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1.
Eur Cell Mater ; 41: 756-773, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34151417

RESUMO

Proper regulation of the innate immune response to bone biomaterials after implantation is pivotal for successful bone healing. Pro-inflammatory M1 and anti-inflammatory M2 macrophages are known to have an important role in regulating the healing response to biomaterials. Materials with defined structural and topographical features have recently been found to favourably modulate the innate immune response, leading to improved healing outcomes. Calcium phosphate bone grafts with submicron-sized needle-shaped surface features have been shown to trigger a pro-healing response through upregulation of M2 polarised macrophages, leading to accelerated and enhanced bone regeneration. The present review describes the recent research on these and other materials, all the way from benchtop to the clinic, including in vitro and in vivo fundamental studies, evaluation in clinically relevant spinal fusion models and clinical validation in a case series of 77 patients with posterolateral and/or interbody fusion in the lumbar and cervical spine. This research demonstrates the feasibility of enhancing biomaterial-directed bone formation by modulating the innate immune response through topographic surface features.


Assuntos
Osso e Ossos/efeitos dos fármacos , Osso e Ossos/imunologia , Consolidação da Fratura/efeitos dos fármacos , Consolidação da Fratura/imunologia , Imunidade Inata/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Materiais Biocompatíveis/farmacologia , Regeneração Óssea/efeitos dos fármacos , Regeneração Óssea/imunologia , Fosfatos de Cálcio/farmacologia , Feminino , Humanos , Imunidade Inata/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Osteogênese/efeitos dos fármacos , Osteogênese/imunologia
2.
J Control Release ; 296: 250-257, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30682444

RESUMO

Rho kinase activity in hepatic stellate cells (HSCs) is associated with activation, transformation and contraction of these cells, leading to extracellular matrix production and portal hypertension in liver cirrhosis. Inhibition of rho kinase activity can reduce these activities, but may also lead to side effects, for instance systemic hypotension. This can be circumvented by liver-specific delivery of a rho kinase inhibitor to effector cells. Therefore, we targeted the rho kinase inhibitor Y27632 to the key pathogenic cells in liver fibrosis, i.e. myofibroblasts including activated HSCs that highly express the PDGFß-receptor, using the drug carrier pPB-MSA. This carrier consists of mouse serum albumin (MSA) covalently coupled to several PDGFßR-recognizing moieties (pPB). We aimed to create a prolonged release system of such a targeted construct, by encapsulating pPB-MSA-Y27632 in biodegradable polymeric microspheres, thereby reducing short-lasting peak concentrations and the need for frequent administrations. Firstly, we confirmed the vasodilating potency of PDGFß-receptor targeted Y27632 in vitro in a contraction assay using HSCs seeded on a collagen gel. We subsequently demonstrated the in vivo antifibrotic efficacy of pPB-MSA-Y27632-loaded microspheres in the Mdr2-/- mouse model of progressive biliary liver fibrosis. A single subcutaneous microsphere administration followed by organ harvest one week later clearly attenuated liver fibrosis progression and significantly suppressed the expression of fibrosis related genes, such as several collagens, profibrotic cytokines and matrix metalloproteinases. In conclusion, we demonstrate that polymeric microspheres are suitable as drug delivery system for the sustained systemic delivery of targeted protein constructs with antifibrotic potential, such as pPB-MSA-Y27632. This formulation appears suitable for the sustained treatment of liver fibrosis and possibly other chronic diseases.


Assuntos
Amidas/administração & dosagem , Portadores de Fármacos/administração & dosagem , Cirrose Hepática/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Piridinas/administração & dosagem , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Linhagem Celular , Preparações de Ação Retardada/administração & dosagem , Feminino , Humanos , Cirrose Hepática/metabolismo , Camundongos Knockout , Microesferas , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATP
3.
J Control Release ; 269: 258-265, 2018 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29170138

RESUMO

Liver fibrogenesis is associated with excessive production of extracellular matrix by myofibroblasts that often leads to cirrhosis and consequently liver dysfunction and death. Novel protein-based antifibrotic drugs show high specificity and efficacy, but their use in the treatment of fibrosis causes a high burden for patients, since repetitive and long-term parenteral administration is required as most proteins and peptides are rapidly cleared from the circulation. Therefore, we developed biodegradable polymeric microspheres for the sustained release of proteinaceous drugs. We encapsulated the drug carrier pPB-HSA, which specifically binds to the PDGFßR that is highly upregulated on activated myofibroblasts, into microspheres composed of hydrophilic multi-block copolymers composed of poly(l-lactide) and poly ethylene glycol/poly(ϵ-caprolactone), allowing diffusion-controlled release. Firstly, we estimated in mice with acute fibrogenesis induced by a single CCl4 injection the half-life of I125-labeled pPB-HSA at 40 min and confirmed the preferential accumulation in fibrotic tissue. Subsequently, we determined in the Mdr2 −/− mouse model of advanced biliary liver fibrosis how the subcutaneously injected microspheres released pPB-HSA into both plasma and fibrotic liver at 24 h after injection, which was maintained for six days. Although the microspheres still contained protein at day seven, pPB-HSA plasma and liver concentrations were decreased. This reduction was associated with an antibody response against the human albumin-based carrier protein, which was prevented by using a mouse albumin-based equivalent (pPB-MSA). In conclusion, this study shows that our polymeric microspheres are suitable as sustained release formulation for targeted protein constructs such as pPB-HSA. These formulations could be applied for the long-term treatment of chronic diseases such as liver fibrosis.


