A Novel Amphotericin B Hydrogel Composed of Poly(Vinyl Alcohol)/Borate Complex for Ophthalmic Formulation.

In this study, we developed a water-soluble complex-hydrogel viscosity-controlled formulation of amphotericin B (AmB). AmB is insoluble in water, but borax makes it soluble by forming a complex with AmB. Borax also forms complexes with poly(vinyl alcohol) (PVA) to produce viscous hydrogels. Furthermore, boric acid interacts with mucin expressed in corneal epithelial cells. Accordingly, by utilizing these properties of borax simultaneously, we prepared a water-soluble AmB complex-hydrogel with poly(vinyl alcohol)/borate (PVA-B-AmB), which is suitable for eye drops. PVA-B-AmB was easily prepared by simply mixing aqueous AmB solution dissolved in borax, PVA solution, and water. The 11B-NMR results suggested that PVA-B-AmB existed by bonding PVA and AmB via boronic acid. PVA-B-AmB (gel ratio = 0.55) has a viscosity of 18.3 ± 0.5 mPa·s and is suitable for ophthalmic formulations. This formulation exhibited sustained release of AmB of approximately 45% at 24 h. It was also shown that this formulation interacts with mucin. These results suggest that PVA-B-AmB can be used as a water-soluble AmB preparation suitable for ophthalmic use.

Amphotericin B nanohydrogel ocular formulation using alkyl glyceryl hyaluronic acid: Formulation, characterization, and in vitro evaluation.

The present study focused on the development of an amphotericin B (AmB) nanoformulation for ophthalmic applications. Accordingly, AmB nanohydrogels (AHA/AmB) using alkyl glyceryl hyaluronic acid (Hyalorepair®, AHA), a hydrophobized hyaluronic acid, were prepared by employing the dialysis method, followed by assessments of physical properties, drug efficacy, and toxicity. In the AHA/AmB formulation, AmB existed in a self-aggregated and amorphous state in the hydrophobic environment of the AHA moiety. AHA/AmB was shown in vitro to interact with mucin, which is known to be expressed in the corneal epithelium and was expected to improve its corneal retention. Compared with the conventional AmB formulation, amphotericin B sodium deoxycholate, AHA/AmB had the same in vitro antifungal activity but significantly lower in vitro toxicity. These findings indicate that nanohydrogels prepared with AHA possess high fungal selectivity and serve as a promising system for ophthalmic AmB delivery.

Development of an amphotericin B micellar formulation using cholesterol-conjugated styrene-maleic acid copolymer for enhancement of blood circulation and antifungal selectivity.

Amphotericin B (AmB) is an effective antifungal agent for life-threatening systemic fungal infections. However, its poor solubility in water and organic solvents makes it difficult to formulate. We previously reported AmB-encapsulated micellar formations using styrene-maleic acid copolymer (SMA) and butylated SMA. These micelles make AmB water-soluble; however, the blood circulation of AmB by these intravenous administrations was as low as that of Fungizone®, a conventional micellar formulation of AmB. The destabilization of SMA micelles by salt in the blood has been suggested to be a cause of low blood circulation. Therefore, in this study, to reduce salt-induced instability and increase blood circulation of the micelles, we covalently attached cholesterol molecules to the SMA backbone because AmB interacts with sterols. This AmB nanoparticle micellar formulation (Cho-SMA/AmB micelles) was water-soluble, stable in the presence of salts, and formed a complex with albumin. Compared with Fungizone®, this formulation had equal antifungal activity and markedly improved blood circulation and lower toxicity. Its toxicity was further reduced in the presence of albumin. Taken together, our results indicate that Cho-SMA/AmB micelles could be an intravenous formulation with high antifungal selectivity, and drug interactants-conjugated SMA system could be applied to a variety of drug-loaded nanomicellar systems.

[Nanoparticle Formation and Delivery of Poorly Water-soluble Drugs by Hydrophobized Polymers].

