Generation and Characterization of CYP2E1-Overexpressing HepG2 Cells to Study the Role of CYP2E1 in Hepatic Hypoxia-Reoxygenation Injury.

The mechanisms of hepatic ischemia/reperfusion (I/R) injury, which occurs during liver transplantation or surgery, are poorly understood. The purpose of the current study was to generate and characterize a HepG2 cell line with a stable overexpression of CYP2E1 to investigate the role of the enzyme in hypoxia/reperfusion (H/R) injury in an ex vivo setting. GFP-tagged CYP2E1 and control clones were developed, and their gene expression and protein levels of GFP and CYP2E1 were determined using RT-PCR and ELISA/Western blot analysis, respectively. Additionally, the CYP2E1 catalytic activity was determined by UPLC-MS/MS analysis of 6-hydroxychlorzoxazone formed from the chlorzoxazone substrate. The CYP2E1 and control clones were subjected to hypoxia (10 h) and reoxygenation (0.5 h), and cell death and reactive oxygen species (ROS) generation were quantitated using LDH and flow cytometry, respectively. Compared with the control clone, the selected CYP2E1 clone showed a 720-fold increase in CYP2E1 expression and a prominent band in the western blot analysis, which was associated with a 150-fold increase in CYP2E1 catalytic activity. The CYP2E1 clone produced 2.3-fold more ROS and 1.9-fold more cell death in the H/R model. It is concluded that the constitutive CYP2E1 in the liver may play a detrimental role in hepatic I/R injury.

Effects of hepatic ischemia-reperfusion injury on the blood-brain barrier permeability to [14C] and [13C]sucrose.

Hepatic encephalopathy that is associated with severe liver failure may compromise the blood-brain barrier (BBB) integrity. However, the effects of less severe liver diseases, in the absence of overt encephalopathy, on the BBB are not well understood. The goal of the current study was to investigate the effects of hepatic ischemia-reperfusion (IR) injury on the BBB tight junction permeability to small, hydrophilic molecules using the widely used [14C]sucrose and recently-proposed alternative [13C]sucrose as markers. Rats were subjected to 20 min of hepatic ischemia or sham surgery, followed by 8 h of reperfusion before administration of a single bolus dose of [14C] or [13C]sucrose and collection of serial (0-30 min) blood and plasma and terminal brain samples. The concentrations of [14C] and [13C]sucrose in the samples were determined by measurement of total radioactivity (nonspecific) and LC-MS/MS (specific), respectively. IR injury significantly increased the blood, plasma, and brain concentrations of both [14C] and [13C]sucrose. However, when the brain concentrations were corrected for their respective area under the blood concentration-time curve, only [14C]sucrose showed significantly higher (30%) BBB permeability values in the IR animals. Because [13C]sucrose is a more specific BBB permeability marker, these data indicate that our animal model of hepatic IR injury does not affect the BBB tight junction permeability to small, hydrophilic molecules. Methodological differences among studies of the effects of liver diseases on the BBB permeability may confound the conclusions of such studies.

Effects of Pringle maneuver and partial hepatectomy on the pharmacokinetics and blood-brain barrier permeability of sodium fluorescein in rats.

Liver diseases are known to affect the function of remote organs. The aim of the present study was to investigate the effects of Pringle maneuver, which results in hepatic ischemia-reperfusion (IR) injury, and partial hepatectomy (Hx) on the pharmacokinetics and brain distribution of sodium fluorescein (FL), which is a widely used marker of blood-brain barrier (BBB) permeability. Rats were subjected to Pringle maneuver (total hepatic ischemia) for 20 min with (HxIR) or without (IR) 70% hepatectomy. Sham-operated animals underwent laparotomy only. After 15 min or 8h of reperfusion, a single 25-mg/kg dose of FL was injected intravenously and serial (0-30 min) blood and bile and terminal brain samples were collected. Total and free (ultrafiltration) plasma, total brain homogenate, and bile concentrations of FL and/or its glucuronidated metabolite (FL-Glu) were determined by HPLC. Both IR and HxIR caused significant reductions in the biliary excretions of FL and FL-Glu, resulting in significant increases in the plasma AUC of the marker. Additionally, the free fraction of FL in plasma was significantly increased by HxIR. Although the brain concentrations of FL were increased by almost twofold in both IR and HxIR animals, the brain concentrations corrected by the free FL AUC (and not the total AUC) were similar in both groups at either time points. It is concluded that Pringle maneuver and/or partial hepatectomy substantially alters the hepatobiliary disposition, plasma AUC, plasma free fraction, and brain accumulation of FL without altering the BBB permeability to the marker.

