Disposition Characteristics of Plasmid DNA in the Single-pass Rat Liver Perfusion System

Purpose. To define the hepatic uptake mechanism of a plasmid DNA, we quantitated the uptake of pCAT (plasmid DNA encoding chloramphenicol acetyltransferase reporter gene fused to simian virus 40 promoter), a model plasmid, after a single pass through the perfused rat liver using albumin- and erythrocyte-free Krebs-Ringer bicarbonate buffer (pH 7.4). Methods. [³²P]pCAT was introduced momentarily into this system from the portal vein as a bolus input or constant infusion mode, and the outflow patterns and hepatic uptake were evaluated using statistical moment analysis. Results. The venous outflow samples had electrophoretic bands similar to that of the standard pCAT, suggesting that the plasmid is fairly stable in the perfusate during liver perfusion. In bolus experiments, pCAT was largely taken up by the liver and the uptake was decreased with increase in injected dose. Statistical moment analysis against outflow patterns demonstrated that the apparent volume of distribution of pCAT was greater than that of human serum albumin, indicating a significant reversible interaction with the tissues. The results of collagenase perfusion experiments suggest that the hepatic accumulation of pCAT occurred preferentially in the nonparenchymal cells (NPC). The amount of total recovery in the liver decreased substantially by preceding administration of polyinosinic acid, dextran sulfate, succinylated bovine serum albumin, but not by polycytidylic acid. This suggests that pC AT is taken up by the liver via scavenger receptors for polyanions on the NPC. In constant infusion experiments, the presence of 2,4-dinitrophenol and NH₄C1 caused a significant increase in the outflow concentration of [³²P]pCAT and decrease by half in the total hepatic recovery than that of plasmid DNA administered alone, suggesting that plasmid DNA may undergo internalization by the NPC. Conclusions. The liver plays an important role in the elimination of plasmid DNA and a successful delivery system will be required to avoid its recognition by the scavenger receptors on the liver NPC.     

Significant Role of Liver Sinusoidal Endothelial Cells in Hepatic Uptake and Degradation of Naked Plasmid DNA After Intravenous Injection

Purpose. Uptake and degradation of naked plasmid DNA (pDNA) by liver sinusoidal endothelial cells (LSECs) were investigated. Methods. Tissue distribution and intrahepatic localization were determined after an intravenous injection of ¹¹¹In- or ³²P-labeled pDNA into rats. Cellular uptake and degradation of fluorescein- or ³²P-labeled pDNA were evaluated using primary cultures of rat LSECs. Results. Following intravenous injection, pDNA was rapidly eliminated from the circulation and taken up by the liver. Fractionation of liver-constituting cells by centrifugal elutriation revealed a major contribution of LSECs to the overall hepatic uptake of pDNA. Confocal microscopic study confirmed intracellular uptake of pDNA in cultured LSECs. Apparent cellular association of pDNA was similar at 37°C and 4°C. However, trichloroacetic acid (TCA) precipitation experiments showed the TCA-soluble radioactivity in the culture medium increased in an accumulative manner at 37°C. Involvement of a specific mechanism was demonstrated, as the uptake of pDNA was significantly inhibited by excess unlabeled pDNA and some polyanions (polyinosinic acid, dextran sulfate, heparin) but not by others (polycytidylic acid, dextran). These inhibitors also reduced the amount of TCA-soluble radioactivity in the culture medium. Conclusion. These results suggest that LSECs efficiently ingested and rapidly degraded naked pDNA in vivo and in vitro and released the degradation products into the extracellular space.     