When a hydrophobic group is introduced into a water-soluble polymer, self-assembly with the hydrophobic group as nucleus occurs in water. In the 1990s, many researchers focused on this phenomenon and various self-aggregates were prepared. Among them, a block copolymer consisting of a hydrophilic chain and a hydrophobic chain is associated in water, producing polymer micelles with the hydrophilic chain oriented in the outer shell and the hydrophobic chain as core. Meanwhile, many studies were conducted to create polymer self-associates by introducing hydrophobic groups into water-soluble polymers. In this review, the author describes hydrophobized polymers with polysaccharides and synthetic polymers that are frequently used as pharmaceutical raw materials. In addition are outlined the usefulness of hydrophobized polymers as carriers with the function of encapsulating and solubilizing poorly water-soluble drugs, along with the results of our research.

Coenzyme Q10-Polyethylene Glycol Monostearate Nanoparticles: An Injectable Water-Soluble Formulation.

Therapeutic applications of coenzyme Q10 (CoQ10) are greatly limited by its lack of solubility in aqueous media. In this study, polyethylene glycol monostearate (stPEG) was used to construct micelles containing CoQ10 as a new formulation. The micellar formulations (stPEG/CoQ10) were prepared using five types of stPEG with 10, 25, 40, 55, and 140 PEG repeat units, respectively. The micellar preparation was simple, consisting of only stPEG and CoQ10. Next, we compared the physical properties and blood circulation of these micelles. The CoQ10 load of this formulation was approximately 15 w/w%. Based on the dynamic light scattering method, the average molecular size of the stPEG/CoQ10 micelles was approximately 15 to 60 nm. The zeta potentials of these micelles were approximately -10 to -25 mV. The micelles using stPEG25, 40, and 55 demonstrated high solubility in water. Furthermore, these micelles had in vitro antioxidant activity. On comparing the blood circulation of micelles using stPEG25, 40, 55, and 140, micelles using stPEG55 had a significantly higher circulation in blood. The stPEG55/CoQ10 micelle demonstrated a protective effect against acetaminophen-induced liver injury in mice. In conclusion, these data indicate that the intravenous administration of the stPEG/CoQ10 micellar aqueous formulation is of great value against oxidant stress.

Synthesis and evaluation of styrene-maleic acid copolymer conjugated amphotericin B.

Amphotericin B (AmB), which plays a central role in the treatment of systemic fungal infections, is difficult to formulate because it's sparingly soluble in water and organic solvents. We previously prepared AmB-loaded micelles using styrene-maleic acid copolymer (SMA). Although solubilization was achieved by this formulation, stability in the blood circulation was as insufficient as that of Fungizone®, which is a conventional formulation of AmB. Meanwhile, it is well known that polymer-drug conjugates are more stable in circulation than drug-loaded micelles. Therefore, in this study, we developed covalently conjugated SMA-AmB (SMA-AmB conjugate). The SMA-AmB conjugate was found to be soluble and present as micelles in aqueous solution. Furthermore, it was revealed that this micelle behaves as a larger molecule by forming a complex with albumin. The circulation in the blood increased significantly compared to that of Fungizone®, which was suggested to be due to this complex-forming ability. Although in vitro and in vivo antifungal activity of the SMA-AmB conjugate against Saccharomyces cerevisiae was reduced by 1/3 compared to that of Fungizone®, hemolysis decreased to 1/40 or less, and the LD50 decreased to 1/10. In conclusion, it is expected that the SMA-AmB conjugate can be a polymer-therapeutic agent with high antifungal selectivity.

Synthesis and evaluation of water-soluble poly(vinyl alcohol)-paclitaxel conjugate as a macromolecular prodrug.

