RANKL confers protection against cell death in precision-cut lung slices.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death globally and constitutes a major health problem. The disease is characterized by airflow obstructions due to chronic bronchitis and/or emphysema. Emerging evidence suggests that COPD is the result of impaired epithelial repair. Motivated by the need for more effective treatments, we studied whether receptor activator of nuclear factor κ-Β ligand (RANKL) contributed to epithelial repair, as this protein has been implicated in epithelial regeneration of breast and thymus. To do so, we used precision-cut lung slices prepared from mouse tissue-viable explants that can be cultured ex vivo for up to a few days while retaining features of lung tissue. Slices were cultured with 10, 100, or 500 ng/ml of mouse RANKL for 24 h. We first found RANKL activated nuclear factor κ-Β signaling, which is involved in cellular stress responses, without affecting the general viability of slices. Cell proliferation, however, was not altered by RANKL treatment. Interestingly, RANKL did reduce cell death, as revealed by TUNEL stainings and profiling of apoptosis-related proteins, indicating that it contributes to repair by conferring protection against cell death. This study improves our understanding of lung repair and could create new opportunities for developing COPD treatments.

Fibrocytes are increased in lung and peripheral blood of patients with idiopathic pulmonary fibrosis.

BACKGROUND: Fibrocytes are implicated in Idiopathic Pulmonary Fibrosis (IPF) pathogenesis and increased proportions in the circulation are associated with poor prognosis. Upon tissue injury, fibrocytes migrate to the affected organ. In IPF patients, circulating fibrocytes are increased especially during exacerbations, however fibrocytes in the lungs have not been examined. Therefore, we sought to evaluate if fibrocytes can be detected in IPF lungs and we compare percentages and phenotypic characteristics of lung fibrocytes with circulating fibrocytes in IPF. METHODS: First we optimized flow cytometric detection circulating fibrocytes using a unique combination of intra- and extra-cellular markers to establish a solid gating strategy. Next we analyzed lung fibrocytes in single cell suspensions of explanted IPF and control lungs and compared characteristics and numbers with circulating fibrocytes of IPF. RESULTS: Using a gating strategy for both circulating and lung fibrocytes, which excludes potentially contaminating cell populations (e.g. neutrophils and different leukocyte subsets), we show that patients with IPF have increased proportions of fibrocytes, not only in the circulation, but also in explanted end-stage IPF lungs. These lung fibrocytes have increased surface expression of HLA-DR, increased intracellular collagen-1 expression, and also altered forward and side scatter characteristics compared with their circulating counterparts. CONCLUSIONS: These findings demonstrate that lung fibrocytes in IPF patients can be quantified and characterized by flow cytometry. Lung fibrocytes have different characteristics than circulating fibrocytes and represent an intermediate cell population between circulating fibrocytes and lung fibroblast. Therefore, more insight in their phenotype might lead to specific therapeutic targeting in fibrotic lung diseases.

Extracellular ATP decreases trophoblast invasion, spiral artery remodeling and immune cells in the mesometrial triangle in pregnant rats.

INTRODUCTION: Preeclampsia is characterized by deficient trophoblast invasion and spiral artery remodeling, a process governed by inflammatory cells. High levels of the danger signal extracellular adenosine triphosphate (ATP) have been found in women with preeclampsia and infusion of ATP in pregnant rats induced preeclampsia-like symptoms such as albuminuria and placental ischemia. We hypothesized that ATP inhibits trophoblast invasion and spiral artery remodeling and affects macrophages and natural killer (NK) cells present in the rat mesometrial triangle. METHODS: Pregnant rats were infused with ATP or saline (control) on day 14 of pregnancy. Rats were sacrificed on day 15, 17 or 20 of pregnancy and placentas with mesometrial triangle were collected. Sections were stained for trophoblast cells, α-smooth muscle actin (spiral artery remodeling), NK cells and various macrophage populations. Expression of various cytokines in the mesometrial triangle was analyzed using real-time RT-PCR. RESULTS: ATP infusion decreased interstitial trophoblast invasion on day 17 and spiral artery remodeling on day 17 and 20, increased activated tartrate resistant acid phosphatase (TRAP)-positive macrophages on day 15, decreased NK cells on day 17 and 20, and decreased inducible nitric oxide synthase (iNOS)-positive and CD206-positive macrophages and TNF-α and IL-33 expression at the end of pregnancy (day 20). DISCUSSION: Interstitial trophoblast invasion and spiral artery remodeling in the rat mesometrial triangle were decreased by infusion of ATP. These ATP-induced modifications were preceded by an increase in activated TRAP-positive macrophages and coincided with NK cell numbers, suggesting that they are involved. CONCLUSION: Trophoblast invasion and spiral artery remodeling may be inhibited by ATP-induced activated macrophages and decreased NK cells in the mesometrial triangle in rat pregnancy.

