RESUMO
Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). More effective strategies to increase HbF are needed because the current standard of care, hydroxyurea, is not effective in a significant proportion of patients. Treatment of the millions of patients projected worldwide would best be accomplished with an orally administered drug therapy that increased HbF. LSD1 is a component of corepressor complexes that repress γ-globin gene expression and are a therapeutic target for HbF reactivation. We have shown that subcutaneous administration of RN-1, a pharmacological LSD1 inhibitor, increased γ-globin expression in SCD mice and baboons, which are widely acknowledged as the best animal model in which to test the activity of HbF-inducing drugs. The objective of this investigation was to test the effect of oral administration of a new LSD1 inhibitor, ORY-3001. Oral administration of ORY-3001 to SCD mice (nâ¯=â¯3 groups) increased γ-globin expression, Fetal Hemoglobin (HbF)-containing (F) cells, and F reticulocytes (retics). In normal baboons (nâ¯=â¯7 experiments) treated with ORY-3001, increased F retics, γ-globin chain synthesis, and γ-globin mRNA were observed. Experiments in anemic baboons (nâ¯=â¯2) showed that ORY-3001 increased F retics (PA8695, predoseâ¯=â¯24%, postdoseâ¯=â¯66.8%; PA8698: predoseâ¯=â¯13%, postdoseâ¯=â¯93.6%), γ-globin chain synthesis (PA8695: predoseâ¯=â¯0.07 γ/γ+ß, postdoseâ¯=â¯0.20 γ/γ+ß; PA8698: predoseâ¯=â¯0.02 γ/γ+ß, postdoseâ¯=â¯0.44 γ/γ+ß), and γ-globin mRNA (PA8695: predoseâ¯=â¯0.06 γ/γ+ß, postdoseâ¯=â¯0.18 γ/γ+ß; PA8698: predoseâ¯=â¯0.03 γ/γ+ß, postdoseâ¯=â¯0.33 γ/γ+ß). We conclude that oral administration of ORY-3001 increases F retics, γ-globin chain synthesis, and γ-globin mRNA in baboons and SCD mice, supporting further efforts toward the development of this drug for SCD therapy.
Assuntos
Anemia Falciforme/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Hemoglobina Fetal/biossíntese , Histona Desmetilases/antagonistas & inibidores , gama-Globinas/biossíntese , Administração Oral , Anemia/sangue , Anemia/tratamento farmacológico , Anemia Falciforme/sangue , Animais , Contagem de Células Sanguíneas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/administração & dosagem , Feminino , Hemoglobina Fetal/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Papio , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reticulócitos/metabolismo , gama-Globinas/genéticaRESUMO
Increased levels of fetal hemoglobin are associated with decreased symptoms and increased lifespan in patients with sickle cell disease (SCD). Hydroxyurea, the only drug currently approved for SCD, is not effective in a large fraction of patients, and therefore, new agents are urgently needed. Recently it was found that lysine demethylase 1, an enzyme that removes monomethyl and dimethyl residues from the lysine 4 residue of histone H3, is a repressor of γ-globin gene expression. In this article, we have compared the ability of tranylcypromine (TCP) and a more potent TCP derivative, RN-1, to increase γ-globin expression in cultured baboon erythroid progenitor cells and in the SCD mouse model. The results indicate that the ability of RN-1 to induce F cells and γ-globin mRNA in SCD mice is similar to that of decitabine, the most powerful fetal hemoglobin-inducing drug known, and greater than that of either TCP or hydroxyurea. We conclude that RN-1 and other lysine demethylase 1 inhibitors may be promising new γ-globin-inducing agents for the treatment of SCD that warrant further studies in other preclinical models, such as nonhuman primates.
