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1.
Bioorg Med Chem Lett ; 28(23-24): 3676-3680, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30554630

RESUMO

Beta-hemoglobinopathies such as sickle cell disease represent a major global unmet medical need. De-repression of fetal hemoglobin in erythrocytes is a clinically validated approach for the management of sickle cell disease, but the only FDA-approved medicine for this purpose has limitations to its use. We conducted a phenotypic screen in human erythroid progenitor cells to identify molecules with the ability to de-repress fetal hemoglobin, which resulted in the identification of the benzoxaborole-containing hit compound 1. This compound was found to have modest cellular potency and lead-like pharmacokinetics, but no identifiable SAR to enable optimization. Systematic deconstruction of a closely related analog of 1 revealed the fragment-like carboxylic acid 12, which was then optimized to provide tetrazole 31, which had approximately 100-fold improved cellular potency compared to 1, high levels of oral exposure in rats, and excellent solubility.


Assuntos
Benzoxazóis/química , Hemoglobina Fetal/metabolismo , Animais , Benzoxazóis/farmacocinética , Benzoxazóis/farmacologia , Disponibilidade Biológica , Ácidos Borônicos/química , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Meia-Vida , Humanos , Ratos , Ratos Sprague-Dawley , Solubilidade
2.
Biol Blood Marrow Transplant ; 19(12): 1745-52, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24120380

RESUMO

Stem cell transplantation can be associated with significant periods of thrombocytopenia, necessitating platelet transfusions and contributing to the risk of bleeding. Thrombopoietin receptor agonists have been shown to enhance platelet counts in other clinical settings, and so a phase 1 clinical trial was conducted to assess the safety, pharmacokinetics, and maximum tolerated dose of once-daily eltrombopag in patients undergoing stem cell transplantation with conditioning regimens containing total body irradiation ≥400 cGy. Eltrombopag was examined at dosage levels of 75, 150, 225, and 300 mg given orally once daily for 27 days, starting at 24 to 48 hours post-transplantation. Pharmacokinetic sampling was performed over a 24-hour period after the first dose of eltrombopag, as well as during the second week of treatment (steady-state). Nineteen patients were enrolled, 15 of whom completed protocol treatments. Three patients completed each dose level up to 225 mg, and 6 completed treatment at the highest dose of 300 mg. Four patients were replaced because drug compliance was <75% of planned doses. No dose-limiting toxicities were observed in this heterogeneous post-transplantation patient population. Common adverse events were related to standard stem cell transplantation. One episode of pulmonary embolus occurred 9 days after discontinuation of eltrombopag, and the only other thromboembolic episode was a grade 2 catheter-related clot. We conclude that up to 27 days of once-daily dosing of eltrombopag after stem cell transplantation is well tolerated.


Assuntos
Benzoatos/efeitos adversos , Benzoatos/uso terapêutico , Hidrazinas/efeitos adversos , Hidrazinas/uso terapêutico , Pirazóis/efeitos adversos , Pirazóis/uso terapêutico , Transplante de Células-Tronco/métodos , Condicionamento Pré-Transplante/métodos , Irradiação Corporal Total/métodos , Adulto , Idoso , Benzoatos/farmacocinética , Relação Dose-Resposta a Droga , Feminino , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/radioterapia , Neoplasias Hematológicas/cirurgia , Neoplasias Hematológicas/terapia , Humanos , Hidrazinas/farmacocinética , Masculino , Pessoa de Meia-Idade , Pirazóis/farmacocinética , Transplante de Células-Tronco/efeitos adversos , Trombocitopenia/tratamento farmacológico , Trombocitopenia/etiologia , Condicionamento Pré-Transplante/efeitos adversos , Transplante Autólogo , Irradiação Corporal Total/efeitos adversos , Adulto Jovem
3.
Am J Physiol Renal Physiol ; 302(9): F1172-9, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22262480

RESUMO

Acute kidney injury (AKI) due to ischemia is an important contributor to the progression of chronic kidney disease (CKD). Key mediators of cellular adaptation to hypoxia are oxygen-sensitive hypoxia-inducible factors (HIF), which are regulated by prolyl-4-hydroxylase domain (PHD)-containing dioxygenases. While activation of HIF protects from ischemic cell death, HIF has been shown to promote fibrosis in experimental models of CKD. The impact of HIF activation on AKI-induced fibrosis has not been defined. Here, we investigated the role of pharmacologic HIF activation in AKI-associated fibrosis and inflammation. We found that pharmacologic inhibition of HIF prolyl hydroxylation before AKI ameliorated fibrosis and prevented anemia, while inhibition of HIF prolyl hydroxylation in the early recovery phase of AKI did not affect short- or long-term clinical outcome. Therefore, preischemic targeting of the PHD/HIF pathway represents an effective therapeutic strategy for the prevention of CKD resulting from AKI, and it warrants further investigation in clinical trials.


