This is the continuation of an ongoing research program on factors that effect the behavior of adenosine and its analogs both as cytostatic agents and as mediators of physiological functions. The studies are in three interrelated areas. 1. Inhibitors of adenosine deaminase (ADA) as chemotherapeutic agents. The objective is to test """"""""second generation"""""""" ADA inhibitors with therapeut- ic potential for the treatment of B-lymphocyte-related diseases, e.g. hairy-cell leukemia, CLL, B-cell lymphomas. Analogs and putative metaboli- tes of (+)EHNA, synthesized by medicinal chemistry colleagues are subjected to kinetic analyses with human and calf intestinal ADA. Analytical methodology including fluorescence HPLC, is under development. As the first stage of a long-term collaborative study of the three dimensional structure of human ADA, methods will be devised to isolate the human enzyme cloned in E. coli. Synthetic and natural mutants of the human enzyme will be studied. 2. The effects of adenosine and its analogs on cellular functions of blood platelets, human leukemia cells, and isolated vascular endothelial cells. Since the clinical toxicity of ADA inhibitors may be due to actions on adenosine receptors,, the effects of nucleoside transport and ADA in- hibitors, A A2 receptor agonists and antagonists and cAMP phosphodiesterase inhibitors will be examined with human blood platelets and proliferating and differentiated HL-60 promyelocytic leukemia cells. Studies will be initiated on factors that affect adenosine synthesis and catabolism by cultured vascular endothelial cells. 3. The role of nucleoside transport and adenosine action. Transport mechanisms will be examined with (+)EHNA, its analogs and putative metabol- ites. A clinical assay for nucleoside transport capacity and inhibitor efficacy will be developed and employed to evaluate donor-dependent variations (as great as 5-fold) identified earlier. Differences will be correlated with effects of adenosine on platelet aggregation. The proper- ties and surface expression of the nucleoside carrier protein of human HL- 60 promyelocytic leukemia cells will be examined with the use of recently developed specific monoclonal antibodies.
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