Abstract
The in vitro deamination, cytotoxicity, cellular drug uptake, distribution and cellular pharmacology in HL-60 cells of N4-hexadecyl-1-beta-D-arabinofuranosylcytosine (NHAC), a lipophilic derivative of arabinofuranosylcytosine (ara-C), were studied. Compared with ara-C, NHAC in liposomal formulations was highly resistant to deamination, resulting in levels of formation of arabinofuranosyluracil 42 and ten times lower in plasma and liver microsomes respectively. The cytotoxicity of NHAC was independent of both the nucleoside transporter mechanism and the deoxycytidine (dCyd) kinase activity as demonstrated by co-incubating NHAC with dipyridamole and/or dCyd. In ara C-resistant HL-60 cells NHAC was still cytotoxic, requiring drug concentration only 1.6 times higher than sensitive cells. Uptake of NHAC was six times higher and was not inhibited by dipyridamole. The pharmacokinetics of NHAC revealed that its intracellular half-life is 4.8 times longer than that of ara-C. Ara-CTP formation and incorporation into DNA was up to 25-50 times lower than that of ara-C and contributed only marginally to the cytotoxic effects of NHAC. These results indicate that, because of the significantly increased stability, the transporter-independent uptake and the dCyd-kinase-independent cytotoxicity, NHAC might be active in ara-C-resistant cells.
Highlights
We evaluated the properties of NHAC compared with ara-C by studying in vitro its cytotoxicity in the human myeloid leukaemia cell line HL-60 and, in ara-C-resistant HL-60/ara-C cells, the cellular uptake, the intracellular drug distribution and pharmacokinetics, the rate of ara-CP formation and incorporation into DNA
Lipid mixtures composed of soy phosphatidylcholine (SPC), cholesterol, DL-a-tocopherol and NHAC at a molar ratio of 1:0.2:0.01:0.1 were hydrated with phosphate-buffered saline (PBS) and sequentially filtrated through Nuckepore (Costar, Sterico, Dietikon, Switzerland) filters of decreasing pore size (I pum, 400 nm, 100 nm)
Cells were incubated with araC (U) or NHAC-liposomes (0) for 24h at 37C (5% carbon dioxide) at a concentration range from I to 200 FM
Summary
Ara-C, dipyridamole and 2'-deoxycytidine (dCyd) were purchased from Sigma (Buchs, Switzerland). [5-3H-]Ara-C (30 Ci i) (5.1 Ci mmol') mmol and custom-synthesised [5-3HNHAC were purchased from Amersham (Amersham, UK). Ara-C, dipyridamole and 2'-deoxycytidine (dCyd) were purchased from Sigma (Buchs, Switzerland). [5-3H-]Ara-C (30 Ci i) (5.1 Ci mmol') mmol and custom-synthesised [5-3HNHAC were purchased from Amersham (Amersham, UK). Ara-C was dissolved in phosphate-buffered saline (PBS; 8 mM sodium phosphate, 1.5 mM potassium dihydrogen phosphate, 0.14 M sodium chloride, 2.6 mM potassium chloride) with trace amounts of [53Hlara-C. NHAC was given in a liposomal formulation as described in the Liposome preparation section. NHAC was synthesised as previously described (Schwendener and Schott, 1992). Tetrahydrouridine was a gift from the Drug Development Branch of the National Cancer Institute (Bethesda, MD, USA). HL-60 promyelocytic leukaemia cells were obtained from the American Type Tissue Culture Collection (ATCC CCL 240)
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