PubMed Health⌕ Search

Biomedical subjects

J Reichen

Publications and source records attributed to J Reichen.

At least 37 records · Page 2Linked to original sources
PubMed2000

Cloning of the rat ADAMTS-1 gene and its down regulation in endothelial cells in cirrhotic rats.

AIMS: This study was undertaken in order to identify genes which are regulated during the process of liver fibrosis. METHODS: The differential display method and RNA from rat endothelial cells before and after induction of cirrhosis were used. RESULTS: A 496 bp fragment, which was down regulated in liver endothelial cells from a cirrhotic animal, was cloned. The cloned fragment showed a 95% homology with the newly cloned mouse ADAMTS-1 gene (a disintegrin and metalloproteinase with thrombospondin motifs), which is implicated in inflammation. The fragment was found to span the 3' of exon 6, the whole exon 7 and the 5' of exon 8. Sequencing of the entire coding region of the rat gene showed a 94% homology at the nucleic acid level and 96% homology at the amino acid level. The sequences responsible for the function of the protein were conserved. Northern blot analysis, using the cloned fragment as a probe, confirmed the finding that the gene was down-regulated in endothelial cells derived from livers of cirrhotic animals. In situ PCR analysis localised the ADAMTS-1 gene in the liver endothelial cells from normal animals. CONCLUSIONS: Regulation of the expression of genes which belong to the metalloproteinase family in liver endothelial cells might be important in the development of liver cirrhosis.

ADAM Proteins↗
PubMed1999

Molecular mimicry of human cytochrome P450 by hepatitis C virus at the level of cytotoxic T cell recognition.

Hepatitis C virus (HCV) is thought to be involved in the pathogenesis of autoimmune hepatitis (AIH) type 2, which is defined by the presence of type I antiliver kidney microsome autoantibodies directed mainly against cytochrome P450 (CYP)2D6 and by autoreactive liver infiltrating T cells. Virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) that recognize infected cells and contribute to viral clearance and tissue injury during HCV infection could be involved in the induction of AIH. To explore whether the antiviral cellular immunity may turn against self-antigens, we characterized the primary CTL response against an HLA-A*0201-restricted HCV-derived epitope, i.e., HCV core 178-187, which shows sequence homology with human CYP2A6 and CYP2A7 8-17. To determine the relevance of these homologies for the pathogenesis of HCV-associated AIH, we used synthetic peptides to induce primary CTL responses in peripheral blood mononuclear cells of healthy blood donors and patients with chronic HCV infection. We found that the naive CTL repertoire of both groups contains cross-reactive CTLs inducible by the HCV peptide recognizing both CYP2A6 and CYP2A7 peptides as well as endogenously processed CYP2A6 protein. Importantly, we failed to induce CTLs with the CYP-derived peptides that showed a lower capacity to form stable complexes with the HLA-A2 molecule. These findings demonstrate the potential of HCV to induce autoreactive CD8(+) CTLs by molecular mimicry, possibly contributing to virus-associated autoimmunity.

Amino Acid Sequence↗
PubMed1999

Inhibition of 11beta-hydroxysteroid dehydrogenase by bile acids in rats with cirrhosis.

Renal sodium retention and potassium loss occur early, in many instances in the preascitic state of cirrhosis, an observation that cannot be fully explained by increased aldosterone concentrations. We therefore hypothesize that 11beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2), which protects mineralocorticoid receptors (MR) from glucocorticosteroids, is down-regulated in cirrhosis. Cirrhosis was induced by bile duct ligation in rats. The urinary ratio of (tetrahydrocorticosterone + 5alpha-tetrahydrocorticosterone)/ 11-dehydro-tetrahydrocorticosterone [(THB+5alpha-THB)/THA] was measured by gas chromatography. Cortical collecting tubules (CCT) were isolated by microdissection and used for measurements of the activity of 11beta-HSD2 by assessing the conversion of corticosterone to dehydrocorticosterone. The mRNA content of 11beta-HSD2 was determined by reverse-transcription polymerase chain reaction (RT-PCR) in CCTs. The urinary ratio of (THB+5alpha-THB)/THA increased concomitantly with the urinary excretion of bile acids following bile duct ligation. Chenodeoxycholic acid (CDCA) dose-dependently inhibited 11beta-HSD2 in CCT with a Ki of 19.9 micromol/L. Four weeks after bile duct ligation, 11beta-HSD2 activity was decreased in CCT, an observation preceded by a reduced mRNA content at weeks 2 and 3. In cirrhosis, the MR-protecting effect by 11beta-HSD2 is diminished, and therefore, endogenous glucocorticoids can induce MR-mediated sodium retention and potassium loss.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1999

Early acute cellular rejection: no effect on late hepatic allograft function in man.

