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1.
J Gastroenterol Hepatol ; 35(12): 2241-2247, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32386240

RESUMO

BACKGROUND AND AIM: Mitochondrial damage is commonly involved in liver injury. We have previously shown that normal mitochondria can be coated with a carrier protein to form complexes that are specifically taken up by liver cells in culture. The aim of the current study was to determine whether mitochondrial complexes could be specifically delivered to the livers of living rats by intravenous injection. METHODS: Mitochondria were harvested from fresh mouse liver, mixed with an asialoglycoprotein-based carrier, asialoorosomucoid-polylysine (AsOR-PL), and purified to form complexes. To facilitate the release of internalized mitochondria from endosomes, an endosomolytic peptide, listeriolysin O (LLO), was coupled to AsOR to form AsOR-LLO. Mitochondria alone, mitochondrial complexes with AsOR-PL, and mitochondrial complexes plus AsOR-LLO conjugate all containing the same number of mitochondria were injected intravenously. Animals were killed, and organs were removed and analyzed by quantitative polymerase chain reaction of mouse mitochondrial DNA, electron microscopy (EM), and in situ polymerase chain reaction and hybridization followed by immunohistochemical analyses. RESULTS: Calculations revealed that approximately 27% of the total injected mitochondria was detected in the liver, while less than 2% was found in spleen, and < 1% in lungs. Immunohistochemistry showed that mouse mitochondrial DNA staining was minimal with mitochondrial complexes alone, strong periportal with mitochondrial complexes co-injected with AsOR-LLO, and absent with mitochondria alone. CONCLUSIONS: Targetable mitochondrial complexes can be delivered to rat liver, and the efficiency of that process is greatly enhanced by co-injection of a targetable endosomal release agent, AsOR-LLO.


Assuntos
Assialoglicoproteínas/administração & dosagem , Toxinas Bacterianas/administração & dosagem , Transplante de Células/métodos , Proteínas de Choque Térmico/administração & dosagem , Proteínas Hemolisinas/administração & dosagem , Fígado , Mitocôndrias Hepáticas/transplante , Orosomucoide/análogos & derivados , Polilisina/administração & dosagem , Animais , Proteínas de Transporte , Endossomos , Feminino , Hepatócitos/citologia , Injeções Intravenosas , Camundongos Endogâmicos , Orosomucoide/administração & dosagem , Ratos Sprague-Dawley
2.
Int J Pharm ; 473(1-2): 366-74, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25051113

RESUMO

Gene therapy is considered a promising approach for the treatment of hepatocellular carcinoma (HCC). In this regard, the main goal of this work was to develop a specific and efficient gene delivery nanosystem to HCC based on 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine:cholesterol cationic liposomes and asialofetuin (ASF), a specific ligand to the asialoglycoprotein receptor (ASGP-R) that is overexpressed in HCC. Our results show that association of ASF to lipoplexes promotes a substantial increase in their biological activity in HCC cells, not only in vitro, but also in an animal model. The transfection activity obtained with this novel nanosystem (ASF-lipoplexes) was much higher than that observed with a highly efficient commercial formulation. On the other hand, the presence of high concentrations of galactose substantially reduced the cell uptake and biological activity of the ASF-lipoplexes. These results, together with those obtained in the presence of inhibitors of endocytosis, show that the potentiation induced by the association of ASF to lipoplexes is due to its specific interaction with the ASGP-R. The physicochemical properties of the generated nanosystem also reinforce this observation. Overall, our results demonstrate for the first time that the novel ASF-lipoplexes present a noticeable ability to specifically and efficiently deliver genetic material into HCC cells.


Assuntos
Receptor de Asialoglicoproteína/metabolismo , Assialoglicoproteínas/administração & dosagem , Carcinoma Hepatocelular/metabolismo , DNA/administração & dosagem , Fetuínas/administração & dosagem , Técnicas de Transferência de Genes , Neoplasias Hepáticas/metabolismo , Colesterol/química , Células HeLa , Células Hep G2 , Humanos , Lipossomos , Nanoestruturas , Fosfatidilcolinas/química , Plasmídeos
3.
J Drug Target ; 21(5): 487-96, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23570609

RESUMO

To develop a novel hepatocyte-selective gene carrier, we prepared polyamidoamine starburst dendrimer (generation 3, G3) conjugates with three functional molecules, i.e. α-cyclodextrin, polyethylene glycol (PEG, molecular weight = 2170) and lactose (PEG-LαCs), and evaluated gene delivery efficiency of these conjugates in vitro and in vivo. PEG-LαC (G3, degrees of substitution of the PEG moiety (DSP) 2.1) showed higher gene transfer activity than other PEG-LαCs (G3, DSP4.0, 6.2) in HepG2 cells, expressing asialoglycoprotein receptor, and the activity decreased in HeLa cells, non-expressing the receptor and in the presence of asialofetuin. High gene transfer activity of PEG-LαC (G3, DSP2.1) was retained even in the presence of 50% serum, although the activity of α-cyclodextrin/lactosylated dendrimer (G3) conjugate (Lac-α-CDE (G3)), which is lacking a PEG moiety, was severely decreased in the presence of 20% serum. PEG-LαC (G3, DSP2.1) provided negligible cytotoxicity up to a charge ratio of 50 (carrier/pDNA) in HepG2 cells and less acute organ toxicity. PEG-LαC (G3, DSP2.1) showed selective gene transfer activity to hepatic parenchymal cells rather than hepatic non-parenchymal cells. These results suggest that PEG-LαC (G3, DSP2.1) is useful as a hepatocyte-selective gene carrier in vitro and in vivo.


