adriamycin-aglycone and adriamycinol

Doxorubicin (DOX) is one of the most effective cytotoxic agents against malignant diseases. However, the clinical application of DOX is limited, due to dose-related toxicity. The development of DOX nanoformulations that significantly reduce its toxicity and affect the metabolic pathway of the drug requires improved methods for the quantitative determination of DOX metabolites with high specificity and sensitivity. This study aimed to develop a high-throughput method based on high-performance liquid chromatography with fluorescence detection (HPLC-FD) for the quantification of DOX and its metabolites in the urine of laboratory animals after treatment with different DOX nanoformulations. The developed method was validated by examining its specificity and selectivity, linearity, accuracy, precision, limit of detection, and limit of quantification. The DOX and its metabolites, doxorubicinol (DOXol) and doxorubicinone (DOXon), were successfully separated and quantified using idarubicin (IDA) as an internal standard (IS). The linearity was obtained over a concentration range of 0.05-1.6 μg/mL. The lowest limit of detection and limit of quantitation were obtained for DOXon at 5.0 ng/mL and 15.0 ng/mL, respectively. For each level of quality control (QC) samples, the inter- and intra-assay precision was less than 5%. The accuracy was in the range of 95.08-104.69%, indicating acceptable accuracy and precision of the developed method. The method was applied to the quantitative determination of DOX and its metabolites in the urine of rats treated by novel nanoformulated poly(lactic-co-glycolic acid) (DOX-PLGA), and compared with a commercially available DOX solution for injection (DOX-IN) and liposomal-DOX (DOX-MY).

Topics: Animals; Doxorubicin; Female; Male; Naphthacenes; Polyethylene Glycols; Rats; Rats, Wistar; Urine

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.

Topics: Animals; Antibiotics, Antineoplastic; Biocompatible Materials; Biodegradation, Environmental; Chitosan; Doxorubicin; Drug Carriers; Drug Delivery Systems; Drug Stability; Feasibility Studies; Female; Liver; Mice; Naphthacenes; Peritoneal Cavity; Permeability; Time Factors

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.

Topics: Blood; Calibration; Chromatography, High Pressure Liquid; Doxorubicin; Humans; Naphthacenes; Polymethacrylic Acids; Quality Control; Spectrometry, Fluorescence; Urine

The effect of tumor cell density on the cellular pharmacokinetics of doxorubicin (DXR) and cisplatin (CDDP) was studied using MOLT-3 human acute lymphoblastic leukemia cells. As determined by the MTT assay, the growth-inhibitory effect of DXR was approx. 40 times lower when cell density was increased from 10(6) to 10(8) cells/ml (positive inoculum effect), whereas little or no influence of cell density was observed in CDDP-induced cell-growth inhibition. As measured by high-performance liquid chromatography using a fluorescence detector, the cellular accumulation of DXR showed 6- and 18-fold decreases after 1 h incubation when the cells were concentrated from 10(6) to 10(7) and 10(8) cells/ml, respectively. Only at low cell density (10(6) cells/ml) did the amount of DXR in the cells increase with increasing exposure times of up to 6 h. The DXR concentration in the supernatant that was separated from a cell suspension showing a density of 10(8) cells/ml fell to 20% of that obtained at 10(6) cells/ml. The metabolites of DXR, including Adriamycinol and Adriamycinone, were not detectable in the cell extracts or supernatants at any cell density examined. In contrast, the cellular accumulation of CDDP calculated from the platinum concentration, which was measured with a flameless atomic absorption spectrophotometer, was essentially identical at all cell densities examined; moreover, extension of the exposure period resulted in a linear increase in the amount of CDDP in the cells. CDDP concentrations in the supernatants were equally retained, irrespective of cell densities. These observations indicate that the positive inoculum effect shown in DXR-induced cell-growth inhibition results from the decreased cellular accumulation of the drug at high cell densities. We found no influence for cell density on the cellular accumulation of CDDP that might be relevant to the therapeutic potentiation of this drug at high tumor-cell density.