Effects of hepatic ischemia-reperfusion injury on the P-glycoprotein activity at the liver canalicular membrane and blood-brain barrier determined by in vivo administration of rhodamine 123 in rats.

PURPOSE: To investigate the effects of normothermic hepatic ischemia-reperfusion (IR) injury on the activity of P-glycoprotein (P-gp) in the liver and at the blood-brain barrier (BBB) of rats using rhodamine 123 (RH-123) as an in vivo marker. METHODS: Rats were subjected to 90 min of partial ischemia or sham surgery, followed by 12 or 24 h of reperfusion. Following intravenous injection, the concentrations of RH-123 in blood, bile, brain, and liver were used for pharmacokinetic calculations. The protein levels of P-gp and some other transporters in the liver and brain were also determined by Western blot analysis. RESULTS: P-gp protein levels at the liver canalicular membrane were increased by twofold after 24 h of reperfusion. However, the biliary excretion of RH-123 was reduced in these rats by 26%, presumably due to IR-induced reductions in the liver uptake of the marker and hepatic ATP concentrations. At the BBB, a 24% overexpression of P-gp in the 24-h IR animals was associated with a 30% decrease in the apparent brain uptake clearance of RH-123. The pharmacokinetics or brain distribution of RH-123 was not affected by the 12-h IR injury. CONCLUSIONS: Hepatic IR injury may alter the peripheral pharmacokinetics and brain distribution of drugs that are transported by P-gp and possibly other transporters.

Hepatic immunosuppressive effects of systemically administered novel dextran-methylprednisolone prodrugs with peptide linkers in rats.

The hepatic immunosuppressive activities of two novel dextran prodrugs of methylprednisolone (MP) containing one (DMP1) or five (DMP5) amino acids as linkers were studied in rats. At various times (0-2 weeks) after intravenous administration of single 5 mg/kg (MP equivalent) doses of each prodrug or MP succinate (MPS), livers were isolated and immunologically stimulated ex vivo with lipopolysaccharide. The concentrations of tumor necrosis factor (TNF)-α in the outlet perfusate were then quantitated to assess immune response. Additionally, the concentrations of DMP1, DMP5, and/or MP were measured in the liver. MPS, DMP5, or DMP1 injections caused a maximum of 48.9%, 63.5%, or 85.7% decrease in the TNF-α secretion into the perfusate, with the time above the 50% inhibitory effect being <5, <24, or 120 h, respectively. Additionally, the area under the effect-time curve for DMP1 was 11-fold or fourfold higher than that after the administration of MPS or DMP5, respectively. Relatively high concentrations of DMP1 were present in the liver even at the last sampling time of 2 weeks. These data suggest that a single intravenous dose of DMP1 produces an intense and sustained immunosuppression in the liver for a relatively long time, which may be useful in liver transplantation.

Effects of transient overexpression or knockdown of cytochrome P450 reductase on reactive oxygen species generation and hypoxia reoxygenation injury in liver cells.