Assuntos
Dendrímeros/administração & dosagem , Hepatócitos/metabolismo , Lactose/administração & dosagem , Poliaminas/administração & dosagem , Polietilenoglicóis/administração & dosagem , alfa-Ciclodextrinas/administração & dosagem , Animais , Receptor de Asialoglicoproteína/genética , Assialoglicoproteínas/administração & dosagem , Linhagem Celular Tumoral , Dendrímeros/química , Dendrímeros/farmacocinética , Fetuínas/administração & dosagem , Técnicas de Transferência de Genes , Terapia Genética/métodos , Células HeLa , Células Hep G2 , Humanos , Lactose/química , Lactose/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Poliaminas/química , Poliaminas/farmacocinética , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , alfa-Ciclodextrinas/química , alfa-Ciclodextrinas/farmacocinética
4.
Cardiovasc Drugs Ther ; 27(2): 117-24, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23314760

RESUMO

PURPOSE: Although erythropoietin (EPO) is known to express angiogenic and cardioprotective effects, it also induces hypertension, polycythemia, and platelet activation, which may cause serious adverse effects in patients with cardiovascular diseases. We compared the angiogenic effects of EPO and its nonerythropoietic derivative, asialo-EPO (AEPO). METHODS: Lower limb ischemia was induced in ICR and C57/BL mice. Mice were injected intramuscularly with 2 µg/kg of EPO derivatives for 6 or 7 days. To assess biological differences, the tissue affinity of both EPO derivatives was analyzed in vitro using heparin affinity column chromatography. Tissue affinity was also analyzed in vivo using an intramuscular pharmacokinetic study. RESULTS: The survival of ischemic legs was better in the AEPO group than that in the EPO group (5/13 = 38.5 % vs 1/13 = 7.7 %, p < 0.05), and an increase in regenerated vessels was observed in the AEPO group, but not in the EPO group in ICR mice. Vessel/muscle ratios in control, EPO, and AEPO groups were 0.50 ± 0.34, 0.61 ± 0.32, and 2.83 ± 1.13, respectively (p < 0.0001). On the other hand, regenerated vessels were observed in both EPO and AEPO groups (p < 0.001) in C57/BL mice. AEPO, but not EPO, expressed heparin affinity in vitro. Intramuscularly injected EPO gradually decreased in muscle tissue, while AEPO was maintained at 2.5 ng/muscle for 1 day after several hours of a rapid clearance phase in vivo. CONCLUSIONS: AEPO exerts stronger angiogenic effects than those of EPO presumably via its tissue affinity. Administration of AEPO is a promising option for the treatment of patients with critical limb ischemia.


Assuntos
Assialoglicoproteínas/administração & dosagem , Eritropoetina/análogos & derivados , Isquemia/tratamento farmacológico , Animais , Assialoglicoproteínas/farmacocinética , Eritropoetina/administração & dosagem , Eritropoetina/farmacocinética , Heparina/metabolismo , Injeções Intramusculares , Isquemia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Neovascularização Fisiológica/efeitos dos fármacos , Ligação Proteica
5.
Int J Pharm ; 439(1-2): 269-74, 2012 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-22989979

RESUMO

Modification of the liposomal surface with a targeting molecule is a promising approach for the targeted delivery of therapeutics. Asialo-erythropoietin (AEPO) is a potent tool for targeting an ischemic region by binding to the EPO receptors on neuronal cells. Additionally, it shows a strong cytoprotective effect against programed cell death. Hence, AEPO-modified liposomes appear likely to have both a neuronal-targeting character and a neuroprotective effect on cerebral ischemic injury. In this study, we assessed the targeting ability of AEPO-modified PEGylated liposomes (AEPO-liposomes) to ischemic region and their improvement effect on neurological deficits induced by ischemia/reperfusion (I/R) in transient middle cerebral artery occlusion (t-MCAO) rats. Immunohistological analysis showed that the AEPO-liposomes given immediately after reperfusion extravasated into the ischemic region and attached strongly to neuronal cells. Also, neuronal nuclei (NeuN) staining was clearly visible only in the AEPO-liposome-treated group, suggesting that AEPO-liposomes protected neuronal cells from ischemia/reperfusion-induced damage. Moreover, a single administration of low-dose AEPO-liposomes significantly improved the neurological deficit compared to vehicle and free-AEPO treatment at 7 days after injection. In conclusion, AEPO-liposomes have clear potential as a neuroprotectant after stroke and as a DDS device targeting ischemic regions.


