Liquid Chromatography-Tandem Mass Spectrometry for the Simultaneous Determination of Doxorubicin and its Metabolites Doxorubicinol, Doxorubicinone, Doxorubicinolone, and 7-Deoxydoxorubicinone in Mouse Plasma.

Doxorubicin, an anthracycline antitumor antibiotic, acts as a cancer treatment by interfering with the function of DNA. Herein, liquid chromatography-tandem mass spectrometry was for the first time developed and validated for the simultaneous determination of doxorubicin and its major metabolites doxorubicinol, doxorubicinone, doxorubicinolone, and 7-deoxydoxorubicinone in mouse plasma. The liquid-liquid extraction of a 10 µL mouse plasma sample with chloroform:methanol (4:1, v/v) and use of the selected reaction monitoring mode led to less matrix effect and better sensitivity. The lower limits of quantification levels were 0.5 ng/mL for doxorubicin, 0.1 ng/mL for doxorubicinol, and 0.01 ng/mL for doxorubicinone, doxorubicinolone, and 7-deoxydoxorubicinone. The standard curves were linear over the range of 0.5-200 ng/mL for doxorubicin; 0.1-200 ng/mL for doxorubicinol; and 0.01-50 ng/mL for doxorubicinone, doxorubicinolone, and 7-deoxydoxorubicinone in mouse plasma. The intra and inter-day relative standard deviation and relative errors for doxorubicin and its four metabolites at four quality control concentrations were 0.9-13.6% and -13.0% to 14.9%, respectively. This method was successfully applied to the pharmacokinetic study of doxorubicin and its metabolites after intravenous administration of doxorubicin at a dose of 1.3 mg/kg to female BALB/c nude mice.

Feasibility of simple chitosan sheet as drug delivery carrier.

Chitosan, a biodegradable and biocompatible polysaccharide, is a potentially useful material in various fields. We developed a simple chitosan sheet and examined the possibility of using an adriamycin-containing chitosan sheet as a drug carrier for controlled release. To prepare a carrier consisting only of chitosan, a chitosan suspension was subjected to acid-alkaline treatment, mixed with adriamycin, frozen and freeze-dried. The adriamycin-containing chitosan sheet was inserted into the peritoneal cavity of mice in order to investigate its biodegradation. The appearance of decomposition of chitosan was observed using scanning electron microscopy, and adriamycin in urine and liver was detected for 1 and 2 weeks, respectively. Adriamycin metabolites were detected in plasma for 2 weeks. Furthermore, adriamycin remained in the chitosan sheet without being metabolized after 2 months. These results suggested that the chitosan sheet prepared in this study might improve therapeutic efficacy in topical lesions as a carrier of sustained-release drugs.

Quantitation by flow cytometry of anthracycline drug uptake by peripheral blood and bone marrow cells in human leukemias.

The inherent fluorescence of the anthracycline drugs can be combined with flow cytometry to obtain a convenient and rapid method for the quantitation of anthracycline drug uptake by human leukemic cells. A good correlation exists between the average cellular fluorescence intensity and the amount of drug associated with the cell as measured by extraction. Cellular incorporation of adriamycin and daunomycin was determined in peripheral blood and bone marrow cells of human leukemic patients after standardized in vitro exposures. Differences in uptake were found between the different cell types, with cells of lymphatic origin incorporating less of the drugs than nonlymphatic cells. Preliminary observations made in two patients with nonlymphatic leukemia showed a correlation between the in vitro uptake of adriamycin and the clinical response.

Plasma kinetics of aclacinomycin A and its major metabolites in man.