Contribution of Parenchymal and Non-Parenchymal Liver Cells to the Clearance of Hepatocyte Growth Factor From the Circulation in Rats

Purpose. The distribution of ¹²⁵I-hepatocyte growth factor (HGF) to either liver parenchymal cells (PC) or non-parenchymal cells (NPC) was investigated in rats. Methods. After injection of a trace amount of ¹²⁵I-HGF, the distribution of radioactivity determined by microautoradiography closely resembled that of ¹²⁵I-epidermal growth factor which distributes mainly to PC. Results. The uptake clearance of ¹²⁵I-HGF estimated by determining the radioactivity of isolated liver cells was three times higher for PC than for NPC. This suggests that HGF distributes mainly to PC at relatively low doses. On the other hand, the uptake clearance by PC fell on coadministering an excess (80 µg/kg) of unlabeled HGF, while no change was observed for NPC, indicating that a saturable process for the hepatic handling of HGF exists only in PC where the HGF receptor is expressed. Conclusions. At such a dose the uptake clearance was comparable for both PC and NPC showing that HGF distributes to both cell types although NPC have few HGF receptors. Since the distribution to NPC was relatively non-specific and heparin-sensitive, it may be that heparin-like substances, which are believed to exist on PC and/ or the extracellular matrix, also exist on NPC.     

In Vivo Disposition Characteristics of Plasmid DNA Complexed with Cationic Liposomes

To control disposition and hence gene expression, we investigated the disposition characteristics of plasmid DNA complexed with the cationic liposomes Lipofectin_® and LipofectACE_® after intravenous injection in mice via the tail vein. The optimum ratios of DNA and liposome complexes were selected through in vitro cytotoxicity and transfection studies. The highest transfection was found at the DNA:liposome ratio of 1:5 w/w. Hence, this ratio was used for in vivo disposition studies, and the distribution patterns were compared with that of naked pCAT. Following intravenous injection of [₃₂P] pCAT, radioactivity was rapidly eliminated from plasma and approximately 60% of the dose was taken up by the liver within 1.5 min. In the case of LipofectACE_® samples, radioactivity elimination from plasma was equally rapid, but its accumulation was observed in both the liver (35%) and the lung (45%). For Lipofectin_® samples, radioactivity was initially accumulated in both the liver (55%) and the lung (25%), but lung accumulation was not sustained beyond 5 min after administration. Both liposomal samples showed in vivo gene expression in the lung, heart, kidney and spleen, but not in the liver. Thus, the present study demonstrated the disposition and gene expression of pCAT can be controlled by complex formation with liposomes.     

Development and Pharmacokinetics of Galactosylated Poly-L-Glutamic Acid as a Biodegradable Carrier for Liver-Specific Drug Delivery

Purpose. A biodegradable carrier for the liver-specific delivery of drugs was developed using poly-L-glutamic acid (PLGA) modified with galactose (galactosylated PLGA or Gal-PLGA), and its feasibility was investigated in mice. Methods. ¹¹¹In-PLGA and ¹¹¹In-Gal-PLGAs were injected in mice and their distribution and biodegradation properties were studied. Results. After intravenous injection, ¹¹¹In-PLGA was rapidly eliminated from the plasma and recovered mainly in the kidneys and urine. Approximately 15% of the dose was recovered in the liver, predominantly in the nonparenchymal cells. ¹¹¹In-Gal-PLGAs were taken up by the liver parenchymal cells. Derivatives having 16 or more galactose residues were taken up by the liver to a higher extent (>60% of the dose). The hepatic clearance of ¹¹¹n-Gal-PLGAs correlated with their number of galactose residues. ¹¹¹In-Gal₁₈-PLGA was degraded into low-molecular weight products in the liver. Conclusions. The advantageous in vivo properties of Gal-PLGA as a liver-specific biodegradable carrier of drugs were demonstrated in mice.     

Relative Contribution of Parenchymal and Non-parenchymal Cells to the Hepatic Distribution and Metabolism of Iron-poly(sorbitol-gluconic acid) Complex in the Rat

Abstract The hepatic cellular distribution in rats of radioiron and radiocarbon after [¹⁴C-gluconic acid] — and ⁵⁹Fe-labelled iron-poly(sorbitol-gluconic acid) complex, glusoferron (Ferastral®), has been investigated. Following administration of the ⁵⁹Fe-iron complex at a dose level corresponding to 10 mg of iron per kg bodyweight, the ratio of radioactivity per 10⁸ cells in parenchymal (P) and non-parenchymal (NP) cell fractions, the P/NP ratio, was 3.0 after 24 hours and rose to 10.4 at day 14. After 100 mg/kg the ratio was 0.9, 10.5 and 5.7 at day 1,4 and 14, respectively. Radiocarbon content in the different cell compartments fell steadily throughout. Radioiron from ⁵⁹Fe-iron-poly(sorbitol-gluconic acid) complex was shown to be incorporated into whole liver ⁵⁹Fe-ferritin. In vitro uptake studies with the different liver cells were also performed. Parenchymal cells were found to be more active than non-parenchymal cells with regard to cellular uptake of radiolabel from both iron complex and polymeric ligand without iron.     