Shifted T-cell polarisation after agricultural dust exposure in mice and men.

RATIONALE: A low prevalence of asthma and atopy has been shown in farmers and agricultural workers. However, in these workers, a higher prevalence of respiratory symptoms has been reported, in which T helper 1 (Th1) and/or Th17 responses may play a role. AIM: We investigated the effect of exposure to dust extracts (DEs) from different farms on airway inflammation and T-cell polarisation in a mouse model and assessed T-cell polarisation in agricultural workers from the same farms. METHODS: DEs were prepared from settled dust collected at cattle and pig farms and bulb and onion industries. Mice were exposed to phosphate-buffered saline (PBS), DEs, house dust mite (HDM) or HDM+DE via nasal instillation, four times per week during 5 weeks. Hyperresponsiveness, airway inflammation, IgE levels and T-cell polarisation were assessed. Th-cell and T cytotoxic (Tc)-cell subsets were investigated in peripheral blood samples from 33 agricultural workers and 9 non-exposed controls. RESULTS: DEs induced interleukin(IL)-17, IL-1ß and IL-6 in mouse lung homogenates. DE-exposed mice had more mixed inflammatory infiltrates in the lungs, and more neutrophils compared with PBS-exposed mice. DEs protected against the HDM-induced Th2 response and methacholine hyperresponsiveness. Interestingly, occupationally exposed humans had higher frequencies of Th cells spontaneously expressing IL-17 and interferon γ compared with controls. CONCLUSION: Chronic exposure to different types of farm dust induces a Th/Tc-17 inflammatory response in mice and agricultural workers. This may contribute to the low prevalence of Th2-related diseases but may constitute a risk for other chronic respiratory diseases.

Altered expression of immune-associated genes in first-trimester human decidua of pregnancies later complicated with hypertension or foetal growth restriction.

During pregnancy the maternal immune system has to coordinate uterine spiral-artery remodelling, trophoblast invasion, and acceptance of the semi-allogenic fetus simultaneously. As dysregulation of the immune system is associated with adverse pregnancy outcomes, we analysed first-trimester deciduas of pregnancies for immune parameters in later complicated pregnancies. Higher IL6 and macrophage mRNA expression, and lower ratios of regulatory macrophages were found in first-trimester deciduas of pregnancies later complicated with pregnancy-induced hypertension. Lower Gata3 (Th2) mRNA expression was found in deciduas of pregnancies with later foetal growth restriction. Our results suggest that adverse pregnancy outcomes are associated with immunological disturbances in first-trimester deciduas.

OS053. Trophoblast invasion in the mesometrial triangle of atp infused pregnant rats.