The plasma pharmacokinetics of the antineoplastic anthracycline antibiotic aclacinomycin A (Acm) and its metabolites were studied in 12 patients treated with 60-120 mg/m2 during a phase I clinical trial. Total plasma drug fluorescence initially declined very rapidly, but from 2 to 24 h after injection, fluorescence rose progressively to intensities greater than those measured 1 min after Acm injection. Plasma total drug fluorescence slowly declined from 24 to 72 hours after Acm administration. These events reflected the rapid disappearance of Acm and the subsequent appearance of two highly fluorescent metabolites. One metabolite co-chromatographed with and had a fluorescence spectrum identical to known metabolite F1 (bisanhydroaklavinone). The other metabolite did not co-chromatograph with any previously described Acm metabolite. This metabolite had a fluorescence spectrum unlike any previously described Acm metabolite and was not altered by treatment for 60 min with 0.2 N HCl at 100 degrees C or by treatment for 24 h at 37 degrees C with bacterial beta-glucuronidase or limpet aryl sulfatase.

Sensitive enzyme immunoassay for the quantification of aclacinomycin A using beta-D-galactosidase as a label.

A sensitive enzyme immunoassay method (EIA) for an anticancer drug, aclacinomycin A (ACM), has been developed. With a double-antibody technique, ACM at a concentration as low as 100 pg/tube can be detected. An antibody to ACM was obtained by immunizing rabbits with an antigen prepared by coupling ACM with mercaptosuccinylated bovine serum albumin via N-maleoyl aminobutyric acid (MABA) as a coupling agent. Enzyme labeling of ACM was performed with beta-D-galactosidase (beta-Gal; EC 3.2.1.23) via m-maleoyl benzoic acid (MBA). The standard curve of the assay was linear on a logit-log plot over a concentration range of 30 pg to 10 ng. The antibody detected ACM and its metabolites, MA144 M1 (M1), MA144 N1 (N1), MA144 S1 (S1), and aklavin (T1) equally well, but was only minimally reactive with aklavinone (D1) and 7-deoxyaklavinone (C1), thus suggesting that this EIA can detect the total amounts of ACM and its biologically active glycosides among metabolites of ACM. This EIA is practically free from interference by any other anticancer drugs. Using this assay, serum levels of ACM equivalents can be determined accurately after administration of the drug to rats at a single dose of 10 mg/kg. Since ACM is now undergoing clinical trial, the EIA of the drug will be a valuable tool in clinical pharmacological studies.

Occurrence of circulating 7-deoxyaglycone metabolites of 4'-deoxydoxorubicin in man.

In five cancer patients we have determined the pharmacokinetics of 4'-deoxydoxorubicin (4'-DOX), its alcoholic metabolite 4'-deoxydoxorubicinol and the occurrence of circulating 7-deoxyaglycone metabolites. The 7-deoxyaglycone of the alcohol metabolite, the major aglycone of Adriamycin (ADR) present in man, was not detected in any serum sample. The 7-deoxyaglycone of the parent drug, which appears in concentrations in excess of 30 ng/ml after ADR administration, was detected in only 2/5 patients in trace amounts. These preliminary data indicate a difference in biotransformation between ADR and 4'-DOX despite their close structural similarities.

Comparative murine metabolism and disposition of class II anthracycline antibiotics.

The metabolism and tissue distribution of aclacinomycin A (ACL), marcellomycin (MCM), and musettamycin (MST), three new anthracycline antibiotics, were compared after IV administration to mice. In plasma, total MCM- and ACL-derived fluorescence declined according to first-order kinetics, whereas an initial decline followed by a rebound was observed for MST. In plasma, MCM remained the predominant compound. ACL was eliminated more quickly, and was replaced by two metabolites, the reduced glycoside M1, and an aglycone. In the case of MST, two unidentified metabolites were observed in concentrations equivalent to that of the parent drug. The three drugs were distributed widely to organs, but only ACL achieved measurable concentrations in the brain. Initially, high concentrations of all three drugs were present in the lungs, but these decreased quickly to values similar to those present in the liver and kidneys. Intermediate concentrations of the three drugs were measured in heart and skeletal muscle. Splenic concentrations of all three drugs rose progressively, reaching a maximum at 8 h after injection in the case of ACL and MST, and at 24 h after injection in the case of MCM. Concentrations of the metabolites of MCM and MST were low in all organs except liver and kidney, where the aglycones 7-deoxypyrromycinone and bisanhydropyrromycinone were seen. The metabolism of ACL was extensive. Aglycones were dominant in the liver and kidneys, whereas reduced glycosides predominated in the spleen. These observations indicate that the murine pharmacology of these three structurally similar drugs differs markedly.