Assuntos
Injúria Renal Aguda/prevenção & controle , Fator 1 Induzível por Hipóxia/metabolismo , Rim/patologia , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Fibrose , Hidroxilação/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Xilazina/efeitos adversos
4.
Blood ; 116(16): 3039-48, 2010 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-20628150

RESUMO

The kidney is the main physiologic source of erythropoietin (EPO) in the adult and responds to decreases in tissue oxygenation with increased EPO production. Although studies in mice with liver-specific or global gene inactivation have shown that hypoxia-inducible factor 2 (Hif-2) plays a major role in the regulation of Epo during infancy and in the adult, respectively, the contribution of renal HIF-2 signaling to systemic EPO homeostasis and the role of extrarenal HIF-2 in erythropoiesis, in the absence of kidney EPO, have not been examined directly. Here, we used Cre-loxP recombination to ablate Hif-2α in the kidney, whereas Hif-2-mediated hypoxia responses in the liver and other Epo-producing tissues remained intact. We found that the hypoxic induction of renal Epo is completely Hif-2 dependent and that, in the absence of renal Hif-2, hepatic Hif-2 takes over as the main regulator of serum Epo levels. Furthermore, we provide evidence that hepatocyte-derived Hif-2 is involved in the regulation of iron metabolism genes, supporting a role for HIF-2 in the coordination of EPO synthesis with iron homeostasis.


Assuntos
Anemia/metabolismo , Eritropoese , Hipóxia/metabolismo , Rim/metabolismo , Fatores de Transcrição/metabolismo , Anemia/patologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Eritropoetina/metabolismo , Ferro/metabolismo , Rim/patologia , Fígado/metabolismo , Camundongos , Camundongos Knockout , Fatores de Transcrição/genética
5.
BMC Cancer ; 12: 405, 2012 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-22967017

RESUMO

BACKGROUND: Numerous efficacious chemotherapy regimens may cause thrombocytopenia. Thrombopoietin receptor (TPO-R) agonists, such as eltrombopag, represent a novel approach for the treatment of chemotherapy-induced thrombocytopenia. The TPO-R MPL is expressed on megakaryocytes and megakaryocyte precursors, although little is known about its expression on other tissues. METHODS: Breast, lung, and ovarian tumor samples were analyzed for MPL expression by microarray and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and for TPO-R protein expression by immunohistochemistry (IHC). Cell line proliferation assays were used to analyze the in vitro effect of eltrombopag on breast, lung, and ovarian tumor cell proliferation. The lung carcinoma cell lines were also analyzed for TPO-R protein expression by Western blot. RESULTS: MPL mRNA was not detectable in 118 breast tumors and was detectable at only very low levels in 48% of 29 lung tumors studied by microarray analysis. By qRT-PCR, low but detectable levels of MPL mRNA were detectable in some normal (14-43%) and malignant (3-17%) breast, lung, and ovarian tissues. A comparison of MPL to EPOR, ERBB2, and IGF1R mRNA demonstrates that MPL mRNA levels were far lower than those of EPOR and ERBB2 mRNA in the same tissues. IHC analysis showed negligible TPO-R protein expression in tumor tissues, confirming mRNA analysis. Culture of breast, lung, and ovarian carcinoma cell lines showed no increase, and in fact, showed a decrease in proliferation following incubation with eltrombopag. Western blot analyses revealed no detectable TPO-R protein expression in the lung carcinoma cell lines. CONCLUSIONS: Multiple analyses of breast, lung, and ovarian tumor samples and/or cell lines show no evidence of MPL mRNA or TPO-R protein expression. Eltrombopag does not stimulate growth of breast, lung, or ovarian tumor cell lines at doses likely to exert their actions on megakaryocytes and megakaryocyte precursors.