Whereas early acute cellular rejection, even if successfully treated, seems to have an impact on late function and survival of kidney and heart transplants, little quantitative data are available on its effect(s) on liver transplants. Routine liver function tests, the functioning liver cell mass (galactose elimination capacity) and microsomal metabolic capacity (aminopyrine breath test) were determined prospectively in 37 consecutive patients 1 year after liver transplantation. Of these, 19 (7 females and 12 males, 32-69 years of age) had previously required treatment for at least one biopsy proven acute cellular rejection episode occuring a median 7 days after grafting, while 18 (6 females and 12 males, 30-67 years of age) had not. The functioning liver cell mass and microsomal metabolic capacity were both within normal limits for the majority of patients and did not differ significantly between patients with and without previous acute cellular rejection episodes. In contrast to other solid organ transplants, early acute cellular rejection episodes do not affect late function of liver allografts in man.

Acute Disease↗
PubMed1999

Primary liver disease as a determinant for acute rejection after liver transplantation.

BACKGROUND: Graft rejection and infection remain major problems following liver transplantation; both are heavily influenced by the immunosuppressive regimen. Despite the disparity in the primary disease leading to transplantation, all patients receive the same posttransplant immunosuppressive treatment in a given center. The aim of this study is to detect a possible effect of the underlying disease on the incidence of early acute rejection episodes after orthotopic liver transplantation (OLT). PATIENTS AND METHODS: Retrospective analysis on all 101 consecutive liver transplants performed in 95 patients between 1983 and March 1998; five of these patients, surviving less than 30 days, were not included. The immunosuppressive regimen was based on conventional triple therapy during the whole study period. The diagnosis and treatment of acute rejection within the first 30 days post-OLT was uniform throughout the whole study period. RESULTS: Though there were no differences with respect to patients' characteristics [age, child classification, number of HLA-mismatches or cytomegalovirus (CMV)-serocompatibility], patients with primary biliary cirrhosis (PBC) showed a significant increase of acute rejection after OLT compared with the other patients transplanted for other liver diseases (P = 0.024). The incidence of infection was not elevated in patients transplanted for PBC when compared with other diagnoses. CONCLUSION: Our results indicate that primary liver disease may be a determinant for acute graft rejection in PBC. Furthermore, these results suggest that immunosuppressive regimens based on the underlying disease should be considered.

Acute Disease↗
PubMed1999

The interaction of Bcl-2 and Bax regulates apoptosis in biliary epithelial cells of rats with obstructive jaundice.

A complex molecular network controls cell homeostasis by inducing apoptosis or proliferation. The balance of Bcl-2 and Bax, members of a protein family, determines whether a cell will become immortal (Bcl-2) or will undergo apoptosis (Bax). To determine the role of Bcl-2 and Bax during proliferation of biliary epithelial cells (BEC) after bile duct ligation (BDL) and their regression after biliary decompression we induced hyperplasia of BEC by BDL in male rats. Regression of hyperplastic BEC by way of apoptosis was induced by biliary decompression through a Roux-en-Y biliodigestive anastomosis. To quantify apoptosis a modified TUNEL assay was used. Expression of Bcl-2 and Bax was visualized by immunohistochemistry and quantified stereologically. BEC increased from <1% to >20% after BDL; this increase was associated with overexpression of Bcl-2 in up to 30% of hyperplastic BEC. After biliodigestive anastomosis, apoptotic BEC increased from <0.1% to a peak of 5.4% after 1 day to reach baseline again 1 week after decompression. This was associated with de novo appearance of Bax. The interaction between Bcl-2 and Bax triggers apoptosis in BEC and acts as a cell rheostat in BEC hyperplasia and its involution after biliary decompression.

