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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.

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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.

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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.

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The key pathogenic event in liver fibrosis is the activation of hepatic stellate cells (HSC). Consequently, new antifibrotic therapies are directed toward an inhibition of HSC activities. The aim of the present study was to develop a drug carrier to HSC, which would allow cell-specific delivery of antifibrotic drugs thus enhancing their effectiveness in vivo. We modified human serum albumin (HSA) with 10 cyclic peptide moieties recognizing collagen type VI receptors (C*GRGDSPC*, in which C* denotes the cyclizing cysteine residues) yielding pCVI-HSA. In vivo experiments showed preferential distribution of pCVI-HSA to both fibrotic and normal rat livers (respectively, 62 +/- 6 and 75 +/- 16% of the dose at 10 min after intravenous injection). Immunohistochemical analysis demonstrated that pCVI-HSA predominantly bound to HSC in fibrotic livers (73 +/- 14%). In contrast, endothelial cells contributed mostly to the total liver accumulation in normal rats. In vitro studies showed that pCVI-HSA specifically bound to rat HSC, in particular to the activated cells, and showed internalization of pCVI-HSA by these cells. In conclusion, pCVI-HSA may be applied as a carrier to deliver antifibrotic agents to HSC, which may strongly enhance the effectiveness and tissue selectivity of these drugs. This approach has the additional benefit that such carriers may block receptors that play a putative role in the pathogenesis of liver fibrosis.

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The hallmark of liver fibrosis is an increased extracellular matrix deposition, caused by an activation of hepatic stellate cells (HSC). Therefore, this cell type is an important target for pharmacotherapeutic intervention. Antifibrotic drugs are not efficiently taken up by HSC or may produce unwanted side-effects outside the liver. Cell-specific delivery can provide a solution to these problems, but a specific drug carrier for HSC has not been described until now. The mannose 6-phosphate/insulin-like growth factor II (M6P/IGF-II) receptor, which is expressed in particular upon HSC during fibrosis, may serve as a target-receptor for a potential carrier. The aim of the present study was to examine if human serum albumin (HSA) modified with mannose 6-phosphate (M6P) is taken up by HSC in fibrotic livers. A series of M6Px-modified albumins were synthetized: x = 2, 4, 10, and 28. Organ distribution studies were performed to determine total liver uptake. The hepatic uptake of M6Px-HSA increased with increasing M6P density. M6Px-HSA with a low degree of sugar loading (x = 2-10) remained in the plasma and accumulated for 9% +/- 0.5% or less in fibrotic rat livers. An increase in the molar ratio of M6P:HSA to 28:1 caused an increased liver accumulation to 59% +/- 9% of the administered dose. Furthermore, we determined quantitatively the in vivo intrahepatic distribution of M6Px-HSA using double-immunostaining techniques. An increased substitution of M6P was associated with an increased accumulation in HSC; 70% +/- 11% of the intrahepatic staining for M6P28-HSA was found in HSC. We also demonstrate that M6P-modified bovine serum albumin (BSA) accumulates in slices of normal and cirrhotic human livers. After incubation of this neoglycoprotein with human tissue, the protein is found in nonparenchymal liver cells. Because M6P-modified albumins are taken up by HSC in fibrotic livers, this neoglycoprotein can be applied as a selective drug carrier for HSC. This technology may create new opportunities for the pharmacological intervention of liver fibrosis.

