Lapdesf 1 a Compound Hybrid of Hydroxyrea and Thalidomide Increases Gamma Globin Expression in the Human CD34⁺ Cultures and Reduces Chemotactic Activity in Neutrophils

Abstract 2124

Sickle cell anemia (SCA), a disorder in which the inheritance of the gene codifies abnormal hemoglobin S (HbS), leads to Hb polymerization, causing a series of cellular alterations in the red cell. Fetal hemoglobin (HbF) is a modulator of clinical and hematologic features. Higher HbF levels are associated with a reduced rate of acute painful episodes, less frequent acute chest syndromes and protection against morbidity and mortality. Previous results showed that Lapdesf1(2-[4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)phenyl]ethyl nitrate, a novel compound that links hydroxyurea and thalidomide's phtalimide, increases HbF and has an anti-inflammatory effect in sickle mice. The aim of this study to evaluate whether Lapdesf1 induces HbF gene expression in CD34⁺ cell cultures. We also evaluated the chemotaxis and production of reactive oxygen species (ROS) in neutrophils treated in vitro with Lapdesf1.CD34⁺cell cultures from 8 healthy volunteers were treated with Lapdesf1 on day 9 and the gama-globin (γ-globin) gene expression was evaluated on day 13 by Real Time PCR. We used, as a positive control, Hydroxyurea (HU), thalidomide and both together. Neutrophils, platelets, mononuclear cells and red blood cells were isolated from peripheral blood samples of healthy controls and patients with SCA (Transfusion-independent patients, not on hydroxyurea treatment). ROS measurement was performed by the incubation with 2'-7'-dichlorofluorecin diacetate (DCFH) and analyzed by flow cytometry. Spontaneous and IL-8-induced neutrophils chemotaxis were assessed using a 96-well chemotaxis chamber assay (ChemoTXNeuroprobe). Data were analyzed statistically using ANOVA followed by Dunnett's test, where a P value of less than 0.05 was considered to be significant. The study was approved by the Research Ethics Committee of the Faculty of Medical Sciences of University of Campinas (UNICAMP).Lapdesf1 (5μM) increased γ-globin gene expression, compared with that of the control (1.85 ± 0.54 vs 0.66 ± 0.16, P<0.05, n=8). HU and thalidomide also increased at 100μM (1.81 ± 0.13; 1.85 ± 0.06, P<0.05, n=3), however treatment with HU and thalidomide together did not increase γ-globin gene expression (0.99 ± 0.04, P<0.05, n=3). There was no significant difference in the ROS production in platelets, red blood cells, mononuclear cells and neutrophils, treated with Lapdesf1. In addition, no significant differences were observed in the chemotaxis of controls and SCA neutrophils. For IL-8-induced chemotaxis, treatment with Lapdesf1 reduced the chemotactic activity at 300 and 600μM (3.94 ± 1.17; 3.86 ± 1.06 respectivelyvs 21.76 ± 6.06, P<0.05, n=3) in controls and 300μM (4.96 ± 0.59 vs 17.54 ± 7.12, P<0.05, n=3) in SCA compared with IL-8 induced control. Our results showed that the Lapdesf1 is capable of inducing CD34⁺ cell γ-globin gene expression at a low concentration and reducing chemotactic activity. Even though further studies are needed, these results suggest that Lapdesf1 may be a promising drug candidate that may provide multiple beneficial actions in the treatment of sickle cell disease symptoms and offering an alternative drug therapy. This work was supported by FAPESP, INCT and CNPq.