Doxorubicinolone formation and efflux: a salvage pathway against epirubicin accumulation in human heart.

Secondary alcohol metabolites and reactive oxygen species mediate cardiomyopathy induced by cumulative doses of antitumor anthracyclines, such as doxorubicin and epirubicin. Epirubicin exhibits a defective conversion to both toxic species, thereby inducing cardiotoxicity at doses higher than equiactive to doxorubicin; however, the gain in cardiac tolerability seems to be marginal compared with the magnitude of the metabolic defects of epirubicin. Cardiomyopathy may occur independent of toxic metabolites if a given anthracycline tends to accumulate in the heart; therefore, we characterized whether epirubicin showed an unusual accumulation in human myocardial strips incubated in plasma. Epirubicin exhibited a higher uptake and reached myocardial levels 2 times higher than those of doxorubicin. Epirubicin also showed a unique metabolization to doxorubicinolone, the product of epirubicin deglycosidation and carbonyl reduction. In diffusing from the strips to plasma, doxorubicinolone caused membrane permeation effects that augmented epirubicin elimination. Experiments with purified doxorubicinolone showed that the efflux of 1 mol doxorubicinolone promoted the concomitant elimination of as many as approximately 40 mol epirubicin. Doxorubicinolone could also diffuse from plasma back to the strips, causing a permeation effect that promoted epirubicin reuptake; however, this reverse process was slower and less potent. On balance, doxorubicinolone efflux diminished the epirubicin to doxorubicin accumulation ratio to approximately 1.5. These results suggest that the cardiac tolerability of epirubicin is limited by its accumulation in the heart and that such accumulation would be even higher in the absence of doxorubicinolone formation and efflux. These results may also serve guidelines for developing noncardiotoxic anthracyclines.

Involvement of neutrophilic granulocytes in the uptake of biodegradable non-stealth and stealth nanoparticles in guinea pig.

The in vivo behavior of monomethoxypoly(ethylene oxide)-poly(lactic acid) (MPEO20-PLA45/PLA (75/25)) nanoparticles in comparison with PLA ones was studied in guinea pig. Indeed, the aim of this study was to bring to the fore the in vivo stealth character of these copolymer nanoparticles and to identify the phagocytic circulating cells involved in their uptake. After the intravascular administration of fluorescent nanoparticles (rubrene), their phagocytosis by granulocytes and monocytes was assayed by flow cytometry. At the same time, the evolution of the number of these phagocytic cells was realized in order to identify their function in the nanoparticle uptake. Finally, a histological study of the spleen (30 h after the nanoparticle administration) was investigated to highlight the splenic trapping of these stealth nanoparticles. This study has shown that the phagocytic circulating cells involved in the nanoparticle uptake were mainly neutrophilic granulocytes and some of them were found in the spleen.

The influence of tumor cell density on cellular accumulation of doxorubicin or cisplatin in vitro.

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.

Comparative pharmacokinetics of escalating doses of doxorubicin in patients with metastatic breast cancer.

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.

Cytotoxic drug penetration studies in multicellular tumour spheroids.

1. The three-dimensional structure of human lung tumour spheroids conferred a degree of resistance to the anthracyclines adriamycin, 4'-deoxydoxorubicin, daunomycin and daunomycin-low density lipoprotein complex in comparison with cells grown as a monolayer, as assessed by delayed growth and clonogenic cell survival. 2. 4'-Deoxydoxorubicin induced a longer growth delay and greater clonogenic cell kill than adriamycin in spheroids, although it was no more cytotoxic in monolayer. 3. Fluorescent microscopy demonstrated that the more lipophilic analogues partitioned into the spheroid more rapidly and to a greater degree than adriamycin. 4. The spheroid model demonstrated that penetration is an important aspect of resistance to anthracycline drugs, and this approach may represent a better in vitro system for testing lipophilic analogues of cytotoxic drugs.

Effects of verapamil on uptake and in vitro toxicity of anthracyclines in human leukemic blast cells.

The effect of a calcium channel blocker, verapamil, on intracellular uptake and cytotoxicity of anthracyclines in vitro was studied on leukemic cells from 32 patients with acute non-lymphoblastic leukemia. Cells were isolated from peripheral blood or bone-marrow and incubated with a concentration of 0.2 mumol/l, 0.5 mumol/l and/or 1.0 mumol/l of doxorubicin or daunorubicin in the absence and presence of verapamil at a concentration of 2 mumol/l and/or 10 mumol/l. Intracellular uptake was determined at the end of the incubations by photofluorometer and the in vitro cytotoxicity was determined after 5 days culturing in liquid medium by dye exclusion according to Weisenthal. Verapamil significantly increased the intracellular uptake of anthracyclines 0.5 mumol/l and 1.0 mumol/l and the cytotoxic effect of anthracyclines 0.2 mumol/l and 0.5 mumol/l and affected doxorubicin and daunorubicin equally. There were no significant differences between the two concentrations of verapamil. Cells from different FAB-groups were equally affected by verapamil. The effect on intracellular uptake was higher in cells from patients who were resistant to therapy compared to those who achieved a complete remission. We conclude that verapamil has an effect on intracellular uptake and cytotoxicity of anthracyclines on tumor cells from patients with acute non-lymphoblastic leukemia. The prognostic and therapeutic relevance of this has to be further evaluated.