Assuntos
Portadores de Fármacos/administração & dosagem , Cirrose Hepática/metabolismo , Polímeros/administração & dosagem , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Albumina Sérica/administração & dosagem , Animais , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/farmacocinética , Portadores de Fármacos/farmacocinética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microesferas , Polímeros/farmacocinética , Albumina Sérica/farmacocinética
4.
Int J Pharm ; 534(1-2): 229-236, 2017 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-29038068

RESUMO

Injectable sustained release drug delivery systems are an attractive alternative for the intravenous delivery of therapeutic proteins. In particular, for chronic diseases such as fibrosis, this approach could improve therapy by reducing the administration frequency while avoiding large variations in plasma levels. In fibrotic tissues the platelet-derived growth factor receptor beta (PDGFßR) is highly upregulated, which provides a target for site-specific delivery of drugs. Our aim was to develop an injectable sustained release formulation for the subcutaneous delivery of the PDGFßR-targeted drug carrier protein pPB-HSA, which is composed of multiple PDGFßR-recognizing moieties (pPB) attached to human serum albumin (HSA). We used blends of biodegradable multi-block copolymers with different swelling degree to optimize the release rate using the model protein HSA from microspheres produced via a water-in-oil-in-water double emulsion evaporation process. The optimized formulation containing pPB-HSA, showed complete release in vitro within 14days. After subcutaneous administration to mice suffering from renal fibrosis pPB-HSA was released from the microspheres and distributed into plasma for at least 7days after administration. Furthermore, we demonstrated an enhanced accumulation of pPB-HSA in the fibrotic kidney. Altogether, we show that subcutaneously administered polymeric microspheres present a suitable sustained release drug delivery system for the controlled systemic delivery for proteins such as pPB-HSA.


Assuntos
Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacologia , Fibrose/tratamento farmacológico , Nefropatias/tratamento farmacológico , Polímeros/química , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Albumina Sérica Humana/química , Animais , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Fibrose/metabolismo , Humanos , Nefropatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microesferas
5.
PDA J Pharm Sci Technol ; 65(2): 116-30, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21502073

RESUMO

AIM: Interleukin-10 (IL-10) is a cytokine with potent antifibrotic and anti-inflammatory properties. However, IL-10 has a very short plasma half-life in vivo. This prompted the question whether a short intravenous treatment might have prolonged effects on more chronic processes like sclerosis. METHODS: Glomerulosclerosis was induced by anti-Thymocyte 1 (Anti-Thy 1) antibody. Four days after induction, an intravenous injection of recombinant human IL-10 (rhIL-10) was given for 3 consecutive days. Untreated rats received vehicle only (phosphate-buffered saline). Parameters of inflammation and fibrosis were assessed at protein and mRNA levels. Untreated rats showed renal histopathological changes as compared to normal rats. RESULTS: Glomerular matrix expansion and inflammatory cell influx was observed and an increase in glomerular-inducible nitric oxide synthetase and α-smooth muscle actin (α-SMA) were found on the protein level, factors that were clearly attenuated by IL-10 treatment. In particular, the decrease of matrix metalloproteinase-13 levels between days 4 and 7 was completely prevented by IL-10. In contrast, IL-10 did not significantly reduce mRNA levels for procollagen α1(1), α-SMA, and transforming growth factor 1. CONCLUSION: A short-term treatment with rhIL-10 after induction of Anti-Thy 1 antibody nephritic rats attenuated intraglomerular inflammation, and at the protein level also influenced the parameters reflecting matrix deposition and degradation. Despite in fact that IL-10 was shown to be effective in the inhibition of matrix deposition, it had no beneficial effect on proteinuria. LAY ABSTRACT: Interleukin-10 is a cytokine with potent antifibrotic and anti-inflammatory properties. Its short plasma half-life raises the question whether a short intravenous treatment might have prolonged effects on chronic disease like sclerosis. To confirm this, recombinant human interleukin-10 was used to treat glomerulosclerosis in rats. The disease was induced by Anti-Thy 1 antibody. Four days after induction, an intravenous injection of IL-10 was given for 3 consecutive days. Untreated rats received vehicle only (phosphate-buffered saline). Parameters of inflammation and fibrosis were assessed at protein and mRNA levels. In this study, untreated rats showed renal histopathological changes as compared to normal rats. Glomerular matrix expansion and inflammatory cell influx was observed, and increases in glomerular nitric oxide synthetase and α-smooth muscle actin α-SMA were found on the protein level. In contrast, treated rats clearly showed reduction of all these parameters. In particular, the decrease of anti-matrix metalloproteinase-13 (MMP-13) levels between days 4 and 7 was completely prevented by IL-10. However, IL-10 did not significantly reduce mRNA levels for procollagen α1(1), α-SMA, and TGFß-1. Based on these results, it can be concluded that a short-term treatment with rhIL-10 after induction of Anti-Thy 1 antibody in nephritic rats attenuated intraglomerular inflammation, and at the protein level also influenced the parameters reflecting matrix deposition and degradation. Despite in fact that IL-10 was shown to be effective in the inhibition of matrix deposition, it had no beneficial effect on proteinuria.


