Novel Hybrids of Hydroxyurea and Thalidomide Based Pharmacophores Induce Fetal Hemoglobin and Block Monocyte Activation

Fetal hemoglobin induction is an effective strategy for the treatment of sickle cell disease (SCD). In addition, targeting systemic inflammation, which consists of activated and injured vascular endothelium, elevated pro-inflammatory cytokines, and activated WBC, promises to reduce vaso-occlusion and disease severity. We therefore aimed to develop novel compounds that combine potent HbF-inducing and anti-inflammatory activities with the goal to improve treatment outcomes. In principle, our rational drug design used molecular hybridization to synthesize three compounds (Lapdesf 1, 2, 3) that link Hydroxyurea's nitric oxide-donating nitrate ester subunit and thalidomide's phthalimide ring with three different intermolecular spacers. Hydroxyurea is to date the only FDA approved drug for the treatment of adult patients with SCD that induces HbF and reduces clinical complications. The release of nitric oxide by HU is an important mechanism of its HbF-inducing properties. On the other hand, thalidomide and its recently developed IMiD derivatives potently inhibit cytokine release from activated monocytes and suppress adhesion molecule expression on vascular endothelium. These properties are critically linked to the phthalimide ring in the thalidomide molecule. The aim of this study was to evaluate the effects of three novel hybrid compounds Lapdesf 1 (2-[4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)phenyl]ethyl nitrate), Lapdesf 2 (4-[1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl]-N-hydroxybenzenesulfonamide), and Lapdesf 3 (3-[1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl]benzyl nitrate) on γ-globin gene expression and cytokine release from activated monocytes.

1. Gamma-globin gene expression. Human K562 cells were maintained in DMEM with 10% FBS, Pen/Strep, in humidified air (5% CO_2, 37°C). Cells (1×10⁷cells/100mL) were incubated with compounds at different concentrations (5, 30, 60, and 100μM) for 24, 48, 72 and 96h. Gamma-globin gene expression was analyzed by qRT-PCR and quantified using the Gnorm program. Results were expressed as arbitrary units. 2. Monocyte stimulation. Animals: Adult knockout-transgenic sickle cell mice were anesthetized with Ketamine/Xylazine and blood collected by intracardiac puncture. Mononuclear cells were purified from peripheral blood using Ficoll gradient separation. Mononuclear cells were placed in plastic dishes with DMEM with 10% calf serum and incubated for 2 h in humidified air (5% CO2, at 37°C). Purity of monocytes was > 90% as determined by May-Giemsa staining. Lapdesf 1, 2, and 3 were added to the monocytes cultures at different concentrations (100μM, 300μM and 600μM) 30 minutes before LPS (1 μg/ml). After 20 h incubation supernatants were collected and stored at −80°C. Cytokines were determined by ELISA.

1. Gamma-globin gene expression. Lapdesf 1 and 2 were equipotent and demonstrated inverse dose-response relationships achieving the highest levels of γ-globin induction at 5 and 30 μM; in contrast, Lapdesf 2 achieved maximal γ-globin induction as early as 24h after treatment, whereas Lapdesf 1 required 72h of incubation (Lapdesf 2 [24h; 5 μM]: 1.48±0.06 AU; control 0.78±0.1 AU; P<0.05; Lapdesf 1 [72h; 5 μM] 1.78±0.5 AU; control 0.64±0.26; P<0.05). Testing of Lapdesf 3 is currently underway. 2. Monocyte stimulation. Monocytes from sickle mice produced significantly higher levels of TNF-α (1.9 fold), IL-6 (3.48 fold), IL-8 (1.95 fold) and IL-1β (3.42 fold) after LPS stimulation compared to hemizygous controls (P<0.05). Lapdesf 1 and 3 (300 μM) significantly suppressed cytokine production compared to vehicle in LPS-stimulated sickle monocytes and were more potent inhibitors than dexamethasone (Lapdesf 1; Lapdesf 3; Dex: TNF-α : −14.71 fold; −4.64 fold; −4.44 fold; n ≥5; P<0.001; IL-6: −35.68 fold; −12.46 fold, −6.13 fold; n ≥5; P<0.001; IL-8: −7.83 fold; −3.10 fold; −2.55 fold; n ≥5; P<0.001; IL-1β: −14.34 fold; −5.26 fold; −8.9 fold; n ≥5; P<0.01). Lapdesf 2 (300 μM) failed to block cytokine release.

These results provide proof-of-concept that our hybrid compounds utilizing critical hydroxyurea and thalidomide based pharmacophores are capable of potent HbF stimulation and anti-inflammatory activities. Testing of our lead compound Lapdesf 1in a preclinical mouse model of SCD is currently underway to evaluate its efficacy in the treatment of this hemoglobinopathy.

No relevant conflicts of interest to declare.