Topics: Cell Count; Cell Division; Chromatography, High Pressure Liquid; Cisplatin; Doxorubicin; Humans; Naphthacenes; Platinum; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Spectrophotometry, Atomic; Tumor Cells, Cultured

Recombinant human granulocyte colony-stimulating factor (G-CSF) has been shown to reduce neutropenia following cytotoxic therapy, thereby enabling dose escalation to improve the response rate. It is important to know whether drug kinetics change as doses are increased. Doxorubicin was selected because of its broad spectrum of activity and its known efficacy in metastatic breast cancer. Doses of 75, 100, 125 and 150 mg/m2 were given to 11 patients with metastatic breast cancer by infusion over 30 min. Serum concentrations of parent drug and metabolites were determined during the first 48 h following the infusion by high-performance liquid chromatography (HPLC). The serum concentration vs time curve decayed as a triple exponential function in four patients and as a double exponential function in seven. A four-compartment model, one central and three peripheral, would predict concentrations to within 1 SE of the observed values. Doxorubicinol was the principal metabolite, and doxorubicinone and 7-deoxydoxorubicinone were clearly identified. There was a linear increase in the AUC infinity with dose. In addition, a small and transient increase in circulating levels of doxorubicinol and other important metabolites was observed 6 h following the administration of doxorubicin, which suggests the existence of an enterohepatic, or other, re-circulation mechanism. We conclude that in the dose range selected the kinetics of doxorubicin are linear and that the increase in toxicities seen with the higher doses of doxorubicin, following the second and third fortnightly administration, may be due to intracellular drug accumulation in tissues.

Topics: Adult; Breast Neoplasms; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Doxorubicin; Female; Half-Life; Humans; Metabolic Clearance Rate; Middle Aged; Naphthacenes

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.

Topics: Chromatography, High Pressure Liquid; Daunorubicin; Doxorubicin; Humans; Naphthacenes; Neoplasms; Reference Values

A rapid and sensitive isocratic technique is described for the determination of concentrations of adriamycin and two of its metabolites, adriamycinol and adriamycinone, in freshly isolated rat hepatocytes. The drugs are easily and efficiently extracted from the cells with an organic mixture (chloroform-n-butanol) after proteolytic digestion with trypsin. Mean recoveries from spiked culture medium cell suspension are greater than 96%. The within run and day-to-day coefficients of variation are less than 7.5%.

Topics: Animals; Cells, Cultured; Chromatography, High Pressure Liquid; Doxorubicin; Liver; Naphthacenes; Rats; Rats, Inbred Strains; Spectrometry, Fluorescence

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.

Topics: Chromatography, High Pressure Liquid; Doxorubicin; Humans; Naphthacenes; Spectrometry, Fluorescence

Topics: Buffers; Cells, Cultured; Doxorubicin; Epirubicin; HeLa Cells; Humans; Liver; Naphthacenes; Time Factors

Adriamycin has been detected by high-pressure liquid chromatography in ascites and pleural fluid within 30 min after intravenous injection of Adriamycin (50 mg/m2). The concentration of Adriamycin was approximately one-half of that in plasma. Significant concentrations of Adriamycin metabolites were seen in the ascites fluid of one patient 4 hr after intravenous drug injection. Individual variability in the achievable ascites and pleural concentrations of cytotoxic drugs may be important in determining the responsiveness of patients to chemotherapy and the described analytical technique should be useful in assessing the concentrations of Adriamycin and its metabolites in malignant ascites and pleural effusions.

Topics: Ascitic Fluid; Chromatography, High Pressure Liquid; Cystadenocarcinoma; Daunorubicin; Doxorubicin; Female; Humans; Kinetics; Middle Aged; Naphthacenes; Ovarian Neoplasms; Pleural Effusion; Reference Standards

The effect of adriamycin on human and mouse CFUGM was examined at concentrations and times suggested by plasma clearance data derived from the results of a number of published studies. Our results suggest that the high concentrations of drug present in the plasma for short periods of time following infusion are only weakly cytotoxic towards the CFUGM when incubated for similar times. In contrast, there was a considerably greater cytotoxic effect when the drug was examined at low concentrations for periods similar to those described for the terminal phase of adriamycin clearance. The principal metabolite, adriamycinol, was poorly cytotoxic.