1. Literature data suggest that the electron-donating enzyme, cytochrome P450 reductase (CPR), might act as a source of reactive oxygen species (ROS). However, the role of CPR in pathophysiological conditions associated with oxidative stress is unknown. The aim of the present study was to study the role of CPR in the generation of ROS and cellular injury under basal conditions, and after simulated in vitro ischaemia-reperfusion (IR). 2. Plasmid DNA or siRNA approaches were used to transiently overexpress or knockdown the human CPR gene in rat liver epithelial (WB-F344) or human hepatoblastoma (HepG2) cells, respectively. The generation of ROS and/or cellular injury was then studied under the basal conditions and after simulated IR (4 h of ischaemia plus 30 min of reoxygenation). 3. Under the basal conditions, transient overexpression of CPR protein in WB-F344 cells caused a 90% increase in the CPR activity, which was associated with a 100% increase in the ROS production. In contrast, after simulated IR, a 2.5-fold higher CPR activity did not significantly affect the magnitude of ROS generation or cell death. Similarly, although the knockdown of CPR protein resulted in a significant reduction (∼30%) in the CPR activity, the ROS production was not substantially altered after simulated IR in HepG2 cells. 4. Our data suggest that CPR plays a major role in the ROS generation by liver cells under the basal conditions. However, the role of CPR in the ROS generation during simulated in vitro IR injury in these cells is minimal, if any.

Divergent effects of nitric oxide donors on the biliary efflux transporters in isolated perfused rat livers: nitric oxide-independent inhibition of ABC transporters by sodium nitroprusside.

Rhodamine 123 (RH-123) and its glucuronidated metabolite (RH-Glu) are excreted into the bile via the ABC efflux transporters P-glycoprotein (P-gp) and multidrug resistance-related protein type 2 (Mrp2), respectively. In this study, we investigated the short-term (2 h) effects of a low or high concentration of nitric oxide (NO) donors sodium nitroprusside (SNP) and isosorbide dinitrate (ISDN) on the hepatobiliary disposition of RH-123 and its metabolite in an isolated perfused rat liver model. Additionally, the effects of ISDN on the hepatobiliary disposition of 5 (and 6)-carboxy-2', 7'- dichlorofluorescein (CDF), a specific marker of Mrp2, were investigated in the same model. Whereas SNP caused a substantial (85-90%) reduction in the P-gp- and Mrp2-mediated transport of RH-123 and RH-Glu, respectively, ISDN did not affect either of these transporters. However, ISDN reduced the biliary recovery of RH-Glu, most likely because of inhibition of the formation of the metabolite. Further studies showed that the effects of SNP on these transporters are due to a substantial (88%) depletion of hepatic ATP levels by this NO donor. Additionally, studies using CDF revealed an almost identical hepatobiliary disposition of this Mrp2 marker in the presence or absence of ISDN. It is concluded that short-term exposure of rat livers to NO does not affect the functions of the efflux transporters P-gp and Mrp2. The observed inhibitory effects of SNP on the functions of both P-gp and Mrp2 are via an NO-independent mechanism.

Cytochrome P450 induction by phenobarbital exacerbates warm hepatic ischemia-reperfusion injury in rat livers.

Recent studies have shown that cytochrome P450 inhibitors reduce oxidative stress and injury to the liver following warm ischemia-reperfusion (IR). The aim here was to test the effect of P450 induction by phenobarbital on the IR injury in rat livers. Rats were pre-treated with saline or phenobarbital and subjected to IR or sham operation. IR significantly increased the plasma alanine aminotransferase concentrations. Phenobarbital further exacerbated the injury by an additional 50% increase in the alanine aminotransferase levels. Phenobarbital also caused an approximately 40% increase in the total P450 content of the liver, which was also associated with a 75% increase in the reactive oxygen species (ROS) generation in the IR group. There was a strong correlation between the microsomal ROS generation and total P450 content, CYP3A2 activity or CYP2B1 activity. It is concluded that the induction of P450 by phenobarbital significantly increases hepatic production of ROS, leading to significantly higher hepatic IR injury.

Plasma pharmacokinetics and tissue disposition of novel dextran-methylprednisolone conjugates with peptide linkers in rats.