INTRODUCTION: Poor placentation (disturbed and decreased trophoblast invasion) is a hallmark of preeclampsia (PE), which is a major complication of pregnancy. Unfortunately, the cause and mechanism of disturbed trophoblast invasion is still unknown. OBJECTIVES: The pro-inflammatory agent ATP has been shown to induce PE-like signs, after a single infusion in pregnant rats. These PE-like characteristics include proteinuria and decreased fetal weight. Since purinergic ATP receptors are expressed on trophoblast cells, we aimed to study the effect of ATP infusion on trophoblast invasion in pregnant rats in this pilot study. METHODS: Pregnant rats received a single ATP (n=4) or saline (control,ni=5) infusion via a permanent jugular vein cannula on day 14 of pregnancy. At the time of maximal trophoblast invasion (day 17 of pregnancy) rats were sacrificed and placentas with mesometrial triangle were collected, fixed in zinc-buffer and embedded in paraffin. 4 µm sections were stained with monoclonal α-cytokeratin antibodies. In the mesometrial triangle, the maternal part of the rat placenta, the percentage of surface area of trophoblast invasion was evaluated using computerized image analysis. Also, the depth and width of invasion were analyzed by subdividing the mesometrial triangle in three concentric depth levels of equal width. In addition, trophoblast invaded versus non-invaded spiral arteries in the mesometrial triangle were quantified. RESULTS: In the mesometrial triangle, no changes in percentage of surface area of trophoblast invasion and percentage of invaded spiral arteries were observed after ATP infusion. However, the pattern of trophoblast invasion appeared to be disturbed in ATP infused rats, with a decreased depth of invasion and an increased width of invasion, resulting in a trend towards a decreased depth/width ratio of trophoblast invasion in ATP infused rats. CONCLUSION: In this (pilot) study we showed an altered trophoblast invasion pattern in the mesometrial triangle of the placenta, although no significant differences in the total surface area of trophoblast invasion were seen in experimental versus control pregnant animals. e mechanism by which ATP induces this altered trophoblast invasion pattern and its potential contribution to the pathophysiology of this experimental PE in the pregnant rat awaits further investigation.

Effects of acute cytomegalovirus infection on rat islet allograft survival.

Transplantation of pancreatic islets is a promising therapy for the treatment of type 1 diabetes mellitus. However, long-term islet graft survival rates are still unsatisfactory low. In this study we investigated the role of cytomegalovirus (CMV) in islet allograft failure. STZ-diabetic rats received an allogenic islet graft in combination with either an acute CMV infection or control infection. A third group received ganciclovir treatment in addition to the CMV infection. Graft function was assessed by measuring basal blood glucose levels. After sacrifice, the islet grafts were retrieved for analysis of infection and leukocyte infiltration. CMV-infected recipients demonstrated accelerated islet graft failure compared to noninfected controls. CMV infection of the graft was only observed prior to complete graft failure. Quantification of the leukocyte infiltration demonstrated increased CD8(+) T-cell and NK cell infiltration in the CMV-infected grafts compared to the controls. This suggests that CMV infection accelerates immune-mediated graft destruction. Antiviral ganciclovir treatment did not prevent accelerated graft failure, despite effectively decreasing the grade of infection. Our data confirm the recently published CITR data, which state that CMV is an independent risk factor for failure of islet grafts. Also, our data demonstrate that new approaches for preventing virus-induced islet allograft failure may be required.

Airway inflammation and remodeling in two mouse models of asthma: comparison of males and females.

BACKGROUND: Asthma and especially severe asthma affect women more frequently than men. Since asthma severity correlates with remodeling changes in the lung, a female propensity to remodeling could be expected. We studied whether our previous observation that female mice have more pronounced airway inflammation than males is associated with more pronounced remodeling in two models of chronic allergic asthma. METHODS: Male and female BALB/c mice were (1) sensitized and subsequently challenged with ovalbumin (OVA) for 4 weeks, or (2) exposed to house dust mite (HDM) for 5 weeks. In both models, allergic inflammation, remodeling, antigen-specific IgE and methacholine (MCh) responsiveness were assessed. RESULTS: Females had higher antigen-specific serum IgE levels, higher numbers of eosinophils and were more responsive to MCh. In the OVA model, females also had higher levels of Th2 cytokines in lung tissue than males. Both sexes developed similar airway remodeling (smooth muscle layer thickness, collagen III deposition and goblet cell hyperplasia) in the two models. CONCLUSIONS: Combining results of an OVA- and a HDM-induced mouse model of allergic airway inflammation, we have shown that more severe allergic inflammation in females is not accompanied with more pronounced airway remodeling.