Assuntos
Anemia Falciforme/genética , Hemoglobina Fetal/biossíntese , Histona Desmetilases/antagonistas & inibidores , Reticulócitos/efeitos dos fármacos , Tranilcipromina/farmacologia , gama-Globinas/biossíntese , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/enzimologia , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/metabolismo , Eritropoese/efeitos dos fármacos , Hemoglobina Fetal/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Histonas/metabolismo , Humanos , Hidroxiureia/farmacologia , Metilação , Camundongos , Camundongos Transgênicos , Papio , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transgenes/efeitos dos fármacos , Tranilcipromina/análogos & derivados , Tretinoína/farmacologia , Células U937 , gama-Globinas/genéticaRESUMO
Fetal swallowing of amniotic fluid, which contains numerous cytokines and growth factors, plays a key role in gut mucosal development. Preterm birth interrupts this exposure to amniotic fluid-borne growth factors, possibly contributing to the increased risk of necrotizing enterocolitis (NEC) in premature infants. We hypothesized that supplementation of formula feeds with amniotic fluid can provide amniotic fluid-borne growth factors and prevent experimental NEC in rat pups. We compared NEC-like injury in rat pups fed with infant formula vs. formula supplemented either with 30% amniotic fluid or recombinant hepatocyte growth factor (HGF). Cytokines/growth factors in amniotic fluid were measured by immunoassays. Amniotic fluid and HGF effects on enterocyte migration, proliferation, and survival were measured in cultured IEC6 intestinal epithelial cells. Finally, we used an antibody array to investigate receptor tyrosine kinase (RTK) activation and immunoblots to measure phosphoinositide 3-kinase (PI3K) signaling. Amniotic fluid supplementation in oral feeds protected rat pups against NEC-like injury. HGF was the most abundant growth factor in rat amniotic fluid in our panel of analytes. Amniotic fluid increased cell migration, proliferation, and cell survival in vitro. These effects were reproduced by HGF and blocked by anti-HGF antibody or a PI3K inhibitor. HGF transactivated several RTKs in IEC6 cells, indicating that its effects extended to multiple signaling pathways. Finally, similar to amniotic fluid, recombinant HGF also reduced the frequency and severity of NEC-like injury in rat pups. Amniotic fluid supplementation protects rat pups against experimental NEC, which is mediated, at least in part, by HGF.
Assuntos
Líquido Amniótico/metabolismo , Enterocolite Necrosante/prevenção & controle , Fator de Crescimento de Hepatócito/administração & dosagem , Líquido Amniótico/química , Ração Animal , Animais , Células Cultivadas , Citocinas/metabolismo , Suplementos Nutricionais , Células Epiteliais/efeitos dos fármacos , Feminino , Regulação Enzimológica da Expressão Gênica , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Lactente , Fórmulas Infantis , Mucosa Intestinal/citologia , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. In tissue samples of NEC, we identified numerous macrophages and a few neutrophils but not many lymphocytes. We hypothesized that these pathoanatomic characteristics of NEC represent a common tissue injury response of the gastrointestinal tract to a variety of insults at a specific stage of gut development. To evaluate developmental changes in mucosal inflammatory response, we used trinitrobenzene sulfonic acid (TNBS)-induced inflammation as a nonspecific insult and compared mucosal injury in newborn vs. adult mice. Enterocolitis was induced in 10-day-old pups and adult mice (n = 25 animals per group) by administering TNBS by gavage and enema. Leukocyte populations were enumerated in human NEC and in murine TNBS-enterocolitis using quantitative immunofluorescence. Chemokine expression was measured using quantitative polymerase chain reaction, immunoblots, and immunohistochemistry. Macrophage recruitment was investigated ex vivo using intestinal tissue-conditioned media and bone marrow-derived macrophages in a microchemotaxis assay. Similar to human NEC, TNBS enterocolitis in pups was marked by a macrophage-rich leukocyte infiltrate in affected tissue. In contrast, TNBS-enterocolitis in adult mice was associated with pleomorphic leukocyte infiltrates. Macrophage precursors were recruited to murine neonatal gastrointestinal tract by the chemokine CXCL5, a known chemoattractant for myeloid cells. We also demonstrated increased expression of CXCL5 in surgically resected tissue samples of human NEC, indicating that a similar pathway was active in NEC. We concluded that gut mucosal injury in the murine neonate is marked by a macrophage-rich leukocyte infiltrate, which contrasts with the pleomorphic leukocyte infiltrates in adult mice. In murine neonatal enterocolitis, macrophages were recruited to the inflamed gut mucosa by the chemokine CXCL5, indicating that CXCL5 and its cognate receptor CXCR2 merit further investigation as potential therapeutic targets in NEC.