The plasma and tissue disposition of two novel dextran prodrugs of methylprednisolone (MP) containing one (DMP-1) or five (DMP-5) amino acids as linkers were studied in rats. Single 5-mg/kg doses (MP equivalent) of each prodrug or MP were administered intravenously, and blood and tissue samples were collected. Prodrug and drug concentrations were quantitated using HPLC, and noncompartmental pharmacokinetic parameters were estimated. Whereas conjugation of MP with dextran in both prodrugs substantially decreased the clearance of the drug by approximately 200-fold, the accumulations of the drug in the liver, spleen, and kidneys were significantly increased by conjugation. However, the extent of accumulation of DMP-1 in these tissues was substantially greater than that for DMP-5. Substantial amounts of MP were regenerated from both prodrugs in the liver and spleen, with the rate of release from DMP-5 being twice as fast as that from DMP-1. However, the AUCs of MP regenerated from DMP-1 in the liver and spleen were substantially higher than those after DMP-5. In contrast, in the kidneys, the AUC of MP regenerated from DMP-5 was higher than that after DMP-1 administration. These data suggest that DMP-1 may be more suitable than DMP-5 for targeting immunosuppression to the liver and spleen.

Effects of cytochrome p450 inhibition by cimetidine on the warm hepatic ischemia-reperfusion injury in rats.

BACKGROUND: Cimetidine is an H(2)-antagonist with cytochrome P450 (P450) inhibitory activity. Recent studies showed that cimetidine improves warm ischemia-reperfusion (IR) injury in isolated rat heart and rabbit lung and in primary cultures of rat proximal tubule epithelial cells by inhibiting P450-mediated reactive oxygen species generation. Here, we studied the effects of cimetidine on the warm IR injury in the liver. METHODS: Three groups of rats were treated with a single i.p. dose (0.6 mmol/kg) of cimetidine or ranitidine (an H(2) antagonist without a significant P450 inhibitory activity) or with saline 1.5 h before surgery. Livers were then subjected to 1 h of in vivo ischemia, followed by 1 h of ex vivo reperfusion using a physiologic buffer in a recirculating manner. A fourth group of animals, receiving saline pretreatment underwent sham operation instead of ischemia. Perfusate and bile samples were collected during the reperfusion, and the liver tissue was collected at the end of reperfusion period for measurement of various biochemical markers. RESULTS: Warm IR resulted in a significant increase in the perfusate concentrations of liver enzymes (3- to 4.5-fold) and hepatic concentrations of lipid hydroperoxides (2-fold). Whereas the glutathione concentrations in the liver tissue were not affected by IR injury, the injury caused a significant decrease ( approximately 40%) in the biliary glutathione excretion. Cimetidine treatment completely or partially reversed all the IR-mediated changes, while ranitidine was ineffective. The protective effects of cimetidine were associated with a 60% decline in the microsomal CYP2C11 activity. CONCLUSIONS: Whereas cimetidine, an H(2) blocker with substantial P450 inhibitory activity, is protective in warm IR injury, ranitidine, a similar drug with no significant P450 inhibitory activity, is devoid of any protective effects. Therefore, P450 inhibition appears to be the underlying mechanism in the protective effects of cimetidine in this model of IR injury.

Protective effects of diallyl sulfide, a garlic constituent, on the warm hepatic ischemia-reperfusion injury in a rat model.

PURPOSE: The aim of this study is to evaluate the effects of diallyl sulfide (DAS) on the warm hepatic ischemia-reperfusion (IR) injury in a rat model. METHODS: Rats (n = 8-10/group) were subjected to sham operation or warm ischemia (1 h)-reperfusion (3 h) preceded by a single intraperitoneal dose (1.75 mmol/kg) of DAS or vehicle, and relevant biochemical parameters were monitored. RESULTS: Warm IR injury caused a significant increase in the plasma markers of liver injury, which was attenuated by DAS. The hepatoprotective effects of DAS were associated with significant reductions in lipid peroxidation markers and in situ generation of superoxide in the liver and increases in the glutathione levels of the liver and bile, suggestive of an antioxidant effect for DAS. Additionally, DAS caused an almost twofold increase in the protein expression of the liver heme oxygenase-1, an enzyme that confers cytoprotection against oxidative stress. Whereas the total cytochrome P450 remained unchanged, the protein levels and activity of CYP2E1, which plays an important role in the generation of reactive oxygen species, significantly decreased by DAS pretreatment. CONCLUSIONS: DAS protects the liver from warm IR injury by reducing oxidative stress through, at least in part, induction of heme oxygenase-1 and inhibition of CYP2E1.