[Phase I study of SM-5887, a new anthracycline derivative].

SM-5887, a new totally synthetic anthracycline derivative, was studied in a phase I setting. Twenty-nine evaluable courses of treatment were conducted in groups at doses increasing from 10 to 130 mg/m2. At 130 mg/m2 the median lowest WBC count was 0.7 x 10(3)/mm3 (range 0.3-1.8) and the median lowest platelet count was 57 x 10(3)/mm3 (range 4-176). Nonhematological side effects were mild gastrointestinal symptoms and hair loss. The recommended dose and schedule for a phase II setting is 100 mg/m2 every 3 weeks.

Aclacinomycin tissue distribution in the rat.

This study was undertaken to investigate aclacinomycin distribution in the rat, using a method based on the histofluorescence of tissues treated in vivo with anthracyclines. The target organs were the kidney, lung and pancreas; a fainter fluorescence was also detected in the heart compared with adriamycin due to a quantitatively different fixing of the two anthracyclines. Our findings revealed a preferential uptake into the cell nucleus in all tissues examined except the adrenal gland medulla where a slight fluorescence appeared only in the cytoplasm.

Selective axonal transport of anthracycline antibiotics.

The retrograde intraaxonal transport of several anthracycline antibiotics was assessed in the peripheral nervous system of adult mice. Despite certain chemical similarities, the intraaxonal transport of these molecules was variable and could be correlated with the structure of the aminosugar at the R-2 position. One particularly interesting compound, 4'-deoxydoxorubicin, was transported by dorsal root ganglia axons, but not by the axons of ventral horn neurons. These observations suggest that the sugar component of a molecule may be important for its intraaxonal transport in the peripheral nervous system.

[Pharmacokinetic study of aclarubicin. The pharmacokinetics of the preparation and its biologically active metabolites in the blood of rats]. / Farmakokineticheskoe izuchenie aklarubitsina. Farmakokinetika preparata i ego biologicheski aktivnykh metabolitov v krovi krys.

Blood pharmacokinetics of the antitumor antibiotic aclarubicin and its metabolites was studied in rats with high performance liquid chromatography. The drug was administered intravenously in single doses of 5 and 10 mg/kg and orally in a single dose of 10 mg/kg. Aclarubicin pharmacokinetics was shown to be nonlinear. However, within every dose level it obeyed a two-compartment model. The nonlinearity could be due to saturation of aclarubicin binding to blood plasma proteins. The blood concentrations of metabolites MA144 N1 and MA144 T1 were close and after 12-18 hours exceeded those of unchanged aclarubicin. The half-lives of aclarubicin and its metabolites ranged from 16 to 21 hours. The MA144 T1 content was not significant. Following oral administration aclarubicin was rapidly absorbed and its bioavailability amounted to 35 per cent. Total bioavailability of aclarubicin, MA144 N1 and MA144 T1 was equal to 89 per cent. This enabled to consider the oral route of aclarubicin administration promising in tumor therapy.

[Pharmacokinetic study of aclarubicin. The distribution of the preparation and its biologically active metabolites in rat tissues]. / Farmakokineticheskoe izuchenie aklarubitsina. Raspredelenie preparata i ego biologicheski aktivnykh metabolitov v tkaniakh krys.

Tissue pharmacokinetics of aclarubicin and its active metabolites was studied with high performance liquid chromatography. The drug was administered to rats intravenously in single doses of 5 and 10 mg/kg and orally in a single dose of 10 mg/kg. With both the administration routes the highest concentrations of the drug and its metabolites were attained in the lymph nodes. Then followed the spleen and lungs. The lowest content of the drug was detected in the heart. The total values of the areas under the concentration/time curves for aclarubicin and its metabolites in the tissues of the heart, lungs, lymph nodes and spleen after oral administration were respectively 2, 3, 4 and 7 times lower than those after the drug intravenous administration in the same dose. The concentrations of the active metabolites MA144N1 and MA144T1 exceeded those of aclarubicin and were detected in the tissues within a longer period as compared to the unchanged drug. With repeated administration preferential accumulation of the metabolites in the tissues and their increased contribution to the aclarubicin antitumor effect could be suspected.

Phase I and pharmacokinetic study of high volume intraperitoneal aclacinomycin-A (Aclarubicin).

Aclacinomycin-A (Aclarubicin) is a relatively new anthracycline antibiotic with potential activity against ovarian cancer. Eight patients with various malignancies (4 ovary, 1 breast and ovary, 1 breast, 1 colon, 1 leiomyosarcoma) and intraperitoneal disease were treated in a Phase I trial with escalating doses of intraperitoneal Aclacinomycin. Drug treatments were administered through a peritoneal catheter in a 2 liter fluid volume (1.5% Dianeal). Seventeen cycles were administered with doses ranging from 25 to 75 mg of Aclacinomycin. Pharmacokinetic studies were carried out in 7 patients. Although high concentrations of Aclacinomycin could be obtained in the peritoneal cavity no drug was detected in the plasma. The major dose-limiting toxicity was chemical peritonitis. Two patients had reduction in the amount of ascites. The recommended dose for Phase II trials is Aclacinomycin 50 mg in 2 liters given every 2 weeks.