Pharmacokinetics and metabolism of doxorubicin after short-term infusions in lymphoma patients.

PURPOSE/METHODS: Twenty-four patients (17 males and 7 females with a mean age of 54 years) with malignant lymphoma participated in a study of doxorubicin pharmacokinetics after 50 mg/m(2) as 10-min infusions. In addition to plasma samples, serial leukocyte samples and - in one subject - serial biopsy specimens from lymphoma infiltrates were obtained. The samples were analysed by reversed-phase high-performance liquid chromatography. RESULTS: In contrast to several previous studies, the data suggested that 7-deoxydoxorubicinolone, and not doxorubicinone, is a metabolite of doxorubicin in humans. Doxorubicin, but no metabolites, was present in significant and fairly constant concentrations in circulating leukocytes. These levels may reflect the drug levels in lymphoma infiltrates. The data further suggest that metabolism to 7-deoxydoxorubicinone is subject to large interindividual variation, possibly due to a genetic polymorphism, and that significant levels of a metabolic product which may be a doxorubicin glucuronide can be recovered from plasma of patients treated with doxorubicin.

Anthracycline assay by high-pressure liquid chromatography.

A general method of analysis of anthracycline concentrations was developed. Drug is extracted from plasma with organic solvent and separated from metabolites by high-pressure liquid chromatography on an aminocyanosilica column. Detection and quantitation are by the endogenous fluorescence of compounds having an intact tetracyclic ring structure. Limits of sensitivity are 5, 1, and 5 ng/ml of plasma for doxorubicin, carubicin, and marcellomycin, respectively. The assay can be used for studying the aldo-keto reductase and reductive glycosidase reactions with anthracyclines as the substrates and for the evaluation of the clinical pharmacology or pharmacodynamics of various doxorubicin analogs.

A sensitive procedure for the quantitation of free and N-(2-hydroxypropyl) methacrylamide polymer-bound doxorubicin (PK1) and some of its metabolites, 13-dihydrodoxorubicin, 13-dihydrodoxorubicinone and doxorubicinone, in human plasma and urine by reversed-phase HPLC with fluorimetric detection.

A high-performance liquid chromatographic assay has been developed and validated for the determination in plasma and urine of doxorubicin (DXR) and some of its metabolites released in vivo from an N-(2-hydroxypropyl)methacrylamide (HPMA) polymer containing DXR linked through its aminosugar moiety to the polymer via an oligopeptide spacer (PK1). The method also allows measurement of the DXR still bound to the polymer. Following addition of two internal standards, the free compounds were extracted twice with isopropanol-chloroform (25:75, v/v). The first extraction was performed at physiological pH and the second after buffering at pH 8.4, in order to extract the aglycones and the glycosides, respectively. Determination of total DXR (polymer-bound plus free DXR) was performed, after quantitative acid hydrolysis to release doxorubicinone from free or polymer-bound DXR, by extraction with the same solvent mixture at pH 7.4. In both cases the organic phase was evaporated to dryness; the compounds were then separated by reversed-phase high-performance liquid chromatography (HPLC) under isocratic conditions and quantitated by fluorimetric detection. In the chromatograms all the analytes appeared to be separated at the baseline and no interference from blank human plasma and urine was observed. The suitability of the method for in vivo samples was checked by the analysis of plasma and urine samples obtained from a cancer patient who had received a single intravenous dose of the test compound.

Rapid quantitative determination of doxorubicin and its metabolites in biological samples.