Synthesis and in vitro characterization of novel dextran-methylprednisolone conjugates with peptide linkers: effects of linker length on hydrolytic and enzymatic release of methylprednisolone and its peptidyl intermediates.

To control the rate of release of methylprednisolone (MP) in lysosomes, new dextran-MP conjugates with peptide linkers were synthesized and characterized. Methylprednisolone succinate (MPS) was attached to dextran 25 kDa using linkers with 1-5 Gly residues. The release characteristics of the conjugates in pH 4.0 and 7.4 buffers, blood, liver lysosomes, and various lysosomal proteinases were determined using a size-exclusion and/or a newly developed reversed-phase HPLC method capable of simultaneous quantitation of MP, MPS, and all five possible MPS-peptidyl intermediates. We synthesized conjugates with >or=90% purity and 6.9-9.5% (w/w) degree of MP substitution. The conjugates were stable at pH 4.0, but released MP and intact MPS-peptidyl intermediates in the pH 7.4 buffer and rat blood, with faster degradation rates for longer linkers. Rat lysosomal fractions degraded the conjugates to MP and all the possible intermediates also at a rate directly proportional to the length of the peptide. Whereas the degradation of the conjugates by cysteine peptidases (papain or cathepsin B) was relatively substantial, no degradation was observed in the presence of aspartic (cathepsin D) or serine (trypsin) proteinases, which do not cleave peptide bonds with Gly. These newly developed dextran conjugates of MP show promise for controlled delivery of MP in lysosomes.

Metabolic bioactivation and toxicity of ethyl 4-hydroxybenzoate in human SK-MEL-28 melanoma cells.

The metabolism and toxicity of ethyl 4-hydroxybenzoate (4-HEB) were investigated in vitro using tyrosinase enzyme, a melanoma molecular target, and CYP2E1 induced rat liver microsomes, and in human SK-MEL-28 melanoma cells. The results were compared to 4-hydroxyanisole (4-HA). At 90 min, 4-HEB was metabolized 48% by tyrosinase and 26% by liver microsomes while the extent of 4-HA metabolism was 196% and 88%, respectively. The IC50 (day 2) of 4-HEB and 4-HA towards SK-MEL-28 cells were 75 and 50 microM, respectively. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased 4-HEB toxicity towards SK-MEL-28 cells indicating o-quinone formation played an important role in 4-HEB induced cell toxicity. Addition of ascorbic acid and GSH to the media was effective in preventing 4-HEB cell toxicity. Cyclosporin A and trifluoperazine, inhibitors of permeability transition pore in mitochondria, were significantly potent in inhibiting 4-HEB cell toxicity. 4-HEB caused time-dependent decline in intracellular GSH concentration which preceded cell death. 4-HEB also led to reactive oxygen species (ROS) formation in melanoma cells which exacerbated by dicoumarol and 1-bromoheptane whereas cyclosporin A and trifluoperazine prevented it. Our findings suggest that the mechanisms of 4-HEB toxicity in SK-MEL-28 were o-quinone formation, intracellular GSH depletion, ROS formation and mitochondrial toxicity.

Effects of methylprednisolone and its liver-targeted dextran prodrug on ischemia-reperfusion injury in a rat liver transplantation model.

