RESUMO
The therapeutic pathways that modulate transcription mechanisms currently include gene knockdown and splicing modulation. However, additional mechanisms may come into play as more understanding of molecular biology and disease etiology emerge. Building on advances in chemistry and delivery technology, oligonucleotide therapeutics is emerging as an established, validated class of drugs that can modulate a multitude of genetic targets. These targets include over 10,000 proteins in the human genome that have hitherto been considered undruggable by small molecules and protein therapeutics. The approval of five oligonucleotides within the last 2 years elicited unprecedented excitement in the field. However, there are remaining challenges to overcome and significant room for future innovation to fully realize the potential of oligonucleotide therapeutics. In this review, we focus on the translational strategies encompassing preclinical evaluation and clinical development in the context of approved oligonucleotide therapeutics. Translational approaches with respect to pharmacology, pharmacokinetics, cardiac safety evaluation, and dose selection that are specific to this class of drugs are reviewed with examples. The mechanism of action, chemical evolution, and intracellular delivery of oligonucleotide therapies are only briefly reviewed to provide a general background for this class of drugs.
Assuntos
Terapia Genética/métodos , RNA Mensageiro/genética , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Ensaios Clínicos como Assunto , Aprovação de Drogas , Sistemas de Liberação de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Oligorribonucleotídeos Antissenso/administração & dosagem , Oligorribonucleotídeos Antissenso/genética , Oligorribonucleotídeos Antissenso/farmacocinética , Interferência de RNA , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/agonistas , RNA Mensageiro/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinética , Transcrição Gênica/efeitos dos fármacosRESUMO
Friedreich's Ataxia (FA) is an inherited neurologic disorder caused by an expanded GAA repeat within intron 1 of the frataxin (FXN) gene that reduces expression of FXN protein. Agents that increase expression of FXN have the potential to alleviate the disease. We previously reported that duplex RNAs (dsRNAs) and antisense oligonucleotides (ASOs) complementary to the GAA repeat could enhance expression of FXN protein. We now explore the potential of a diverse group of chemically modified dsRNAs and ASOs to define the breadth of repeat-targeted synthetic nucleic acids as a platform for therapeutic development for FA. ASOs and dsRNAs can activate FXN protein expression in FA patient-derived cell lines that possess varied numbers of GAA repeats. Increased FXN protein expression was achieved by ASOs incorporating diverse chemical modifications with low nanomolar potencies, suggesting substantial flexibility in choosing compounds for further chemical optimization and animal studies. Our data encourage further development of ASOs as agents to treat FA.
Assuntos
Proteínas de Ligação ao Ferro/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos/genética , RNA de Cadeia Dupla/genética , RNA Mensageiro/genética , Expansão das Repetições de Trinucleotídeos , Adolescente , Adulto , Linhagem Celular , Criança , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/patologia , Ataxia de Friedreich/terapia , Regulação da Expressão Gênica , Terapia Genética/métodos , Humanos , Íntrons , Proteínas de Ligação ao Ferro/agonistas , Proteínas de Ligação ao Ferro/metabolismo , Masculino , Oligonucleotídeos/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Cultura Primária de Células , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/agonistas , RNA Mensageiro/metabolismo , Triazóis/química , FrataxinaRESUMO
Methylprednisolone sodium succinate (MPSS) has been suggested as a treatment for spinal cord injury (SCI), but its use has been limited due to its adverse effects. Erythropoietin (EPO) has been suggested as a promising candidate for limiting SCI in mammals. The aim of the present study was to investigate the effects of EPO in combination with MPSS on astrocytes following ischemic injury in vitro. Astrocytes were isolated from the cerebral cortex of postnatal day 3 SpragueDawley rats and cultured in vitro. Astrocyte ischemic injury was induced by oxygen and glucose deprivation for 4 h, and reperfusion was simulated by subsequent culture under normoxic conditions. The effects of EPO and MPSS on the expression of aquaporin4 (AQP4) were investigated. Ischemic astrocytes were treated with EPO (10 U/ml), MPSS (10 µg/ml), or EPO (10 U/ml) in combination with MPSS (10 µg/ml) during reperfusion. The cell viability of astrocytes was assessed using an MTT assay. The mRNA and protein expression levels of AQP4 were determined using reverse transcriptionquantitative polymerase chain reaction and western blot analysis, respectively. The role of the protein kinase C (PKC) signaling pathway in the molecular mechanisms underlying the effects of EPO and MPSS was also investigated. The present results demonstrated that following treatment with EPO and MPSS, the mRNA expression levels of AQP4 were upregulated and cell viability was enhanced. EPO and MPSS effectively inhibited the oxygen and glucose deprivationmediated downregulation of AQP4 following reperfusion. In addition, the combined treatment with EPO and MPSS exhibited higher AQP4 expression levels and cell viability compared with each treatment alone. Finally, the effects of EPO and MPSS on AQP4 expression were partially reversed by pretreatment with the PKC inhibitor Ro 318220. The present study indicated that EPO and MPSS had a synergistic effect on AQP4 expression following reperfusion, and suggest that they may be combined in the treatment of SCI.