Maternal smoking during pregnancy induces airway remodelling in mice offspring.

Children from smoking mothers have an increased risk of developing asthma for reasons largely unknown. The effects of maternal smoking during pregnancy on remodelling, allergic airway inflammation and hyperresponsiveness in offspring were investigated in an experimental asthma model. Mice were exposed to fresh air or cigarette smoke from 3 weeks prior to conception until birth. Offspring were exposed to house dust mite (HDM) or PBS intranasally four times per week from week 5 to week 10 after birth onwards. Maternal smoking increased airway smooth muscle layer, collagen III deposition and HDM-induced goblet cell numbers in offspring. It additionally increased methacholine responsiveness, which correlated significantly with increased airway smooth muscle layer and collagen deposition. Maternal smoking increased HDM-induced numbers of neutrophils and mast cells in lung tissue. No further effects were observed. Smoking during pregnancy induces airway remodelling in mice offspring, which may contribute to increased methacholine responsiveness. This takes place irrespective of allergen exposure but may worsen the outcome of the allergic stimulus, resulting in higher methacholine responsiveness in house dust mite-exposed offspring from smoking mothers when compared to nonsmoking mothers. The results provide a possible mechanism behind the association between maternal smoking and asthma.

Effects of 4 months of smoking in mice with ovalbumin-induced airway inflammation.

BACKGROUND: The effects of smoking on asthma pathogenesis are complex and not well studied. We have shown recently that 3 weeks of smoking attenuates ovalbumin (OVA)-induced airway inflammation in mice and that 4-6 months of smoking induces emphysema in mice without airway inflammation. Effects of combined long-term smoking and OVA exposure have not been investigated so far. OBJECTIVE: To study whether long-term smoking affects progression of allergic airway inflammation and/or enhances the development of emphysema in mice. METHODS: Mice were sensitized to OVA and challenged with saline or OVA aerosols for 6 months. From 2 months onwards, mice were also exposed to air or smoke. Lung tissue was analysed for extent of inflammation, emphysema, remodelling and for cytokine levels, and serum for OVA-specific IgE levels. RESULTS: Chronic OVA exposure of 6 months resulted in a T helper type 2 (Th2)-type inflammation with increased levels of IL-4, IL-5, IL-6 and infiltration of eosinophils, CD4(+) T cells, macrophages and plasma cells. Smoking induced a Th17-type of airway inflammation, characterized by neutrophils, macrophages, B cells and increased levels of IL-17, IL-6, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor and monocyte chemoattractant protein-1. Concomittant smoking and OVA exposure resulted in inflammation similar to OVA exposure alone. OVA exposure increased IgE levels compared with saline exposure, and smoking did not further increase these levels. CONCLUSION: We did not find evidence for increased inflammation, IgE levels or emphysema in mice with allergic airway inflammation after 4 months of smoking compared with non-smoking. However, a 4-month exposure to smoke alone did enhance neutrophilic airway inflammation characterized by high pulmonary IL-17 levels. A Th2 inflammatory environment due to OVA exposure may be one explanation as to why no further detrimental effects of smoking on allergic airway inflammation were found.

Female mice are more susceptible to the development of allergic airway inflammation than male mice.