Assuntos
Assialoglicoproteínas/administração & dosagem , Eritropoetina/análogos & derivados , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fármacos Neuroprotetores/administração & dosagem , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Eritropoetina/administração & dosagem , Infarto da Artéria Cerebral Média/fisiopatologia , Lipossomos , Masculino , Atividade Motora/efeitos dos fármacos , Neurônios/metabolismo , Polietilenoglicóis/química , Ratos , Ratos Wistar , Traumatismo por Reperfusão/fisiopatologia
6.
Circ Heart Fail ; 5(2): 274-85, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22343033

RESUMO

BACKGROUND: We investigated the effects of asialoerythropoietin (asialoEPO), a nonerythrogenic erythropoietin derivative, on 3 murine models of heart failure with different etiologies. METHODS AND RESULTS: Doxorubicin (15 mg/kg) induced heart failure within 2 weeks (toxic cardiomyopathy). Treatment with asialoEPO (6.9 µg/kg) for 2 weeks thereafter attenuated the associated left ventricular dysfunction and dilatation. In addition, the asialoEPO-treated heart showed less myocardial fibrosis, inflammation, and oxidative damage, and diminished atrophic cardiomyocyte degeneration, which was accompanied by restored expression of GATA-4 and sarcomeric proteins. Mice with large 6-week-old myocardial infarctions exhibited marked left ventricular dysfunction with adverse remodeling (ischemic cardiomyopathy). AsialoEPO treatment for 4 weeks significantly mitigated progression of the dysfunction and remodeling and reduced myocardial fibrosis, inflammation, and oxidative damage. Finally, 25-week-old δ-sarcoglycan-deficient mice (genetic cardiomyopathy) were treated with asialoEPO for 5 weeks. AsialoEPO mitigated the progressive cardiac remodeling and dysfunction through cardiomyocyte hypertrophy, and upregulated expression of GATA-4 and sarcomeric proteins. AsialoEPO appears to act by altering the activity of the downstream erythropoietin receptor signals extracellular signal-regulated protein kinase, Akt, signal transducer, and activator of transcription 3 and 5 in a model-specific manner. CONCLUSIONS: The findings suggest that asialoEPO exerts broad cardioprotective effects through distinct mechanisms depending on the model, which are independent of the erythrogenic action. This compound may be promising for the treatment of heart failure of various etiologies.


Assuntos
Assialoglicoproteínas/uso terapêutico , Eritropoetina/análogos & derivados , Insuficiência Cardíaca/tratamento farmacológico , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Animais , Assialoglicoproteínas/administração & dosagem , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Eritropoetina/administração & dosagem , Eritropoetina/uso terapêutico , Seguimentos , Insuficiência Cardíaca/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Resultado do Tratamento , Função Ventricular Esquerda/fisiologia
7.
J Control Release ; 160(1): 81-7, 2012 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-22342472

RESUMO

Cerebral ischemia-reperfusion (I/R) injury induces secondary cerebral damage. As drugs for treating this type of injury have shown poor efficacy and adverse side effects in clinical trials, a novel therapeutic strategy has been long awaited. In this study, we focused on the disruption of the blood-brain barrier after stroke, and applied a liposomal drug delivery system (DDS) designed to enhance the pharmacological effect of the neuroprotectant and to avoid side effects. PEGylated liposomes were injected at varying time after the start of reperfusion in transient middle cerebral artery occlusion (t-MCAO) model rats. The results showed PEGylated liposomes accumulated in the ischemic hemisphere at an early stage after reperfusion and were retained in the lesion for at least 24h after injection. We also investigated the effectiveness of asialo-erythropoietin (AEPO)-modified PEGylated liposomes (AEPO-liposomes) in treating the cerebral I/R injury. AEPO-liposome treatment significantly reduced TTC-defined cerebral legion following cerebral I/R injury, and ameliorated motor function compared with vehicle and AEPO treatment. In conclusion, these results indicate that AEPO-liposomes are a promising liposomal formulation for protecting the brain from I/R injury, and that this liposomal DDS has potential as a novel strategy for the treatment of cerebral I/R injury.


Assuntos
Assialoglicoproteínas/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Eritropoetina/análogos & derivados , Ataque Isquêmico Transitório/tratamento farmacológico , Fármacos Neuroprotetores/administração & dosagem , Traumatismo por Reperfusão/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Assialoglicoproteínas/efeitos adversos , Assialoglicoproteínas/farmacocinética , Assialoglicoproteínas/uso terapêutico , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Eritropoetina/administração & dosagem , Eritropoetina/efeitos adversos , Eritropoetina/farmacocinética , Eritropoetina/uso terapêutico , Marcação In Situ das Extremidades Cortadas , Ataque Isquêmico Transitório/complicações , Ataque Isquêmico Transitório/metabolismo , Lipossomos , Masculino , Fármacos Neuroprotetores/efeitos adversos , Fármacos Neuroprotetores/farmacocinética , Fármacos Neuroprotetores/uso terapêutico , Células PC12 , Ratos , Ratos Wistar , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismo , Distribuição Tecidual
8.
Thromb Haemost ; 104(2): 243-51, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20508904

