This project is designed to improve understanding of the action of low-dose 1-Beta-D-arabinofuranosylcytosine (LD-ARA-C) (20 mg/m2 q 24 hrs) and 2,2'-anhydro-1-Beta-D-arabinofuranosyl-5-fluorocytosine (AAFC) in the treatment of human acute non-lymphocytic leukemia. A phase I study will determine the in vivo dose of AAFC equivalent to LD-ARA-C. Studies in patients will be complemented by in vitro investigation using the human promyelocytic cell line HL-60. Specifically, effects on cell cycle, RNA content, chromatin structure in situ, 3H-TdR incoporation will be investigated with a variety of flow cytometric techniques (FCM) and autoradiography and will elucidate cell kinetic effects of ARA-C and AAFC at different dose levels. Pharmacokinetic studies of LD-ARA-C and AAFC will include determination of ARA-C and ARA-U levels in plasma and urine by radioimmunoassay (RIA) and ARA-CTP formation and inhibition of 3H-thymidine incorporation into DNA of bone marrow cells by ARA-CTP. Determination of deoxycytidine kinase and cytidine deaminase in plasma and bone marrow cells will use deoxycytidine and ARA-C and cytidine and ARA-C respectively as substrates. Levels of AAFC and/or its metabolites will be studied by HPLC. If required for very low AAFC levels, a RIA for AAFC will be developed. ARA-C and AAFC will be compared as to clinical, cell kinetic, and pharmacokinetic effects. In vitro studies will evaluate cell kinetic effects, deaminase and kinase in medium and HL-60 cells, and intracellular nucleotide formation at different concentrations and time points. For this purpose, 3H-ARA-C and 14C-AAFC will be used. Concomitant studies of differentiation antigens will investigate the effect of LD-ARA-C and AAFC on the differentiation of leukemic cells. A panel of monoclonal antibodies directed against intracellular onc-gene (ras, myb, mos, myc, sis) products and cell surface antigens will define discrete steps of onc-gene expression and differentiation of myeloid leukemia and will be determined serially in vitro and in vivo. Simultaneous determination of cellular DNA content and antigen expression and of marrow cellularity by multiparameter FCM will clearly distinguish differentiation effects from cytotoxic effects on human leukemic and non-leukemic cells. We expect these studies to improve our understanding of the mode of action of ARA-C and AAFC, to correlate pharmacological, cell biological and clinical effects, to predict response and failure for groups of patients and to optimize therapeutic effects in individual patients.