PURPOSE: To evaluate the effectiveness of a liver-targeted dextran prodrug (DMP) of methylprednisolone (MP) in cold preservation-warm reperfusion injury associated with liver transplantation. METHODS: The effects of donor pretreatment with single 5 mg/kg doses of MP or DMP on ischemia-reperfusion damage to the liver were studied after 8 or 24 h of cold preservation in both isolated perfused rat liver (IPRL) and syngeneic orthotopic rat liver transplantation (OLT) models. RESULTS: In IPRL studies, donor pretreatment with DMP, and to a lesser degree MP, significantly improved the uptake of hyaluronic acid (HA), a marker of endothelial cell function, following 8 h of cold preservation. However, neither pretreatment was protective after 24 h of preservation. In the OLT model using 24-h-preserved livers, the seven-day survival of untreated grafts was 50%. DMP pretreatment of donors significantly improved graft survival to 100%, whereas MP pretreatment was ineffective. Additionally, only DMP significantly increased the blood glucose concentrations and decreased the plasma concentrations of tumor necrosis factor-alpha after OLT. Other measured markers of liver injury were not affected by either pretreatment. CONCLUSIONS: Selective delivery of methylprednisolone to the liver as a donor pretreatment strategy improves 24-h preserved graft survival in the OLT model.

Attenuation of acute rejection in a rat liver transplantation model by a liver-targeted dextran prodrug of methylprednisolone.

BACKGROUND: The use of methylprednisolone (MP) and other corticosteroids for the treatment of acute liver allograft rejection is associated with severe toxicities in nontarget tissues. Therefore, selective delivery of MP to the liver may improve its efficacy and alleviate its side effects. We investigated the effects of a novel liver-targeted dextran prodrug of MP (DMP) in an orthotopic rat liver transplantation (OLT) model. METHODS: The model consisted of a high responder rejection strain combination (Dark Agouti donors and Lewis recipients). Liver recipients were intravenously administered saline or a single subtherapeutic dose of MP (5 mg/kg) as the parent drug (MP) or its prodrug (DMP). Different groups were then monitored for graft survival or euthanized 5 or 9 days posttransplantation. Plasma chemistry, including alkaline phosphatase and bilirubin, allograft histology, and survival duration were determined. RESULTS: Untreated recipients exhibited elevated plasma levels of liver injury markers, progressive portal and venous inflammation and cellular infiltration in liver allografts, and a mean graft survival time (MST) of 10.5 days. MP treatment did not alter any of these parameters. In contrast, a single dose of DMP resulted in a decrease in plasma levels of liver injury markers, a decrease in histological grade of rejection on day 5, and a substantial increase in MST (27.5 days). CONCLUSIONS: These results demonstrate attenuation of acute rejection following local (allograft) immunosuppression with a single subtherapeutic dose of MP delivered as a liver-targeted prodrug. Dextran prodrugs may be useful for selective delivery of immunosuppressants to the liver following liver transplantation.

Rapid determination of reduced and oxidized glutathione levels using a new thiol-masking reagent and the enzymatic recycling method: application to the rat liver and bile samples.

A microtiter plate assay for quantitation of reduced (GSH) and oxidized (GSSG) glutathione in the rat liver tissue and bile is described. The assay is based on the established enzymatic recycling method and a new thiol-masking reagent, 1-methyl-4-vinyl-pyridinium trifluoromethane sulfonate (M4VP). Samples were first processed by homogenization with (liver) or addition of (bile) sulfosalicylic acid. The total glutathione and GSSG were then determined before and after rapid (< or = 2 min) and efficient (100%) masking of the GSH content of the samples with M4VP followed by the enzymatic recycling assay. The percentages of error and coefficient of variation of the assay were within the accepted guidelines, indicating the accuracy and precision of the assay in the range of 6.25-100 pmol GSH per microplate well and 2.17-140 pmol GSSG per well, with lower limit of quantitation of 6.25 and 2.17 pmol per well for GSH and GSSG, respectively. Furthermore, the recoveries of added GSH or GSSG from the liver and bile samples were accurate and precise. The assay was applied to measurement of GSH, GSSG, and GSH:GSSG ratio in the liver and serially collected bile samples in sham-operated and ischemic rat livers, demonstrating a depletion of glutathione and a decrease in the GSH:GSSG ratio as a result of ischemia. The developed assay is rapid, sensitive, accurate, and precise and is suitable for studies of the redox status of liver under physiologic and pathophysiologic conditions.