BACKGROUND: In humans the prevalence of asthma is higher among females than among males after puberty. The reason for this phenomenon is not clear. OBJECTIVE: We tested the hypothesis that female mice are more susceptible to the development of allergic asthma than male mice and studied allergic immune responses in the lung. METHODS: We compared allergic airway inflammation, i.e. methacholine (MCh) responsiveness, serum IgE, and cytokines, and the number of the different leucocytes in lungs of male and female BALB/c mice, twice-sensitized to ovalbumin (OVA) and subsequently challenged with OVA (OVA-mice) or phosphate-buffered saline (PBS-mice) aerosols on days 24-26, 30, and 31. RESULTS: OVA challenge significantly increased MCh responsiveness, numbers of eosinophils, CD4(+) T cells, CD4(+)/CD25(+) T cells, B cells, and levels of Thelper (Th)2 cytokines, total, and OVA-specific IgE. There was, however, also an effect of gender, with female mice responding to OVA challenges with higher numbers of eosinophils, CD4(+) T cells, B cells, and levels of IL-4, IL-13, IFN-gamma, total, and OVA-specific IgE than male mice. In contrast, female PBS-mice had significantly lower percentages of regulatory CD4(+)/CD25(+) T cells than males (females 4.2+/-0.2% vs. males 5.3+/-0.1% of CD4(+) T cells, P<0.05). CONCLUSION: Female mice develop a more pronounced type of allergic airway inflammation than male mice after OVA challenge. The reduced percentage of regulatory T cells in the lungs of female PBS-mice may indicate that the level of these cells in the lung during the sensitization phase is important for the development and/or progression of an allergic immune response after multiple OVA challenges.

Targeting dexamethasone to Kupffer cells: effects on liver inflammation and fibrosis in rats.

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.

Rat liver slices as a tool to study LPS-induced inflammatory response in the liver.

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.

Cellular distribution and handling of liver-targeting preparations in human livers studied by a liver lobe perfusion.

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.

Cellular distribution and handling of liver-targeting preparations in human livers studied by a liver lobe perfusion.

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.

Dexamethasone coupled to albumin is selectively taken up by rat nonparenchymal liver cells and attenuates LPS-induced activation of hepatic cells.

BACKGROUND/AIMS: The human serum albumin (HSA) conjugate Dexa10-HSA was designed to specifically deliver the anti-inflammatory drug dexamethasone (Dexa) to nonparenchymal cells (NPC) in the rat liver. NPC play an important role in the pathogenesis of acute and chronic inflammatory liver diseases like fibrosis. Targeting Dexa to these cells might reduce its adverse effects and increase the efficacy. METHODS: Tissue and intrahepatic distributions of Dexa10-HSA were assessed in normal and fibrotic rats with 125I-labelled conjugate and by immunohistochemistry. The effect of the conjugate on lipopolysaccharide (LPS)-induced inflammation and cell activation was studied in vitro with precision-cut liver slices and in vivo. RESULTS: Ten minutes after i.v. injection 72+/-13% and 65+/-12% of a tracer dose of Dexa10-HSA had been taken up in normal and fibrotic livers, respectively. Unconjugated Dexa also accumulated in livers, but cellular distribution studies revealed an accumulation in parenchymal cells (NPC vs. parenchymal cell (PC) ratio=0.29+/-11, p<0.005) whereas Dexa10-HSA accumulated in nonparenchymal cells (NPC/PC ratio=7.9+/-3.1). Both coupled and uncoupled Dexa showed effective inhibition of LPS-induced NOx and TNFalpha production in precision-cut liver slices. At low concentrations (0.02 microM), however, Dexa10-HSA was more efficient at inhibiting TNFalpha production than uncoupled Dexa. In fibrotic rats Dexa10-HSA (3 mg/kg) and an equimolar amount of uncoupled Dexa (0.22 mg/kg) both significantly promoted survival after LPS-induced acute inflammation. CONCLUSION: Dexa10-HSA was at least as effective as uncoupled Dexa at inhibiting LPS-induced effects, which indicates that HSA-bound Dexa is pharmacologically active. Coupling Dexa to HSA shifted the accumulation of Dexa from the PC to the NPC of livers. Since mediator release from NPC is crucial in the initiation and propagation of the fibrotic process, selective delivery of Dexa in NPC may improve the efficacy and safety of corticosteroid treatment of liver fibrosis.

Targeting of naproxen covalently linked to HSA to sinusoidal cell types of the liver.