Paclitaxel (PTX) is an antitumor agent for the treatment of various human cancers. Cremophor EL and ethanol are used to formulate PTX in commercial injection solutions, because of its poor solubility in water. However, these agents cause severe allergic reaction upon intravenous administration. The aim of this study is to synthesize water-soluble macromolecular prodrugs of PTX for enhancing the therapeutic efficacy. Poly(vinyl alcohol) (PVA, 80 kDa), water-soluble synthetic polymer, was used as a drug carrier which is safe and stable in the body. The 2'-hydroxyl group of PTX was reacted with succinic anhydride and then carboxylic group of the succinyl spacer was coupled to PVA via ethylene diamine spacer, resulting the water-soluble prodrug of poly(vinyl alcohol)-paclitaxel conjugate (PVA-SPTX). The solubility of PTX was greatly enhanced by the conjugation to PVA. The release of PTX from the conjugate was accelerated at the neutral to basic conditions in in vitro release experiment. [125 I]-labeled PVA-SPTX was retained in the blood circulation for several days and was gradually distributed into the tumorous tissue after intravenous injection to the tumor-bearing mice. PVA-SPTX inhibited the growth of sarcoma 180 cells subcutaneously inoculated in mice. It was suggested that the water-solubility of PTX was markedly enhanced by the conjugation to PVA, and PVA-SPTX effectively delivered PTX to the tumorous tissue due to the enhanced permeability and retention (EPR) effect.

Synthesis of poly(vinyl alcohol)-doxorubicin conjugates containing cis-aconityl acid-cleavable bond and its isomer dependent doxorubicin release.

Aconityl-doxorubicin (ADOX) was synthesized by the modified method of Shen and Ryser. Two isomers of cis-ADOX (cis-configuration) and trans-ADOX (trans-configuration) were generated in the reaction of DOX and cis-aconitic anhydride. These products were separated completely by using HPLC and analyzed by TOF-MS spectroscopy and (1)H- and (13)C-NMR experiments. The yields of cis-ADOX and trans-ADOX were 36.3 and 44.8%, respectively. The free gamma-carboxylic group of ADOX molecule was coupled to poly(vinyl alcohol) (PVA) via ethylenediamine spacer, resulting the macromolecular conjugates of PVA-cis-ADOX and PVA-trans-ADOX, respectively. The DOX content of the conjugates estimated by the hydrolysis method detected the aglycone of DOX which can be estimated as the PVA-bound DOX selectively was 4.4 w/w% which was similar to 4.6 w/w% by the ordinary UV method. Both PVA-cis-ADOX and PVA-trans-ADOX were very stable at neutral pH, but the release of DOX was increased markedly under acidic conditions. Half-life of the release of DOX from PVA-cis-ADOX at pH 5.0 was 3 h which was 4.7-fold shorter than that from PVA-trans-ADOX (14 h). The cytotoxicities of PVA-cis-ADOX and PVA-trans-ADOX were evaluated by using J774.1 cells employing a [(3)H]uridine incorporation assay as a measure of RNA synthesis. A significant difference in antitumor activity between PVA-cis-ADOX and PVA-trans-ADOX was observed where the former was much active than the later. It was suggested that the conjugate enters the cells and reaches the lysosomal/endosomal compartment, and that the aconityl spacer releases DOX from the conjugate in the acidic compartment of lysosomes/endosomes due to the participation of a free carboxylic group.

Uptake of pullulan in cultured rat liver parenchymal cells.

Uptake of pullulan, including a binding process followed by internalization, was examined in cultured rat liver parenchymal cells. A tyramine derivative of pullulan was labeled with [125I]iodine and used as a ligand. Pullulan bound to the cell surface was released by EDTA treatment, indicating that pullulan binding requires Ca2+ and a contribution from the asialoglycoprotein receptor. Binding of pullulan reached a steady state and internalization represented a biphasic mode, which included first- and zero-order processes in the initial stage and after 20 min incubation, respectively. The uptake of pullulan could be estimated by a similar model for intracellular disposition of asialofetuin. Kinetic parameters of pullulan constituting both binding and internalization were below those found for asialofetuin. These results suggest that pullulan is taken up by liver parenchymal cells via the asialoglycoprotein receptor; however, uptake availability is lower than that of asialofetuin.

Pharmacokinetics and biodisposition of poly(vinyl alcohol) in rats and mice.

