Search

center dot In vitro studies show that daunorubicin (DNR) cytotoxicity decreases with increasing cell density because of a high cellular uptake and depletion of drug in the medium. center dot It i ...

Anthracyclines like daunorubicin (DNR) are important drugs in the treatment of acute myeloid leukaemia (AML). In vitro studies have shown that cellular metabolism of antrhacyclines could play a role in drug resistance. Currently, it is not known what enzyme is responsible for anthracycline metabolism in leukemic cells. To study C-13 reduction of DNR to daunorubicinol (DOL) in leukemic cells isolated from patients with AML and to determine the most important enzyme involved. Mononuclear blood cells from 25 AML patients were isolated at diagnosis and used in a metabolic assay to determine the % DOL formed. MRNA and western blot analysis were performed on the 2 most likely candidates for anthracycline metabolism; carbonyl reductase 1 (CR1) and aldoketoreductase 1A1 (AKR1A1). DNR and DOL concentrations were determined by HPLC. We found a large interindividual variation (up to 47-fold) in leukemic cell DNR metabolism. The specific CR1 inhibitor zeraleone analogue 5 significantly inhibited DNR metabolism with a mean inhibitory effect of 68 %. No correlation between mRNA levels of the enzymes and metabolism were found. Cellular DNR metabolism correlated significantly with CR1 protein expression, determined by western blot, (p < 0.05, R2 = 0,229) while no significant correlation was found with AKR1A1 protein expression. DNR metabolism in AML cells shows a pronounced interindividual variability. Our results support that CR1 is the most important enzyme for conversion of DNR to DOL in AML cells. This information could in the future be used to genotype CR1 and possibly help to individualise dosing.

To study anthracycline-induced apoptosis in leukemic cells isolated from patients with acute myelogenous leukemia (AML) in vitro and to compare intracellular anthracycline concentrations causing apoptosis in vitro with those obtained in vivo during anthracycline treatment. Mononuclear blood cells from AML patients were isolated before (n = 20) and after anthracycline infusion (n = 24). The pre-treated cells were incubated in vitro with daunorubicin (DNR) and/or idarubicin (IDA). Anthracycline concentrations were determined by high-performance liquid chromatography, and apoptosis was detected by propidium iodine staining using a flow cytometer. There was a clear concentration–response relationship between intracellular anthracycline levels and apoptosis albeit with a large interindividual variation. Intracellular levels >1200 μM always led to high apoptosis development (>60%) in vitro. The intracellular concentrations of DNR in vivo (n = 24) were more than tenfold lower than the concentrations needed to induce effective apoptosis in vitro, although a significant relation between in vivo concentrations and clinical remission was found. We also found a significant relation between apoptosis induction in leukemic cells by IDA in vitro and clinical remission. Our results indicate that intracellular anthracycline levels in vivo are suboptimal and that protocols should be used that increase intracellular anthracycline levels.

The use of low density lipoprotein (LDL) as drug carrier in acute myeloblastic leukemia chemotherapy is attractive due to high LDL uptake by leukemic cells. Lipid-based formulations, such as liposomes or microemulsions are promising alternatives. In the current study, we incorporated N-trifluoroacetyl-adriamycin-14-valerate (AD32), a lipophilic derivative of daunorubicin (DNR), and WB4291, a lipophilic alkylating agent, into LDL or lipid microemulsions and evaluated their cytotoxic activities towards leukemic cell lines using as references DNR and melphalan. The incorporation of AD32 into LDL or emulsion resulted in complexes with poor cytotoxicity. WB4291-LDL and WB4291-emulsion exerted, on the other hand, promising cytotoxic effects towards parental and resistant K562 and HL60 cell lines.

White blood cell count (WBC) is generally accepted as a prognostic risk factor in acute myeloid leukemia (AML) outcome and displays a marked interindividual variation. The dose regimen currently used ignores the size of the tumor burden and the standardization of the dose is generally based on body surface area. In this study we have investigated the effect of cell density on the cytotoxic activity of daunorubicin (DNR) and cytosine arabinoside (AraC) towards HL60 cells and leukemic cells isolated from patients with AML. We demonstrate that drug cytotoxicity decreased with cell density and that apoptosis induction by DNR in isolated leukemic cells was greatly reduced at higher cell density. A marked reduction of the uptake of DNR and AraC in HL60 parental and mitoxantrone resistant cells was observed with increasing cell density. Such a drug depleting effect by cells at high density has been previously described for vincristine, doxorubicin and paclitaxel. By extrapolating the in vitro results to the in vivo situation, one could hypothesize that a high WBC can lower the plasma concentration through high uptake in the tumor burden, leading to a shortage of drug in leukemic blasts. Patients with high WBC might therefore benefit from a dose increase of DNR and/or AraC.

Aiming to determine if a concentration window exists in which apoptosis induction by daunorubicin (DNR) is optimal, we studied the relationship between DNR concentration and apoptosis induction in HL60 and K562 cells and in peripheral leukemic cells isolated from three patients with acute myelogenous leukemia (AML). Cells were incubated for 2hr with increasing DNR concentrations and thereafter for 22hr in drug-free medium. Apoptosis was measured by detection of caspase-3-like activity and DNA fragmentation assayed by propidium iodide and flow cytometry. High DNR concentrations initiated faster apoptosis in HL60 cells and in AML cells, as shown by caspase-3 and DNA fragmentation data. DNA fragmentation into small fragments was preceded by the formation of a narrow peak on the left side of the G1 peak, most likely large DNA fragments, but further studies are required for unequivocal confirmation. This peak could easily be misinterpreted as a G1 peak without careful time monitoring. In K562 cells, no left peak was detected, apoptosis was slow and not related to concentration. In AML cells, large interindividual variations were observed in the time course of DNA fragmentation at 0.25microg DNR/mL. In conclusion, our findings support the concept of dose intensification for optimal apoptosis induction as higher doses correlate with earlier and more rapid caspase-3 induction and DNA fragmentation in leukemic cells. The DNA fragmentation assay may be a valuable tool to determine leukemic cells' chemosensitivity to apoptosis.

The selective targeting of antineoplastic drugs to tumours by incorporation in low density lipoproteins (LDL) is an attractive possibility if the drug-LDL complex remains stable in the circulation and is taken up by the tumour. In previous studies we have shown that vincristine- and N- trifluoroacetyladriamycin-14-valerate-LDL complexes were unstable in vivo. We synthesized five N-substituted lipophilic derivatives of daunorubicin and studied their incorporation into LDL. Three out of five daunorubicin derivatives incorporated successfully into LDL. In vitro these complexes were more cytotoxic towards LDL receptor positive Chinese hamster ovary cells than LDL receptor negative cells. Non-specific cytotoxicity was explained by slow dissociation of the drug-LDL complex in plasma. Our results underline the importance of careful studies of plasma stability when investigating lipoproteins and other carriers in drug targeting.