Assuntos
Interleucina-10 , Glomérulos Renais , Animais , Glomerulonefrite/tratamento farmacológico , Humanos , Infusões Intravenosas , Interleucina-10/farmacologia , Glomérulos Renais/efeitos dos fármacos , Metaloproteinase 2 da Matriz/farmacologia , Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/farmacologia , Proteinúria , Ratos , Ratos Wistar
6.
J Pharmacol Exp Ther ; 337(3): 628-35, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21383021

RESUMO

One of the pathways activated during liver fibrosis is the Rho kinase pathway, which regulates activation, migration, and contraction of hepatic stellate cells (HSC). Inhibition of this kinase by the Rho kinase inhibitor Y27632 [(+)-(R)-trans- 4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] has been shown to reduce fibrosis in animal models. However, kinase expression is ubiquitous, so any inhibitor may affect many cell types. We hypothesize that cell-specific delivery of a kinase inhibitor will be beneficial. Therefore, we conjugated Y27632 to the carrier mannose-6-phosphate (M6P) human serum albumin (HSA), which is taken up specifically in activated HSC through the M6P/insulin-like growth factor II receptor. This conjugate decreased protein expression of phosphorylated myosin light chain 2 (pMLC2) and vinculin, downstream of Rho kinase, in activated primary HSC and decreased the migration and contraction of HSC. In an ex vivo model, free Y27632 decreased contractility of rat aortas, whereas the Y27-conjugate did not, showing that the Y27-conjugate does not affect nontarget tissue. In chronic CCl(4)-induced liver fibrosis, both free drug and conjugate reduced HSC activation; however, only the Y27-conjugate significantly reduced collagen deposition. Treatment with the Y27-conjugate, but not with free drug, reduced pMLC2 expression in livers 24 h after injection, demonstrating prolonged inhibition of the Rho kinase pathway. The Rho kinase inhibitor Y27632 can be specifically targeted to HSC using M6PHSA, decreasing its effects in nontarget tissues. The targeted drug effectively reduced fibrotic parameters in vivo via the inhibition of the Rho kinase pathway.


Assuntos
Amidas/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Amidas/química , Amidas/metabolismo , Animais , Aorta/efeitos dos fármacos , Portadores de Fármacos , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Masculino , Manosefosfatos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Piridinas/química , Piridinas/metabolismo , Ratos , Ratos Wistar , Albumina Sérica/metabolismo , Quinases Associadas a rho/metabolismo
7.
Gut ; 58(3): 379-87, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18852260

RESUMO

BACKGROUND AND AIMS: Crohn's disease (CD) and ulcerative colitis (UC) are chronic multifactorial inflammatory bowel diseases (IBDs) with unknown aetiology, but a deregulated mucosal immune response to gut-derived bacterial antigens is thought to be involved. Toll-like receptor ligands, especially lipopolysaccharide (LPS), contribute to the maintenance of the disease. It has previously been shown that the enzyme alkaline phosphatase (AP) is able to detoxify LPS, and the aim of this study was to examine a possible role in IBDs. METHODS: Intestinal AP (iAP) mRNA expression and LPS dephosphorylation in intestinal biopsies of control subjects and patients with IBD were examined, and the effect of orally administered iAP tablets on the progression of dextran sodium sulfate-induced colitis in rats was subsequently studied. RESULTS: In healthy persons, iAP mRNA and enzyme activity was high in the ileum relative to the colon. In patients with UC and CD, iAP mRNA expression was found to be markedly reduced when inflamed tissue was compared with non-inflamed tissue. Oral administration of iAP tablets to colitic rats resulted in a significant attenuation of colonic inflammation as reflected by reduced mRNA levels for tumour necrosis factor alpha, interleukin 1 beta, interleukin 6 and inducible nitric oxide synthase NOS (iNOS), a reduced iNOS staining and inflammatory cell influx, and a significantly improved morphology of the intestinal wall. CONCLUSIONS: The present study shows that epithelial iAP mRNA expression is reduced in patients with UC and CD. The rat model demonstrates that oral administration of active iAP enzymes in the intestinal tract results in a significant reduction of inflammation. This provides new insight on IBD pathology and a novel treatment approach to this severe inflammatory disease.


