daunorubicinol and adriamycinol

The role of metabolism in daunorubicin (DAUN)- and doxorubicin (DOX)-associated toxicity in cancer patients is dependent on whether the parent drugs or major metabolites, doxorubicinol (DOXol) and daunorubicinol (DAUNol), are the more toxic species. Therefore, we examined whether an association exists between cytotoxicity and the metabolism of these drugs in cell lines from nine different tissues. Cytotoxicity studies using MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] cell viability assays revealed that four cell lines [HepG2 (liver), HCT-15 (colon), NCI-H460 (lung), and A-498 (kidney)] were more tolerant to DAUN and DOX than the five remaining cell lines [H9c2 (heart), PC-3 (prostate), OVCAR-4 (ovary), PANC-1 (pancreas), and MCF-7 (breast)], based on significantly higher LC50 values at incubation times of 6, 24, and 48 hours. Each cell line was also assessed for its efficiency at metabolizing DAUN and DOX. The four drug-tolerant cell lines converted DAUN/DOX to DAUNol/DOXol more rapidly than the five drug-sensitive cell lines. We also determined whether exposure to DAUN or DOX induced an increase in metabolic activity among any of these nine different cell types. All nine cell types showed a significant increase in their ability to metabolize DAUN or DOX in response to pre-exposure to the drug. Western blot analyses demonstrated that the increased metabolic activity toward DAUN and DOX correlated with a greater abundance of eight aldo-keto and two carbonyl reductases following exposure to either drug. Overall, our findings indicate an inverse relationship between cytotoxicity and DAUN or DOX metabolism in these nine cell lines.

Topics: Alcohol Oxidoreductases; Aldehyde Reductase; Aldo-Keto Reductases; Animals; Antibiotics, Antineoplastic; Cell Culture Techniques; Cell Line; Cell Survival; Daunorubicin; Doxorubicin; Humans; Lethal Dose 50; Organ Specificity; Rats; Species Specificity

The anthracycline drugs are important for the treatment of a number of malignancies; however, their clinical use is associated with dose-dependent severe chronic cardiotoxicity. Although the mechanism for this side effect has not yet been identified, the alcohol metabolites formed during daunorubicin (DAUN) and doxorubicin (DOX) therapies have been implicated. The alcohol metabolites of DAUN and DOX, daunorubicinol (DAUNol) and doxorubicinol (DOXol), respectively, are generated through reduction of the C-13 carbonyl function, which is reportedly mediated by members of the aldo-keto reductase and carbonyl reductase families of proteins. In our search for potential biomarkers for the occurrence of this side effect, we examined the activity of recombinant aldo-keto reductase enzymes, aldo-keto reductase (AKR) 1A1 and AKR1C2, with DAUN and DOX as substrates. Using purified histidine-tagged recombinant proteins and the direct measurement of metabolite formation with a high-performance liquid chromatography-fluorescence assay, we did not observe DAUNol or DOXol generation in vitro by AKR1C2, whereas AKR1A1 did catalyze the reduction reactions. DAUNol was generated by AKR1A1 at a rate of 1.71 +/- 0.09 nmol/min/mg protein, and a low level of DOXol was produced by AKR1A1; however, it was below the limits of quantification for the method. These data suggest that the generation of DAUNol or DOXol by AKR1C2 metabolism in vivo is unlikely to occur during anthracycline treatment.

Topics: Alcohol Oxidoreductases; Aldehyde Reductase; Aldo-Keto Reductases; Antibiotics, Antineoplastic; Chromatography, High Pressure Liquid; Daunorubicin; Doxorubicin; Fluorescence; Humans; Hydroxysteroid Dehydrogenases; Recombinant Proteins

Carbonyl reductase (CR) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of several carbonyls. Anthracyclines used to treat cancer are reduced by CR at the C13 carbonyl and the resulting metabolites are implicated in the cardiotoxicity associated with anthracycline therapy. CR also is believed to have a role in detoxifying quinones, raising the question whether CR catalyzes reduction of anthracycline quinones. Steady-state kinetic studies were done with several anthraquinone-containing compounds, including 13-deoxydoxorubicin and daunorubicinol, which lack the C13 carbonyl, thus unmasking the anthraquinone for study. k(cat) and k(cat)/K(m) values for 13-deoxydoxorubicin and daunorubicinol were nearly identical, indicating that that the efficiency of quinone reduction was unaffected by the differences at the C13 position. k(cat) and k(cat)/K(m) values were much smaller for the analogs than for the parent compounds, suggesting that the C13 carbonyl is preferred as a substrate over the quinone. CR also reduced structurally related quinone molecules with less favorable catalytic efficiency. Modeling studies with doxorubicin and carbonyl reductase revealed that methionine 234 sterically hinder the rings adjacent to the quinone, thus accounting for the lower catalytic efficiency. Reduction of the anthraquinones may further define the role of CR in anthracycline metabolism and may influence anthracycline cytotoxic and cardiotoxic mechanisms.