Topics: Animals; Cell Survival; Colony-Forming Units Assay; Doxorubicin; Female; Granulocytes; Hematopoietic Stem Cells; Humans; Macrophages; Male; Mice; Naphthacenes; Time Factors

A reversed-phase isocratic high-performance liquid chromatographic assay is described for the measurement of adriamycin, adriamycinol and their 7-deoxyaglycones in human serum. The lower limit of detection in serum is 3 ng/ml for adriamycin and 1 ng/ml for adriamycinol and the 7-deoxyaglycones with coefficients of variation for k' of less than 5% throughout the day. An extraction technique for serum is described which is capable of an almost equal recovery (greater than 77%) of adriamycin, metabolites and daunorubicin (the internal standard) without interference from endogenous components of serum. Serum concentrations of metabolites 15 min after intravenous bolus administration of 40 mg/m2 adriamycin in two different patients were 26.5 and 16.6 ng/ml for adriamycinol; 109.8 and 5.8 ng/ml for the adriamycinol 7-deoxyaglycone and 21.4 and 17.1 ng/ml for the adriamycin 7-deoxyaglycone. A total of six metabolites of adriamycin were detected in the two patients using this methodology.

Topics: Chemical Phenomena; Chemistry, Physical; Chromatography, High Pressure Liquid; Doxorubicin; Humans; Naphthacenes; Solubility

A sensitive reproducible, nondestructive method for the determination of adriamycin and its metabolites in plasma, leukocytes and tissues has been developed. Apolar substances as adriamycinone (adm-one) were extracted at pH 2 with chloroform, polar ones as adriamycin (adm) and adriamycinol (adm-ol) at pH 8.8 with chloroform: methanol, separated by thin-layer-chromatography and quantitated by fluorescence spectrophotometry. The plasma levels of adm-ol and adm-one were lower in all patients compared to those of adm. Further metabolites were found in the bile. Toxic effects were found in patients with prolonged half-lives in the elimination phase. A delayed elimination was observed in a patient with an elevation of the bilirubin level, but also in patients without overt liver disease. The pharmacokinetics of adm showed considerable inter- and intraindividual fluctuations.

Topics: Chromatography, Thin Layer; Doxorubicin; Half-Life; Humans; Kinetics; Leukocytes; Naphthacenes; Spectrometry, Fluorescence; Tissue Distribution

We have examined the in vitro sensitivity to Adriamycin of human ovarian cancer colonies cloned in soft agar. In the 26 patients tested, 3 different patterns of sensitivity to Adriamycin were observed: (a) in 75% of the previously untreated patients, there was greater than 70% reduction in colony-forming cells after exposure to Adriamycin (1.0 micrograms/ml), a level which approximates the peak plasma level after i.v. therapy; (b) in all the patients who had progressive disease while on a chemotherapy regimen without Adriamycin, a greater than 70% reduction in colony-forming cells was observed only at a concentration of 10 micrograms/ml, a level not achievable by i.v. administration; (c) in 80% of patients with progressive disease after treatment with Adriamycin as part of the primary chemotherapy regimen, a 70% reduction in tumor colony-forming cells could not be achieved even at 10 micrograms/ml. These in vitro results are in agreement with clinical observations regarding the effectiveness of Adriamycin in previously untreated patients (42% response rate) with ovarian cancer as well as its ineffectiveness (0 to 6% response rate) as a second-line therapy in relapsed patients. The results also have provided a rationale for an ongoing Phase I trial of i.p. Adriamycin in patients with ovarian cancer from Group b above since cytotoxic levels can be produced i.p. using large-volume dialysis via a Tenckhoff dialysis catheter. The relative cytotoxicity of Adriamycin to its two major metabolites, adriamycinol and adriamycin aglycone, was also determined in the clonogenic assay. Both derivatives produced suppression of ovarian cancer colony formation; however, Adriamycin was more cytotoxic that was either metabolite.

Topics: Cell Division; Cell Survival; Cells, Cultured; Doxorubicin; Female; Humans; Naphthacenes; Neoplasms, Experimental; Ovarian Neoplasms