Assuntos
Neoplasias da Mama/genética , Neoplasias Pulmonares/genética , Neoplasias Ovarianas/genética , Receptores de Trombopoetina/genética , Benzoatos/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Hidrazinas/farmacologia , Concentração Inibidora 50 , Neoplasias Pulmonares/metabolismo , Masculino , Neoplasias Ovarianas/metabolismo , Pirazóis/farmacologia , Receptores de Trombopoetina/metabolismo
6.
Blood ; 114(18): 3899-908, 2009 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-19710504

RESUMO

Thrombocytopenia is a frequent symptom and clinical challenge in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Eltrombopag is a small molecule thrombopoietin receptor agonist that might be a new option to treat thrombocytopenia in these diseases, provided that it does not stimulate malignant hematopoiesis. In this work, we studied the effects of Eltrombopag on proliferation, apoptosis, differentiation, colony formation, and malignant self-renewal of bone marrow mononuclear cells of patients with AML and MDS. Malignant bone marrow mononuclear cells did not show increased proliferation, or increased clonogenic capacity at concentrations of Eltrombopag ranging from 0.1 to 30 microg/mL. On the contrary, we observed a moderate, statistically nonsignificant (P = .18), decrease of numbers of malignant cells (mean, 56%; SD, 28%). Eltrombopag neither led to increased 5-bromo-2-deoxyuridine incorporation, decreased apoptosis, an increase of malignant self-renewal, nor enhanced in vivo engraftment in xenotransplantations. Furthermore, we found that Eltrombopag was capable of increasing megakaryocytic differentiation and formation of normal megakaryocytic colonies in patients with AML and MDS. These results provide a preclinical rationale for further testing of Eltrombopag for treatment of thrombocytopenia in AML and MDS.


Assuntos
Benzoatos/farmacologia , Hidrazinas/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/metabolismo , Pirazóis/farmacologia , Receptores de Trombopoetina/agonistas , Trombocitopenia/tratamento farmacológico , Trombocitopenia/metabolismo , Animais , Apoptose/efeitos dos fármacos , Benzoatos/uso terapêutico , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hematopoese/efeitos dos fármacos , Humanos , Hidrazinas/uso terapêutico , Leucemia Mieloide Aguda/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Megacariócitos/metabolismo , Megacariócitos/patologia , Camundongos , Camundongos Endogâmicos NOD , Síndromes Mielodisplásicas/patologia , Pirazóis/uso terapêutico , Receptores de Trombopoetina/metabolismo , Trombocitopenia/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Stem Cells ; 27(2): 424-30, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19038790

RESUMO

Eltrombopag is a first-in-class, orally bioavailable, small-molecule, nonpeptide agonist of the thrombopoietin receptor (TpoR), which is being developed as a treatment for thrombocytopenia of various etiologies. In vitro studies have demonstrated that the activity of eltrombopag is dependent on expression of TpoR, which activates the signaling transducers and activators of transcription (STAT) and mitogen-activated protein kinase signal transduction pathways. The objective of this preclinical study is to determine if eltrombopag interacts selectively with the TpoR to facilitate megakaryocyte differentiation in platelets. Functional thrombopoietic activity was demonstrated by the proliferation and differentiation of primary human CD34(+) bone marrow cells into CD41(+) megakaryocytes. Measurements in platelets in several species indicated that eltrombopag specifically activates only the human and chimpanzee STAT pathways. The in vivo activity of eltrombopag was demonstrated by an increase of up to 100% in platelet numbers when administered orally (10 mg/kg per day for 5 days) to chimpanzees. In conclusion, eltrombopag interacts selectively with the TpoR without competing with Tpo, leading to the increased proliferation and differentiation of human bone marrow progenitor cells into megakaryocytes and increased platelet production. These results suggest that eltrombopag and Tpo may be able to act additively to increase platelet production.


