Increased fetal hemoglobin (HbF) in cells of patients with beta- hemoglobinopathies (beta-thalassemia and sickle cell anemia) ameliorates clinical symptoms of the underlined disease. Recently, several pharmacological agents have been used to stimulate HbF synthesis: treatment of patients with 5-azacytidine or hydroxyurea resulted in higher HbF levels; yet, the clinical benefit is still unclear. Since these agents are either toxic and/or have been implicated in carcinogenesis, there is considerable interest in other, less toxic, agents with the potential to increase HbF. So far only a handful of agents have been tested, mainly due to the lack of an appropriate experimental system that allows a rapid and accurate determination of the effect on relevant cells. We have recently developed a novel, 2-phase liquid culture system for growing erythroid progenitors derived from the peripheral blood of normal individuals and patients with hemoglobinopathies. This system has proved to recapitulate in vitro many hematological effects of hydroxyurea in vivo, including stimulation of HbF production. The purpose of the proposed research is to utilize this and modified methods for measuring Hb production developed in Phase I, for mass screening of agents for their HbF stimulating potential and subsequently studying their modes (S) of action to produce an efficient, low toxicity therapy. The latter studies will provide the rationale for further screening of additional agents and their chemical modification in order to increase efficacy. Classification of the drugs according to functional and mechanistic considerations will enhance experiments with combinations of drugs belonging to different groups and, thus, expected to act synergistically. In addition, we will attempt to develop, based on the cell culture system, an assay for evaluating an individual patient's response to a particular drug or drug combination. This will prevent both expensive and potentially risky treatment of patients that do not respond and suggest an alternative treatment (e.g., by other agents).
