Koyo Nishida

Ternary complexes of pDNA, polyethylenimine, and γ-polyglutamic acid for gene delivery systems

We discovered a vector coated by gamma-polyglutamic acid (gamma-PGA) for effective and safe gene delivery. In order to develop a useful non-viral vector, we prepared several ternary complexes constructed with pDNA, polyethylenimine (PEI), and various polyanions, such as polyadenylic acid, polyinosinic-polycytidylic acid, alpha-polyaspartic acid, alpha-polyglutamic acid, and gamma-PGA. The pDNA/PEI complex had a strong cationic surface charge and showed extremely high transgene efficiency although it agglutinated with erythrocytes and had extremely high cytotoxicity. Those polyanions changed the positive zeta-potential of pDNA/PEI complex to negative although they did not affect the size. They had no agglutination activities and lower cytotoxicities but most of the ternary complexes did not show any uptake and gene expression; however, the pDNA/PEI/gamma-PGA complex showed high uptake and gene expression. Most of the pDNA/PEI/gamma-PGA complexes were located in the cytoplasm without dissociation and a few complexes were observed in the nuclei. Hypothermia and the addition of gamma-PGA significantly inhibited the uptake of pDNA/PEI/gamma-PGA by the cells, although l-glutamic acid had no effect. These results strongly indicate that the pDNA/PEI/gamma-PGA complex was taken up by gamma-PGA-specific receptor-mediated energy-dependent process. Thus, the pDNA/PEI/gamma-PGA complex is useful as a gene delivery system with high transfection efficiency and low toxicity.

Hepatic Disposition Characteristics of Electrically Charged Macromolecules in Rat in Vivo and in the Perfused Liver

The effect of electric charge on the hepatic disposition of macromolecules was studied in the rat. Charged derivatives of dextran (T-70) and bovine serum albumin (BSA), mitomycin C–dextran conjugates (MMC-D), and lactosaminated BSA (Lac-BSA) were employed as model macromolecules. After intravenous injection, cationic macromolecules were rapidly eliminated from plasma because of their extensive hepatic uptake, while anionic and neutral macromolecules were slowly eliminated. Cationic macromolecules were recovered from parenchymal and nonparenchymal hepatic cells at a cellular uptake (per unit cell number) ratio of 1.4–3.2, while that of Lac-BSA was 14. During liver perfusion using a single-pass constant infusion mode, cationic macromolecules were continuously extracted by the liver, with extraction ratios at steady-state (Ess) ranging between 0.03 and 0.54, whereas anionic and neutral macromolecules were almost completely recovered in the outflow at steady state. The Ess for cationized BSA (Cat-BSA) and cationic MMC-Dcat were concentration dependent and decreased at low temperatures and in the presence of colchicine and cytochalasin B. The possible participation of the internalization process in the uptake of cationic macromolecules by hepatocytes was suggested.

The development of a gene vector electrostatically assembled with a polysaccharide capsule.

The purpose of this study was to develop a gene vector electrostatically assembled with a polysaccharide capsule. We used pDNA/polyethylenimine (PEI) complexes as efficient non-viral vectors. The pDNA/PEI complex was electrostatically encapsulated with various polysaccharides such as fucoidan, lambda-carrageenan, xanthan gum, alginic acid, hyaluronic acid, and chondroitin sulfate (CS). The pDNA/PEI complex was shown as nanoparticles with positive zeta-potential, although the ternary complexes encapsulated with polysaccharides were shown as nanoparticles with negative zeta-potential. The pDNA/PEI complex showed high agglutination activity and cytotoxicity, although the ternary complexes encapsulated with polysaccharides had no agglutination activities and lower cytotoxicities. The pDNA/PEI complex showed high uptake and high transgene efficiency in B16-F10 cells. On the other hand, most of the ternary complexes show little uptake and gene expression. The ternary complex encapsulated by CS, however, showed comparable transgene efficiency to the pDNA/PEI complex. The uptake and gene expression of the ternary complex encapsulated by CS were significantly inhibited by hypothermia and the addition of CS, suggesting that the ternary complex was taken by CS-specific receptor-mediated energy-dependent process.

Different Effects of Absorption Promoters on Corneal and Conjunctival Penetration of Ophthalmic Beta-Blockers

AbstractPurpose. The purpose of this study was to investigate the improvement in corneal penetration of ophthalmic beta-blockers of various lipophilicities afforded by absorption promoters and to compare the corneal against conjunctival penetration in response to absorption promoters. Methods. The penetration of the beta-blockers, atenolol, carteolol, tilisolol, timolol, and befunolol, in the presence of absorption promoters, across the isolated corneal and conjunctival membranes of albino rabbits was measured using a two-chamber glass diffusion cell. EDTA, taurocholic acid, capric acid, and saponin were used as the absorption promoters. Results. The absorption promoters significantly increased the corneal permeability of most beta-blockers, especially the hydrophilic agents. The absorption promoters also enhanced the conjunctival permeability of beta-blockers, although their effect in promoting conjunctival penetration was less than that on corneal penetration. There was a differing penetration of instilled beta-blockers in the cornea and conjunctiva in response to absorption promoters. Capric acid and saponin showed significant promoting action on corneal penetration, but not on conjunctival penetration. Taurocholic acid had a significant effect on conjunctival penetration but not on corneal penetration. Saponin caused slight irritation. Conclusions. Absorption promoters can improve the ocular delivery of beta-blockers and a selective use of absorption promoter can improve the extent and pathway of drug ocular absorption.