Assuntos
Fosfatase Alcalina/fisiologia , Colite Ulcerativa/enzimologia , Colo/enzimologia , Doença de Crohn/enzimologia , Mucosa Intestinal/enzimologia , Adolescente , Adulto , Idoso , Análise de Variância , Animais , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Imunidade nas Mucosas/fisiologia , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Adulto Jovem
8.
J Pharmacol Exp Ther ; 324(3): 902-10, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18077624

RESUMO

Liver fibrosis is associated with proliferation of hepatic stellate cells (HSCs) and their transformation into myofibroblastic cells that synthesize scar tissue. Several studies indicate that induction of apoptosis in myofibroblastic cells may prevent fibrogenesis. Gliotoxin (GTX) was found to induce apoptosis of hepatic cells and caused regression of liver fibrosis. However, the use of apoptosis-inducing drugs may be limited due to lack of cell specificity, with a risk of severe adverse effects. In previous studies, we found that mannose-6-phosphate-modified human serum albumin (M6P-HSA) selectively accumulated in liver fibrogenic cells. The aim of this study therefore was to couple GTX to M6P-HSA and test its pharmacological effects in vitro and in rats with liver fibrosis. The conjugate GTX-M6P-HSA bound specifically to HSCs and reduced their viability. Apoptosis was induced in cultures of human hepatic myofibroblasts (hMFs) and in liver slices obtained from rats with liver fibrosis. In vivo treatment with GTX or GTX-M6P-HSA in bile duct ligated rats revealed a significant decrease in alpha-smooth muscle actin mRNA levels and a reduced staining for this HSC marker in fibrotic livers. In addition, although GTX also affected hepatocytes, GTX-M6P-HSA did not significantly affect other liver cells. In conclusion, we developed an HSC-specific compound that induced apoptosis in human hMFs, rat HSCs, and in fibrotic liver slices. In vivo, both GTX and GTX-M6P-HSA attenuated the number of activated HSCs, but GTX also affected hepatocytes. This study shows that cell-selective delivery of the apoptosis-inducing agent GTX is feasible in fibrotic livers.


Assuntos
Apoptose/fisiologia , Sistemas de Liberação de Medicamentos/métodos , Gliotoxina/administração & dosagem , Hepatócitos/citologia , Cirrose Hepática/patologia , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Humanos , Cirrose Hepática/tratamento farmacológico , Masculino , Ratos , Ratos Wistar
10.
Biochim Biophys Acta ; 1667(2): 208-14, 2004 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-15581857

RESUMO

Adsorption of serum proteins to the liposomal surface plays a critical role in liposome clearance from the blood. The aim of this study was to investigate the role of liposome-adsorbed serum proteins in the interaction of liposomes with hepatocytes. We analyzed the serum proteins adsorbing to the surface of differently composed small unilamellar liposomes during incubation with human or rat serum, and found that one protein, with a molecular weight of around 55 kDa, adsorbed in a large amount to negatively charged liposomes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). The binding was dependent on the liposomal charge density. The approximately 55-kDa protein was identified as beta2-glycoprotein I (beta2GPI) by Western blotting. Despite the high affinity of beta2GPI for strongly negatively charged liposomes, in vitro uptake and binding experiments with isolated rat hepatocytes, Kupffer cells or liver endothelial cells, and with HepG2 cells showed no enhancing effect of this protein on the association of negatively charged liposomes with any of these cells. On the contrary, an inhibitory effect was observed. We conclude that despite abundant adsorption to negatively charged liposomes, beta2GP1 inhibits, rather than enhances, liposome uptake by liver cells.


Assuntos
Glicoproteínas/metabolismo , Hepatócitos/metabolismo , Lipossomos/metabolismo , Lipossomos/farmacocinética , Adsorção , Animais , Proteínas Sanguíneas/metabolismo , Western Blotting , Linhagem Celular , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Células Endoteliais/metabolismo , Humanos , Células de Kupffer/metabolismo , Lipossomos/química , Fígado/citologia , Masculino , Peso Molecular , Ratos , Ratos Endogâmicos , Propriedades de Superfície , beta 2-Glicoproteína I
11.
Biochem Biophys Res Commun ; 325(3): 908-14, 2004 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-15541376

RESUMO

We investigated the potential role of scavenger receptor B-I (SR-BI) in the selective removal of liposomal markers from blood by hepatocytes. Liposomes were labeled with [(3)H]cholesteryloleyl-ether ([(3)H]COE), 1,2-di[1-(14)C]palmitoyl-phosphatidylcholine ([(14)C]PC), and N-(lissamine rhodamine-B sulfonyl)-phosphatidylethanolamine (N-Rh-PE). The radiolabels were eliminated at identical rates from plasma, while N-Rh-PE was cleared twice as fast. Involvement of SR-BI in the selective removal of N-Rh-PE from liposomes was studied in transfected Chinese hamster ovary cells over-expressing SR-BI. Uptake of N-Rh-PE from liposomes containing phosphatidylserine was higher than [(3)H]COE, and was further enhanced by apolipoprotein A-I, confirming involvement of SR-BI in the selective uptake of liposomal N-Rh-PE by cells.