Poly(vinyl alcohol) (PVA) of various molecular weight (MW=10,560-116,600) was successfully labeled with fluorescein isothiocyanate isomer I (FITC) according to the method of de Belder and Granath. A high-performance size-exclusion chromatographic procedure was developed for the quantitative analysis of FITC-labeled poly(vinyl alcohol) (F-PVA) in biological samples. F-PVA (80 K) disappeared slowly from the blood circulation according to the first-order kinetics (t1/2=7 h) after intravenous injection to rats. A dose-independent behavior of F-PVA (80 K) was observed in the blood circulation, in the tissue distribution and in the urinary and fecal excretions. This suggested that PVAs are eliminated exclusively by the mechanisms that do not involve saturable transport processes. Furthermore, it was found that PVAs are very stable in the body because no degradation product was detected in the urine and feces. 125I-labeled poly(vinyl alcohol) (125I-PVA) was prepared by introducing tyramine residues to the hydroxyl groups of PVA molecules by the 1,1'-cabonyldiimidazole (CDI) activation method. 125I-PVA (80 K) was retained in the blood circulation for several days after intravenous injection to mice. Although the tissue distribution of PVAs was small, a significant accumulation into the liver and the spleen was observed. Fluorescence microscopic examination of paraffin section of the liver revealed that F-PVA (80 K) was endocytosed by the liver parenchymal cells. 125I-PVA (80 K) captured by liver was slowly transported via the bile canaliculi and gall bladder to the intestine and excreted in the feces. It was suggested, therefore, a long time is necessary for 125I-PVA (80 K) to be excreted perfectly from the body.

Intracellular disposition of polysaccharides in rat liver parenchymal and nonparenchymal cells.

Binding and internalization of arabinogalactan, pullulan, dextran, and mannan were examined in rat liver parenchymal and nonparenchymal cells using 125I or fluorescein isothiocyanate (FITC) labeled polysaccharides. Binding and uptake of arabinogalactan and pullulan into parenchymal cells was inhibited by asialofetuin, indicating that the asialoglycoprotein receptor is involved in the intracellular disposition of arabinogalactan and pullulan. Uptake of 125I-labeled dextran to parenchymal cells was unchanged upon addition of excess unlabeled dextran, suggesting that dextran uptake occurs via fluid phase endocytosis. Of the polysaccharides tested, mannan showed the strongest specific association with liver nonparenchymal cells. FITC-labeled polysaccharides showed arabinogalactan and pullulan are internalized to liver parenchymal cells, whereas mannan is internalized to nonparenchymal cells. This study demonstrates that intracellular disposition of polysaccharides in the liver occurs via receptor-mediated endocytosis (RME), indicating that RME plays a role in the biodisposition of these polysaccharides as drug carriers.

Cellular disposition of arabinogalactan in primary cultured rat hepatocytes.

To characterize a targeting property of arabinogalactan (AG) as a carrier to the liver, we examined cellular disposition, such as binding and internalization in primary cultured rat hepatocytes, comparing them to those of asialofetuin (AF). A tyramine derivative of AG was synthesized to allow labeling with 125I. Binding of AG to the cells was concentration-dependent and saturable. The number of binding sites (n) of AG on the cell surface was 4.0 x 10(5) +/- 0.1 x 10(5) sites per cell which was about similar to that of AF. The value of Ka of AG was 2.2 x 10(8) +/- 0.1 x 10(8) M-1 being seven-fold higher than that of AF. The binding of AG was competitively inhibited by AF and was decreased by calcium depletion. These results indicate that AG can bind strongly to hepatocytes probably through the recognition by the asialoglycoprotein receptor (ASGP-R). Both 125I-labeled AG and fluorescein-labeled AG were internalized into the cells. The rate of internalization of AG was faster than that of AF, indicating that AG is effectively endocytosed. Microscopic observations showed that FITC labeled AG accumulated in granules within the primary cultured rat hepatocytes. Subcellular fractionation indicated that the internalized AG was mainly associated with the lysosomal fraction. However, the internalized AG seemed to remain intact in the hepatocytes. In conclusion, we found that AG is effectively internalized in primary cultured rat hepatocytes. Although AG seems a good candidate for targeting to the liver due to its high affinity binding and rapid internalization, it remains to be established whether the apparent lack of biodegradation will result in cytotoxic effects at chronic administration in vivo.