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We recently showed that absence of mdr1-type P-glycoprotein (P-gp) in mice resulted in profoundly reduced hepatic and intestinal clearance of type 1 and type 2 cationic drugs compared with that in wild-type mice. These data strongly support the concept that mdr1-type P-gps are involved in the disposition of cationic amphiphilic drugs from the body. We tested the hypothesis that mdr1-type P-gps are involved in the transmembrane transport of organic cations in epithelial cells expressing various drug-transporting P-gps. Therefore, transepithelial transport of the P-gp substrate vinblastine, the steroidal (type 2) cation vecuronium, the relatively small (type 1) cationic compound azidoprocainamide methoiodide and the aliphatic cation tri-n-butylmethylammonium were measured. Apical expression of the mdr1a, mdr1b or MDR1 gene in confluently grown polarized transformed LLC-PK1 cells resulted in highly enhanced apical directed secretion of all the drugs tested compared with controls. The vectorial transport of tri-n-butylmethylammonium in the apical direction in the P-gp (over)expressing cells could be inhibited by vinblastine. The present observations show that apical secretion of type 1 as well as of type 2 organic cations is enhanced significantly in the presence of apical expressed mdr1-type P-gp. These findings provide evidence for the involvement of drug-transporting P-gp in transmembrane transport of various organic cations, including relatively small molecular weight aromatic and aliphatic compounds.

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1. We have used mice with homozygously disrupted mdr1a and mdr1b genes (mdr1a/1b (-/-) mice) to study the role of the mdr1-type P-glycoprotein (P-gp) in the elimination of cationic amphiphilic compounds from the body. These mice lack drug-transporting P-gps, but show no physiological abnormalities under laboratory conditions and have normal bile flow. 2. 3H-labelled cationic drugs were administered intravenously (i.v.) to mice as a single bolus dose and the disposition of the studied cationic drugs was investigated by focusing on drug secretion into bile, intestinal lumen and urine. 3. Hepatobiliary secretion of the investigated cationic drugs was profoundly reduced in mice devoid of the mdr1-type P-gps. In fact, the cumulative biliary output, measured during 1 h, of the small type 1 compounds tri-butylmethyl ammonium (TBuMA) and azidoprocainamide methoiodide (APM), as well as of the more bulky type 2 cationic drug vecuronium, was reduced by at least 70% in the mdrla/lb (-/-) mice compared to wild-type. 4. The intestinal secretion of TBuMA, APM and vecuronium was also profoundly reduced in mdrla/lb (-/-) mice compared to wild-type mice. The absence of the mdrl-type P-gp resulted in virtual elimination of intestinal secretion of TBuMA and APM (>90% reduced as compared to wild-type (P=0.0001 and 0.0022, respectively)). The intestinal secretion of the type 2 cation drug vecuronium was reduced by 58% (P=0.0004) compared to the wild-type mice. 5. Increased renal clearances of both the type 1 compounds TBuMA and APM and also of the type 2 cationic compound vecuronium in the mdrla/lb (-/-) mice were observed. Furthermore, the balance between hepatic, intestinal and renal clearances of small type 1 organic cations clearly shifted towards a predominant role for renal clearance. Increased renal clearance may be explained by (over)expression of additional mechanisms for renal organic cation secretion, alternatively they may also point to an as yet undefined role of P-glycoprotein in kidney physiology and renal secretory function. 6. We conclude that the elimination from the body of a broad spectrum of cationic amphiphilic drugs via liver and intestine, is largely dictated by the activity of mdrl-type P-glycoproteins.

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In the mouse, both the mdr1a and the mdr1b gene encode drug-transporting P-glycoproteins. The mdr1a P-glycoprotein is expressed in epithelial cells of, among others, the liver and the intestine. Furthermore, the mdr1b gene product is found in the liver but is not detectable in the intestine. To establish the potential involvement of P-glycoprotein in the elimination of cationic amphiphilic drugs from the body, we investigated biliary, intestinal, and urinary excretion in mice with a homozygous disruption of the mdr1a gene (mdr1a(-/-) mice). These mice are fully viable under laboratory conditions and have normal bile flow. Cumulative biliary excretion (expressed as percent of the intravenously administered dose excreted over a 1-hour period) of several cationic compounds was decreased as follows in mdr1a(-/-) mice compared with the wild-type animals: tri-n-butylmethylammonium (TBuMA), 0.7% versus 2.1%; azidoprocainamide methoiodide (APM), 3.8% versus 7.6%; and vecuronium, 22.7% versus 41.3%. The luminal secretion of both TBuMA and APM in the small intestine was profoundly decreased, respectively 4.6-fold (1.8% vs. 8.2% in the wild-type) and 7.9-fold (1.6% vs. 10.3% in the wild-type) in mdr1a(-/-) mice. Thus mdr1a P-glycoprotein contributes substantially to the removal of a wide variety of cationic agents from the body through intestinal and hepatobiliary secretion, but it evidently acts in concert with other transport system(s). These processes probably provide a protective mechanism limiting the overall rate of absorption as well as the bioavailability of potentially toxic organic amines.

