ABSTRACT
In this study, we compared the regulation of CYP3A isozymes by the vitamin D receptor (VDR) ligand 1 alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) against ligands of the pregnane X receptor (PXR), the glucocorticoid receptor (GR) and the farnesoid X receptor (FXR) in precision-cut tissue slices of the rat jejunum, ileum, colon and liver, and human ileum and liver. In the rat, 1,25(OH)(2)D(3) strongly induced CYP3A1 mRNA, quantified by qRT-PCR, along the entire length of the intestine, induced CYP3A2 only in ileum but had no effect on CYP3A9. In contrast, the PXR/GR ligand, dexamethasone (DEX), the PXR ligand, pregnenolone-16 alpha carbonitrile (PCN), and the FXR ligand, chenodeoxycholic acid (CDCA), but not the GR ligand, budesonide (BUD), induced CYP3A1 only in the ileum, none of them influenced CYP3A2 expression, and PCN, DEX and BUD but not CDCA induced CYP3A9 in jejunum, ileum and colon. In rat liver, CYP3A1, CYP3A2 and CYP3A9 mRNA expression was unaffected by 1,25(OH)(2)D(3), whereas CDCA decreased the mRNA of all CYP3A isozymes; PCN induced CYP3A1 and CYP3A9, BUD induced CYP3A9, and DEX induced all three CYP3A isozymes. In human ileum and liver, 1,25(OH)(2)D(3) and DEX induced CYP3A4 expression, whereas CDCA induced CYP3A4 expression in liver only. In conclusion, the regulation of rat CYP3A isozymes by VDR, PXR, FXR and GR ligands differed for different segments of the rat and human intestine and liver, and the changes did not parallel expression levels of the nuclear receptors.
Subject(s)
Cytochrome P-450 CYP3A/metabolism , Intestines/enzymology , Liver/enzymology , Receptors, Calcitriol/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Glucocorticoid/metabolism , Receptors, Steroid/metabolism , Animals , Base Sequence , Cytochrome P-450 CYP3A/genetics , DNA Primers , Humans , In Vitro Techniques , Isoenzymes/genetics , Isoenzymes/metabolism , Ligands , Male , Polymerase Chain Reaction , Pregnane X Receptor , Rats , Rats, WistarABSTRACT
In fibrotic livers, collagen producing hepatic stellate cells (HSC) represent a major target for antifibrotic therapies. We designed liposomes with surface-coupled mannose 6-phosphate (M6P) modified human serum albumin (HSA) to target HSC via the M6P receptor. In this study we determined the pharmacokinetics and target specificity of M6P-HSA-liposomes in a rat model of liver fibrosis. Ten minutes after injection of [(3)H]-M6P-HSA-liposomes 90% of the dose has cleared the circulation. The blood elimination of these liposomes was counteracted by free M6P-HSA and polyinosinic acid, a competitive inhibitor of scavenger receptors. The M6P-HSA-liposomes accumulated in HSC. However, also Kupffer cells and endothelial cells contributed to the uptake of M6P-HSA-liposomes in the fibrotic livers. Polyinosinic acid inhibited the accumulation of the liposomes in Kupffer cells and liver endothelial cells, but not in HSC. PCR analysis revealed that cultured HSC express scavenger receptors. This was confirmed by Western blotting, although activation of HSC diminishes scavenger receptor protein expression. In conclusion, in a rat model for liver fibrosis M6P-HSA-liposomes can be efficiently targeted to non-parenchymal cells, including HSC. M6P receptors and scavenger receptors are involved in the cellular recognition of these liposomes, allowing multiple pharmacological interference in different pathways involved in the fibrosis.
