The objective of this application is to establish a reference laboratory and standard protocols for evaluation of potential therapeutic agents for sickle cell disease. This proposed reference laboratory is designed to facilitate rapid, efficient, and economical evaluation of agents that may be beneficial in the treatment of patients with sickle cell disease. This proposal represents the combined efforts of three different laboratories that are actively engaged in research related to sickle cell disease. We will first examine the effect of candidate agents on cell sickling, functional properties of hemoglobin, and the existence of adverse effects, such as hemoglobin oxidation or red blood cell hemolysis. Additional studies will be carried out to determine effects of candidate drugs on the membrane transport properties and cell volume control, and on the interaction between sickle cells and endothelial cells. The information gathered from the studies in vitro will be used to determine which compounds to evaluate in vivo using various lines of transgenic mice which produce human sickle hemoglobin (Hb S mouse models) including the recently developed Hb S-F mice, which produce human Hb S and Hb F exclusively. Transgenic Hb S mouse models are particularly useful to study effects of new therapeutic approaches for patients with sickle cell disease, such as gene therapy, stimulation of Hb F synthesis, hydration of red blood cells by membrane-acting agents and inhibition of endothelial adhesion. The availability of transgenic sickle mice will allow testing in vivo of these potential approaches and provide important information for the feasibility and design of human trials. Evaluation of therapeutic agents in vivo is essential since many drugs that prevent sickling in vitro can not be used in vivo because of toxicity or inability to reach a therapeutic concentration. This reference laboratory will also evaluate the effects of combined therapy of drugs with different mechanisms of action, such as hydroxyurea and membrane active agents. We will study several agents in combination with hydroxyurea to determine the optimal dosage of each drug required to achieve a significant therapeutic effect with the least toxicity. This reference laboratory will offer its services to any investigator with a potential candidate drug for treatment of sickle cell disease and will provide an economical and efficient system to evaluate new therapies for patients with sickle cell disease.
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