Assuntos
Benzoatos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Hidrazinas/farmacologia , Pirazóis/farmacologia , Receptores de Trombopoetina/agonistas , Animais , Antígenos CD34/metabolismo , Benzoatos/administração & dosagem , Western Blotting , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Hidrazinas/administração & dosagem , Megacariócitos/citologia , Megacariócitos/metabolismo , Camundongos , Estrutura Molecular , Pan troglodytes , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Pirazóis/administração & dosagem , Receptores de Trombopoetina/química , Transdução de Sinais/efeitos dos fármacos
8.
J Cardiovasc Pharmacol ; 56(2): 147-55, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20714241

RESUMO

BACKGROUND: Hypoxia inducible factors (HIFs) are transcription factors that are regulated by HIF-prolyl 4-hydroxylases (PHDs) in response to changes in oxygen tension. Once activated, HIFs play an important role in angiogenesis, erythropoiesis, proliferation, cell survival, inflammation, and energy metabolism. We hypothesized that GSK360A, a novel orally active HIF-PHD inhibitor, could facilitate local and systemic HIF-1 alpha signaling and protect the failing heart after myocardial infarction (MI). METHODS AND RESULTS: GSK360A is a potent (nanomolar) inhibitor of HIF-PHDs (PHD1>PHD2 = PHD3) capable of activating the HIF-1 alpha pathway in a variety of cell types including neonatal rat ventricular myocytes and H9C2 cells. Male rats treated orally with GSK360A (30 mg x kg x d) had a sustained elevation in circulating levels of erythropoietin and hemoglobin and increased hemoxygenase-1 expression in the heart and skeletal muscle. In a rat model of established heart failure with systolic dysfunction induced by ligation of left anterior descending coronary artery, chronic treatment with GSK360A for 28 days prevented the progressive reduction in ejection fraction, ventricular dilation, and increased lung weight, which were observed in the vehicle-treated animals, for up to 3 months. In addition, the microvascular density in the periinfarct region was increased (>2-fold) in GSK360A-treated animals. Treatment was well tolerated (survival was 89% in the GSK360A group vs. 82% in the placebo group). CONCLUSIONS: Chronic post-myocardial infarction treatment with a selective HIF PHD inhibitor (GSK360A) exerts systemic and local effects by stabilizing HIF-1 alpha signaling and improves long-term ventricular function, remodeling, and vascularity in a model of established ventricular dysfunction. These results suggest that HIF-PHD inhibitors may be suitable for the treatment of post-MI remodeling and heart failure.


Assuntos
Vasos Coronários/efeitos dos fármacos , Glicina/análogos & derivados , Fator 1 Induzível por Hipóxia/metabolismo , Infarto do Miocárdio/tratamento farmacológico , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Quinolonas/farmacologia , Remodelação Ventricular/efeitos dos fármacos , Animais , Linhagem Celular , Vasos Coronários/metabolismo , Vasos Coronários/fisiopatologia , Glicina/farmacologia , Hemodinâmica/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley
9.
Exp Hematol ; 33(1): 85-93, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15661401

RESUMO

OBJECTIVE: Peptide and other small molecule agonists have been described for several cytokines and growth factors. Hydrazone compounds described here as thrombopoietin receptor agonists were identified as activating STAT proteins in a Tpo responsive cell line. METHODS: STAT activation and analysis of signal transduction pathways in cell lines and normal human platelets was elucidated by Western blot and electrophoretic mobility shift assays. Proliferation assays in cell types responsive to other cytokines determined specificity for Tpo receptor. Flow cytometry quantified differentiation of CD34(+) cells into CD41(+) megakaryocytes and platelet production in vitro. RESULTS: Activation of STAT5, mitogen-activated protein kinase, p38, and early response genes by SB 394725 was similar to that induced by Tpo. SB 394725 induced a reporter gene response under a STAT activation promoter as well as the megakaryocyte-specific gpIIb promoter. The compound induced proliferation of Tpo responsive lines but demonstrated no activity in cell lines responding to other cytokines, i.e., erythropoietin, granulocyte-colony stimulating factor, interleukin-3, interferon-gamma. The response of normal human Tpo receptors was elucidated by measuring growth and differentiation of human bone marrow in vitro. Activation of endogenous Tpo receptors by SB 394725 was demonstrated in human and chimp platelets, but not in platelets of other species including mouse, dog, rabbit, or cynomolgus monkey. CONCLUSIONS: SB 394725, a small molecule with a molecular weight of 452 Da, is capable of activating Tpo-specific signal transduction, proliferation, and differentiation responses similar to the responses and functions of the protein growth factor, Tpo.