Assuntos
Aquaporina 4/genética , Astrócitos/efeitos dos fármacos , Eritropoetina/farmacologia , Hemissuccinato de Metilprednisolona/farmacologia , RNA Mensageiro/genética , Animais , Animais Recém-Nascidos , Aquaporina 4/agonistas , Aquaporina 4/metabolismo , Astrócitos/citologia , Astrócitos/metabolismo , Hipóxia Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica , Glucose/deficiência , Glucose/farmacologia , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Indóis/farmacologia , Fármacos Neuroprotetores/farmacologia , Oxigênio/farmacologia , Cultura Primária de Células , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/agonistas , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Transdução de SinaisRESUMO
Diabetes patients associated with liver disease carry a significant risk of morbidity and mortality. Cinnamon has been reported to reduce fructose-induced oxidative stress in the rat liver. However, the mechanism by which cinnamon protects the liver in a high-saccharide environment remains to be investigated. HepG2 cells were cultured with 30 mM D-ribose to mimic the high-oxidative-stress environment, typical of a liver in a diabetic patient. Three different chemical types of C. osmophloeum ethanol extracts (CEEs) were added in HepG2 culture media and the administration of all three CEEs protected HepG2 cells from D-ribose damage and increased cell survival by approximately 20 percent. Exclusively, the transcript variant 1 of the ghrelin gene, but not variant 3, was 2-3 times induced by the addition of these CEEs. Moreover, the mRNAs of ghrelin processing enzyme, furin, and mboat4 were detected in HepG2 cells. The ghrelin hormones in the culture media were increased 4-9 times by the addition of CEEs. The protective effects of ghrelin on HepG2 cells in D-ribose environment were further confirmed by recombinant ghrelin transfection. We conclude that the CEEs induce ghrelin gene expression and protect HepG2 cells from D-ribose-induced oxidative damage through ghrelin signalling.
Assuntos
Antioxidantes/farmacologia , Cinnamomum/química , Grelina/agonistas , Extratos Vegetais/farmacologia , RNA Mensageiro/agonistas , Aciltransferases/genética , Aciltransferases/metabolismo , Antioxidantes/química , Sobrevivência Celular/efeitos dos fármacos , Etanol/química , Furina/genética , Furina/metabolismo , Regulação da Expressão Gênica , Grelina/genética , Grelina/metabolismo , Células Hep G2 , Humanos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/química , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribose/antagonistas & inibidores , Ribose/farmacologia , Transdução de Sinais , Solventes/químicaRESUMO
We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor-α by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-κB cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages.