Assuntos
Hepatócitos/metabolismo , Lipossomos/química , Lipossomos/farmacocinética , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/farmacocinética , Receptores Imunológicos/metabolismo , Rodaminas/química , Rodaminas/farmacocinética , Animais , Antígenos CD36 , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Sistemas de Liberação de Medicamentos/métodos , Lipossomos/sangue , Masculino , Fosfatidiletanolaminas/análise , Ratos , Receptores Depuradores , Rodaminas/análise , Receptores Depuradores Classe B , Temperatura , Distribuição Tecidual
12.
Int J Pharm ; 257(1-2): 273-81, 2003 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-12711182

RESUMO

In this study the possibility to deliver the acid-sensitive enzyme alkaline phosphatase (AP) from calf intestine (CIAP) to the intestinal system by oral administration was investigated. Tablets were prepared and in vitro evaluated. Final proof of concept studies were performed in rats. This acid labile enzyme is potentially useful in the treatment of sepsis, a serious condition during which endotoxins can migrate into the blood stream. The CIAP was freeze-dried with inulin and subsequently compacted into round biconvex tablets with a diameter of 4mm and a weight of 25-30 mg per tablet. The tablets were coated with an enteric coating in order to ensure their survival in the stomach. In vitro evaluation of tablets containing alkaline phosphatase from bovine intestine (BIAP) was the first step in the development. It was found that tablets without enteric coating dissolved rapidly in 0.10 M HCl with total loss of enzymatic activity of the alkaline phosphatase. Tablets that were coated were stable for at least 2 h in 0.10 M HCl, but dissolved rapidly when the pH was increased to 6.8. Furthermore, it was shown that the enzymatic activity of the released BIAP was fully preserved. The in vivo test clearly showed that the oral administration of enteric coated tablets resulted in the release of enzymatically active CIAP in the intestinal lumen of rats. The location of the enhanced enzymatic activity of AP in the intestines varied with the time that had passed between the administration of the tablets and the sacrificing of the rats. Also, the level of enzymatic activity increased with an increasing number of tablets that were administered.


Assuntos
Fosfatase Alcalina/administração & dosagem , Mucosa Intestinal/metabolismo , Inulina/administração & dosagem , Administração Oral , Fosfatase Alcalina/química , Animais , Diálise , Estabilidade Enzimática , Liofilização , Masculino , Ratos , Ratos Wistar , Solubilidade , Comprimidos
13.
J Hepatol ; 35(2): 187-94, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11580140

RESUMO

BACKGROUND/AIMS: Inflammation in the liver is a complex interaction between parenchymal and non-parenchymal cells, and therefore can not be studied in vitro in pure cultures of these cells. METHODS: We investigated whether Kupffer cells in the liver slice are still responsive to an inflammatory stimulus of lipopolysaccharide (LPS), and evoke an inflammatory response in the hepatocytes. RESULTS: TNFalpha, IL-1beta and IL-10 were significantly elevated in culture medium of LPS-stimulated rat liver slices. Nitric oxide (NO) production of LPS-treated slices gradually increased from 5 to 24 h (24 h: 81+/-5 microM vs. 14+/-2 microM in control P < 0.05), paralleled by inducible nitric oxide synthase (iNOS) in the hepatocytes, iNOS mRNA was induced after 3 h. NO production but not iNOS induction was significantly inhibited by NOS inhibitors S-methylisothiourea and N(G)-nitro-L-arginine methylester. Both pentoxifylline and dexamethasone inhibited TNFalpha and IL-1beta production, albeit to a different extent, iNOS induction and, as a result thereof, NO production. CONCLUSIONS: These results imply that non-parenchymal cells in liver slices are viable and can be activated by LPS. In addition, it is concluded that the upregulation of iNOS in hepatocytes by LPS is caused by cytokines produced by Kupffer cells because inhibition of TNFalpha and IL-1beta production attenuated iNOS induction.