Topics: Alcohol Oxidoreductases; Animals; Anthraquinones; Daunorubicin; Doxorubicin; Humans; Models, Molecular; Oxidation-Reduction; Protein Structure, Tertiary; Recombinant Proteins

Recognition and analysis of distinct mechanisms by which primaquine and other hemolytic drugs activate the hexose monophosphate shunt (HMS) have suggested a hitherto unsuspected pharmacogenetic interaction between daunorubicin metabolism and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Because this deficiency is very common, and because anthracyclines are indispensable antitumor antibiotics that are biotransformed mainly by carbonyl reductase, we have compared the reductase-mediated conversion of daunorubicin to daunorubicinol and the conversion of doxorubicin to doxorubicinol in G6PD-deficient and nondeficient erythrocytes. We found that even without G6PD deficiency, the HMS dehydrogenases selectively limited daunorubicin metabolism, as contrasted with that of doxorubicin. The milder GdA- variety of G6PD deficiency restricted the biotransformation of daunorubicin at therapeutic levels, in hemolysates and intact erythrocytes, within 15 minutes, for at least 24 hours. The bioconversion defect was even more severe in Gd Mediterranean G6PD deficiency. Primaquine aldehyde competed with daunorubicin as a substrate for carbonyl reductase. These studies show that HMS dehydrogenase activity controls carbonyl reductase-dependent biotransformation. New issues arise concerning possible effects of G6PD deficiency on the oncolytic and toxic properties of anthracyclines that are effective substrates for carbonyl reductase and also on non-xenobiotic reactions catalyzed by this enzyme.

Topics: Adult; Alcohol Oxidoreductases; Binding, Competitive; Biotransformation; Daunorubicin; Doxorubicin; Erythrocytes; Glucosephosphate Dehydrogenase Deficiency; Humans; Primaquine

We have studied the growth inhibition, DNA synthesis inhibition and cell incorporation of five 13-dihydrometabolites of anthracyclines in a model of doxorubicin-sensitive and -resistant rat C6 glioblastoma cells. These compounds were major metabolites for doxorubicin, epirubicin, daunorubicin, idarubicin and the new anthracycline 4'-deoxy-4'-iododoxorubicin and are known to be present in appreciable amounts in the plasma of patients treated with these drugs. We have shown that in vitro growth inhibition in sensitive cells was either much lower than that of the parent drug (doxorubicinol, epirubicinol, daunorubicinol), or similar to it (idarubicinol, 4'-iodoxorubicinol). In resistant cells, growth inhibition was about 100 times lower than in wild cells, and was always lower than that of the parent anthracycline. DNA synthesis inhibition occurred in sensitive cells for doses about 100 times higher than those required for growth inhibition, but in resistant cells, similar doses provided growth inhibition and DNA synthesis inhibition. Metabolite incorporation was always lower than that of the corresponding parent anthracycline; it was greatly reduced in resistant cells as compared to sensitive ones. The calculated intracellular concentrations obtained for the same growth inhibition are higher in resistant cells than in sensitive cells; in contrast, the calculated intracellular concentrations obtained for the same DNA synthesis inhibition are similar in resistant and sensitive cells, and similar for all the metabolites studied. These results suggest that the amount of drug incorporated is primarily responsible for DNA synthesis inhibition, which is directly correlated to growth inhibition in resistant cells, but not in sensitive cells.

Topics: Animals; Antibiotics, Antineoplastic; Cell Division; Daunorubicin; DNA, Neoplasm; Doxorubicin; Drug Resistance; Epirubicin; Glioma; Rats; Tumor Cells, Cultured

A rapid chromatographic procedure for the quantitative determination of the anthracycline antibiotics adriamycin and daunorubicin and their chief metabolites adriamycinol and daunorubicinol in plasma and urine is described. The extraction is performed using SEP-PAK silica cartridges. After filtration the eluate is chromatographed on a reversed-phase column.

Topics: Adult; Chromatography, Gel; Chromatography, High Pressure Liquid; Daunorubicin; Doxorubicin; Humans

The tissue distribution mechanism of adriamycin (ADR), its relatives, daunomycin (DNR), adriamycinol (ADR-ol), daunorubicinol (DNR-ol) and actinomycin D (ACT-D) has been studied in rats and rabbits. The following evidences with respect to tissue distribution of ADR were obtained: 1) remarkable binding of ADR to tissue homogenate, 2) significant difference in the tissue binding of ADR among tissues, 3) exclusive localization of ADR in cell nucleus, 4) good correlation between the tissue binding of ADR and the tissue desoxyribonucleic acid (DNA) concentration, 5) comparatively good coincidence between the experimentally determined tissue binding of ADR and that calculated from in vitro nuclear binding parameters reported and the tissue DNA concentration, and 6) no correlation between the concentration of tissue phospholipids (i.e. cardiolipin, acidic phospholipids and total phospholipids) and the Kp value of ADR in rats. From these findings, it was confirmed that the nuclear binding is a determinant of the extensive tissue distribution of ADR and that a remarkable variation in the tissue concentration of ADR is due mainly to the difference in the tissue DNA concentration. Furthermore, good correlations were demonstrated between the DNA concentration and Kpapp values of DNR and ACT-D in rats and DNR, ADR-ol and DNR-ol in rabbits. Hence, it is suggested that there is a common mechanism of in vivo tissue distribution of ADR and its relatives which can intercalate to DNA and the determinant of characteristic tissue distribution is nuclear binding of these antibiotics.

Topics: Animals; Cell Nucleus; Dactinomycin; Daunorubicin; DNA; Doxorubicin; In Vitro Techniques; Rabbits; Rats; Tissue Distribution

Adriamycin and daunorubicin and their metabolites adriamycinol and daunorubicinol were separated by reversed-phase liquid chromatography using LiChrosorb RP-2, RP-8 and RP-18 as supports and acetone, acetonitrile and alcohols as organic modifiers in the mobile phase. The highest separation selectivity was obtained using a mobile phase containing low concentrations (less than 20%) of acetonitrile. The length of the hydrocarbon chains of the surface-modified silica supports had no significant influence on the selectivity. The lowest capacity factor was obtained with 40-60% of organic solvent in the mobile phase. Increasing the length of the hydrocarbon chains of the supports increased the retention of the solutes.

Topics: Chromatography, Liquid; Daunorubicin; Doxorubicin