Anastomosis, Roux-en-Y↗
PubMed1999

Endothelium-dependent blunted membrane potential responses to ATP-sensitive K+ channel modulators in aortae from rats with cirrhosis.

BACKGROUND/AIMS: In vivo studies have shown that arterial vasodilation induced by synthetic openers of ATP-sensitive K+ (K(ATP)) channels is decreased in rats with cirrhosis. Since vasodilation induced by these substances is mediated by membrane potential hyperpolarization in arterial smooth muscle cells, membrane potential hyperpolarization in response to K(ATP) channel openers may be altered in cirrhotic smooth muscle cells. The aim of the present study was to investigate the effects of K(ATP) channel modulators (i.e. openers and blockers of these channels) on the membrane potential in smooth muscle cells in isolated aortae from cirrhotic and normal rats. The influence of endothelin-1 production by endothelial cells on smooth muscle cells membrane potential responses to K(ATP) channel modulators was also studied. METHODS: Cells were impaled in situ (in intact and endothelium-denuded aortae) with a microelectrode that was used to measure membrane potentials. K(ATP) channel openers were diazoxide or cromakalim; blockers were glibenclamide or tolbutamide. Bosentan (a mixed endothelin receptor antagonist) and exogenous endothelin-1 were also used. Preproendothelin-1 mRNA was assayed in aortae by RNase protection assay. Aortic wall endothelin-1 concentration was measured by double antibody radioimmunoassay technique. RESULTS: As expected, in smooth muscle cells in intact normal aortae, K(ATP) channel openers induced membrane potential hyperpolarization and K(ATP) channel blockers membrane potential depolarization. In smooth muscle cells in intact cirrhotic aortae, K(ATP) channel openers and blockers did not significantly change the membrane potential. Endothelium removal or exposure of intact aortae to bosentan restored normal membrane potential responses to K(ATP) channel modulators in cirrhotic smooth muscle cells and did not alter the effects of these substances in normal smooth muscle cells. In endothelium-denuded aortae, exposure to exogenous endothelin-1 suppressed membrane potential responses to K(ATP) channel modulators. In intact aortae, the abundance of preproendothelin-1 mRNA and endothelin-1 did not significantly differ between normal and cirrhotic rats. CONCLUSIONS: K(ATP) channel opener-induced membrane hyperpolarization and K(ATP) channel blocker-elicited membrane depolarization are blunted in smooth muscle cells in intact cirrhotic aortae. This blunting is due to the activation of the endothelin-1 pathway in the aortic wall, downstream to the endothelial production of endothelin-1.

Adenosine Triphosphate↗
PubMed1999

Myofibroblasts in the cirrhotic rat liver reflect hepatic remodeling and correlate with fibrosis and sinusoidal capillarization.

BACKGROUND/AIMS: Myofibroblasts are essential in fibrogenesis during development of cirrhosis. In the present study we stereologically quantitated MFB's and correlated them with fibrosis and sinusoidal capillarization. METHODS: Male SD rats were rendered cirrhotic by chronic exposure to phenobarbital/CCl4, (CIR; n = 16); untreated littermates served as controls (CTR; n = 10). Sinusoidal capillarization was assessed by a multiple indicator dilution technique as previously described. The volume fractions of myofibroblasts and other liver components were estimated by morphometry. RESULTS: Myofibroblasts averaged 15.7 +/- SD 0.7% in CIR as compared to 6.7% +/- SD 0.4% in CTR (p < 0.01). An extra-littoral compartment of myofibroblasts was found in portal tracts and within fibrous septa. In CIR, hepatocytes showed a bimodal distribution of volume fractions, and hepatocyte volume distribution disclosed a mirror image of that of myofibroblasts. Connective tissue was markedly increased in CIR, averaging 13.2 +/- 1.2% in CIR vs. 1.2 +/- 0.3% in CTR (p < 0.0001). Extravascular albumin space--a measure of sinusoidal capillarization--was reduced by 44% in CIR (0.028 +/- 0.017 vs. 0.050 +/- 0.010 ml/g; p < 0.001). The volume fraction of myofibroblasts correlated best with extravascular albumin space (r = -0.84, p < 0.001). Multiple regression analysis selected only extravascular albumin space and connective tissue to be determined by the volume fraction of myofibroblasts (r = 0.923; p < 0.001). CONCLUSION: We conclude that increased myofibroblasts reflect the degree of hepatic remodeling rather than cirrhosis inasmuch as myofibroblast volume fraction inversely reflects that of hepatocyte bimodality. Myofibroblasts form an extra-littoral compartment in this model of CIR and correlate with hepatic fibrosis and sinusoidal capillarization.