Assuntos
Inflamação/induzido quimicamente , Inflamação/fisiopatologia , Lipopolissacarídeos/toxicidade , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Indução Enzimática/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Inflamação/patologia , Interleucina-1/metabolismo , Interleucina-10/metabolismo , Células de Kupffer/efeitos dos fármacos , Células de Kupffer/patologia , Células de Kupffer/fisiologia , L-Lactato Desidrogenase/metabolismo , Fígado/patologia , Masculino , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismo
14.
Hepatology ; 34(4 Pt 1): 719-28, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11584368

RESUMO

Kupffer cells (KC) play an important role in the pathogenesis of inflammatory liver diseases leading to fibrosis. Anti-inflammatory drugs are only effective when administered at high doses that may cause side effects. Therefore, dexamethasone coupled to mannosylated albumin (Dexa(5)-Man(10)-HSA) was designed by us to selectively deliver this anti-inflammatory drug to the KC. The effectiveness of Dexa(5)-Man(10)-HSA was studied both in organ cultures and fibrosis induced by bile duct ligation (BDL) in rats. Dexa(5)-Man(10)-HSA accumulated in livers of both healthy and fibrotic rats (67% +/- 5% and 70% +/- 9% of the dose, respectively) and uptake was found almost exclusively in KC. Active dexamethasone was liberated from its carrier, because Dexa(5)-Man(10)-HSA could effectively inhibit nitric oxide (NO) and tumor necrosis factor alpha (TNF-alpha) release in endotoxin-activated liver slices. In vivo, however, this was associated with increased collagen I and III depositions and enhanced tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression. This was accompanied by a decreased influx of reactive oxygen species (ROS) producing cells in the livers of BDL animals treated with Dexa(5)-Man(10)-HSA as compared with untreated BDL rats. Dexa(5)-Man(10)-HSA treatment also replenished the depleted glycogen stores in hepatocytes of BDL livers. In conclusion, our studies showed selective delivery of dexamethasone to KC with Dexa(5)-Man(10)-HSA. This conjugate reduced intrahepatic ROS in vivo and TNF-alpha production in vitro and prevented glycogen depletion in vivo, indicating effective pharmacologic targeting. Dexa(5)-Man(10)-HSA, however, also accelerated fibrogenesis, which was paralleled by TIMP-1 mRNA induction. Targeting of dexamethasone to KC provides evidence for a dual role of this cell type in fibrogenesis of BDL rats.


Assuntos
Dexametasona/administração & dosagem , Hepatite/tratamento farmacológico , Células de Kupffer/efeitos dos fármacos , Cirrose Hepática Experimental/tratamento farmacológico , Animais , Colágeno/metabolismo , Dexametasona/metabolismo , Interleucina-10/biossíntese , Glicogênio Hepático/metabolismo , Masculino , Óxido Nítrico/biossíntese , Ratos , Ratos Wistar , Albumina Sérica/administração & dosagem , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Fator de Necrose Tumoral alfa/biossíntese
15.
Liver ; 21(5): 320-8, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11589768

RESUMO

BACKGROUND/AIMS: Drug targeting to hepatic stellate cells (HSC) may improve the pharmacological effects of antifibrotic drugs. Recently, albumin substituted with 28 mannose 6-phosphate moieties (M6P(28)-HSA) was found to distribute selectively to HSC in fibrotic rat livers. To assess whether this albumin can be used as a carrier for intracellular drug delivery, we explored the cellular handling of M6P(28)-HSA in HSC. METHODS/RESULTS: Application of competitive substrates for the M6P/IGFII receptor or other receptors showed that the binding of M6P-HSA to the M6P/IGFII receptor is specific. Binding was strong to activated HSC, but not to quiescent HSC. Furthermore, M6P(28)-HSA was extensively internalized by these cells. Using monensin, a specific inhibitor of the lysosomal pathway, proof was obtained that M6P-HSA is endocytosed via this route. The experiments performed with tissue slices, prepared from rat and human livers, revealed a specific binding and uptake of M6P(28)-HSA in both normal and cirrhotic livers. In livers from cirrhotic patients, HSC contributed predominantly to the uptake of this neoglycoprotein. CONCLUSIONS: Based on our in vivo data demonstrating the HSC-selectivity and on our in vitro data demonstrating binding and rapid internalization in activated HSC, we conclude that M6P(28)-HSA is applicable as a stellate cell-selective carrier for antifibrotic drugs that act intracellularly. This may have implications for the design of new strategies for the treatment of liver fibrosis.


Assuntos
Portadores de Fármacos/farmacocinética , Hepatócitos/metabolismo , Cirrose Hepática Experimental/metabolismo , Manosefosfatos/farmacocinética , Albumina Sérica/farmacocinética , Animais , Células Cultivadas , Técnicas de Cultura , Imuno-Histoquímica , Radioisótopos do Iodo , Cirrose Hepática Experimental/tratamento farmacológico , Masculino , Ratos , Ratos Wistar
16.
J Control Release ; 72(1-3): 157-64, 2001 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-11389994