Cellular localization of stable solid liposomes in the liver of rats

Small solid liposomes made from distearoylphosphatidyl choline and cholesterol (molar ratio 2 : 1) showed significant stability in plasma, with a half-life of about 24 hr after intravenous injection in rats. The major cellular uptake of intact liposomes was found in the liver and spleen, peaking after 2–4 hr in the liver and after 24 hr in the spleen. Isolation of parenchymal and non-parenchymal cells from rat livers at various intervals after injection of liposomes showed that both cell types adsorbed liposomal membranes and took up the liposomal contents. Our study has shown that most of the liposomal markers found in the liver shortly (< 40 min) after administration stemmed from the liposomes adsorbed to extracellular binding sites, and that uptake into the cells took place subsequently. In non-parenchymal cells, uptake was rapid and the intracellular level remained rather constant after 40 min and for up to 4 hr. The uptake of liposomes by parenchymal cells was slower, it showed a lag-phase of approx. $\text{1}\text{2}$ hr and peaked at 2 hr, whereupon the radioactivity in parenchymal cells dropped. The contents of liposomes behaved in a manner similar to the membranes. It is concluded that, in addition to a rapid uptake of liposomes in non-parenchymal liver cells, there is a significant degree of association with parenchymal cells, provided that the liposomes administered are small (< 100 nm in diameter) and stable.     

Targeting of stabilized plasmid lipid particles to hepatocytes in vivo by means of coupled lactoferrin

For non-viral gene delivery we prepared stabilized plasmid lipid particles (SPLPs), to which lactoferrin (LF) was coupled as a hepatocyte specific targeting ligand. LF–SPLPs and untargeted SPLPs labeled with [³H]cholesteryloleyl-ether were injected into rats. About 87% of the LF–SPLPs were eliminated from the blood within 5 min, while 80% of untargeted SPLPs were still circulating after 2 h. Fifty-two percent of the LF–SPLPs were taken up by hepatocytes, while non-parenchymal liver cells accounted for 16% of the uptake. Despite the efficient targeting of LF–SPLPs to hepatocytes and their capacity to transfect HepG2 and COS-7 cells in vitro, expression of a reporter gene was not detected in vivo. Overall, covalent coupling of LF to SPLPs leads to massive delivery in hepatocytes after systemic administration. However, these LF–SPLPs are not able to transfect these cells in vivo.     

Hepatitis B surface protein docked vesicular carrier for site specific delivery to liver

The intrinsic liver tropism of liposomes can be augmented by the addition of targeting features such as the incorporation of hepatotropic elements of the hepatitis viruses. Hepatitis B virus is known to infect hepatocytes after viremia by asialoglycoprotein receptor mediated uptake. However, the specificity of hepatitis B virus surface protein (HBsAg) towards hepatocytes has confronting reports. In the present study, we evaluated the functional ability of HBsAg to be employed as a ligand for targeting hepatocytes. We prepared ¹⁴C labeled small unilamellar vesicles (SUVs) composed of egg PC/Cholesterol/N-glutarylphosphatidylethanolamine (NGPE) in a 60:30:10 molar ratio. HBsAg was covalently linked to SUVs using a water-soluble carbodiimide (EDC) mediated conjugation with NGPE. In vitro cell binding and uptake studies revealed that bioprotein docked carrier system was efficiently taken up by HepG2 cells by the receptor mediated endocytosis. The biodistribution behaviour of plain and HBsAg coated liposomes was also examined followed by intravenous injection. The study revealed that almost 75% of the radioactivity was recovered in the liver after 4 h of injection that was nearly three-fold greater in magnitude than the plain liposomes. Further, fractionation of liver into liver parenchymal cells (PC) and non-parenchymal cells confirmed the preferential localization of the HBsAg coated liposomal carrier in the parenchymal cells.     

