In vitro studies of cytostatic drugs and growth factors on leukemic and normal hematopoietic cells : based on in vivo intracellular pharmacokinetics

Abstract: The aims were to develop, evaluate and apply in vitro methods for cytostatic drug sensitivity, based on intracellular concentrations found after in vivo infusions. The intracellular pharmacokinetics of anthraquinones and ara-C were compared during and after infusion to leukemic patients. Drug concentrations were evaluated with HPLC. All anthraquinones had a rapid uptake in the leukemic cells. Daunorubicin and idarubicin reached the highest levels. With lOmg/m2, the intracellular concentration of idarubicin was 70% of that of daunorubicin 50mg/m2, administered simultaneously to the same patient. Idarubicin had a more rapid initial intracellular elimination but a longer terminal retention. Doxorubicin and mitoxantrone also reached their intracellular peak levels at the end of the infusion, but the peaks were lower than those of daunorubicin and idarubicin. Both doxorubicin and mitoxantrone had a marked retention in leukemia cells. Continuous infusion of ara-C was needed to maintain adequate intracellular concentrations of the active metabolite ara-CTP. Based on these results we established in vitro incubations to reflect the in vivo intracellular concentrations of the drugs. For daunorubicin and mitoxantrone incubations during one hour at a concentration of 0.2 and 0.05myM, respectively were optimal. The corresponding conditions for doxorubicin, that is less lipid soluble were 0.211M during three hours. Ara-C, an S-phase specific drug, required a continuous incubation of 0.5myM to reach intracellular concentrations of ara-CTP mimicking those in vivo. The toxicity to leukemic cells of these incubations was tested with the DiSC-assay to gain feasible effects in vitro. For anthracyclines 60% and for ara-C 35% surviving cells were the limits that best discriminated drug resistant from sensitive cells. When both drugs were analysed together, the predictive accuracy was 91% for in vitro drug sensitivity and 92% for resistance. The probability to predict a complete remission was 95% and the probability to predict resistant disease was 85%. Toxicity to normal hematopoietic stem cells is dose limiting for most cytostatic drugs. Bone marrow cells from healthy donors were incubated with anthraquinones, AMSA and ara-C during one and three hours and continuously. The cytotoxicity was evaluated with the clonogenic assay for CFU-GM. There was a 50-fold interindividual difference in toxicity. Drugs with rapid intracellular uptake and high peak concentrations, like daunorubicin, idarubicin, aclarubicin, mitoxantrone and AMSA, exerted a high toxicity to the colony forming cells already after one hour. Doxorubicin and epirubicin were less toxic after a one hour incubation, but showed an increased toxicity after a three hours incubation. The differences were smoothened out by continuous incubation. Ara-C, usually administered as continuous infusion, had a weak effect after short time incubations but a pronounced toxicity with continuous incubation. Bone marrow cells from donors over 40 years of age were significantly more sensitive to doxorubicin and epirubicin. The in vitro techniques were used to evaluate how cytokines affected the toxicity of cytostatic drugs to leukemic and normal hematopoietic cells. We adopted the ATP bioluminescence assay for testing cytotoxicity to leukemic cells and used the clonogenic assay for the normal stem cells. Growth factors stimulated both the leukemic and the normal hematopoietic cells to an increased proliferation. G-CSF increased the cytotoxic effect of daunorubicin, mitoxantrone and etoposide to leukemic cells, while GM-CSF and IL-3 enhanced the cytotoxic effect of ara-C. However, this effects were offset by the stimulating effect of growth factors on the malignant clone. Normal hematopoietic stem cells became significantly more sensitive to daunorubicin after stimulation with G-CSF. GM-CSF and IL-3, on the other hand decreased the toxic effects exerted by cytostatic drugs to the normal bone marrow cells. We conclude that in vitro assays, based on in vivo intracellular pharmacokinetics of cytostatics, have a high clinical relevance and can be used to study the effects of these drugs on both leukemic and normal hematopoietic stem cells.