RESUMO

UNLABELLED: Small therapeutic oligopeptides (two to 12 amino acids), designed for interaction with cytokine and growth factor receptors, unfortunately, are rapidly removed from the body. Efficient glomerular filtration and carrier-mediated membrane transport processes are involved in their clearance. By coupling of such peptides to macromolecules, elimination via these pathways is prevented and exposure to the particular receptors can be largely improved. Some of these constructs undergo receptor-mediated endocytoses and can be used as carriers to deliver associated drugs to various cell types in the body. It has been shown that, in the case of neo-glycoprotein carriers, down-regulation of the receptors aimed at can occur in the diseased state. We therefore designed a new type of polypeptide carrier, homing on receptors that are known to be highly upregulated in the pathological target tissue. For this purpose we designed ligand peptides (minimized proteins) representing the receptor-recognizing domains of PDGF and collagen type VI, aimed at receptors that are highly expressed, particularly on activated hepatic stellate cells (HSC). This myofibroblast-type of cell largely contributes to connective tissue expansion during liver fibrosis. Drug carriers for the stellate cell have not been reported before. METHODS: Cyclic octapeptide moieties (n10--12) with affinity for the two receptors were coupled to HSA (pPB-HSA and pCVI-HSA, respectively). Receptor binding experiments confirmed binding of these ligand peptides to their receptors in vitro. IN VITRO STUDIES: rat HSC were isolated and purified according to standard techniques. The cells were cultured for 2 days (quiescent phenotype) or for 10 days (activated phenotype). Cell cultures were incubated with the carriers and the binding (at 4 degrees C), uptake (at 37 degrees C), and degradation were determined with radioactive and immunohistochemical methods. The results were compared with data obtained with unmodified HSA. IN VIVO STUDIES: the organ distribution of pCVI-HSA and pPB-HSA was determined 10 min after i.v. injection of tracer doses in normal and fibrotic rats, 3 weeks after bile duct ligation. Hepatocellular distribution was scored after double-immunostaining of the liver sections with an antibody against the designated hepatic cell type in combination with anti-HSA IgG. IN VITRO STUDIES: All three carriers preferentially bound to the activated rather than to quiescent HSC. Binding to cells was inhibitable by an excess of unlabelled pCVI-HSA, endocytosis was inhibitable by 2 mM monensin suggestive of lysosomal routing of the proteins, whereas pPB-HSA, at least partly, remained at the cell surface. Degradation products of the carriers were detected extracellularly after incubation with fibrotic rat liver slices during 2-h experiments. IN VIVO STUDIES: 62+/-6% of the dose of pCVI-HSA accumulated in fibrotic livers at 10 min after injection, of which the major part was taken up in HSC. 48+/-9% of pPB-HSA accumulated in fibrotic rat livers and this carrier was also mainly taken up by HSC (5). Similar amounts of both constructs were taken up in normal rat livers, but predominantly in other cell types. The preferential homing to the stellate cells, only in the fibrotic liver is explained by the marked proliferation of this cell type as well as overexpression of the targeted receptors on these cells in the diseased state. CONCLUSIONS: The in vivo results support the in vitro studies showing accumulation of these modified albumins in HSC in fibrotic rat livers and, in particular, in the stellate cells. The results demonstrate the specificity of the stellate cell targeting and imply applicability of pCVI-HSA as carriers for drugs that act intracellularly. In addition, pPB-HSA may be used to deliver drugs that act extracellularly, such as receptor antagonists. This concept may create new opportunities for delivery of conventional drugs that are not effective enough in vivo and/or display serious extrahepatic side-effects. Minimized proteins attached to soluble or particle type of macromolecules represent a novel carrier modality of which selective body distribution is induced by the disease process to be targeted. They can be utilized as receptor antagonists and at the same time can deliver therapeutic agents to the desired site of action (dual targeting).


Assuntos
Albuminas/farmacocinética , Sistemas de Liberação de Medicamentos , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Oligopeptídeos/farmacocinética , Animais , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Células de Kupffer/efeitos dos fármacos , Ligantes , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/patologia , Ratos , Regulação para Cima/efeitos dos fármacos
17.
Drug Metab Dispos ; 29(6): 361-7, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11353741

RESUMO

We developed and tested a novel method for perfusing parts of human liver to study uptake and handling of drug-targeting preparations. These preparations, designed for the treatment of liver fibrosis in man, have been extensively studied in animals, but little is known about the uptake and handling by human livers. Human liver tissue was obtained from livers procured from multiorgan donors and from cirrhotic livers of patients. To assess tissue viability, perfusate glutamate-oxalacetate-transaminase (GOT), glutamate-pyruvate-transaminase (GPT), and lactate dehydrogenase (LDH) levels were determined. To assess tissue functionality, the uptake of taurocholic acid and phase I and II metabolism of lidocaine and 7-hydroxycoumarin were determined. Uptake of a drug-targeting preparation was studied with Dexa(10)-HSA, which is designed for targeting of dexamethasone to nonparenchymal cells in the liver. During a 90-min perfusion period, no elevation of either GOT, GPT, or LDH was found. Both healthy control livers and cirrhotic livers showed phase I and II drug metabolism and functional taurocholic acid uptake. Studies with Dexa(10)-HSA revealed that 60 min after administration, 40% of the dose had been taken up by control livers and only 5% by cirrhotic livers. In control livers, Kupffer and endothelial cells had taken up Dexa(10)-HSA, whereas in cirrhotic livers only Kupffer cells were responsible for the uptake. Viability parameters and liver function tests clearly showed the applicability of this method. In the perfusion set-up, we showed uptake of the drug-targeting preparation Dexa(10)-HSA by healthy and cirrhotic human liver tissue, although the distribution patterns differed. This demonstrates the need to study new concepts in (diseased) human tissue.