Assuntos
Adjuvantes Imunológicos/farmacologia , Interleucina-6/agonistas , Macrófagos Peritoneais/efeitos dos fármacos , Extratos Vegetais/farmacologia , Spinacia oleracea/química , Fator de Necrose Tumoral alfa/agonistas , Animais , Linhagem Celular , Feminino , Expressão Gênica , Interleucina-6/genética , Interleucina-6/imunologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , NF-kappa B/agonistas , NF-kappa B/genética , NF-kappa B/imunologia , Fagocitose/efeitos dos fármacos , Cultura Primária de Células , RNA Mensageiro/agonistas , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Bone homeostasis is maintained by balancing bone formation and bone resorption, but an imbalance between them is associated with various bone-related diseases such as osteoporosis and rheumatoid arthritis. We found that 5,6-dehydrokawain (DK) and dihydro-5,6-dehydrokawain (DDK), which were isolated as promising compounds from Alpinia zerumbet rhizomes, promote differentiation of osteoblastic MC3T3-E1 cells. DK and DDK increased the alkaline phosphatase activity and matrix mineralization of MC3T3-E1 cells. DK exerts larger effects than DDK. The gene expression of runt-related transcription factor 2 and osterix, which are essential transcription factors in the early period of osteoblast differentiation, was significantly increased by DK treatment. The mRNA level of distal-less homeobox 5 was also enhanced by DK treatment, and DK activated the p38 mitogen-activated protein kinase pathway. Therefore, DK may have clinical potential for preventing osteoporosis, and could be considered as a potential anabolic therapeutic agent.
Assuntos
Alpinia/química , Diferenciação Celular/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Pironas/farmacologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/agonistas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/agonistas , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/genética , Extratos Vegetais/química , Pironas/isolamento & purificação , RNA Mensageiro/agonistas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rizoma/química , Fator de Transcrição Sp7 , Fatores de Transcrição/agonistas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The objective of this study was to evaluate the estrogenic effects and mechanisms of three flavonoid components in Xiaoyao powder: quercetin, kaempferol, and isorhamnetin. The drugs were used to treat estrogen receptor (ER)-positive human breast cancer MCF-7 cells, and proliferation was measured using the MTT method. The expression of proteins and mRNA of the ER subtype were measured using western blotting and real time polymerase chain reaction. The quercetin (10(-2) µM, 10(-3) µM), kaempferol (100 µM, 10(-2) µM), and isorhamnetin (10(-3) µM) promoted the proliferation of MCF-7 cells, and the expression of ERα and ERß proteins and mRNA were all increased significantly (P < 0.05). These effects were reversed by treatment with 0.1 µM estrogen antagonist ICI182780. Three flavonoid components in Xiaoyao powder increased the expression of proteins and mRNA of ERα and ERß and promoted the proliferation of MCF-7 cells. These estrogenic effects were mediated by the ER.
Assuntos
Medicamentos de Ervas Chinesas/química , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Quempferóis/farmacologia , Quercetina/análogos & derivados , Quercetina/farmacologia , RNA Mensageiro/genética , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/metabolismo , Ácido Fólico/análogos & derivados , Ácido Fólico/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Quempferóis/antagonistas & inibidores , Células MCF-7 , Pós/química , Quercetina/antagonistas & inibidores , RNA Mensageiro/agonistas , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
Berberine, which is a wellknown drug used in traditional medicine, has been demonstrated to exert diverse pharmacological effects, including antiinflammatory effects. However, whether berberine can affect the production of inflammatory molecules in vascular endothelial cells remains to be elucidated. Therefore, the present study aimed to determine the effects of berberine, and the underlying molecular mechanisms of these effects. The effect of berberine on tumor necrosis factor (TNF)αinduced inflammatory molecule expression was examined in cultured human aortic endothelial cells (HAECs). The HAECs were stimulated with TNFα and incubated with or without berberine. The activation of nuclear factor (NF)κB and adenosine monophosphateactivated protein kinase (AMPK) were analyzed using western blotting, and the protein secretion of intercellular adhesion molecule (ICAM)1 and monocyte chemoattractant protein (MCP)1 was measured using ELISA kits. The mRNA expression levels of ICAM1 and MCP1 were analyzed using reverse transcriptionquantitative polymerase chain reaction. The results of the present study demonstrated that berberine significantly inhibited the TNFαinduced expression of ICAM1 and MCP1, as well as the activation of NFκB in the HAECs. These effects were attenuated following cotreatment with AMPK inhibitor compound C, or specific small interfering RNAs. In conclusion, the results of the present study indicated that berberine inhibits the TNFαinduced expression of ICAM1 and MCP1, and the activation of NFκB in HAECs in vitro, possibly through the AMPKdependent pathway.