The kinetic behaviour of a naproxen human serum albumin conjugate (Nap23-HSA) was investigated in rats and in isolated perfused rat livers (IPRL), as compared to its active metabolite naproxen-lysine (Nap-lysine) and free naproxen. Through covalently linking the anti-inflammatory drug naproxen to HSA, this drug can be selectively delivered to non parenchymal cells of the liver. Liver endothelial and Kupffer cells play an important role in the pathogenesis of inflammatory liver diseases. Targeting naproxen to these cells might increase its efficacy and reduce the side effects. The altered kinetic properties of Nap23-HSA, after i.v. injection of 22 mg x kg(-1), as compared to an equimolar amount of the uncoupled drug, were demonstrated in vivo by a decrease in the steady state volume of distribution (41 +/- 5 vs. 134 +/- 19 ml x kg(-1)), a decrease in its clearance (0.48 +/- 0.05 vs. 0.63 +/- 0.1 ml x min(-1) x kg(-1)), a shorter plasma half life (60 +/- 11 vs. 152 +/- 44 min) and a sustained biliary excretion. Liver targeting of Nap23-HSA was clearly demonstrated: drug content of the liver 180 min after injection was about 30 times higher for Nap23-HSA as compared to naproxen itself. The IPRL experiments showed that the Vmax of hepatic removal of the conjugate was 40 microg x min(-1) x g liver(-1). With doses below receptor saturation a rapid removal of the conjugate (t1/2 = 6 min) from the perfusion medium was found. In conclusion, this study demonstrates the saturable uptake of Nap23-HSA and its lysosomal degradation in both in vivo and IPRL experiments. Covalently linked naproxen is released as Nap-lysine. This active metabolite accumulates in Kupffer and endothelial cells in which it reaches therapeutic concentrations. Release from these cells leads to rapid uptake by hepatocytes and carrier mediated excretion into bile. Levels of Nap-lysine in bile and plasma reflect the slowest step in its generation: the proteolytic release in endothelial and Kupffer cells.

Effect of chronic bile duct obstruction and LPS upon targeting of naproxen to the liver using naproxen-albumin conjugate.

Naproxen covalently linked to human serum albumin (NAP-HSA) is efficiently targeted to endothelial and Kupffer cells of the liver and may offer a new therapeutic approach in the treatment of liver disease associated with inflammatory processes. In the present investigation we explored the pharmacokinetic behaviour of targeted and non-targeted naproxen as well as the pharmacokinetic properties of the active metabolite, Naproxen lysine (Nap lysine), in rats rendered fibrotic by bile duct ligation (BDL) for 4 weeks. Furthermore, we studied the effect of endotoxemia, experimentally induced by intravenous injection of 800 microg/kg lipopolysaccaride (LPS) upon the pharmacokinetics of these agents in order to investigate the feasibility of targeting naproxen to non-parenchymal cells in the inflamed and fibrotic liver. Our studies demonstrate that liver disease altered the pharmacokinetic behaviour of the different naproxen compounds. Thus, initial plasma concentrations of NAP HSA and naproxen were markedly lower in BDL rats accompanied by an increase of the volume of distribution during the terminal elimination phase (Vd(beta) BDL vs control 114 +/- 63 vs 50 +/- 7 and 202 +/- 24 vs 115 +/- 11 ml/kg for naproxen and NAP-HSA, respectively). After injection of LPS, no significant change in the pharmacokinetics of NAP-HSA was found whereas the naproxen treated control animals showed an increase in the terminal volume of distribution (176 +/- 34 vs 115 +/- 11 ml/kg) as well as an elevation of the plasma half-life (171 +/- 27 vs 116 +/- 14 min). The feasibility of targeting naproxen to the chronically diseased liver could be clearly demonstrated: 15 min after administration of the conjugate 46% and 55% of the administered dose was found in the liver of CTR and BDL rats, whereas after injection of free naproxen only 5% and 12% of the dose was detected in liver tissue, respectively. We conclude that targeting albumin-linked naproxen to non-parenchymal cells in the liver is still feasible under the pathological conditions induced in the present study. Liver fibrosis induced significant alterations in the pharmacokinetic behaviour of the studied compounds.