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The aim of this study was to compare human hepatocytes isolated from livers accepted and from livers discarded for transplantation with respect to viability and drug transport function. In addition, the influence of age of the donor and preservation time of the liver on cell viability was determined. Cell viability was assessed by trypan blue exclusion, MTT reduction, morphological integrity and ATP content, and drug transport function by uptake and excretion of taurocholic acid. Hepatocytes could be isolated successfully from livers accepted as well as from livers discarded for transplantation, with a median yield of 5.0 x 10(6) cells/g (range 0.1 to 42.4) and 0.7 x 10(6) cells/g (range 0.0 to 22.7), respectively (not significantly different). These cells were not significantly different with respect to viability and transport rate of taurocholate. Neither the age of the donor nor the duration of liver preservation (6-43 hr in University of Wisconsin solution) significantly influenced cell yield and viability. It is concluded that because of this overlap in cell viability, hepatocytes isolated both from accepted and from discarded livers can in principle be used to investigate drug transport functions in the human liver.

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The uptake and efflux of three categories of substrates were measured in isolated human hepatocytes and compared to those in rat hepatocytes. In addition, the extent to which the in vitro experiments quantitatively reflect liver function in vivo in both species was investigated. The anionic bile acid taurocholic acid was taken up by isolated human hepatocytes at a considerably lower rate than observed in isolated rat hepatocytes. Taurocholic acid uptake both in human hepatocytes and in liver plasma membrane vesicles showed sodium dependency. The uptake rate of taurocholic acid in isolated hepatocytes of both species was quantitatively compatible with the reported liver clearance of the bile acid in vivo. Ouabain uptake rate in isolated human hepatocytes was lower than in rat hepatocytes. This species difference was in accordance with pharmacokinetic studies in vivo on hepatic clearance of ouabain in man and rat. Uptake of vecuronium into human hepatocytes was about a factor of 10 lower than that in rat hepatocytes. Uptake into and efflux from human hepatocytes was comparable for the two short acting muscle relaxants vecuronium and rocuronium. Since distribution to the liver is considered to be a major factor in termination of action of vecuronium and rocuronium these observations were in line with the human pharmacokinetic profiles. In conclusion, the uptake rate of the studied model compounds in human hepatocytes appeared to be lower than that in rat hepatocytes. These observed transport rates reflected the relative hepatic transport rates observed in these species in the intact organism, but the absolute values in both species for some substrates may have been somewhat lower than calculated from in vivo data. It is concluded that transport studies in isolated hepatocytes are suitable for comparative drug transport studies, but are less precise in the prediction of quantitative membrane transport.

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Rat hepatocytes were preserved for 24 hr with high recovery and good maintenance of viability and transport function both in University of Wisconsin (UW) solution and in various simplified UW solutions. Cell quality is somewhat affected after 48 hr of preservation in both the original UW solution and the simplified solutions. ATP content and uptake rate of taurocholic acid are more sensitive markers of cell viability than Trypan blue exclusion or the MTT test. A much less expensive solution than UW, containing only K(+)-lactobionate, KH2PO4, MgSO4 and raffinose, can be used successfully for preservation of rat hepatocytes for 24 hr for drug transport studies.