Targeting Efficiency of Galactosylated Liposomes to Hepatocytes in Vivo: Effect of Lipid Composition

Purpose. To investigate the effects of the lipid composition of galactosylated liposomes on their targeted delivery to hepatocytes. Methods. Several types of liposomes with a particle size of about 90 nm were prepared using distearoyl-L-phosphatidylcholine (DSPC), cholesterol (Chol) and cholesten-5-yloxy-N-(4-((1-imino-2-D-thiogalactosylethyl)amino)butyl)formamide (Gal-C4-Chol), and labeled with [³H]cholesterol hexadecyl ether. Their tissue disposition was investigated in mice following intravenous injection. The binding and internalization characteristics were also studied in HepG2 cells. Results. Compared with [³H]DSPC/Chol (60:40) liposomes, [³H]D-SPC/Chol/Gal-C4-Chol (60:35:5) liposomes exhibit extensive hepatic uptake. Separation of the liver cells showed that galactosylated liposomes are preferentially taken up by hepatocytes, whereas those lacking Gal-C4-Chol distribute equally to hepatocytes and nonparenchymal cells (NPC). Increasing the molar ratio of DSPC to 90% resulted in enhanced NPC uptake of both liposomes, suggesting their uptake via a mechanism other than asialoglycoprotein receptors. DSPC/Chol/Gal-C4-Chol (60:35:5) and DSPC/Chol/Gal-C4-Chol (90:5:5) liposomes exhibited similar binding to the surface of HepG2 cells, but the former were taken up faster by the cells. Conclusions. The recognition of galactosylated liposomes by the asialoglycoprotein receptors is dependent on the lipid composition. Cholesterol-rich galactosylated liposomes, exhibiting less non-specific interaction and greater receptor-mediated uptake, are better for targeting drugs to hepatocytes in vivo.     

Construction of nanostructured DNA harbouring phosphorodiamidate morpholino oligonucleotide for controlled tissue distribution in mice

Phosphorodiamidate morpholino oligonucleotides (PMOs) are a class of antisense oligonucleotides used in the treatment of neuromuscular diseases. Their major drawbacks are high blood clearance and poor cellular delivery. Previously, we demonstrated that tripod-like nanostructured DNA, or tripodna, was efficiently taken up by macrophages and dendritic cells. In this study, we used iodine-125(¹²⁵I)-labelled PMOs, designed a tripodna harbouring an ¹²⁵I-PMO (¹²⁵I-PMO/tripodna), and evaluated whether this tripodna could control the pharmacokinetic properties of PMO. Gel electrophoresis showed that ¹²⁵I-PMO was almost completely incorporated into the tripodna. Compared to ¹²⁵I-PMO, ¹²⁵I-PMO/tripodna was more efficiently taken up by macrophage-like RAW264.7 cells. Moreover, after intravenous injection into mice, the area under the plasma concentration–time curve of ¹²⁵I-PMO/tripodna was significantly larger than that of ¹²⁵I-PMO. The distribution of ¹²⁵I-PMO/tripodna in the liver and spleen at 24?h was 32- and 51-fold higher than that of ¹²⁵I-PMO, respectively. The fractionation of liver cells revealed that non-parenchymal cells were the major cells contributing to the hepatic uptake of ¹²⁵I-PMO/tripodna. These results indicate that tripodna has the potential to deliver PMO, particularly to the liver and spleen.     