Actins↗
PubMed1999

Intraportal administration of glyceryl trinitrate or nitroprusside exerts more systemic than intrahepatic effects in anaesthetised cirrhotic rats.

BACKGROUND/AIMS: Increased intrahepatic vascular tone can be pharmacologically manipulated in isolated cirrhotic livers. Intrahepatic endothelial dysfunction may lead to a decreased production of the potent endogenous vasodilator nitric oxide in cirrhotic livers. The aims of the study were to determine whether portal pressure can be lowered in vivo by injecting nitric oxide donors glyceryl trinitrate or nitroprusside directly in the portal vein and whether this is related to a decrease in intrahepatic resistance. METHODS: In anaesthetised CCl4 cirrhotic rats, intraportal doses of glyceryl trinitrate 0.5, 1 or 5 microg/kg/ min or nitroprusside 1, 5 or 10 microg/kg/min did not decrease portal pressure but only arterial pressure. Systemic and splanchnic haemodynamics were measured before and during 15 min intraportal infusion of glyceryl trinitrate 10 microg/kg/min or nitroprusside 20 microg/kg/min. RESULTS: Glyceryl trinitrate decreased portal pressure from 14.0+/-1.1 to 11.8+/-1.4 mm Hg, splanchnic perfusion pressure from 102+/-10 to 74+/-5 mm Hg and portal sinusoidal flow from 2.11+/-0.38 to 1.70+/-0.35 ml/min/g liver (all p<0.05). Nitroprusside did not decrease portal pressure significantly but led to a reduction of the splanchnic perfusion pressure (104+/-9 to 66+/-7 mm Hg) and the portal sinusoidal flow (2.39+/-0.50 to 1.77+/-0.31 ml/min/g liver; all p<0.05). Portal sinusoidal resistance was not altered by either drug. CONCLUSIONS: Intraportal infusion of nitric oxide donors decreased arterial pressure more than portal pressure. Portal sinusoidal resistance remained unaffected, but the liver parenchyma became less perfused with high doses. The systemic effects of nitric oxide donating drugs prevailed.

Animals↗
PubMed1999

Cells with morphological and immunohistochemical features of hepatic stellate cells (Ito cells) form an extralittoral (extrasinusoidal) compartment in the cirrhotic rat liver.

Systematic studies on hepatic stellate cells and myofibroblasts have so far mainly focused on cells located in the perisinusoidal space of Disse, the so-called littoral compartment. Here, these cells play a key role for intralobular fibrogenesis and sinusoidal capillarization. However, advanced hepatic fibrosis and cirrhosis are characterized by portal tract fibrosis and septal fibrosis, thus involving cells outside the perisinusoidal space. To study the question as to whether hepatic stellate cells occur and are expanded in an extralittoral (extrasinusoidal) compartment in cirrhogenesis, we systematically analyzed the distribution and density of desminreactive stellate cells in a rat model of hepatic fibrosis. Fibrosis and remodeling of the liver were induced by bile duct ligation, and stellate cells were identified by single and double immunohistochemistry. We can show that desmin-reactive cells are reproducibly detectable in extralittoral compartments of the normal and fibrotic rat liver. Periductular extralittoral stellate cells are significantly more frequent in cirrhosis, indicating that extralittoral stellate cells expand in concert with proliferating ductules. The findings suggest that ductular proliferation thought to represent a pacemaker of hepatic remodeling is accompanied by a population of cells exhibiting the same phenotype as perisinusoidal stellate cells.