Short-term inhibitory effects of nitric oxide on cytochrome P450-mediated drug metabolism: time dependency and reversibility profiles in isolated perfused rat livers.

Nitric oxide (NO) is implicated as a mediator in the decreased catalytic activities of cytochrome P450 (P450) enzymes during inflammation or infection. Here, we examined the time course and the reversibility of the NO effect on P450s using isolated perfused rat livers. Livers were perfused at a constant rate with the NO donor sodium nitroprusside (SNP) for 0.5 or 1 h, followed by washout periods of 0 to 2.5 h. At the end of perfusion, microsomes were prepared and analyzed for P450 activities and other metabolic markers. Whereas 0.5 h of NO exposure caused an irreversible decline (approximately 30%) in total P450 content, a greater decline after 1 h of NO (approximately 55%) was mostly (approximately 30%) reversible, a pattern identical to that observed for the microsomal heme content. NO exposure also caused an enzyme-selective and time-dependent decline in P450 activities. Whereas the pattern of decline and reversibility of activities were qualitatively similar for CYP3A2, 2C11, 2E1, and 1A1/2, they differed for 2B1/2 and 2D1 in that the decline in the activity was delayed (1 h) for 2B1/2 and not observed for 2D1. This may be attributed to the accessibility of heme or cysteine thiolate and/or the presence/reactivity of critical cysteinyl amino acid residues in various P450 enzymes. Additionally, for most enzymes, the activity showed a biphasic decline, one within 1 h of SNP perfusion and another after 2 h of washout. This was associated with an identical biphasic decline in the microsomal free thiols, presumably due to the rapid and slow reaction of NO and peroxynitrite, respectively, with critical P450 thiols. The short-term effects of NO on P450 are time-dependent and enzyme-selective, with both reversible and irreversible mechanisms.

Recent trends in the use of polysaccharides for improved delivery of therapeutic agents: pharmacokinetic and pharmacodynamic perspectives.

New and innovative methods of delivery of therapeutic agents using polysaccharides have been recently developed, which target site of action, increase the intensity and/or prolong pharmacologic action, and/or reduce toxicity of small molecule drugs, proteins, or enzymes. This review is focused on the role of dextran, pullulan, and mannan polysaccharides in such applications. While dextran and pullulan are glucose polymers with different glucosidic linkages, mannan is composed of mannose units. In terms of pharmacokinetics of the carriers themselves, molecular weight (MW), electric charge, various chemical modifications, and degree of polydispersity and/or branching would mostly determine their fate in vivo. Generally, large MW polysaccharides (MWs > or = 40 kD) have low clearance and relatively long plasma half life, resulting in accumulation in reticuloendothelial or tumor tissues. The tumor accumulation in most cases is a passive targeting due to "enhanced permeation and retention" of macromolecules by tumors. Additionally, drugs such as anticancer agents may be actively targeted to specific cells by polysaccharides to which appropriate ligands are attached. In terms of mode of use, polysaccharides have been utilized in a variety of innovative ways for improvement of drug delivery. Their most important application has been as carriers for preparation of macromolecular prodrugs that are normally inactive and need to release the active drug at the site(s) of interest. Also, they have been used for preparation of macromolecule-protein conjugates, which may retain the activity of the proteins, in order to increase the duration of effect and decrease the immunogenicity of proteins. Several other new applications, such as polysaccharide-anchored liposomal formulations, have also been gained attention recently and are briefly reviewed here. Finally, four recent examples of polysaccharide-based delivery systems involving specific drugs/imaging agents are reviewed in detail in terms of their development, pharmacokinetics, and pharmacodynamics. Collectively, these data suggest that macromolecular polysaccharides are promising agents for improving drug delivery.

