. In four patients repeated pharmacokinetic analyses gave superimposable results. Thus, there was no enzyme induction during therapy. By contrast, two patients with oedema had a much longer mean residence time (MRT) and AUC for MTO in serum. Despite the altered pharmacokinetics of f-MTD and 1-MTO, no toxic adverse effects occurred in these two patients. CONCLUSIONS: f-MTO and 1-MTO exhibited different distribution patterns in the deep compartment with a significantly increased half-life for 1-MTO. There is no need to monitor MTO for treatment of breast cancer patients with f-MTO. In patients with oedema, the MRT of MTO is prolonged. The clinical relevance of this observation is as yet unclear.

New aspects on the pharmacokinetics of mitoxantrone and its two major metabolites.

In spite of its broad clinical application in the treatment of malignant disorders, the pharmacokinetics of mitoxantrone are still not fully understood and warrant further investigation. Information is also limited about interindividual differences in the plasma AUC infinity (area-under-the-curve concentration to time infinity) and renal elimination of mitoxantrone and its main metabolites, mono- and dicarboxylic acid. In the present study, the plasma concentration of mitoxantrone was measured by HPLC during 120 h after the end of a 30-min infusion of 10 mg/m2 in 18 patients undergoing combination therapy with mitoxantrone and high-dose cytosine arabinoside for acute myeloid leukemia. Plasma kinetics and renal elimination of mono- and dicarboxylic acid were analyzed in addition in eight of these patients, and in five cases with chronic lymphocytic leukemia receiving a 30-min infusion of 5 mg/m2 mitoxantrone weekly for 3 consecutive weeks. Fitting the results to a three compartment model, a substantial interindividual variation was observed for plasma and urine pharmacokinetics. Plasma AUC infinity for mitoxantrone differed approximately 13-fold between individual patients and varied between 80-1030 ngh/ml. The corresponding values for mono- and dicarboxylic acid ranged from 23-147 ngh/ml and 51-471 ngh/ml, respectively. The median terminal half-life for mitoxantrone was similar to that of the mono- and dicarboxylic acid and was 75 h. Cumulative renal elimination ranged from 670-1950 micrograms for mitoxantrone, from 366-852 micrograms for monocarboxylic acid, and from 792-3420 micrograms for dicarboxylic acid. Renal clearance of mitoxantrone reached a median level of 69 ml/min and for the total plasma clearance a median of 1136 ml/min was found. The corresponding values for the mono- and dicarboxymetabolites were 57 and 67 ml/min. In contrast to the great interindividual differences in pharmacokinetic results, a low intraindividual variability was observed upon repeated determinations of renal elimination of mitoxantrone and its metabolites at weekly intervals in five patients. These data provide new insights into the pharmacokinetic of mitoxantrone and its main metabolites revealing substantial differences in drug metabolism and elimination between individual patients. Further studies are needed to explore the potential impact on response and/or toxicity and the requirement of a pharmacokinetic directed adjustment of drug dosage in clinical trials.

Polarographic and absorptive stripping voltammetric determination of mitoxantrone, emodin and chrysarobin using mercury and glassy carbon electrodes.

Polarographic and voltammetric methods were used to study drugs of anthrone derivatives: mitoxandrone (antineoplastic), emodin (cathartic) and chrysarobin (antipsoriatic) on mercury and glassy carbon electrodes. The surface-active properties were exploited for developing a highly sensitive adsorptive stripping voltammetric method for determination of trace amounts of these compounds. Effective pre-concentration was obtained at glassy carbon electrode, with the surface species being measured via its reduction or oxidation, respectively. It was concluded that observed cathodic and anodic currents had the diffusive character. A simple, quick and accurate methods for the determination of all studied compounds in pure form and mitoxantrone in vials have been developed. The acetate buffer pH 4.6 containing 5 to 30% ethanol was used as a supporting electrolyte. The linear calibration range was 0.5 = 100 microg cm-3 on mercury and 25 - 250 ng cm-3 on glassy carbon electrode.

Evidence for oxidative activation of mitoxantrone in human, pig, and rat.

A new metabolite of mitoxantrone in human, rat, and pig urine has been discovered by means of HPLC. The metabolite has been isolated by preparative HPLC from patient urine and is characterized by tandem mass spectrometry and UV-visible spectroscopy as 8,11-dihydroxy-4-(2-hydroxyethyl)-6-[[2-[(2-hydroxyethyl)amino]ethyl] amino]-1,2,3,4,7,12-hexahydronaphtho-[2,3-f]-chinoxaline-7,1 2-dione. Final structural proof has been obtained by independent synthesis. The new metabolite is a product of the enzymatic oxidation of the phenylenediamine substructure of mitoxantrone. An important biological consequence of the oxidative biotransformation is the possibility of covalent binding to intracellular targets via a highly electrophilic intermediate. Thus, alkylation may be an important mode of action of mitoxantrone. Incubation of mitoxantrone with horseradish peroxidase/hydrogen peroxide in the presence of glutathione led to the formation of two glutathione conjugates of mitoxantrone. Their structures have been elucidated by combination of IonSpray (Sciex, Canada) ionization and tandem mass spectrometry. Radioactive mitoxantrone, synthesized from sodium [14C]cyanide, was used to determine interspecies variations between human and rat. The collected rat urine was analyzed by HPLC using a radioactivity monitoring detector and revealed significant differences in the biotransformation of mitoxantrone in rat compared to human. The main metabolites thus far described in human urine are not observed in rat urine.

Isolation and structure elucidation of urinary metabolites of mitoxantrone.