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The hepatic transport mechanism for the fluorescent bivalent hydrophilic organic cation lucigenin (LU) was characterized employing kinetic and morphological methods. The extraction of LU by the perfused rat liver was 50% and uptake was saturable. LU did not inhibit the carrier-mediated hepatic uptake of the model organic cationic compounds tributylmethyl ammonium (type 1) and vecuronium (type 2) in isolated hepatocytes, whereas the uptake of LU in the perfused liver was not affected by either type of cation or by the cardiac glycoside cymarin, a potent type 2 inhibitor. The cytoskeleton-disrupting agents cytochalasin B and nocodazole, however, significantly lowered hepatic uptake of LU. In the intact liver, LU did not stimulate fluid phase endocytosis, as indicated by a lack of effect on the internalization of horseradish peroxidase. These kinetic data point to adsorptive endocytosis as the most probable uptake mechanism. This was confirmed by the inhibitory effect of neomycin and the polycation poly(L-lysine) on LU uptake. Fluorescence microscopy revealed that LU accumulated in the hepatocytes in discrete vesicular structures. Partial co-localization of rhodamine-dextran and acid phosphatase with LU indicated that part of the LU fluorescence was present in lysosomes, although not all lysosomes contained LU. Taken together, we conclude that we identified a novel vesicular pathway for uptake of organic cations by hepatocytes.

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The pharmacokinetics of Indocyanine Green (ICG) has been studied in 15 patients given 0.5, 1.0 and 2.0 mg.kg-1. The plasma disappearance and biliary excretion rate were measured in patients with tightly fitting catheters under slight negative pressure in order to achieve complete collection of bile. Recovery of unchanged ICG in bile over 18 h after the i.v. injection was 80% of the dose in all three dose groups. Plasma disappearance in all 3 groups was biphasic, showing an initial phase with a t1/2 of 3-4 min and a secondary phase with a dose-dependent apparent t1/2 of 67.6, 72.5 and 88.7 min, respectively. After 0.5 and 1.0 mg.kg-1 the biliary excretion rate curves showed an ascending phase with a mean t1/2 of 5 min and a descending phase with a mean t1/2 of 72 min. It was inferred that the secondary component of the plasma-decay mainly reflected the biliary excretion rate. After 2.0 mg.kg-1 in some patients the biliary excretion curve showed features of saturation; the t1/2 of the descending phase ranged from 73 to 440 min, and the time of maximal excretion was increased from 1.3 to 2.7 h after injection, whilst the mean maximal excretion rate was in the same range as the excretion rate after the 1.0 mg.kg-1 dose. The non-linear pharmacokinetics was only moderately reflected in the measured plasma disappearance patterns. Two compartment analysis of the plasma levels indicated a clearance of 230-260 ml.min-1, whereas the clearance conventionally calculated from the initial t1/2 was 475 ml.min-1.(ABSTRACT TRUNCATED AT 250 WORDS)

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In hepatobiliary transport of organic cations some remarkable differences have been reported between the monovalent compounds (prototype procainamidethobromide) and the potentially bivalent cations, containing a second quaternary ammonium group or a protonated tertiary amine function (prototype d-tubocurarine). In order to characterize the hepatic uptake mechanism for such bivalent cations in more detail, we studied the uptake of the steroidal muscle relaxant vecuronium in isolated rat hepatocytes. Uptake occurred by both a saturable (Vmax = 181 pmol/min x 10(6) cells, Km = 15 microM) and a nonsaturable process (rate constant = 1.10 pmol/min/10(6) cells/microM). The uptake of vecuronium was reduced by various metabolic inhibitors and by sulfhydryl-blocking agents. The transport system showed temperature dependency with an activation energy of 85 kJ/mol. Sodium replacement by lithium or choline in the extracellular medium had no effect on the uptake of vecuronium. Replacement of sodium chloride by sucrose led to a decrease of the uptake, whereas chloride replacement by bicarbonate or iodide stimulated the vecuronium uptake. These data point to a significant anion-dependency of the uptake system and indicate electroneutral uptake of vecuronium. The uptake of vecuronium was inhibited by a variety of hepatic transport model compounds, including bile acids, uncharged compounds and high molecular weight organic cations. Low molecular weight monovalent cations had no effect on the uptake of vecuronium.(ABSTRACT TRUNCATED AT 250 WORDS)

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