Attenuation of Kupffer cell activation in cold-preserved livers after pretreatment of rats with methylprednisolone or its macromolecular prodrug.

PURPOSE: Activation of hepatic Kupffer cells (KCs) during organ preservation and subsequent reperfusion causes release of proinflammatory mediators and is responsible, at least in part, for rejection of transplanted livers. Our hypothesis was that donor pretreatment, before liver harvest, with methylprednisolone (MP) or its dextran prodrug (DMP) would reduce KC activation. METHODS: Adult donor rats were administered a single 5-mg/kg (MP equivalent) IV dose of MP or DMP or saline 2 h before liver harvest. The livers were then stored in University of Wisconsin solution for 24, 48, or 96 h (n = 4/treatment/time). A recirculating perfusion model was used to study, for 180 min, the release of KC activation markers, tumor necrosis factor (TNF)-alpha and acid phosphatase, and other biochemical indices from the cold-preserved livers. RESULTS: Cold ischemia-reperfusion resulted in release of substantial levels of TNF-alpha in untreated groups. Pretreatment of rats with MP or DMP caused a significant (p < 0.0001) reduction in TNF-alpha AUC in the perfusate, with no significant differences between MP and DMP. The maximum inhibitory effect of MP (77.5 +/- 10.2%) was observed after 48 h of preservation, whereas DMP showed maximal inhibition of TNF-alpha AUC at both 24 (74.5 +/- 15.8%) and 48 (74.8 +/- 12.6%) h of preservation. Similarly, both MP and DMP resulted in a significant (p < 0.0004) decrease in acid phosphatase levels of cold-preserved livers. However, neither pretreatment had any substantial effect on the levels of other biochemical markers. CONCLUSIONS: Both MP and DMP pretreatments decreased the release of TNF-alpha and acid phosphatase from livers subjected to cold ischemia preservation. Therefore, pretreatment of liver donors with MP or its prodrug decreases KC activation by cold ischemia-reperfusion.

Dextran-methylprednisolone succinate as a prodrug of methylprednisolone: local immunosuppressive effects in liver after systemic administration to rats.

PURPOSE: The purpose of this work was to study the local immunosuppressive effects of systemically administered methylprednisolone (MP) and its prodrug, dextran-methylprednisolone (DMP), in rat livers. METHODS: Single 5 mg/kg (MP equivalent) doses of MP or DMP were injected intravenously to rats, and livers were isolated at different time points (0-72 h; n = 4/time point). Isolated livers were stimulated ex vivo with bacterial lipopolysaccharide, and outlet perfusate and bile samples were analyzed for their concentrations of tumor necrosis factor (TNF)-alpha by enzyme-linked immunosorbent assay. The area under the perfusate TNF-alpha concentration-time curve (AUC) was used as a measure of immune response. Hepatic concentrations of MP and DMP were also measured by high-performance liquid chromatography. RESULTS: Both MP and DMP resulted in a decrease in lipopolysaccharide-induced increase in TNF-alpha AUC. MP injection 8 h before liver isolation resulted in a maximum of 50% decrease in TNF-alpha AUC. Compared with MP, the maximum effect of the prodrug (DMP) was both more intense (approximately 80% reduction in TNF-alpha AUC) and delayed (maximum inhibition at 24 h). Overall, the area under the effect (% inhibition of TNF-alpha)-time (%inhibition-h) for DMP (3,680 +/- 406) was approximately four times more than that for the parent drug (846 +/- 114). Whereas the MP concentrations in the liver were not quantifiable after the injection of the parent drug, relatively large concentrations of DMP and regenerated MP were found in the liver of DMP-injected rats. CONCLUSIONS: After systemic administration to rats, both MP and DMP exhibit local immunosuppressive effects in the liver. The local effects of the prodrug (DMP), however, appear to be more intense and sustained than those of the parent drug (MP).