RESUMO

Clearance mechanisms for recombinant activated human FVII (rFVIIa; NovoSeven), a heterogeneously glycosylated protein, have yet to be fully elucidated, but may involve the liver. The effects of the gamma-carboxy glutamic acid (Gla) domain and the sialic acid content of the protein on rFVIIa clearance were investigated following intravenous administration of rFVIIa lacking the Gla domain, des(1-44) rFVIIa and asialo-rFVIIa in pharmacokinetic (PK) studies and perfused rat livers. PK parameters for both rFVIIa and des(1-44) rFVIIa had similar biphasic clearance profiles, as well as half-lives ([t(1/2)]=80 and 88 minutes, respectively), while asialo-rFVIIa was cleared quickly (t(1/2)=21 minutes) with a linear clearance profile. Perfused liver studies with all proteins (10 nM) mirrored the trends in profiles observed in the PK study. rFVIIa and des(1-44) rFVIIa were cleared to a similar extent, 41% and 35%, respectively, after 1 h, whereas plasma-derived FVII from humans (which has a higher sialylation content than rFVIIa) was cleared to a lesser extent (21%). Asialo-rFVIIa, on the other hand, was almost totally cleared and when an excess of asialo-orosomucoid was added to the perfusate, its clearance was significantly reduced (by 34%) and also for rFVIIa, albeit to a lesser extent (by 14%). Together these data suggest that carbohydrate receptor(s) (e.g. the asialoglycoprotein receptor, ASGPR) play a role in asialo-rFVIIa and rFVIIa clearance. In vivo and liver clearance data correlated well showing similar trends and indicated that rFVIIa clearance is not affected by the Gla domain, but rather by a subpopulation of N-glycosylated structures on rFVIIa.


Assuntos
Assialoglicoproteínas/farmacocinética , Coagulantes/farmacocinética , Fator VIIa/farmacocinética , Fígado/metabolismo , Fragmentos de Peptídeos/farmacocinética , Animais , Receptor de Asialoglicoproteína/metabolismo , Assialoglicoproteínas/administração & dosagem , Assialoglicoproteínas/sangue , Assialoglicoproteínas/farmacologia , Coagulantes/administração & dosagem , Coagulantes/sangue , Fator VIIa/administração & dosagem , Glicosilação , Meia-Vida , Humanos , Injeções Intravenosas , Fígado/efeitos dos fármacos , Masculino , Taxa de Depuração Metabólica , Orosomucoide/análogos & derivados , Orosomucoide/farmacologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/sangue , Perfusão , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/sangue , Proteínas Recombinantes/farmacocinética
9.
Br J Radiol ; 83(986): 129-32, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19546178

RESUMO

To increase the survival rate of patients with acute superior mesenteric artery thromboembolism (ASMAT) treated by catheter thrombolysis, we examined the effects of delivering edaravone and asialoerythropoietin, agents with tissue-protective activities, using a rabbit autologous fibrin clot ASMAT model. Japanese white rabbits (n=32) were randomly separated into four equal groups. 45 min after introducing autologous fibrin clot, Group U received urokinase and heparin; Group E received urokinase and heparin plus edaravone; Group A received urokinase and heparin plus asialoerythropoietin; and Group EA received urokinase, heparin and edaravone plus asialoerythropoietin via a catheter. The intestines were removed 6 h later and intestinal mucosal damage was scored using the Park's injury score. Survival time was assessed. Average mucosal injury was 5.78+/-1.52 (Group U), 2.88+/-0.72 (Group E), 1.90+/-1.23 (Group A) and 1.18+/-1.25 (Group EA). The degree of mucosal injury was significantly lower in Group EA than in Groups U and E (p<0.05). Conversely, there was no significant difference between Group A and Group EA, or between Group A and Group E. The survival times were 31.50+/-13.30 h (Group U), 51.00+/-24.74 h (Group E), 48.00+/-16.97 h (Group A) and 82+/-51.07 h (Group EA); the difference among the four groups was not significant. In conclusion, the concomitant administration of asialoerythropoietin and edaravone reduced mucosal membrane injury significantly compared with edaravone alone. However, to improve the survival of ASMAT rabbit models, the delivery of an appropriate dose of asialoerythropoietin is required, together with the development of methods to assess peripheral recanalisation.


