Yoshiharu Kaneo

Evidence for receptor-mediated hepatic uptake of pullulan in rats

Fluorescein-labeled pullulan (FP-60; MW 58,200) was prepared by reaction with FITC according to the method of de Belder and Granath. The hepatic distribution of FP-60 was examined using a specific high-performance size-exclusion chromatography. Intravenously administered FP-60 was rapidly eliminated from the blood circulation followed by an appreciable distribution to the liver. A marked dose-dependency was seen in the hepatic uptake of FP-60 which was markedly reduced by the coadministration of both asialofetuin and arabinogalactan. Measurement of the hepatocellular localization demonstrated the overwhelming distribution of FP-60 in the parenchymal liver cell fraction. Furthermore, microscopic examination revealed that FP-60 was effectively endocytosed by the parenchymal liver cells. Radiolabeled pullulan ([(125)I]P-60) was prepared by (125)I-labeling the tyramine derivative of pullulan which was synthesized by the cyano-transfer method. [(125)I]P-60 was predominantly accumulated in sliced rat liver tissue at 37 degrees C, which was drastically inhibited by the addition of both asialofetuin and arabinogalactan. The kinetic parameters of the specific binding of [(125)I]P-60 to monolayered hepatocytes at 0 degrees C were almost identical to those for asialofetuin. The binding of [(125)I]P-60 to isolated parenchymal cells was significantly inhibited by arabinogalactan and asialofetuin, however dextran, the same glucan as pullulan, did not affect the binding of [(125)I]P-60. It was found that pullulan, which is bound to the asialoglycoprotein receptor with high affinity, is subsequently internalized to the hepatocyte via receptor-mediated endocytosis.

Pharmacokinetics and biodisposition of fluorescein-labeled arabinogalactan in rats.

Fluorescein-labeled arabinogalactan (FA) was prepared by the reaction with FITC in methyl sulphoxide according to the method of deBelder and Granath. A systemic kinetic analysis of FA in rats was carried out by using a specific high-performance size-exclusion chromatography. Intravenously administered FA was rapidly eliminated from the blood circulation followed by an appreciable distribution to the liver and kidney. FA was accumulated in these organs over a long period whereas negligible levels of FA were detected in the other organs. A marked dose-dependency was seen in the hepatic uptake of FA which was markedly reduced by coinjected asialofetuin whereas the renal uptake of FA was not altered. Measurement of the hepatocellular localization demonstrated the overwhelming distribution of FA in the parenchymal liver cell fraction. Furthermore, the microscopic examination revealed FA that was effectively endocytosed by the parenchymal liver cells. These results suggested that FA which is bound to the asialoglycoprotein receptor with a high affinity is subsequently internalized to the hepatocyte via receptor-mediated endocytosis. FA was partially activated by periodate oxidation in order to acquire aldehyde groups to which guest molecules can be bound. A 12.5% oxidized arabinogalactan keeping a hepatocellular targetability showed a good conjugating reactivity to guest molecules via Schiff-base formation or by reductive amination. It was suggested that arabinogalactan can serve as a potential carrier for the delivery of enzymes and drugs to the parenchymal liver cells via the asialoglycoprotein receptor.

Nanoparticles of hydrophobized cluster dextrin as biodegradable drug carriers: solubilization and encapsulation of amphotericin B

Cluster dextrin (CDex), a highly branched cyclic dextrin, is a novel glucose polymer that is produced from a waxy corn starch by a branching enzyme. Despite its large molecular weight (462 kDa), CDex is highly water-soluble and is easily digested by enzymes, such as α-amylase. Amphotericin B (AmB) is a broad-spectrum fungicidal antibiotic used primarily in the treatment of life-threatening systemic fungal infections. Unfortunately, AmB is insoluble in water; therefore, we have attempted to render it soluble and transportable by encapsulating it in hydrophobized CDex nanoparticles. The amphiphilic polymers of CDex were prepared with hydrophobic groups bonded through ester bonds, and the degrees of substitution were 1.00 mol/mol % for n-octanoyl CDex, 1.80 mol/mol % for stearoyl CDex, 1.07 mol/mol % for oleoyl CDex, and 1.07 mol/ mol % for cholesteryl CDex. To study the formation of the monodispersed nanoparticles of the hydrophobized CDexs by self-assembly and their complex formations with AmB, size-exclusion chromatography and dynamic light scattering were utilized. AmB encapsulated in the cholesteryl CDex nanoparticle was found to be non-hemolytic, even at 30 μg/mL, which suggested that it had a much higher concentration than the minimum inhibitory concentration of 0.78 μg/mL required to inhibit Saccharomyces cerevisiae. Therefore, the biodistributions of the AmB-loaded cholesteryl CDex nanoparticles and AmBisome (liposomal AmB) were further compared in mice. After intravenous injection of the AmB-loaded cholesteryl CDex nanoparticle, prolonged persistence of AmB in the circulating blood was observed.

Preparation and characterization of a soluble glutathione-dextran conjugate

Glutathione (GSH), a naturally occurring antidote, cannot permeate into the liver when given extracellularly and has a very short half-life in the body. A dextran conjugate of GSH was synthesized by coupling GSH covalently to dextran (T-40, Mw = 43,900) by the CNBr activation method in order to improve these disadvantages. We have demonstrated that GSH is delivered effectively into the hepatic cells by the conjugate which protects mice from the acetaminophen hepatotoxicity (Kaneo Y. et al., Int. J. Pharm., 44 (1988) 265–267). The conjugate was a water-soluble white powder containing 10% w/w of GSH. The molecular weight of the conjugate was distributed more widely than the original dextran and that of the peak estimated by size exclusion chromatography was 2.5 × 105. The isoelectric point of the conjugate was estimated to be 2.5 by the cellulose acetate paper electrophoresis. Kinetics of GSH regeneration from the conjugate was examined at various pH values. The conjugate significantly stabilized GSH and liberated it gradually at physiological conditions (t12 = 99 min). Tripeptide GSH has one sulfhydryl, one amino, and two carboxyl groups in the molecule. It was found that the sulfhydryl groups participate in chemical bonding between GSH and CNBr-activated dextran and appear gradually by the cleavage of the bonding according to the determination of sulfhydryl groups by the method of Ellman. It was confirmed that the content of free amino groups of the conjugate is very high by the measurement of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS). These results indicated that at least 80% of the conjugated GSH are attached to dextran via sulfhydryl groups. This may contribute to chemical stability against autoxidation of the thiol group and then to the advantageous features of the conjugate as a macromolecular prodrug.

Incorporation of Amphotericin B Into Self-Assembled Hydrophobized Kollicoat IR Nanoparticles

Kollicoat IR (KOL) is a new poly(ethylene glycol)-poly(vinyl alcohol) graft copolymer that is a promising material for medical applications because of its biocompatibility and hydrophilic nature. Amphotericin B (AmB) is a broad-spectrum fungicidal antibiotic used primarily in the treatment of life-threatening systemic fungal infections. However, AmB is limited in clinical use because of its poor water solubility. In this study, we synthesized AmB-loaded hydrophobized KOL nanoparticles for the first time. These self-assembled, stable nanoparticles exhibited a high AmB content. Among the hydrophobized KOL nanoparticles, a cholesterol-grafted KOL nanoparticle reduced AmB toxicity with respect to hemolysis and effectively increased the overall water solubility of AmB. Furthermore, a relatively high retention of AmB in the plasma was demonstrated in vivo in an animal experiment at early time after injection, suggesting that the cholesterol-grafted KOL nanoparticles could enable new pharmaceutical applications for AmB.