Animals↗
PubMed1999

Differential immunodiagnosis between cystic hydatid disease and other cross-reactive pathologies.

We assessed an Echinococcus granulosus hydatid fluid antigen-ELISA (EgHF-ELISA) as a serologic prescreening test for E. granulosus infections, supplemented by more specific confirmatory tests, including arc-5 immunoprecipitation and antigen B subunit 8-kD immunoblotting. The diagnostic sensitivity of the EgHF-ELISA was 91%. With regard to the test specificity of the EgHF-ELISA (overall = 82%), we observed relatively frequent cross-reactions in tumor patients (6%) and in patients with other parasitic diseases. Cestode-related cross-reactivity can be resolved by the complementary use of E. multilocularis-specific antigens or Taenia solium cysticercosis-specific immunoblotting. Immunoblotting based upon the detection of antibody reactivity to the 8-kD antigen of EgHF, or if appropriately detectable, to the 29-kD and 34-kD bands exhibited a 91% diagnostic sensitivity and an overall specificity of 97% or 94%, respectively. Thus, immunoblotting provided a 99% discrimination between seropositive pre-operative cystic hydatid disease cases and cross-reactive non-cestode parasitic infections or malignancies.

Agglutination Tests↗
PubMed1998

Down-regulation of hepatic and renal 11 beta-hydroxysteroid dehydrogenase in rats with liver cirrhosis.

BACKGROUND & AIMS: 11 beta-Hydroxysteroid dehydrogenase (11 beta-OHSD) enzymes are responsible for the interconversion of active 11 beta-hydroxycorticosteroids into inactive 11-ketoglucocorticosteroids and by that mechanism regulate the intracellular access of the steroids to the cognate receptor. A down-regulation of the shuttle of active to inactive glucocorticoids enhances access of glucocorticosteroids to both the glucocorticoid and the mineralocorticoid receptors. In liver cirrhosis, enhanced mineralocorticoid and glucocorticoid effects are observed. We therefore investigated the impact of liver cirrhosis after bile duct ligation on the transcription and activity of 11 beta-OHSD1 and 11 beta-OHSD2 in the corresponding tissues. METHODS: Messenger RNA from 11 beta-OHSD1 and 11 beta-OHSD2 was assessed by reverse-transcription polymerase chain reaction; activity was assessed by measuring the interconversion of corticosterone to dehydrocorticosterone. The effect of bile and bile salts was determined using COS-1 cells transfected with 11 beta-OHSD1 or 11 beta-OHSD2. RESULTS: In liver tissue, the messenger RNA ratios of 11 beta-OHSD1 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels and, in kidney tissue, the ratios of 11 beta-OHSD2 to GAPDH levels decreased after induction of liver cirrhosis. The 11 beta-OHSD activities were correspondingly reduced. Bile and individual bile salts inhibited 11 beta-OHSD1 and 11 beta-OHSD2 oxidative activity in transfected COS-1 cells. CONCLUSIONS: These findings indicate that in liver cirrhosis the mineralocorticoid and glucocorticoid receptor-protecting effects by the 11 beta-OHSD isoenzymes are down-regulated and that by the same mechanism the glucocorticoid and mineralocorticoid effects are enhanced.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1998

Detection of endothelin-1 and its receptors in rat liver endothelial cells by in situ reverse transcriptase-polymerase chain reaction.

BACKGROUND/AIMS: Endothelin (ET)-1 is a potent vasoconstrictor, also in the liver; it increases intrahepatic resistance by targeting hepatic stellate cells acting via the ET-A receptor. The role of the expression and regulation of ET-1 and its receptors in endothelial liver cells, an important site of ET production in normal liver, is not yet well documented. In the present study, we have developed an in situ reverse transcriptase (RT)-polymerase chain reaction method to elucidate the presence of prepro-ET-1, ET-A and ET-B receptors in isolated rat liver endothelial cells. METHODS: After in situ collagenase-pronase liver perfusion, endothelial cells were isolated using a Nycodenz gradient. RT-polymerase chain reaction and in situ RT-polymerase chain reaction were performed using specific primers for prepro-endothelin, ET-A and ET-B. RESULTS: Trypan blue exclusion test showed a viability of more than 90% in the freshly isolated non-parenchymal cells. RT-polymerase chain reaction showed expression of prepro-endothelin, ET-A and ET-B in RNA isolated from the endothelial cells. After in situ RT-polymerase chain reaction, we detected cytoplasmatic staining representing the presence of mRNA in the endothelial cells, with all sets of primers. About 60-80% of cells were positive. Negative controls, in which the RT step was omitted, did not show any cytoplasmatic staining. CONCLUSIONS: Endothelin 1 and both ET-A and ET-B receptors are expressed in rat endothelial liver cells. The expression of ET and its receptors in liver endothelial cells could be important in the development of liver diseases and the regulation of microvascular exchange.