Subject(s)
Drug Delivery Systems/methods , Liposomes/pharmacokinetics , Liposomes/therapeutic use , Liver Cirrhosis/drug therapy , Animals , Blotting, Western , DNA Primers , Immunohistochemistry , Kupffer Cells/metabolism , Liposomes/metabolism , Mannosephosphates/metabolism , Poly I/metabolism , Polymerase Chain Reaction , Rats , Receptors, Scavenger/metabolism , Serum Albumin/metabolism , TritiumABSTRACT
In the fibrotic liver, hepatic stellate cells (HSC) produce large amounts of collagen and secrete variety of mediators that promote development of fibrosis in this organ. Therefore, these cells are considered an attractive target for antifibrotic therapies. We incorporated the bioactive lipid dilinoleoylphosphatidylcholine (DLPC) into the membrane of liposomes, and then we evaluated its effect on hepatic stellate cell activation and liver fibrosis. To target DLPC-liposomes to HSC, human serum albumin modified with mannose 6-phosphate (M6P-HSA) was coupled to the surface of these liposomes. In vitro, the effects of the carrier were determined in primary cultures of HSC, Kupffer cells, and liver endothelial cells using real-time reverse transcription-polymerase chain reaction. In vivo DLPC-liposomes were tested in bile duct-ligated rats. Targeted M6P-HSA-DLPC-liposomes and DLPC-liposomes significantly reduced gene expression levels for collagen 1alpha1, alpha-smooth muscle actin (alpha-SMA), and transforming growth factor-beta (TGF-beta) in cultured HSC. In fibrotic livers, DLPC-liposomes decreased gene expression for TGF-beta and collagen 1alpha1 as well as alpha-SMA and collagen protein expression. In contrast, M6P-HSA-DLPC-liposomes enhanced expression of profibrotic and proinflammatory genes in vivo. In cultured Kupffer and endothelial cells M6P-HSA liposomes influenced the expression of proinflammatory genes. Both types of liposomes increased hepatocyte glycogen content in fibrotic livers, indicating improved functionality of the hepatocytes. We conclude that DLPC-containing liposomes attenuate activation of cultured HSC. In fibrotic livers, M6P-HSA-mediated activation of Kupffer and endothelial cells probably counteracts this beneficial effect of DLPC-liposomes. Therefore, these bioactive drug carriers modulate the activity of all liver cells during liver fibrosis.
Subject(s)
Liver Cirrhosis, Experimental/drug therapy , Phosphatidylcholines/administration & dosage , Animals , Cells, Cultured , Chemokine CCL2/analysis , Cholestasis/complications , Gene Expression/drug effects , Interleukin-6/genetics , Ligation , Liposomes , Liver/cytology , Liver/drug effects , Liver/metabolism , Liver Cirrhosis, Experimental/metabolism , Male , Rats , Rats, Wistar , Tumor Necrosis Factor-alpha/geneticsABSTRACT
BACKGROUND/AIMS: Inhibition of hepatic stellate cell (HSC) proliferation is a relevant strategy to inhibit liver fibrosis. Coupling of antiproliferative drugs to the HSC-selective drug carrier mannose-6-phosphate-modified human serum albumin (M6PHSA) may lead to cell-selective inhibition of HSC proliferation. We coupled the antiproliferative drug doxorubicin (DOX) to this drug carrier and investigated the pharmacokinetics of this construct in a rat model of liver fibrosis, as well as in cultured HSC. METHODS/RESULTS: M6PHSA-DOX was cleared from the plasma in a biphasic manner. Upon i.v. injection of 4 microg kg(-1) (tracer), 2 and 20 mg kg(-1), the clearance in the distribution phase of drug disposition (CL(d)) significantly decreased from 9.7+/-0.7 to 4.7+/-2.3 and 1.0+/-0.1 ml kg(-1)min(-1), respectively. This indicates that saturation of clearance mechanisms occurs in this phase of drug disposition, likely reflecting saturable receptor-mediated uptake in the target cells. Gamma-camera studies revealed that the majority of the conjugate accumulated in the liver within 5 min, and immunohistochemical double-staining of liver sections demonstrated co-localization of the construct with HSC-markers. Simulation of the release of DOX from the carrier, after cellular uptake by HSC, showed that a gradual release of the drug takes place over a 9h period. Studies in cultured HSC illustrated that after 24h incubation with the conjugate, DOX was associated with the cell nucleus. CONCLUSIONS: The rapid distribution of M6PHSA-DOX from the blood to HSC, in combination with the expected gradual release of DOX within these cells, make this construct a promising tool for achieving sustained and selective inhibition of HSC proliferation.
Subject(s)
Bile Ducts/metabolism , Doxorubicin/pharmacokinetics , Hepatocytes/metabolism , Ligation , Animals , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacokinetics , Bile Ducts/physiopathology , Disease Models, Animal , Doxorubicin/metabolism , Drug Carriers/metabolism , Drug Delivery Systems , Hepatocytes/cytology , Liver/cytology , Male , Rats , Rats, Wistar , Tissue DistributionABSTRACT
Gut-derived lipopolysaccharide (LPS) plays a role in the pathogenesis of liver diseases like fibrosis. The enzyme alkaline phosphatase (AP) is present in, among others, the intestinal wall and liver and has been previously shown to dephosphorylate LPS. Therefore, we investigated the effect of LPS on hepatic AP expression and the effect of AP on LPS-induced hepatocyte responses. LPS-dephosphorylating activity was expressed at the hepatocyte canalicular membrane in normal and fibrotic animals. In addition to this, fibrotic animals also displayed high LPS-dephosphorylating activity around bile ducts. The enzyme was shown to dephosphorylate LPS from several bacterial species. LPS itself rapidly enhanced the intrahepatic mRNA levels for this enzyme within 2 h by a factor of seven. Furthermore, in vitro and in vivo studies showed that exogenous intestinal AP quickly bound to the asialoglycoprotein receptor on hepatocytes. This intestinal isoform significantly attenuated LPS-induced hepatic tumor necrosis factor-alpha and nitric oxide (nitrite and nitrate) responses in vitro. The enzyme also reduced LPS-induced hepatic glycogenolysis in vivo. This study shows that LPS enhances AP expression in hepatocytes and that intestinal AP is rapidly taken up by these same cells, leading to an attenuation of LPS-induced responses in vivo. Gut-derived LPS-dephosphorylating activity or enzyme upregulation within hepatocytes by LPS may therefore be a protective mechanism within the liver.