Assuntos
Hidrazonas/farmacologia , Receptores de Citocinas/agonistas , Animais , Plaquetas , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Megacariócitos , Camundongos , Proteínas do Leite/metabolismo , Proteínas de Neoplasias/agonistas , Proteínas Proto-Oncogênicas/agonistas , Receptores de Trombopoetina , Fator de Transcrição STAT5 , Transdução de Sinais/efeitos dos fármacos , Especificidade da Espécie , Transativadores/metabolismo
10.
Exp Hematol ; 31(5): 382-8, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12763136

RESUMO

OBJECTIVE: The Siglec family of proteins consists of at least 10 members with immunoglobulin and lectin domains and with similar sialic acid-binding properties. Many Siglec family members are expressed on hematopoietic cells and are involved in cell/cell interactions. Some family members are suspected of regulating cellular processes through specific signaling pathways. Monoclonal antibodies were generated against specific epitopes of Siglec-5 (CD170) and were used to determine expression of Siglec-5 on normal blood and marrow cells and cell lines. The antibodies also were used to elucidate functional activity for Siglec-5 on blood neutrophils. METHODS: Flow cytometry and ELISA were used to determine the specificity of the monoclonal antibodies for Siglec-5 and to determine expression patterns. Chemiluminescence assays were employed to measure the oxidative burst activity of whole blood or purified neutrophils following treatment with the anti-Siglec-5 antibodies. RESULTS: Cell surface expression analysis demonstrated that the protein was expressed on gated human neutrophil and monocyte populations, both in the blood and bone marrow. Expression on neutrophils was enhanced by one-hour treatment with fMLP or TNF-alpha. Epitope-specific anti-Siglec-5 monoclonal antibodies did not directly activate human neutrophils; however, antibody binding augmented neutrophil oxidative burst activity as determined by fMLP-induced luminol-dependent chemiluminescence. CONCLUSION: Data demonstrating expression of Siglec-5 on cells of the myelomonocytic lineage and alteration of its expression by inflammatory stimuli suggest a role for this protein in cell/cell interactions following microbial exposure.


Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD/fisiologia , Antígenos de Diferenciação Mielomonocítica/fisiologia , Lectinas/fisiologia , Fagócitos/fisiologia , Animais , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Comunicação Celular , Linhagem Celular , Humanos , Lectinas/análise , Medições Luminescentes , Camundongos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
11.
J Clin Invest ; 124(6): 2396-409, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24789906

RESUMO

The hypoxia-inducible transcription factors HIF-1 and HIF-2 mediate key cellular adaptions to hypoxia and contribute to renal homeostasis and pathophysiology; however, little is known about the cell type-specific functions of HIF-1 and HIF-2 in response to ischemic kidney injury. Here, we used a genetic approach to specifically dissect the roles of endothelial HIF-1 and HIF-2 in murine models of hypoxic kidney injury induced by ischemia reperfusion or ureteral obstruction. In both models, inactivation of endothelial HIF increased injury-associated renal inflammation and fibrosis. Specifically, inactivation of endothelial HIF-2α, but not endothelial HIF-1α, resulted in increased expression of renal injury markers and inflammatory cell infiltration in the postischemic kidney, which was reversed by blockade of vascular cell adhesion molecule-1 (VCAM1) and very late antigen-4 (VLA4) using monoclonal antibodies. In contrast, pharmacologic or genetic activation of HIF via HIF prolyl-hydroxylase inhibition protected wild-type animals from ischemic kidney injury and inflammation; however, these same protective effects were not observed in HIF prolyl-hydroxylase inhibitor-treated animals lacking endothelial HIF-2. Taken together, our data indicate that endothelial HIF-2 protects from hypoxia-induced renal damage and represents a potential therapeutic target for renoprotection and prevention of fibrosis following acute ischemic injury.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Isquemia/fisiopatologia , Rim/lesões , Rim/fisiopatologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Modelos Animais de Doenças , Células Endoteliais/fisiologia , Fibrose , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Integrina alfa4beta1/antagonistas & inibidores , Integrina alfa4beta1/fisiologia , Isquemia/patologia , Isquemia/prevenção & controle , Rim/irrigação sanguínea , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Traumatismo por Reperfusão/prevenção & controle , Obstrução Ureteral/complicações , Molécula 1 de Adesão de Célula Vascular/fisiologia
12.
J Biomol Screen ; 18(10): 1212-22, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24163393