Assuntos
Dexametasona/farmacocinética , Fígado/metabolismo , Anestésicos Locais/farmacocinética , Colagogos e Coleréticos/farmacocinética , Dexametasona/química , Humanos , Lidocaína/metabolismo , Fígado/enzimologia , Testes de Função Hepática , Perfusão , Albumina Sérica/química , Ácido Taurocólico/farmacocinética , Umbeliferonas/farmacocinética
18.
Drug Metab Dispos ; 29(4 Pt 1): 361-7, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11259317

RESUMO

We developed and tested a novel method for perfusing parts of human liver to study uptake and handling of drug-targeting preparations. These preparations, designed for the treatment of liver fibrosis in man, have been extensively studied in animals, but little is known about the uptake and handling by human livers. Human liver tissue was obtained from livers procured from multiorgan donors and from cirrhotic livers of patients. To assess tissue viability, perfusate glutamate-oxalacetate-transaminase (GOT), glutamate-pyruvate-transaminase (GPT), and lactate dehydrogenase (LDH) levels were determined. To assess tissue functionality, the uptake of taurocholic acid and phase I and II metabolism of lidocaine and 7-hydroxycoumarin were determined. Uptake of a drug-targeting preparation was studied with Dexa10-HSA, which is designed for targeting of dexamethasone to nonparenchymal cells in the liver. During a 90-min perfusion period, no elevation of either GOT, GPT, or LDH was found. Both healthy control livers and cirrhotic livers showed phase I and II drug metabolism and functional taurocholic acid uptake. Studies with Dexa10-HSA revealed that 60 min after administration, 40% of the dose had been taken up by control livers and only 5% by cirrhotic livers. In control livers, Kupffer and endothelial cells had taken up Dexa10-HSA, whereas in cirrhotic livers only Kupffer cells were responsible for the uptake. Viability parameters and liver function tests clearly showed the applicability of this method. In the perfusion set-up, we showed uptake of the drug-targeting preparation Dexa10-HSA by healthy and cirrhotic human liver tissue, although the distribution patterns differed. This demonstrates the need to study new concepts in (diseased) human tissue.


Assuntos
Dexametasona/farmacocinética , Fígado/fisiologia , Anestésicos Locais/metabolismo , Colagogos e Coleréticos/farmacocinética , Dexametasona/química , Glucocorticoides/química , Glucocorticoides/farmacocinética , Humanos , Lidocaína/metabolismo , Fígado/metabolismo , Perfusão , Albumina Sérica , Ácido Taurocólico/farmacocinética , Umbeliferonas/metabolismo
20.
Spine (Phila Pa 1976) ; 25(17): 2176-9, 2000 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-10973399

RESUMO

STUDY DESIGN: Accurate determination of the three-dimensional coordinates of paraspinal muscles is presented. OBJECTIVES: To determine the precise position and the size of the paraspinal muscles. SUMMARY OF BACKGROUND DATA: The accurate measurements of the muscle moment arms are important in the evaluation of computer models for spinal movement. It has been reported that a change in the modelling of the erector spinae muscles can alter the compressive forces in the spinal column by 20% and can increase the offsetting shear forces. Classic studies used measurements from cross-sectional anatomic diagrams of human cadavers, scaled to the width and depth of the trunk of the subject, to calculate the moment that produces the joint torque. METHODS: Computed tomography scans of two male cadavers provided data on the bony elements. Three-dimensional coordinates of the origins and insertions of the muscle-fascicles of the paraspinal muscles were obtained with the use of 3-Space Isotrak equipment. The bony and muscle coordinates were combined in the ANSYS (version 5.4; Swanson Analysis Systems, Canonsberg, PA) program. RESULTS: Results of this combination and the three-dimensional coordinates of the paraspinal muscles as acquired by the 3-Space Isotrak are given. CONCLUSION: The position and the moment arms of paraspinal muscles were accurately determined. This is important for further evaluation of mathematical models.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Movimento/fisiologia , Músculo Esquelético/anatomia & histologia , Coluna Vertebral/anatomia & histologia , Tomografia Computadorizada por Raios X/métodos , Idoso , Fenômenos Biomecânicos , Cadáver , Humanos , Masculino , Modelos Biológicos , Músculo Esquelético/fisiologia , Coluna Vertebral/fisiologia
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