Three 13C-labeled 1,4-dihydroxy-5,8-bis(2-[(2-hydroxyethyl)amino]-ethyl)amino-9,10- anthracenedione dihydrochloride (mitoxantrone) isotopomers were synthesized to prove the proposed chemical structures of human urinary metabolites by means of nuclear magnetic resonance spectroscopy. After application of labeled mitoxantrone to an anesthetized pig, urine was collected by way of a vesicourethral catheter. The urinary metabolites were isolated by liquid chromatography using a new procedure developed for extraction of mitoxantrone metabolites. Structural elucidation by nuclear magnetic resonance spectroscopy and tandem mass spectrometry confirmed the proposed mono- and dicarboxylic acid structures of the metabolites. High-performance liquid chromatography of native pig urine showed an additional metabolite detected by its UV-visible absorption. The new metabolite was identified as a glucuronic acid conjugate of mitoxantrone by means of nuclear magnetic resonance spectroscopy and tandem mass spectrometry. Incubation with beta-glucuronidase under high-performance liquid chromatography control revealed mitoxantrone as the sole product. Quantitative high-performance liquid chromatography analyses showed that the new urinary metabolite represents the main biotransformational pathway of mitoxantrone in pigs, indicating significant interspecies variation in mitoxantrone biotransformation. Expressed in equivalents of mitoxantrone, the new metabolite amounts to 25% and 31%, respectively, of urinary excreted drug-related material. Extraction of patient urine using the same procedure led to the isolation of pure metabolite B. Tandem mass spectrometric data delivered definitive evidence for the structure of metabolite B as monocarboxylic acid of mitoxantrone.

Excretion and metabolism of mitoxantrone in rabbits.

The hepatic clearance of mitoxantrone was evaluated in rabbits using both bile-duct cannulated animals and freshly isolated hepatocytes in suspension or in primary culture. Mitoxantrone metabolic behavior was assessed by high-performance liquid chromatography using a method which specifically resolved mitoxantrone from its mono- and dicarboxylic acid derivatives. Excretion of mitoxantrone in bile and urine was studied over a 6-h period of observation following i.v. bolus injection of 0.04, 0.20, and 1.0 mg [14C]mitoxantrone/kg. Bile route represented the main excretion pathway for mitoxantrone and its metabolites--mainly the monocarboxylic acid derivative. Biliary excretion was very rapid (maximum biliary concentration achieved 9 to 18 min following drug administration) and amounted to 29.5 +/- 9.3%, 27.6 +/- 7.9%, and 28.3 +/- 3.8% of administered drug, respectively. Urinary excretion amounted to 7.3 +/- 0.2%, 7.1 +/- 4.6%, and 6.0 +/- 1.5%, respectively. Both biliary and urinary excretions of mitoxantrone and its metabolites remained linear over the range of concentrations routinely used in clinic. Metabolism of mitoxantrone was first studied using rabbit hepatocytes in suspension. Since metabolic rate was slow under these incubation conditions (observation period, 1 h), mitoxantrone metabolism was investigated in primary cultures of rabbit hepatocytes. Mitoxantrone was rapidly accumulated within the cells and metabolized to its various metabolites which rapidly effluxed in the extracellular medium. After a 48-h exposure of hepatocytes to a broad range of mitoxantrone concentrations (1 to 20 microM), it could be seen that (a) drug accumulation and metabolism did not exhibit saturation processes, (b) mitoxantrone was the main intracellular form, while (c) metabolites rapidly effluxed in the extracellular compartment and (d) the monocarboxylic acid derivative represented the main extracellular metabolite. This data demonstrates the important role played by the liver in the pharmacokinetic behavior of mitoxantrone and suggests a careful drug monitoring in patients with severe liver dysfunction.

Direct determination of mitoxantrone and its mono- and dicarboxylic metabolites in plasma and urine by high-performance liquid chromatography.

The simultaneous isolation and determination of mitoxantrone (Novantrone) and its two known metabolites (the mono- and dicarboxylic metabolites) were carried out using a high-performance liquid chromatographic (HPLC) system equipped with an automatic pre-column-switching system that permits drug analysis by direct injection of biological samples. Plasma or urine samples were injected directly on to an enrichment pre-column flushed with methanol-water (5:95, v/v) as the mobile phase. The maximum amount of endogenous water-soluble components was removed from biological samples within 9 min. Drugs specifically adsorbed on the pre-column were back-flushed on to an analytical column (Nucleosil C18, 250 X 4.6 mm I.D.) with 1.6 M ammonium formate buffer (pH 4.0) (2.5% formic acid) containing 20% acetonitrile. Detection was effected at 655 nm. Chromatographic analysis was performed within 12 min. The detection limit of the method was about 4 ng/ml for urine and 10 ng/ml for plasma samples. The precision ranged from 3 to 11% depending on the amount of compound studied. This technique was applied to the monitoring of mitoxantrone in plasma and to the quantification of the unchanged compound and its two metabolites in urine from patients receiving 14 mg/m2 of mitoxantrone by intravenous infusion for 10 min.

Pharmacokinetics of ametantrone acetate (NSC-287513).

Ametantrone is the second anthracene derivative to enter clinical trials. The pharmacokinetic parameters for ametantrone acetate (CI-881) were characterized in six patients concurrently with the phase I clinical trial. Biological samples were assayed by a specific and sensitive high-performance liquid chromatography procedure. Plasma levels of ametantrone declined in a triexponential fashion, with a mean terminal half-life (t 1/2 gamma) of 25 h. The estimated mean total-body plasma clearance was 25.9 +/- 14.7 1 h-1 m-2. The steady-state volume of distribution (Vdss) was large, averaging 568 +/- 630 l/m2. Excretion of unchanged ametantrone in the urine over 48 h averaged 5.7% of the total dose, indicating that there is another major route of elimination.