Pharmacokinetics and Biodistribution of a Nucleotide-Based Thrombin Inhibitor in Rats

Purpose. To characterize the pharmacokinetic and tissue distribution profiles of a nucleotide-based thrombin inhibitor (GS522, phosphodiester oligonucleotide, GGTTGGTGTGGTTGG) following intravenous administration to rats. Methods. Pharmacokinetic study: 10 mg/kg, 20 mg/kg, 30 mg/kg (6 animals/dose) were administered to rats by rapid injection into the femoral vein. Blood samples were collected over a 45 minute period. Plasma concentrations of GS522 were determined using capillary gel electrophoresis with laser-induced fluorescence detection. Biodistribution Study: l0mg/kg (400l, 31.46 Ci/ml) of ³H-GS522 was administered to rats by rapid injection into the femoral vein. The animals were sacrificed by decapitation at 1, 5, 10, 30, 60, 360 minutes post-dose (3 rats/point). Brain, blood, duodenum, eyes, heart, kidney, liver, lungs, muscle, pancreas, skin, spleen and vein samples were collected, processed and quantitated using liquid scintillation counting. Results. The pharmacokinetic profile declines in multiexponential manner, exhibiting extremely fast distribution and elimination (t_1/2 = 7.6–9.0 min, Cl = 22.0–28.0 ml/min, V = 83.9–132.4 ml/kg). GS522 follows linear pharmacokinetics, with the area under the curve being proportional to the dose (Rsq = 0.9744). Highest radioactivity levels were detected in kidney, liver and blood (39.7, 15.7 and 15.3% dose/ respective organ). Less than 1% of the dose was detected in the heart, spleen and lungs, and >0.3% of the dose was found in the brain and eyes. The oligonucleotide associated radioactivity was uniformly distributed between the brain regions (left and right lobe and cerebellum). Six hours following the dose administration a statistically significant increase (p < 0.05) in radioactivity levels was observed in the brain, eyes, skin, liver, pancreas and vein. Conclusions. The pharmacokinetic and biodistribution profiles of GS522 following intravenous administration to rats at three doses were characterized. The oligonucleotide associated radioactivity was widely distributed in tissues. The amount of radioactivity sharply decreased with time in most tissues. Kidney, liver and muscle were the main sites of accumulation. The oligonucleotide associated radioactivity did not cross the blood brain barrier to an appreciable extent. In addition, a statistically significant increase (p < 0.05) in the radioactivity levels observed in select tissues suggested a re-uptake mechanism for intact oligonucleotide or its degradation products.     

Importance of mechanistic drug metabolism studies in support of drug discovery: A case study with an N -sulfonylated dipeptide VLA-4 antagonist in rats

N-(1-(3,5-dichlorobenzenesulfonyl)-2S-methyl-azetidine-2-carbonyl)-L-4-(2′,6′-dimethoxyphenyl)phenylalanine (1) is a potent antagonist of the very late activating (VLA) antigen-4. During initial screening, 1 exhibited an apparent plasma clearance (CL) of 227 ml min^?1 kg^?1 in Sprague–Dawley rats following an intravenous bolus dose formulated in an aqueous solution containing 40% polyethylene glycol. Such a high CL value led to speculation that the elimination of compound 1 involved extra-hepatic tissues. However, the apparent plasma CL was reduced to 97 ml in^?1 kg^?1 when a 2-min time point was added to sample collections, and further decreased to 48 ml min^?1 kg^?1 after the dose was formulated in rat plasma. The lung extraction of 1 in rats was negligible whereas the hepatic extraction was ≥90%, based on comparison of area under the curve (AUC) values derived from intra-artery, intravenous, and portal vein administration. In rats dosed intravenously with [¹⁴C]-1, approximately 91% of the radioactivity was recovered in bile over 48 h, with 85% accounted for in the first 4-h samples. The biliary radioactivity profile consisted of approximately 30% intact parent compound, 20% 1-glucuronide, and 50% oxidation products resulting from O-demethylation or hydroxylation reactions. When incubated with rat liver microsomes, oxidative metabolism of 1 was inhibited completely by 1-aminobenzotriazole (ABT), whereas the oxidation and glucuronidation reactions were little affected in the presence of cyclosporin A (CsA). In contrast, the hepatic extraction of 1 in rats was unperturbed in animals pre-dosed with ABT, but was reduced approximately 60% following treatment with CsA. In vitro, 1 was a substrate of the rat organic anion transporter Oatp1b2, and its cellular uptake was inhibited by CsA. In addition, the hepatic extraction of 1 was approximately 30% lower in Eisai hyperbilirubinaemic rats which lack functional multidrug resistant protein-2 (MRP2). Collectively, these data suggest that the clearance of 1 in rats likely is a result of the combined processes of hepatic oxidation, glucuronidation and biliary excretion, all of which are facilitated by active hepatic uptake of parent compound and subsequent active efflux of both unchanged parent and its metabolites into bile. It was concluded, therefore, that multiple mechanisms contribute to the clearance of 1 in rats, and suggest that appropriate pharmacokinetic properties might be difficult to achieve for this class of compounds. This case study demonstrates that an integrated strategy, incorporating both rapid screening and mechanistic investigations, is of particular value in supporting drug discovery efforts and decision-making processes.     