A rapid sensitive and selective method is developed for the plasma analysis of doxorubicin and its metabolites, doxorubicinol and doxorubicinone with daunorubicin as the internal standard, by using high performance liquid chromatography (HPLC) with fluorescence detection and a "zorbax ODS" column. An eluent containing tetrahydrofuran and trietylamine afforded improved efficiency and resolution and was used to resolve the four anthracycline derivatives in a sole isocratic run. An example of the plasma levels obtained in a cancerous patient after two different administrations is shown.

[Studies on the absorption, excretion and distribution of aclacinomycin A: absorption, excretion and distribution of 14C- or 3H-aclacinomycin A in mice, rats and rabbits (author's transl)].

A new anthracycline antitumor antibiotic, aclacinomycin A, was labeled with 3H uniformly or with 14C simultaneously at the anthracycline nucleus and L-rhodosamine. These labeled drugs were administered intravenously to normal dd mice, solid type Sarcoma 180 tumor-bearing ICR mice, normal or pregnant Wistar rats and normal rabbits, respectively. 14C-Aclacinomycin A given to rabbits (5 mg/kg) was rapidly cleared from the blood and transferred to tissues. But low level of radioactivity (equivalent to about 0.5 mcg/ml) was remained in the blood even 8 approximately 10 hours after administration. About 45% of the radioactivity were recovered from the urine and 20% from the feces by 72 hours after administration. Tissue levels of 3H-14C-aclacinomycin A given to normal and tumor-bearing mice were highest in the lungs and spleen. Higher distribution was observed also in the liver and kidneys 2 hours after administration. Bioassay revealed that the drug was present in the lungs and spleen in biologically active form and in the liver and kidneys in inactive form, respectively. In the tumor tissue the radioactivity was low but it persisted for 48 hours. Autoradiography with 14C-aclacinomycin A in rats demonstrated that radioactivity due to the drug distributed in the lungs, spleen, kidneys, thymus, intestine, lymph nodes, bone marrow, salivary gland, hypophysis and pineal body but it was rapidly cleared. About 0.2% of radioactivity given to a pregnant rat were transferred to a fetus when 14C-aclacinomycin A was administered intravenously on the 18 approximately 19th day of pregnancy.

[Studies on the absorption, excretion and distribution of aclacinomycin A: absorption, excretion and distribution of aclacinomycin A in mice, rabbits and dogs by photometric assay (author's transl)].

An anthracycline antitumor antibiotic, aclacinomycin A, was given to mice, rabbits or dogs intravenously to study the pharmacokinetics by photometric assay based on the absorption of anthracycline ring. The drug was rapidly eliminated from the blood in these animals. Drug levels were much higher in the blood cells than in the plasma. Tissue levels in dogs were 50 approximately 100 times higher than the blood levels, which showed the drug was rapidly transferred from the blood to tissues after administration. Higher levels were observed in the lungs, spleen and lymph nodes, where the drug was present as aclacinomycin A itself and the glycoside-type metabolites that were biologically active. The active form was also detected in the pancreas, heart, thymus, bone marrow and gastrointestinal tract. In the liver and kidneys, biologically inactive aglycone-type metabolites were observed. About 2 approximately 4% of the drug given to rabbits or dogs was recovered in the urine by 72 hours after administration, in which only 10% of the excreted drug was active form in rabbits but about 65% in dogs. The rest was inactive aglycone-type metabolites that were excreted almost in the conjugated form. Biliary excretion also contributed to the total clearance of the drug. Aclacinomycin A was absorbed even by oral administration in rabbits and dogs. Tissue distribution of the drug orally given to dogs was similar to that in intravenous administration, except that higher levels of active form were detected in the gastrointestinal tract and of inactive form in the liver.

Determination of adriamycin and its fluorescent metabolites in human plasma by HPLC.

We describe a method for measuring adriamycin and its metabolites, adriamycinol and adriamycinone in plasma, using reversed phase HPLC and fluorescence detection. The lower limit of detection is approximately 1 ng/mL for each compound. An extraction technique for serum is described which is capable of an almost equal recovery (greater than 93%) of adriamycin and metabolites without interference from endogenous components of plasma and from other common drugs. Within-day and day to day coefficients of variation are estimated.