Assuntos
Antipirina/análogos & derivados , Assialoglicoproteínas/administração & dosagem , Eritropoetina/análogos & derivados , Sequestradores de Radicais Livres/administração & dosagem , Oclusão Vascular Mesentérica/complicações , Traumatismo por Reperfusão/prevenção & controle , Tromboembolia/complicações , Animais , Antipirina/administração & dosagem , Antipirina/farmacologia , Assialoglicoproteínas/farmacologia , Cateterismo , Modelos Animais de Doenças , Combinação de Medicamentos , Edaravone , Eritropoetina/administração & dosagem , Eritropoetina/farmacologia , Fibrina , Fibrinolíticos/uso terapêutico , Sequestradores de Radicais Livres/farmacologia , Heparina/uso terapêutico , Injeções Intra-Arteriais , Mucosa Intestinal/patologia , Artéria Mesentérica Superior , Oclusão Vascular Mesentérica/tratamento farmacológico , Oclusão Vascular Mesentérica/mortalidade , Coelhos , Distribuição Aleatória , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/patologia , Taxa de Sobrevida , Tromboembolia/tratamento farmacológico , Tromboembolia/mortalidade , Ativador de Plasminogênio Tipo Uroquinase/uso terapêutico
10.
Clin Exp Pharmacol Physiol ; 37(2): 185-92, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19650797

RESUMO

1. Asialoerythropoietin (aEPO), a derivative of cytokine erythropoietin, has been shown to have neuroprotective effects without haematological complications when administered in single or repeated doses. The present study examines our hypothesis that aEPO may provide neuroprotection against programmed apoptotic cell death when administered in a continuous low dose. 2. Focal cerebral ischaemia was introduced by occlusion of the middle cerebral artery using a surgically placed intraluminal filament in young male Sprague Dawley rats (9 weeks old). After 90 min ischaemia, reperfusion was established by filament removal. Both study and control groups had implanted osmotic minipumps through which they received either aEPO (1 microL/h; 20 microg/kg per 24 h) or normal saline (1 microL/h) for 4 days. On Day 4, infarct volume, the number of apoptotic cells and concentrations of activated caspase 3 and 9 were evaluated in the penumbra region. 3. Asialoerythropoietin was detected in the cerebrospinal fluid of the study group, whereas none was detected in the control group. Although there were no significant changes in haematocrit levels or behaviour scores (on Days 1 and 4) between the study and control groups, aEPO administration significantly reduced infarct volume in the study group compared with the control group (168 +/- 19 vs 249 +/- 28 mm(3), respectively; P < 0.05). 4. The number of terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL)-positive cells and the concentration of activated caspase 3 and 9 in the penumbra region were significantly lower in the study group compared with the control group. 5. In conclusion, our data suggest that aEPO provides a short-term, possibly histological, protection in young adult male rats when administered immediately after reperfusion.


Assuntos
Apoptose/efeitos dos fármacos , Assialoglicoproteínas/administração & dosagem , Infarto Cerebral/tratamento farmacológico , Eritropoetina/análogos & derivados , Fármacos Neuroprotetores/administração & dosagem , Animais , Isquemia Encefálica/tratamento farmacológico , Caspase 3/metabolismo , Caspase 9/metabolismo , Infarto Cerebral/patologia , Eritropoetina/administração & dosagem , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Masculino , Ratos , Ratos Sprague-Dawley
11.
ScientificWorldJournal ; 9: 967-9, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19768353

RESUMO

The hematopoietic growth factor erythropoietin (EPO) and its neuroprotective, but not hematopoietic, variants asialoEPO, carbamylated EPO (CEPO), and low sialic acid EPO (Neuro-EPO) are attractive candidates for stroke treatment. Due to their large molecular weight, these proteins enter the brain only to a minor extent when intravenously administered, which has raised the question for alternative delivery strategies, among which intranasal delivery may certainly be an attractive choice, as the review by Garcia Rodriguez and Sosa Teste in this journal points out. Before this strategy may be considered clinically applicable, however, more and, in particular, quantitative information is needed about (a) the temporospatial accumulation of EPO and its variants in the brain tissue both in animals and nonhuman primates, and (b) the accumulation of EPO and its variants in the human cerebrospinal fluid.


Assuntos
Assialoglicoproteínas/administração & dosagem , Eritropoetina/análogos & derivados , Eritropoetina/administração & dosagem , Administração Intranasal , Barreira Hematoencefálica , Isquemia Encefálica/tratamento farmacológico , Humanos , Proteínas Recombinantes , Acidente Vascular Cerebral/tratamento farmacológico
12.
ScientificWorldJournal ; 9: 970-81, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19768354

RESUMO

Intranasal delivery provides a practical, noninvasive method of bypassing the blood-brain barrier (BBB) in order to deliver therapeutic agents to the brain. This method allows drugs that do not cross the BBB to be delivered to the central nervous system in a few minutes. With this technology, it will be possible to eliminate systemic administration and its potential side effects. Using the intranasal delivery system, researchers have demonstrated neuroprotective effects in different animal models of stroke using erythropoietin (EPO) as a neuroprotector or other different types of EPO without erythropoiesis-stimulating activity. These new molecules retain their ability to protect neural tissue against injury and they include Asialoerythropoietin (asialoEPO) carbamylated EPO (CEPO), and rHu-EPO with low sialic acid content (Neuro-EPO). Contrary to the other EPO variants, Neuro-EPO is not chemically modified, making it biologically similar to endogenous EPO, with the advantage of less adverse reactions when this molecule is applied chronically. This constitutes a potential benefit of Neuro-EPO over other variants of EPO for the chronic treatment of neurodegenerative illnesses. Nasal administration of EPO is a potential, novel, neurotherapeutic approach. However, it will be necessary to initiate clinical trials in stroke patients using intranasal delivery in order to obtain the clinical evidence of its neuroprotectant capacity in the treatment of patients with acute stroke and other neurodegenerative disorders. This new therapeutic approach could revolutionize the treatment of neurodegenerative disorders in the 21st century.


