Leukemia chemotherapy : Experimental studies on pharmacological optimisation

University dissertation from Stockholm : Karolinska Institutet, Department of Medicine

Abstract: Our main goal has been to identify new means to improve chemotherapy of acute leukaemia: 1. By using low-density lipoprotein (LDL) as a drug carrier to increase the selectivity of antileukemic drugs, based on high LDL uptake in acute myeloid leukemia (AML) cells. Our first concern was to investigate the importance of chemical structures to obtain a stable anchorage of the drug into LDL. The only stable complex was obtained when incorporating cholesteryl-linoleat in LDL as shown by dialysis and autoradiography data. With N-trifluoroacetyl-adriamycin-14-valerat-LDL (AD32-LDL) the drug leaked slowly into the plasma. In AML patients a rapid plasma dissociation of AD-32-LDL was observed, illustrating a much higher in vivo instability of this complex. We thereafter synthesised five lipophilic derivatives of daunorubicin (DNR) for incorporation into LDL. Three incorporated successfully into LDL: 2 benzyloxy and the isonicotinoyl derivatives. In vitro these complexes were more cytotoxic towards a LDL receptor positive cell line than to LDL receptor negative cells, but non-specific cytotoxicity was quite high and was explained by slow dissociation of the drug-LDL complexes in plasma. These results underline the difficulty in obtaining a stable LDL complex. Finally we studied the cytotoxicity of WB4291, a lipophilic alkylating agent, after incorporation in LDL or lipid microemulsions towards sensitive and resistant myeloid cell lines. The complexes exerted a better activity than melphalan and DNR towards all the resistant sublines expressing Pgp, K562/Vcr and/Dnr. 2. By studying the relation between DNR concentration and apoptosis induction in leukemic cells. We studied the time course of induction of apoptosis by DNR in HL60 and K562 cells and in isolated leukemic cells from patients with AML, after a pulse incubation with increasing drug concentrations. Caspase-3-like activity correlated positively with DNR concentrations, appearing faster at high DNR concentrations in all the cells. DNA fragmentation occured in two steps, an intranucleosomal cleavage producing high MW DNA fragments, followed by an internucleosomal cleavage and the apparition of small fragments observed in a typical DNA ladder. The high MW fragments were observed in all the cells with the exception of K562 cells. DNA fragmentation was faster at high DNR concentrations in all the cells except K562 cells. In leukemic cells from patients with AML, the time course of DNA fragmentation at 0.25?g/ml showed large interindividual variations in in vitro chemosensitivity that could reflect variations in in vivo sensitivity. The results support the concept of dose intensification in induction therapy with DNR. 3. By studying of the impact of cell density on drug cytotoxicity in order to optimise individual treatment. White blood cell count (WBC) is generally accepted as a significant prognostic risk factor in acute leukemia outcome and shows a marked variation at diagnosis. Actually the dose regimen currently used ignores the size of the tumor burden and the standardization of the dose is generally based only on body surface area. In this study we investigated the effect of cell density on the cytotoxic activity of DNR and Ara-C in HL60 cells and in leukemic cells isolated from patients with AML. We showed that their cytotoxicity decreased with cell density and that apoptosis induction in isolated leukemic cells by DNR was reduced at higher cell density. This correlated with the marked reduction of DNR and AraC uptake in HL60 cells at high cell density. We hypothesised that a high WBC will lower the plasma concentration through a high uptake in the tumor cells and in this way decrease the drug concentration in leukemic blasts. In patients with high WBC, a dose increase and an optimal administration schedule should be evaluated for treatment with DNR and/or AraC.

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