Pharmacokinetic and Tissue Distribution Mechanism of Mouse Recombinant Heat Shock Protein 70 in Mice

No HeadingPurpose. To investigate the in vivo pharmacokinetics and uptake mechanisms of recombinant mouse heat shock protein 70 (Hsp70) by hepatocytes in mice.Methods. The tissue distribution and intrahepatic localization of Hsp70 were determined after an intravenous injection of ¹¹¹In-Hsp70 (¹¹¹In-Hsp70) into mice. Ligands of CD91 or scavenger receptors were injected prior to Hsp70 to examine the involvement of these molecules on the distribution of ¹¹¹In-Hsp70. The uptake of ¹¹¹In-Hsp70 by primary mouse hepatocytes was also examined.Results. After intravenous injection, ¹¹¹In-Hsp70 was rapidly eliminated from the circulation and taken up mainly by the liver. The hepatic uptake was significantly inhibited by preinjection of ligands for CD91 or scavenger receptors. The separation of liver-constituting cells revealed a major contribution of hepatocytes to the overall hepatic uptake of ¹¹¹In-Hsp70. The uptake of ¹¹¹In-Hsp70 by cultured hepatocytes was inhibited by a CD91 ligand or anti-CD91 anibody. In addition, after subcutaneous injection, ¹¹¹In-Hsp70 gradually disappeared from the injection site and accumulated in primary lymph nodes.Conclusions. These results indicate for the first time that intravenous Hsp70 is, at least partially, recognized by CD91 and eliminated by hepatocytes, whereas subcutaneous Hsp70 is efficiently delivered to regional lymph nodes.     

Quantitative investigation of hepatobiliary transport of [11C]telmisartan in humans by PET imaging

The pharmacokinetics of telmisartan are nonlinear within the clinical dose range. To identify the underlying mechanism of this nonlinearity, we conducted a PET study in healthy subjects using [¹¹C]telmisartan. Eight healthy male subjects were enrolled in a 2-way crossover study. PET imaging was performed after intravenous administration of [¹¹C]telmisartan with or without a 1-h oral predose of two 40 mg Micardis® tablets. About 60% of the injected [¹¹C]telmisartan accumulated in the liver within 10 min after injection. With predosing of 80 mg telmisartan, the systemic elimination of [¹¹C]telmisartan was slightly delayed, but the liver exposure started to decrease earlier and biliary excretion was greatly enhanced. Hepatic uptake clearance of the radioactivity was not changed by telmisartan predosing, whereas the biliary clearance of radioactivity from the liver was significantly increased. Thus, the alteration in the pharmacokinetics of the radioactivity could not be explained simply by the saturation of hepatic uptake. Therefore, other mechanisms, such as the saturation of intracellular binding of telmisartan and/or its glucuronide, and the glucuronidation of telmisartan by uridine 5′-diphospho-glucuronosyltransferases, should be considered. This is the first reported human PET study using [¹¹C]telmisartan, the results of which can assist understanding of the hepatobiliary transport of telmisartan in humans.     