RESUMO

Sickle cell anemia (SCA) is a genetic disorder of the ß-globin gene. SCA results in chronic ischemia with pain and tissue injury. The extent of SCA symptoms can be ameliorated by treatment with drugs, which result in increasing the levels of γ-globin in patient red blood cells. Hydroxyurea (HU) is a Food and Drug Administration-approved drug for SCA, but it has dose-limiting toxicity, and patients exhibit highly variable treatment responses. To identify compounds that may lead to the development of better and safer medicines, we have established a method using primary human bone marrow day 7 erythroid progenitor cells (EPCs) to screen for compounds that induce γ-globin production. First, human marrow CD34(+) cells were cultured and expanded for 7 days and characterized for the expression of erythroid differentiation markers (CD71, CD36, and CD235a). Second, fresh or cryopreserved EPCs were treated with compounds for 3 days in 384-well plates followed by γ-globin quantification by an enzyme-linked immunosorbent assay (ELISA), which was validated using HU and decitabine. From the 7408 compounds screened, we identified at least one new compound with confirmed γ-globin-inducing activity. Hits are undergoing analysis in secondary assays. In this article, we describe the method of generating fit-for-purpose EPCs; the development, optimization, and validation of the ELISA and secondary assays for γ-globin detection; and screening results.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Células Precursoras Eritroides/metabolismo , Ativação Transcricional/efeitos dos fármacos , gama-Globinas/genética , Anemia Falciforme/tratamento farmacológico , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Ácido Butírico/farmacologia , Sobrevivência Celular , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Decitabina , Ensaio de Imunoadsorção Enzimática , Epigênese Genética/efeitos dos fármacos , Células Precursoras Eritroides/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Cultura Primária de Células , gama-Globinas/metabolismo
13.
Leuk Res ; 34(9): 1224-31, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20202683

RESUMO

Leukemia cell lines were treated with eltrombopag or thrombopoietin and their proliferative response was determined. Eltrombopag did not increase proliferation of cell lines that did not express high levels of megakaryocyte markers. Instead, treatment with eltrombopag alone inhibited proliferation of many cell lines (IC(50) range=0.56-21 microg/mL). The addition of other cytokines, such as G-CSF, Epo or Tpo, did not affect the decrease in proliferation. The decrease in proliferation appears to be through a TpoR-independent, nonapoptotic mechanism. These findings suggest that eltrombopag does not enhance, but rather inhibits, proliferation of leukemia cell lines in vitro.


Assuntos
Benzoatos/farmacologia , Hidrazinas/farmacologia , Leucemia Mieloide Aguda/patologia , Leucemia/patologia , Linfoma/patologia , Pirazóis/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Primers do DNA , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
J Oncol ; 2010: 135354, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21318160

RESUMO

Thrombopoietin (TPO) receptor agonists represent a new approach for the treatment of thrombocytopenia, which may develop as a consequence of immune thrombocytopenia, chemotherapy treatment, chronic hepatitis C infection, or myelodysplastic syndromes. There are concerns that use of certain growth factors can hasten disease progression in some types of hematologic malignancies and solid tumors. In this study, expression of MPL (TPO-R) mRNA was examined in tumor cell lines, patient tumor samples (renal cell carcinoma, prostatic carcinoma, soft tissue and bony/cartilage sarcoma, colon cancer, and lymphoma), and normal tissues using microarray analysis and qRT-PCR. MPL mRNA is expressed at very low or undetectable levels compared with erythropoietin receptor (EPOR), human epidermal growth factor (ERBB2; HER2), and insulin-like growth factor-1 receptor (IGF1R) in these patient samples. These data suggest TPO-R agonists will likely preferentially stimulate proliferation and differentiation of cells of megakaryocytic lineage, potentially demonstrating their utility for correcting thrombocytopenia in clinical settings.