Animals↗
PubMed1998

The effect of endothelin and its antagonist Bosentan on hemodynamics and microvascular exchange in cirrhotic rat liver.

BACKGROUND/AIMS: To characterize the effects of endothelin-1 and of Bosentan, a mixed endothelin antagonist, on hepatic hemodynamics in cirrhotic animals in vivo and on hepatic microvascular exchange in the perfused rat liver. METHODS: Biliary cirrhosis was induced by bile duct ligation, and micronodular cirrhosis by chronic exposure to phenobarbital/CCl4 in male rats. Hepatic hemodynamics were studied under basal conditions and after administration of Bosentan (3-30 mg/kg) by the microsphere technique. Microvascular exchange was assessed in the in situ perfused rat liver by the multiple indicator dilution technique. RESULTS: Bosentan lowered portal pressure in a dose-dependent fashion; at the highest dose tested, this decrease averaged -29+/-11 and -26+/-8% in biliary and micronodular cirrhosis, respectively (p<0.01). This was achieved mainly via a marked decrease in hepatic arterial flow. In the perfused liver, endothelin-1 induced a dose-dependent vasoconstriction; up to 10(-9) mol/l; this was not associated with any effect on viability. At this dose, endothelin-1 markedly decreased extravascular albumin space in both controls and micronodular cirrhosis; this could be antagonized by Bosentan 10(-5) mol/l. CONCLUSIONS: Endothelin-1 affects hepatic microvascular exchange, presumably by a direct effect on hepatic sinusoidal endothelial cells. A mixed endothelin antagonist lowers portal pressure in vivo, presumably by acting on hepatic stellate cells, and counteracts the microvascular effects of endothelin-1 in vitro. These properties could prove useful in treatment of portal hypertension.

Animals↗
PubMed1998

False positive staining in the TUNEL assay to detect apoptosis in liver and intestine is caused by endogenous nucleases and inhibited by diethyl pyrocarbonate.

BACKGROUND: The terminal transferase uridyl nick end labelling (TUNEL) assay allows the easy demonstration of cell death as a result of apoptosis. However, when this assay is applied to liver tissue, the number of TUNEL positive cells is dependent on the time of incubation with proteinase K. AIM: To test whether false positive results are the result of the release of endogenous endonucleases by proteinase K and can be abolished by pretreatment with diethyl pyrocarbonate (DEPC). METHODS: Involution of hyperplastic ductules in bile duct ligated rats after biliary decompression by Roux-en-Y anastomosis and acute CCl4 intoxication were studied as models of apoptosis and necrosis, respectively. A standard TUNEL assay was applied to formalin fixed tissue sections mounted with cement. To inhibit putative endogenous endonucleases, tissue slides were pre-incubated with DEPC. RESULTS: In the standard TUNEL assay, the number of positive nuclei was highly dependent upon the length of time that sections were incubated with proteinase K. After pretreatment with DEPC, only cells that also exhibited morphological features of apoptosis stained positive. DEPC pretreatment abolished false positive staining in CCl4 induced hepatocyte necrosis and blocked interference by endogenous alkaline phosphatase in intestine. The method of gluing the tissue section to the glass slide was found to be of utmost importance because the effect of DEPC was abolished on silanised slides. CONCLUSIONS: False positive staining in the TUNEL assay in the liver is caused by the release of endogenous endonucleases as a result of proteinase treatment. This can be abolished by pretreatment of tissue slides with DEPC.

Animals↗
PubMed1998

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.

Animals↗