Assuntos
Administração Intranasal , Eritropoetina/administração & dosagem , Eritropoetina/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Assialoglicoproteínas/administração & dosagem , Barreira Hematoencefálica , Isquemia Encefálica/tratamento farmacológico , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Eritropoetina/análogos & derivados , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Receptores da Eritropoetina/metabolismo , Proteínas Recombinantes
13.
J Gene Med ; 11(1): 38-45, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19021130

RESUMO

BACKGROUND: Transgene expression in vivo for therapeutic purposes will require methods that allow for efficient gene transfer into cells. Although current vector technologies are being improved, the development of novel vector systems with improved targeting specificity, higher transduction efficiencies and improved safety is necessary. METHODS: Asialoglycoprotein receptor-targeted cationic nanoparticles for interleukin (IL)-12 encapsulation (NP1) or adsorption (NP2) have been formulated by blending poly(D,L-lactic-co-glycolic) acid (PLGA) (50 : 50) with the cationic lipid 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and the ligand asialofetuin (AF), by using a modified solvent evaporation process. RESULTS: We present a novel targeted lipopolymeric vector, which improves significantly the levels of luciferase gene expression in the liver upon i.v. administration. Targeted-NP2 particles showed a five- and 12-fold higher transfection activity in the liver compared to non-targeted (plain) complexes or naked pCMV DNA, respectively. On the other hand, BNL tumor-bearing animals treated with AF-NP1 containing the therapeutic gene IL-12, showed tumor growth inhibition, leading to a complete tumor regression in 75% of the treated mice, without signs of recurrence. High levels of IL-12 and interferon-gamma were detected in the sera of treated animals. Mice survival also improved considerably. Tumor treatment with AF-NP2 formulations lead only to a retardation in the tumor growth. CONCLUSIONS: In the present study, we have developed an efficient targeted non-viral vector for IL-12 gene transfer in hepatocellular carcinoma in vivo, by employing non-toxic cationic PLGA/DOTAP/AF nanoparticles. These results demonstrate for the first time that this cationic system could be used successfully and safely for delivery of therapeutic genes with antitumor activity into liver tumors with targeting specificity.


Assuntos
Carcinoma Hepatocelular/terapia , Interleucina-12/genética , Neoplasias Hepáticas/terapia , Nanopartículas/química , Animais , Assialoglicoproteínas/administração & dosagem , Assialoglicoproteínas/química , Ácidos Graxos Monoinsaturados/administração & dosagem , Ácidos Graxos Monoinsaturados/química , Feminino , Fetuínas , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos , Ácido Láctico/administração & dosagem , Ácido Láctico/química , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Tamanho da Partícula , Ácido Poliglicólico/administração & dosagem , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Compostos de Amônio Quaternário/administração & dosagem , Compostos de Amônio Quaternário/química , Transgenes , Células Tumorais Cultivadas , alfa-Fetoproteínas/administração & dosagem , alfa-Fetoproteínas/química
14.
Mech Ageing Dev ; 125(5): 375-80, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15130755

RESUMO

Dietary restriction (DR) is known to prolong life in laboratory animals. Intermittent (alternate-day) fasting or short-term repeated fasting has also been reported to increase the life span of animals. In the present study, we investigated the changes or induction of abnormalities of protein metabolism in rats during fasting, and measured asialoglycoprotein uptake and cell death/proliferation in the liver of rats receiving fasting and refeeding. In the results, liver weight decreased significantly after 48 h of fasting and increased during the refeeding period, returning to the pre-fasting level by 12 h of refeeding. Cell death, determined by single stranded DNA (ssDNA) staining method, increased during the fasting period, and returned to the pre-fasting level during the refeeding period. Cell proliferation, determined using antibodies (Ab) against proliferating cell nuclear antigen, decreased during the fasting period, and increased during the refeeding period. Changes in cell death and cell proliferation were inversely related. However, there was no significant difference in asialoglycoprotein uptake by the whole liver between the ad libitum (AL)-fed rats and 48 h fasted rats. Thus, neither the changes in liver weight nor cell death/proliferation affected asialoglycoprotein uptake on a living body. These results suggest that episodes of 48 h fasting do not induce protein metabolism abnormalities in the liver.