15.
Exp Hematol ; 37(9): 1030-7, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19631713

RESUMO

OBJECTIVE: The thrombopoietin receptor (TPOR) is a therapeutic target for treatment of thrombocytopenia because stimulation of this receptor results in enhanced megakaryocyte proliferation, differentiation, and ultimately platelet production. In addition to effects on megakaryocytes, TPOR stimulation also impacts platelet function. The present study examined platelet function following stimulation with the small molecule TPOR agonist eltrombopag. MATERIALS AND METHODS: Platelets were obtained from healthy volunteers, and signal transduction pathway activation was examined in washed platelet preparations. Platelet aggregation was examined in both washed platelet preparations and platelet-rich plasma. Platelet alpha-granule release was determined via fluorescein-activated cell sorting measurement of CD62P. RESULTS: In signal transduction studies of washed human platelets, eltrombopag induced the phosphorylation signal transducers and activators of transcription (STAT) proteins with no phosphorylation of Akt, whereas recombinant human TPO (rhTPO) induced the phosphorylation of Akt as well as STAT-1, -3, and -5. In studies conducted at subthreshold/submaximal concentrations of adenosine diphosphate (ADP) or collagen, eltrombopag pretreatment did not result in platelet aggregation. In contrast, rhTPO acted in synergy with submaximal concentrations of ADP or collagen to induce maximal aggregation under all conditions examined. Similarly, platelet activation as examined via surface expression of CD62P was not enhanced by eltrombopag pretreatment as compared to rhTPO. CONCLUSIONS: These results demonstrate that the nonpeptidyl TPOR agonist eltrombopag stimulates platelet signal transduction with little or no effect on overall platelet function, in contrast to TPO, which significantly primes platelet activation. These data demonstrate that effects of TPOR ligands on platelet function can vary depending on the specific mechanism utilized to stimulate the TPOR.


Assuntos
Benzoatos/farmacologia , Plaquetas/metabolismo , Hidrazinas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Pirazóis/farmacologia , Receptores de Trombopoetina/agonistas , Transdução de Sinais/efeitos dos fármacos , Trombopoetina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Selectina-P/biossíntese , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Trombopoetina/metabolismo , Fatores de Transcrição STAT/metabolismo , Trombocitopenia/tratamento farmacológico , Trombocitopenia/metabolismo
16.
J Biol Chem ; 283(52): 36665-75, 2008 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-18854306

RESUMO

During anemia erythropoiesis is bolstered by several factors including KIT ligand, oncostatin-M, glucocorticoids, and erythropoietin. Less is understood concerning factors that limit this process. Experiments performed using dual-specificity tyrosine-regulated kinase-3 (DYRK3) knock-out and transgenic mice reveal that erythropoiesis is attenuated selectively during anemia. DYRK3 is restricted to erythroid progenitor cells and testes. DYRK3-/- mice exhibited essentially normal hematological profiles at steady state and reproduced normally. In response to hemolytic anemia, however, reticulocyte production increased severalfold due to DYRK3 deficiency. During 5-fluorouracil-induced anemia, both reticulocyte and red cell formation in DYRK3-/- mice were elevated. In short term transplant experiments, DYRK3-/- progenitors also supported enhanced erythroblast formation, and erythropoietic advantages due to DYRK3-deficiency also were observed in 5-fluorouracil-treated mice expressing a compromised erythropoietin receptor EPOR-HM allele. As analyzed ex vivo, DYRK3-/- erythroblasts exhibited enhanced CD71posTer119pos cell formation and 3HdT incorporation. Transgenic pA2gata1-DYRK3 mice, in contrast, produced fewer reticulocytes during hemolytic anemia, and pA2gata1-DYRK3 progenitors were compromised in late pro-erythroblast formation ex vivo. Finally, as studied in erythroid K562 cells, DYRK3 proved to effectively inhibit NFAT (nuclear factor of activated T cells) transcriptional response pathways and to co-immunoprecipitate with NFATc3. Findings indicate that DYRK3 attenuates (and possibly apportions) red cell production selectively during anemia.


Assuntos
Eritropoese , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Alelos , Anemia/metabolismo , Animais , Antígenos CD/metabolismo , Transplante de Medula Óssea , Linhagem Celular , Fluoruracila/farmacologia , Humanos , Células K562 , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Receptores da Transferrina/metabolismo , Transgenes
17.
Blood ; 109(11): 4739-41, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17327409

RESUMO

Eltrombopag (SB-497 115) is a first-in-class, oral, small-molecule, nonpeptide agonist of the thrombopoietin receptor (TpoR), being developed as a treatment for thrombocytopenia of various etiologies. In this phase 1 placebo-controlled clinical trial in 73 healthy male subjects, eltrombopag was administered as once-daily oral capsules for 10 days at doses of 5, 10, 25, 30, 50, and 75 mg. The pharmacokinetics of eltrombopag were dose dependent and linear, and eltrombopag increased platelet counts in a dose-dependent manner. There were no apparent differences in the incidence or severity of adverse events in subjects receiving active or placebo study medication. These observations indicate that eltrombopag is a once-daily, oral TpoR agonist with demonstrated thrombopoietic activity in human subjects, encouraging further studies in patients with thrombocytopenia.