Cellular handling of a dexamethasone-anti-E-selectin immunoconjugate by activated endothelial cells: comparison with free dexamethasone

Purpose. For selective inhibition of endothelial cell activation in chronic inflammation, we have developed a dexamethasone-anti-E-selectin immunoconjugate. The present study was performed to evaluate the cellular handling of this immunoconjugate by activated primary endothelial cells and to compare its drug delivery capacity with free dexamethasone. Methods. The binding, uptake, and degradation of ¹²⁵I-radiolabeled dexamethasone-anti-E-selectin immunoconjugate by TNF-activated endothelial cells were studied for different time periods and at different concentrations, as well as in the presence of inhibitors for E-selectin binding and lysosomal degradation. Its drug delivery capacity was compared with the uptake of unconjugated ³H-labeled dexamethasone. Results. The immunoconjugate was internalized by E-selectin expressing activated endothelial cells and degraded in the lysosomal compartment. The receptor-mediated binding and uptake was saturable, implying a maximal attainable intracellular concentration of the drug. In contrast, free dexamethasone entered both resting and activated endothelial cells by passive diffusion. Conclusions. The dexamethasone-anti-E-selectin immunoconjugate is capable of selective delivering the coupled drug into activated endothelial cells. This targeting concept enables disease-induced drug delivery in which intracellular concentrations can be reached comparable with those obtained after incubation with 3 M dexamethasone.     

Synthetic Glycopeptide-Based Delivery Systems for Systemic Gene Targeting to Hepatocytes

Purpose. To design, synthesize, and test synthetic glycopeptide-baseddelivery systems for gene targeting to hepatocytes by systemicadministration.Methods. All peptides were synthesized by the solid phase methoddeveloped using Fmoc chemistry on a peptide synthesizer. The bindingof galactosylated peptides to HepG2 cells and accessibility of thegalactose residues on particle surface was demonstrated by acompetition assay using ¹²⁵I-labeleld asialoorosomucoid and RCA lectinagglutination assay, respectively. DNA plasmid encoding chloramphenicolacetyl transferase (CAT) gene was complexed with a tri-galactosylatedpeptide (GM245.3) or tri-galactosylated lipopeptide (GM246.3) in thepresence of an endosomolytic peptide (GM225.1) or endosomolyticlipopeptide (GM227.3) to obtain DNA particles of 100–150 nm insize. The plasmid/peptide complexes were added to HepG2 cell culturesor intravenously administered by tail vein injection into normal miceor rats. Plasmid uptake and expression was quantified by qPCR andELISA, respectively.Results. Multiple antennary glycopeptides that have the ability tocondense and deliver DNA plasmid to hepatocytes were synthesized andcomplexed with DNA plasmid to obtain colloidally stable DNA/peptidecomplexes. Addition of DNA/GM245.3/GM225.1 peptide complexes(1:3:1 (–/+/–)) to HepG2 cell cultures yielded CAT expression intransfected cells. The transfection efficiency was significantly reducedin the absence of galactose ligand or removal of endosomolytic peptide.Intravenous administration of DNA/GM245.3 peptide complexes (1:0.5(–/+)) into the tail vein of normal rats yielded DNA uptake in theliver. Substitution of GM245.3 by galactosylated lipopeptide GM246.3resulted in more stable DNA particles, and a 10-fold enhancement inliver plasmid uptake. CAT expression was detectable in liver followingintravenous administration of DNA/GM246.3 complexes. Addition ofendosomolytic lipopeptide GM227.3 into the complexes(DNA/GM246.3/GM227.3 (1:0.5:1 (–/+/–))) yielded a 5-fold increase inCAT expression. Liver expression was 8-fold and 40-fold higher thanlung and spleen, respectively, and localized in the hepatocytes only.The transfection efficiency in liver was enhanced by increasing DNAdose and injection volume. The plasmid uptake and expression in liverusing DNA/GM246.3/GM227.3 complexes was 100-200-fold higherthan DNA formulated in glucose. Tissue examination and serumbiochemistry did not show any adverse effect of the DNA/GM246.3/GM227.3 (1:0.5:1 (–/+/–)) complexes after intravenous delivery.Conclusions. Gene targeting to hepatocytes was achieved by systemicadministration of a well-tolerated synthetic glycopeptide-baseddelivery system. The transfection efficiency of this glycopeptide deliverysystem was dependent on peptide structure, endosomolytic activity,colloidal particle stability, and injection volume.     

