Hydroxyurea is a cell-cycle specific agent that blocks DNA synthesis by inhibiting ribonucleotide reductase, the enzyme that converts ribonucleotides to deoxyribonucleotides. Hydroxyurea has been shown to induce the production of fetal hemoglobin (HbF), initially in non-human primates, and now in patients with sickle cell anemia. The majority of patients with sickle cell disease respond to the drug with a more than two-fold increase in HbF levels;in some patients the percent of HbF exceeds 10 or 15 percent. We have found in our laboratories that hydroxyurea exerts many of its effects via the production of nitric oxide gas. In this study we treated patients chronically with hydroxyurea to determine hematological changes longitudinally. Once a maximal Hb-F raising effect of hydroxyurea had been established, oral L-arginine (the substrate for NO synthase) and sildenafil (Viagra, a phosphodiesterase inhibitor that potentates cGMP dependent signaling) was added to determine the ability of other agents to enhance HbF synthesis, especially in hydroxyurea non-responders or partial-responders. We began enrolling patients in May 2003 and have treated 37, 13 with hydroxyurea and L-arginine (13 subjects have completed this arm of the study), 14 with hydroxyurea and sildenafil (13 subjects completed this arm of the study) , 11 with hydroxyurea alone and 2 with sildenafil alone. The results are now published in the British Journal of Haematology, indicating that sildenafil therapy decreased the estimated pulmonary artery systolic pressure by 9 mmHg (p=0.04) and increased 6-minute walk distance by 78 m (p=0.001). We have found that in patients with sickle cell disease, 1) pulmonary hypertension, though relatively mild, is associated with severe impairments in cardiopulmonary function, 2) traditional markers of functional capacity such as six-minute walk test can be utilized in this population as a therapeutic endpoint for clinical trials, 3) and therapy with sildenafil seems to have a favorable impact on pulmonary pressures and functional capacity. We are no longer enrolling patients and are continuing with our formal data analysis. Fetal hemoglobin (HbF) induction involves NO-cGMP signaling pathways. L-arginine, an NO precursor, and the phosphodiesterase (PDE) 5 inhibitor sildenafil, which potentiates cGMP, were studied in adults with sickle cell disease (SCD) who were stably on HU. Twenty four courses of L-arginine (0.1-0.2 g/kg divided TID) or sildenafil (25-100 mg TID), assigned based on gender due to concerns about sildenafil-related priapism, were successfully completed. Biochemical assays, pulmonary pressures, and cardiopulmonary exercise capacity are reported from patients in whom serial values are available. Hematologic responses are reported in 14 subjects with HbSS who had stable baseline HbF levels. L-arginine increased plasma arginine and ornithine, but not citrulline, suggesting diversion by plasma arginase from NO, and citrulline, generation. Glutathione increased only in patients on L-arginine. Sildenafil increased plasma cGMP and citrulline, but not other amino acids. Pulmonary pressures and 6-min walk distances improved only in patients on sildenafil. In subjects with stable baseline HbF levels, HbF levels changed little from a normalized baseline on l-arginine, decreasing by 2.9 +/- 16.1%, n = 6;P = n.s., but increased on sildenafil, by 7.5 +/- 11.7%, n = 8, P <0.05. Absolute reticulocyte counts initially decreased in patients on sildenafil. L-arginine, at doses that increase plasma arginine levels, altered redox potential in red cells. The lack of clinically detectable efficacy of L-arginine may be due to increased arginine metabolism in SCD patients. In vivo augmentation of the cyclic nucleotide pathway by PDE inhibition may induce HbF slightly, but strikingly improves hemodynamic and functional status in SCD.

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National Heart, Lung, and Blood Institute
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