Daunorubicin EP Impurity D (Doxorubicin) - CAS 23214-92-8 Catalog number: BADC-00039

In Vitro

Peritoneal macrophages from BD IX rats collected 24 hr after an i.p. injection of Adriamycin (10 mg/kg) were cytotoxic to syngeneic cancer cells in culture. In contrast, incubation in vitro in Adriamycin solutions did not evoke tumoricidal activity in peritoneal macrophages, whatever the incubation time (from 1 to 24 hr) and the Adriamycin concentration (from 1 ng to 100 micrograms/ml).

In Vivo

Macrophages incubated with Adriamycin in vitro accumulated the drug in their nuclei, whereas macrophages from animals receiving Adriamycin in vivo accumulated it is cytoplasmic vacuoles. Early observation of peritoneal cells after in vivo exposure to Adriamycin shows that Adriamycin is concentrated in mast cell granules which are released and then phagocytosed by peritoneal macrophages. Adriamycin fluorescence appears in nuclei of cancer cells incubated with in vivo-labeled macrophages, suggesting that macrophages can directly transfer the drug into cancer cells and therefore play a role in the Adriamycin antitumor effect.

Assay

Dispense 160 μL cell suspension (3×10 4 cells/mL) into three 96-well U-bottom microplates and incubate for 24 h at 37 °C, 5% CO2 in a completely humidified atmosphere. In plate 1, add doxorubicin (20 μL; Final concentration, 0.1-2 μM) and simvastatin (20 μL; Final concentration, 0.25-2 μM) to a final volume of 200 μL and incubate for another 72 h. Then, add serial dilutions of other drugs (40 μL) and replenish the medium to a final volume of 200 μL and incubate for 48 h. Doxorubicin and simvastatin are used as positive controls alone (40 μL per well), and solvent-only cells are considered negative controls. To assess cell survival, add 20 μL of TTT solution (5 mg/mL in PBS) to each well and incubate for 3 h. The medium is then replaced with 150 μLDMSO and complete dissolution of the crystals is achieved by repeating the pipetting solution. The absorbance is then determined at 540 nm by ELISA plate reader. Determine the concentration of each drug in 4 or 8 wells and repeat 3 times. The cytotoxic/cytostatic effect of doxorubicin is expressed as relative survival (% control) and calculated.

Mechanism Of Action

Doxorubicin has antimitotic and cytotoxic activity through a number of proposed mechanisms of action: Doxorubicin forms complexes with DNA by intercalation between base pairs, and it inhibits topoisomerase II activity by stabilizing the DNA-topoisomerase II complex, preventing the religation portion of the ligation-religation reaction that topoisomerase II catalyzes.

Pharmacology

Doxorubicin is an antineoplastic in the anthracycline class. General properties of drugs in this class include: interaction with DNA in a variety of different ways including intercalation (squeezing between the base pairs), DNA strand breakage and inhibition with the enzyme topoisomerase II. Most of these compounds have been isolated from natural sources and antibiotics. However, they lack the specificity of the antimicrobial antibiotics and thus produce significant toxicity. The anthracyclines are among the most important antitumor drugs available. Doxorubicin is widely used for the treatment of several solid tumors while daunorubicin and idarubicin are used exclusively for the treatment of leukemia. Doxorubicin may also inhibit polymerase activity, affect regulation of gene expression, and produce free radical damage to DNA. Doxorubicin possesses an antitumor effect against a wide spectrum of tumors, either grafted or spontaneous. The anthracyclines are cell cycle-nonspecific.

Toxicity (LD50)

LD50=21800 ug/kg (rat, subcutaneous).