Systemic Administration of TerplexDNA System: Pharmacokinetics and Gene Expression

Purpose. The aim of this study is to extend our previous studies to investigate the TerplexDNA synthetic gene carrier system in pharmacokinetics, biodistribution, and gene expression in major organs after systemic administration. Methods. The stability of the TerplexDNA system was analyzed in vitro with a serum incubation assay. The TerplexDNA PK/PD studies were conducted by quantitation of Terplex/radiolabeled DNA [CTP -³²P] complexes after rat-tail vein injection. The effect of the TerplexDNA system on gene expression in mouse major organs was analyzed by measuring luciferase activities after systemic administration. Results. The TerplexDNA gene carrier showed significantly longer retention in the vascular space than naked plasmid DNA alone. At early time points (1 h postvenous injection), the lung was the major organ of the TerplexDNA distribution, followed by the liver as a major distribution organ at later time points (24 h postinjection). The major organs of transgene expression after intravenous injection were the liver and heart. Conclusion. The TerplexDNA system has the potential for in vivo applications due to its higher bioavailability of plasmid DNA in the tissues, and due to its organ specific distribution.     

Distribution and Fate of the Insect Repellent 14C-N,N-diethyl-m–toluamide in the Animal Body. I. Distribution and Excretion after Injection into Mice

Abstract: The tissue distribution of ¹⁴C-labelled N,N-diethyl-m-toluamide (DEET), a widely used mosquito repellent, was studied in mice basically by means of whole-body autoradiography. After intravenous injection of the substance high tissue concentrations of radioactivity were found mainly in the liver, kidney, lacrimal gland, and nasal mucosa. At short time intervals after injection concentrations above the blood level were also seen in the thyroid and brown fat. Quantitative measurements revealed that the highest uptake was present in the lacrimal gland where radioactivity was also retained longer than in other tissues. Rapid excretion of the substance was found to take place mainly through the kidney. Thus, by 4 hours after injection, very little radioactivity remained in all tissues except the lacrimal gland. A placental barrier for the substance was observed, with much lower concentration in the foetuses of pregnant mice than in the mother.     

Pharmacokinetics of Differently Designed Immunoliposome Formulations in Rats with Or Without Hepatic Colon Cancer Metastases

Purpose. Compare pharmacokinetics of tumor-directed immunoliposomes in healthy and tumor-bearing rats (hepatic colon cancer metastases). Methods. A tumor cell-specific monoclonal antibody was attached to polyethyleneglycol-stabilized liposomes, either in a random orientation via a lipid anchor (MPB-PEG-liposomes) or uniformly oriented at the distal end of the PEG chains (Hz-PEG-liposomes). Pharmacokinetics and tissue distribution were determined using [³H]-cholesteryloleylether or bilayer-anchored 5-fluoro[³H]deoxyuridine-dipalmitate ([³H]FUdR-dP) as a marker. Results. In healthy animals clearance of PEG-(immuno)liposomes was almost log-linear and only slightly affected by antibody attachment; in tumor-bearing animals all liposomes displayed biphasic clearance. In normal and tumor animals blood elimination increased with increasing antibody density; particularly for the Hz-PEG-liposomes, and was accompanied by increased hepatic uptake, probably due to increased numbers of macrophages induced by tumor growth. The presence of antibodies on the liposomes enhanced tumor accumulation: uptake per gram tumor tissue (2-4% of dose) was similar to that of liver. Remarkably, this applied to tumor-specific and irrelevant antibody. Increased immunoliposome uptake by trypsin-treated Kupffer cells implicated involvement of high-affinity Fc-receptors on activated macrophages. Conclusions. Tumor growth and immunoliposome characteristics (antibody density and orientation) determine immunoliposome pharmacokinetics. Although with a long-circulating immunoliposome formulation, efficiently retaining the prodrug FUdR-dP, we achieved enhanced uptake by hepatic metastases, this was probably not mediated by specific interaction with the tumor cells, but rather by tumor-associated macrophages.