Assuntos
Administração Oral , Benzoatos/farmacocinética , Benzoatos/uso terapêutico , Plaquetas/efeitos dos fármacos , Hidrazinas/farmacocinética , Hidrazinas/uso terapêutico , Pirazóis/farmacocinética , Pirazóis/uso terapêutico , Receptores de Trombopoetina/agonistas , Trombocitopenia/tratamento farmacológico , Adulto , Relação Dose-Resposta a Droga , Humanos , Masculino , Placebos , Contagem de Plaquetas , Trombopoese , Fatores de Tempo
18.
Bioorg Med Chem Lett ; 16(5): 1212-6, 2006 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-16376078

RESUMO

A novel benzimidazole series of small-molecule thrombopoietin receptor agonists has been discovered. Herein, we discuss the preliminary exploration of structure-activity relationships within this chemotype.


Assuntos
Benzimidazóis/química , Benzimidazóis/farmacologia , Receptores de Citocinas/agonistas , Receptores de Citocinas/metabolismo , Trombopoetina/metabolismo , Ácidos/química , Benzimidazóis/síntese química , Estrutura Molecular , Naftóis/química , Relação Estrutura-Atividade
19.
Genomics ; 85(1): 117-30, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15607427

RESUMO

DYRKs are an emerging family of dual-specificity kinases that play key roles in cell proliferation, survival, and development. Up to seven mammalian DYRK isoforms have been reported, but only the DYRK1A gene (and its products) has been well characterized. Defined here are the genomic structures (and phylogenetics) of DYRK3, four additional murine and/or human DYRKs, and two related HIPK genes. For murine DYRK3, direct BAC sequences are provided, a basis for differential processing of murine versus human transcripts is defined, and tissue expression profiles plus a functional DYRK3 promoter are characterized. Unlike complex DYRKs 1A, 1B, and 4A, DYRK3 and DYRK2 possess simple 4-exon structures. For DYRK3, expression is strong in erythroid cells and testis, but is also detected in adult kidney and liver in situ. In addition, a 1930-bp DYRK3 promoter drives erythroid expression and transcript expression is inhibited sharply on GATA1-induced G1E cell differentiation.


Assuntos
Eritropoese/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Tirosina Quinases/biossíntese , Animais , Eritropoese/genética , Éxons/genética , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Filogenia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Homologia de Sequência do Ácido Nucleico , Quinases Dyrk
20.
Microvasc Res ; 63(1): 10-8, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11749068

RESUMO

Recently, we showed that TNF enhances the susceptibility of endothelial cells from murine liver sinusoids (LEC) to Fas-mediated apoptosis, suggesting that signals transduced by Fas and TNF receptors may synergistically increase intracellular death signals in these cells. In this work we evaluated whether caspase-3 and p38 are involved in LEC apoptosis induced by Fas and TNF. Here we show that LEC treated with Fas agonist (Jo2 mAb at 0.1 microg/ml) and TNF had a greater caspase-3 activity (twofold increase) than cells treated with each factor alone. There was a strong correlation between caspase-3 activity and cell killing induced by Jo2/TNF, indicating that this caspase plays a critical role in this process. Likewise, there was a significant increase in caspase-8 activity in LEC treated with Jo2 and TNF, compared with untreated cells or cells treated with each factor alone. Apoptosis of LEC induced by Jo2/TNF was partially reversed by SB203580, a p38 inhibitor, suggesting that p38 is involved in apoptosis of these cells. To our knowledge, this is the first report that apoptosis induced by Fas/TNF in LEC is associated with coactivation of both caspase-3 and p38. Potentially, both caspase-3 and p38 may be of great importance in endothelial cell pathology as molecular targets for preventing vascular damage due to endothelial cell apoptosis.


Assuntos
Apoptose , Caspases/fisiologia , Endotélio/citologia , Fígado/citologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Receptor fas/biossíntese , Animais , Caspase 3 , Caspases/biossíntese , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Imidazóis/farmacologia , Fígado/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Piridinas/farmacologia , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno
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