Subject(s)
Alkaline Phosphatase/metabolism , Lipopolysaccharides/metabolism , Liver/metabolism , Animals , Collagen Type III/metabolism , Enterocytes/enzymology , Gluconeogenesis/drug effects , Hepatocytes/enzymology , Hepatocytes/metabolism , Hepatocytes/ultrastructure , Immunohistochemistry , In Vitro Techniques , Kidney/enzymology , Kidney/metabolism , Kidney/ultrastructure , Lipopolysaccharide Receptors/metabolism , Liver/enzymology , Liver/ultrastructure , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Male , Nitric Oxide/metabolism , Phosphorylation , RNA/biosynthesis , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Tumor Necrosis Factor-alpha/metabolismABSTRACT
BACKGROUND/AIMS: Hepatic stellate cell (HSC) activation and proliferation are key events in the pathology of liver fibrosis. Inhibiting these parameters, therefore, is a relevant option to treat liver fibrosis pharmacologically. The immunosuppressive drug mycophenolic acid (MPA) has been shown to inhibit proliferation and activation of various types of fibroblasts. In an effort to circumvent the immunosuppression and at the same time enhance this antifibrotic effect, we coupled MPA to the HSC-selective drug carrier mannose-6-phosphate modified human serum albumin and evaluated this conjugate for its specificity and antifibrotic activity. METHODS/RESULTS: We found that MPA inhibited proliferation of HSC in vitro. The drug coupled to the drug carrier bound specifically to HSC and reduced HSC proliferation in vitro. In vivo studies demonstrated that our conjugate accumulated selectively in the liver with significant uptake in HSC apart from Kupffer and endothelial cells, whereas primary and secondary lymphoid tissues were avoided. Treatment of bile duct-ligated rats with this conjugate reduced hepatic inflammation and hepatic alpha-beta-Crystallin mRNA expression, a marker for HSC activation. CONCLUSIONS: This study shows that targeted delivery of MPA to HSC results in a decrease in HSC activation, making it the first drug that is successfully delivered to this cell type.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Cell Proliferation/drug effects , Hepatocytes/drug effects , Liver/pathology , Mycophenolic Acid/pharmacology , Animals , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/metabolism , Antibiotics, Antineoplastic/therapeutic use , Collagen/metabolism , Crystallins/genetics , Crystallins/metabolism , Desmin/metabolism , Drug Carriers , Fibrosis/drug therapy , Fibrosis/pathology , Glial Fibrillary Acidic Protein/metabolism , Hepatocytes/cytology , Humans , Liver/drug effects , Liver/metabolism , Male , Mannosephosphates/chemistry , Mannosephosphates/metabolism , Molecular Structure , Mycophenolic Acid/chemistry , Mycophenolic Acid/metabolism , Mycophenolic Acid/therapeutic use , Protein Isoforms/genetics , Protein Isoforms/metabolism , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , Tissue DistributionABSTRACT
PURPOSE: Because interleukin-10 (IL-10) seems a promising new antifibrotic drug, we investigated the pharmacokinetic and biodistribution profile of this potent therapeutic cytokine in rats with extensive liver fibrosis (BDL-3). IL-10 receptor expression was also determined in relation to these aspects. METHODS: To study the pharmacokinetic and biodistribution of IL-10, rhIL-10 was labeled with 125-iodine. Plasma samples of 125IrhIL-10 were obtained over a 30-min time period after administration of radiolabeled-cytokine to BDL-3 and normal rats. The tissue distribution was assessed 10 and 30 min after i.v. administration of 125IrhlL-10. IL-10 receptor expression was determined by immurohistochemical staining and RT-PCR technique. RESULTS: . The 125IrhIL-10 plasma curves followed two-compartment kinetics with a lower AUC in BDL-3 rats as compared to control. Plasma clearance and distribution volume at steady state were larger in BDL-3 rats. Tissue distribution analysis in normal rats showed that 125IrhIL-10 highly accumulated in kidneys. In BDL-3 rats, the liver content of 125IrhIL-10 increased by a factor of 2, whereas kidney accumulation did not significantly change. Immunohistochemical staining and RT-PCR analysis showed that IL-10 receptor was clearly upregulated in BDL-3 rat livers. CONCLUSIONS: . In normal rats, 125IrhIL-10 rapidly disappears from the circulation, and the kidney is predominantly responsible for this. In BDL-3 rats, the liver largely contributes to this rapid plasma disappearance, probably due to an increase in IL-10 receptor expression. The extensive renal clearance of IL-10 in vivo may limit a clinical application of this cytokine for the treatment of chronic liver diseases. To optimize the therapeutic effects of IL-10 in hepatic diseases, alternative approaches that either decrease renal disposition or that further enhance hepatic delivery should be considered.