Assuntos
Proliferação de Células , Proteínas Alimentares/administração & dosagem , Jejum/fisiologia , Fígado/fisiologia , Animais , Assialoglicoproteínas/administração & dosagem , Morte Celular/fisiologia , Fígado/patologia , Masculino , Tamanho do Órgão/fisiologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos
15.
Vaccine ; 18(15): 1522-30, 2000 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-10618550

RESUMO

Plasmid DNA encoding herpes simplex virus type-1 glycoprotein D (gD-1) was complexed with asialoorosomucoid conjugated to poly-L-lysine. Following its intravenous injection into BALB/c mice, this complex was targeted to the liver. Liver cells expressing gD-1 were detected immunohistochemically through day 6 post-immunization, while gD-1 DNA was detectable through 14 days post-immunization. Decline of gD-1 expression and detectable gD-1 DNA in the liver correlated with influx of T cells, predominantly CD4(+). The ASOR-poly-L-lysine DNA carrier system promotes hepatic expression of gD-1 and may be useful in vaccination against herpes simplex virus type-1.


Assuntos
Herpesvirus Humano 1/imunologia , Fígado/metabolismo , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/genética , Vacinas Virais/imunologia , Animais , Assialoglicoproteínas/administração & dosagem , Feminino , Imunização , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Orosomucoide/administração & dosagem , Orosomucoide/análogos & derivados , Polilisina/administração & dosagem , Proteínas do Envelope Viral/análise , Proteínas do Envelope Viral/imunologia
16.
Arch Dermatol Res ; 292(11): 542-9, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11194892

RESUMO

Intravenous injection of plasmid DNA encoding herpes simplex virus type-1 glycoprotein D (gD-1) complexed with asialoorosomucoid-poly-L-lysine (gD-ASOR) targets foreign DNA to the liver, leading to hepatic expression of gD-1. BALB/c mice were given two intravenous injections of gD-ASOR, pBK-ASOR (plasmid lacking the gD-1 gene but complexed with ASOR), or PBS. The skin was inoculated with 1 x 10(4) PFU of HSV-1 or sham-inoculated, and analyzed for infectious virus and cellular infiltration 1, 3, and 5 days after inoculation. Prior immunization with gD-ASOR led to significantly lower (P < 0.05) viral titers in the skin 5 days after inoculation compared with controls. Infiltration of the skin at the site of inoculation by polymorphonuclear neutrophils (PMNs), T cells, B cells, dendritic cells, and macrophages was monitored immunohistochemically. Significantly higher numbers (P < 0.05) of CD4+ and CD8+ T cells, dendritic cells, and macrophages responded to HSV-1 challenge in mice immunized with gD-ASOR than in mice immunized with pBK-ASOR or PBS. The response by PMNs and B cells was indistinguishable among the treatment groups. These results suggest that BALB/c mice sensitized to gD-1 following gD-ASOR immunization develop an enhanced T-cell response to primary HSV-1 infection.


Assuntos
Quimiocinas CX3C , DNA Viral/administração & dosagem , Proteínas do Envelope Viral/imunologia , Viroses/prevenção & controle , Animais , Antígenos de Diferenciação/análise , Assialoglicoproteínas/administração & dosagem , Assialoglicoproteínas/química , Antígenos CD4/análise , Antígenos CD8/análise , Quimiocina CX3CL1 , Quimiocinas CXC/análise , DNA Viral/química , DNA Viral/genética , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Feminino , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Antígenos de Histocompatibilidade Classe II/análise , Imunidade Celular/imunologia , Imuno-Histoquímica , Antígenos Comuns de Leucócito/análise , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos BALB C , Orosomucoide/administração & dosagem , Orosomucoide/análogos & derivados , Orosomucoide/química , Plasmídeos/administração & dosagem , Plasmídeos/química , Plasmídeos/genética , Polilisina/administração & dosagem , Polilisina/análogos & derivados , Polilisina/química , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteínas S100/análise , Pele/química , Pele/imunologia , Fatores de Tempo , Proteínas do Envelope Viral/genética , Viroses/imunologia , Viroses/virologia
17.
Bioconjug Chem ; 8(6): 935-40, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9404669

RESUMO

Antisense oligonucleotides have been covalently attached to asialoglycoprotein (ASGP) via disulfide bond conjugation chemistry. These conjugates were characterized extensively by an array of chemical, chromatographic, and spectroscopic means. Multiple (approximately six) oligonucleotides can be conjugated to each ASGP molecule. The molecular conjugates were used to deliver antisense oligonucleotides complementary to the mRNA of the interleukin 6 signal transduction protein (gp130) to modulate the acute phase response of hepatoma (HepG2) cells in vitro. These conjugates were biologically active, as measured by inhibition of the cytokine-stimulated up-regulation of the acute phase protein haptoglobin. The level of inhibition was comparable to that found with previous technology featuring noncovalent complexes of ASGP-poly(L-lysine) and oligonucleotide. Because of the ability to control the stoichiometry of the conjugate and its unimolecular nature (as opposed to bimolecular for the noncovalent conjugates), this methodology holds great promise for further development and application.