Ophthalmic Preservatives as Absorption Promoters for Ocular Drug Delivery

The effects of ophthalmic preservatives on the drug permeability through isolated ocular membranes of albino rabbits were investigated using a two‐chamber glass diffusion cell. Tilisolol and fluorescein isothiocyanate (FITC)‐dextrans (average molecular weights 4400 and 9400 Da; FD‐4 and FD‐10, respectively) were used as model penetrants of ophthalmic β‐blockers and peptide drugs. Preservatives significantly enhanced the corneal penetration of not only tilisolol but also FITC‐dextrans. Especially, benzalkonium chloride increased the corneal permeability of FD‐4 and FD‐10 by 28·8 and 37·1 times, respectively. These results indicate the usefulness of ophthalmic preservatives as absorption promoters for the ocular delivery of β‐blockers and hydrophilic macromolecules. Preservatives also enhanced the conjunctival permeability of tilisolol, FD‐4 and FD‐10. The promoting effect of preservatives on the conjunctival drug penetration was smaller than that on the corneal one. Preservative increased the ratio of corneal to conjunctival permeability of tilisolol, FD‐4 and FD‐10.

Statistical Moment Analysis of Hepatobiliary Transport of Phenol Red in the Perfused Rat Liver

A new experimental system was applied to study hepatobiliary transport of drugs. Rat livers were perfused using a single-pass technique, and phenol red was momentarily introduced to this system from the portal side. Outflow dilution patterns of phenol red were analyzed using statistical moment theory, and kinetic parameters of hepatic distribution and elimination of phenol red were calculated from moments, namely, the hepatic extraction ratio (Ei) and elimination rate constant (kel,i). A larger distribution volume (Vi) was obtained for phenol red than for 131I-human serum albumin (HSA) and 51Cr-red blood cells (RBC), indicating its extravascular diffusivity. The biliary excretion of conjugated phenol red was delayed relative to that of the free agent. The larger biliary mean transit time (tbile,conj.) represents the processes of biliary transport and intrahepatic metabolism. Further, the effects of dose and perfusion temperature on the hepatobiliary transport of phenol red were determined. With high doses or low perfusion temperatures (20 and 27°C), Ei, kel,i, and intrinsic clearance (CLint,i) of phenol red and biliary recovery of free and conjugated phenol red (Fbile,free, Fbile,conj) significantly decreased. The temperature-dependent and saturable processes in hepatic uptake, metabolism, and biliary excretion of phenol red were assessable to moment analysis.

Absorption of Organic Anions as Model Drugs Following Application to Rat Liver Surface In‐vivo

Abstract— Absorption of organic anions (phenol red, bromphenol blue and bromosulphonphthalein) has been studied after their application to rat liver surface in‐vivo, employing a cylindrical glass cell (i.d. 9 mm, area 0·64 cm2). Each drug appeared gradually in the blood with the peak level at about 1 h, after which its concentration declined slowly. Absorbed model drug was efficiently excreted into the bile. These observations appear to indicate the possibility of drug absorption from liver surface membrane. Absorption of model drugs was estimated to be more than 59% in 6 h. The biliary recovery and metabolism of phenol red did not change as compared with that after intravenous administration.

One-side-coated insert as a unique ophthalmic drug delivery system

We newly prepared a unique one-side-coated insert that releases drug from only uncoated side. The purpose of this study is to determine whether ocular and systemic absorption of ophthalmic drug could be altered by an inserting direction of the insert in rabbit eyes. One-side-coated insert was prepared by attaching a polypropylene tape on the one side of the polymer disc of poly(2-hydroxypropyl methacrylate) (HPM) containing tilisolol as a model ophthalmic drug. The insert was applied in the lower conjunctival cul-de-sac of albino rabbits with the uncoated side facing bulbar conjunctiva/sclera (SC insert) or palpebral conjunctiva (CJ insert). At the adequate intervals, the tear fluid, plasma, aqueous humor, conjunctiva, and sclera were collected and the drug concentrations were determined by an HPLC. A release of tilisolol from the one-side-coated insert was twice slower than from the uncoated insert. Ocular application of the one-side-coated insert produced the constant concentrations of tilisolol in the tear fluid over 180 min. SC insert showed higher drug concentrations in the aqueous humor and sclera, and lower drug concentrations in the plasma and conjunctiva than CJ insert.The one-side-coated insert can alter the ocular and systemic absorption of drug by an inserting direction.

Effect of Preservatives on Systemic Delivery of Insulin by Ocular Instillation in Rabbits

Abstract— The effects of absorption promoters and ophthalmic preservatives on the systemic absorption of insulin through the ocular route were investigated in albino rabbits. Insulin absorption was evaluated by its hypoglycaemic response. Although ocular instillation of insulin alone did not decrease the serum glucose concentration, instillation of insulin with absorption promoters such as EDTA and saponin decreased it. The promoting effect depended on the concentration of the absorption promoters. Of ophthalmic preservatives investigated (benzalkonium chloride, paraben, 2‐phenylethanol, benzyl alcohol and sorbic acid), benzal‐konium, chloride and paraben showed promoting effects. The promoting effect for benzalkonium chloride was reversible and dependent on concentration of both benzalkonium chloride and insulin in the formulation.

Mechanism for drug absorption from rat-liver surface membrane: effect of dose and transport inhibitors on the pharmacokinetics of phenol red

We examined the effect of dose and transport inhibitors on the pharmacokinetics of phenol red as a model drug after application to rat liver surface in‐vivo, employing a cylindrical glass cell (i.d. 9 mm, area 0·64 cm2), to elucidate the mechanism for drug absorption from liver surface membrane.