Subject(s)
Interleukin-10/pharmacokinetics , Liver Cirrhosis/metabolism , Animals , Area Under Curve , Dose-Response Relationship, Drug , Humans , Injections, Intravenous , Interleukin-10/metabolism , Iodine Radioisotopes , Isotope Labeling , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Receptors, Interleukin/biosynthesis , Receptors, Interleukin/metabolism , Recombinant Proteins/pharmacokinetics , Reverse Transcriptase Polymerase Chain Reaction , Tissue DistributionABSTRACT
Lactoferrin is an antimicrobial agent, that, amongst other viruses, inhibits cytomegalovirus (CMV). In this study, we addressed the mechanism(s) by which lactoferrin interacts with CMV and its target cells to inhibit infection. We also studied the antiviral activity of lactoferrin in vivo in rat CMV models with and without immune suppression. We cationized a protein of similar molecular weight, i.e. human serum albumin (HSA), as well as a protein with a smaller molecular weight (beta-lactoglobulin). While HSA itself displayed no anti-CMV activity in vitro, cationic HSA inhibited CMV replication to a similar extent as lactoferrin. Time-of-addition assays indicated that all cationic proteins interacted with an early event in the infection and pre-incubation of cells rather than of virus significantly reduced CMV replication. Rats were treated with lactoferrin (4, 40 or 160 mg/kg, intravenously), beginning at 6h after CMV administration. Subsequently, the rats were treated three times a week. As a positive control, CMV-infected rats were treated with cidofovir, and this agent proved to be highly active in the rat models for CMV. Treatment with lactoferrin was beneficial when infection was initiated with cell-free virus, but not with virus-infected leukocytes. Lactoferrin treatment led to a 10-fold reduction in the final virus titers (salivary glands) at 4 weeks after infection in the immunocompromised rats. Lactoferrin exerted its effects via inhibition of cell entry rather than via stimulation of the immune system.
Subject(s)
Antiviral Agents/therapeutic use , Cytomegalovirus Infections/drug therapy , Cytomegalovirus/drug effects , Lactoferrin/therapeutic use , Spleen/physiopathology , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/blood , Cell Line , Lactoferrin/administration & dosage , Lactoferrin/blood , Rats , Spleen/drug effectsABSTRACT
1 In previous studies on the renal targeting of the ACE inhibitor captopril, we demonstrated that a 6 fold increased concentration of this drug could be obtained in the kidney after conjugation to the low-molecular-weight protein lysozyme. In this study, we investigated in unrestrained rats whether systemic administration of captopril-lysozyme also results in an enhanced effect on renal parameters, relative to the systemic effects. 2 Renal effects: intravenous infusion of captopril-lysozyme for 6 h resulted in a more pronounced increment of renal blood flow (31+/-2% vs 17+/-4% at 0.5 mg kg(-1) 6h(-1), P<0.01) and an approximately 5 fold enhanced natriuresis (167+/-17% vs 36+/-7% at 1 mg kg(-1) 6 h(-1), P<0.001) in comparison with equimolar amounts of captopril as a free drug. In correspondence with these findings, renal ACE inhibition was potentiated approximately 5 fold (-50+/-4% vs -22+/-3% at 1 mg kg(-1) 6 h(-1), P<0.001). 3 Systemic effects: conjugated captopril did not affect blood pressure in dosages up to 5 mg kg(-1) 6 h(-1). This effect coincided with a less pronounced inhibition of the pressor response to intravenously administered angiotensin I (-12+/-3% vs -66+/-5% at 1 mg kg(-1) 6 h(-1), P<0.001), and a markedly attenuated plasma ACE inhibition (-19+/-2% vs -37+/-3% at 1 mg kg(-1) 6 h(-1), P<0.001) compared to an equivalent dose of free captopril. 4 An experiment of continued intravenous administration of captopril-lysozyme for 7 days in nephrotic syndrome demonstrated that the conjugate is also active in renal disease: the antiproteinuric response was substantially augmented (-67+/-5% vs -15+/-7% at 4 mg kg(-1) 24 h(-1), P<0.001) compared to the free drug, in the absence of blood pressure reduction. 5 These data demonstrate that intravenous administration of a captopril-lysozyme conjugate leads to more selective renal ACE inhibition and enhanced renal effects as well as less systemic effects compared to captopril itself.