Assuntos
Assialoglicoproteínas/administração & dosagem , Assialoglicoproteínas/química , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/química , Assialoglicoproteínas/síntese química , Carcinoma Hepatocelular/metabolismo , Reagentes de Ligações Cruzadas/química , Dissulfetos/química , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Humanos , Oligonucleotídeos Antissenso/síntese química , Piridinas/química , Succinatos/química , Células Tumorais Cultivadas/efeitos dos fármacos
18.
Blood Coagul Fibrinolysis ; 8 Suppl 2: S31-8, 1997 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9607111

RESUMO

The development of non-viral gene therapy has been hampered by an inability to reproducibly manufacture and characterize delivery system components and final formulations. Formation of interpolyelectrolyte complexes as the basis of various gene delivery methods has been approached as the first step towards development of synthetic viruses. We have found that preparation of interpolyelectrolyte complexes from disperse reagents gives a more homogeneous gene delivery vehicle than other methods. Methods which increase homogeneity also result in higher transfection efficiency in vivo. Expression levels of human growth hormone and other reporter proteins in mice confirm the potential of parenteral non-viral gene delivery for some therapeutic applications. Serum is demonstrated to inhibit transfection efficiency in vivo. Our results suggest that further development of methods to manufacture homogeneous disperse non-viral delivery vehicles with stealth characteristics may enhance both the potency and reproducibility of gene transfer in vivo.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Animais , Assialoglicoproteínas/administração & dosagem , Assialoglicoproteínas/uso terapêutico , Centrifugação com Gradiente de Concentração , Regulação da Expressão Gênica , Hormônio do Crescimento Humano/biossíntese , Hormônio do Crescimento Humano/genética , Injeções Intravenosas , Ligantes , Luciferases/genética , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos/genética , Orosomucoide/administração & dosagem , Orosomucoide/análogos & derivados , Orosomucoide/uso terapêutico , Polilisina/administração & dosagem , Polilisina/análogos & derivados , Polilisina/uso terapêutico
19.
Bioconjug Chem ; 7(1): 131-7, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8742001

RESUMO

A derivative of colchicine was synthesized, in a manner that preserved its important structural features, and conjugated to an asialoglycoprotein. The conjugate was characterized by ultraviolet-visible spectrophotometry and protein analysis. An average coupling ratio of 2 mol of colchicine per mole of asialoglycoprotein was achieved. The conjugate was stable to incubation in serum but was split into its separate components under chemically reducing conditions. Incubation with cells in culture revealed that the conjugate had antiproliferative activity similar to that of colchicine, but only in asialoglycoprotein receptor-containing cells. There was no effect at all on asialoglycoprotein receptor (-) cells. Furthermore, the antiproliferative effect of the conjugate on receptor (+) cells was blocked by addition of a large molar excess of free asialoglycoprotein. Immunofluorescence microscopy revealed disruption of microtubules in cell cultures that were pretreated with the conjugate. These results indicate that a colchicine conjugate that is taken up specifically into cells by asialoglycoprotein receptors and released intracellularly in a biologically active form can be prepared.


Assuntos
Assialoglicoproteínas/administração & dosagem , Colchicina/análogos & derivados , Colchicina/administração & dosagem , Receptor de Asialoglicoproteína , Assialoglicoproteínas/metabolismo , Carcinoma Hepatocelular , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Colchicina/metabolismo , Colchicina/farmacologia , Portadores de Fármacos , Humanos , Indicadores e Reagentes , Neoplasias Hepáticas , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Receptores de Superfície Celular/metabolismo , Espectrofotometria , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Células Tumorais Cultivadas
20.
J Viral Hepat ; 2(6): 273-8, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-8732172

RESUMO

An asialoglycoprotein-based DNA delivery system containing an antisense oligo DNA against the polyadenylation region and adjacent upstream sequences of woodchuck hepatitis virus (WHV) was prepared. Experimental woodchucks were inoculated neonatally with the woodchuck virus 23 weeks before initiating the study, and all animals subsequently developed hepatitis as evidenced by the presence of measurable levels of circulating viral DNA. Animals were injected intravenously (i.v.) with asialoorosomucoid (AsOR)-poly-L-lysine complexes containing 0.1 mg kg-1 antisense DNA for five consecutive days. Levels of surface antigen did not differ substantially between treated and control animals. However, intravenous administration of complexed antisense DNA significantly decreased viraemia, as shown by a five- to 10-fold decrease in circulating viral DNA 25 days post treatment. The decline lasted for at least 2 weeks, after which there was a gradual increase in DNA levels. Antisense DNA alone or a complex containing a random oligo DNA of the same size and linkage failed to have any significant effect on viral DNA levels. We conclude that antisense oligo DNA can be targeted to the liver in vivo, resulting in a substantial and prolonged decrease in viral DNA levels in WHV-infected woodchucks.


Assuntos
DNA Antissenso/administração & dosagem , DNA Antissenso/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , Vírus da Hepatite B da Marmota , Vírus da Hepatite B/química , Hepatite B/tratamento farmacológico , Hepatite B/virologia , Animais , Assialoglicoproteínas/administração & dosagem , Sequência de Bases , DNA Viral/análise , Feminino , Regulação Viral da Expressão Gênica , Antígenos de Hepatite/análise , Masculino , Marmota , Dados de Sequência Molecular , Replicação Viral
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