Shigeo Takemura

Targeted delivery of plasmid DNA complexed with galactosylated poly(L-lysine).

Galactose was introduced to poly(L-lysine) (PLL) with an average molecular weight of 13,000 to develop a hepatocyte-specific carrier for gene drugs. The pharmacokinetic characteristics of a model plasmid, pCAT (plasmid DNA encoding chloramphenicol acetyltransferase reporter gene), complexed with galactosylated PLL (Gal-PLL) was studied in mice in relation to its physicochemical properties. pCAT/Gal-PLL complex at a ratio of 1:0.6 (w/w) has a zeta potential of -20 mV and a mean particle size of about 180 nm. After intravenous injection, [32P]pCAT/Gal-PLL was rapidly eliminated from the circulation and preferentially taken up by the livers parenchymal cells. The hepatic uptake of [32P]pCAT/Gal-PLL was significantly inhibited by prior administration of Gal-bovine serum albumin, suggesting that the uptake was mediated by the asialoglycoprotein receptors on hepatocytes. In vitro transfection experiments using a hepatoma cell line expressing the asialoglycoprotein receptor revealed that pCAT/Gal-PLL gave a high CAT gene expression whereas pCAT complexed with unmodified PLL failed to transfect the cells.

Physicochemical and disposition characteristics of antisense oligonucleotides complexed with glycosylated poly(l-lysine)

The disposition characteristics of a 20 mer antisense phosphodiester oligonucleotide (PO) and its fully phosphorothioated derivative (PS) alone or complexed with glycosylated poly(L-lysine) (galactosylated polylysine, Gal-PLL; mannosylated polylysine, Man-PLL) were studied in mice in relation to their physicochemical characteristics. Good complex formation was obtained at a ratio of 1:0.6, w/w [oligonucleotides (ODNs)/carrier]. The 1:0.6 weight ratio of ODNs/Gal-PLL and ODNs/Man-PLL complexes had zeta potentials of -27 to -31 mV and mean particle size of 100 to 160 nm. After intravenous injection, 35S-labeled ODNs were eliminated rapidly from the circulation; however, their organ disposition characteristics depended on their type. Complex formation with glycosylated PLL increased the hepatic uptake and decreased the urinary clearance of these ODNs to a great extent. These complexes were taken up by both liver parenchymal cells (PC) and nonparenchymal cells (NPC). However, ODNs/Gal-PLL complexes showed a fairly high PC concentration, whereas ODNs/Man-PLL complexes distributed equally to both PC and NPC. The hepatic uptakes of PS/Gal-PLL and PS/Man-PLL complexes were partially inhibited by prior administration of Gal-BSA and Man-BSA, respectively, suggesting their hepatic uptake via the respective receptor-mediated endocytosis. However, uptake by galactose receptors of Kupffer cells, zeta potential, particle size, and Kupffer cell phagocytosis also seem to influence their uptake process. In conclusion, this study illustrates that ODNs can be delivered to hepatocytes and macrophages via galactose and mannose receptors, respectively.

Studies on lactulose formulations for colon-specific drug delivery

A novel, colon-targeted delivery system (CODES), which uses lactulose, was investigated in this study. Lactulose is not absorbed in the upper GI tract, but degraded to organic acids by enterobacteria in the lower gastrointestinal tract, especially the colon. A CODES consists of three components: a core containing lactulose and the drug, an inner acid-soluble material layer, and an outer layer of an enterosoluble material. When a CODES containing a pigment was introduced into the rat cecum directly after shaking in JP 2nd fluid for 3 h, pigment release was observed 1 h after introduction. A CODES containing 5-aminosalicylic acid (5-ASA) was orally administered to fasting and fed dogs to evaluate its pharmacokinetic profiles. 5-ASA was first detected in plasma after 3 h, which is the reported colon arrival time for indigestible solids, after dosing to fasting dogs. The T(max) in fed dogs was delayed by 9 h when compared to fasting dogs. This corresponds to the gastric emptying time. However, the C(max) and AUC under fed conditions were almost as same as those under fasting conditions. The results of this study show that lactulose can act as a trigger for drug release in the colon, utilizing the action of enterobacteria.

Pharmacokinetics and In Vivo Gene Transfer of Plasmid DNA Complexed with Mannosylated Poly(L-Lysine) in Mice

Abstract To achieve mannose receptor-mediated, cell-specific, in vivo gene transfer by intravenous injection of plasmid DNA, mannosylated poly(L-lysine) (Man-PLL) was synthesized as a carrier molecule, and mixed with a plasmid DNA encoding chloramphenicol acetyl-transferase (CAT) gene to form DNA/Man-PLL complex. The particle size and zeta potential of DNA/Man-PLL (prepared at 1:0.7 on a weight basis) were determined to be 220 nm and +12 mV, respectively. The pharmacokinetics of the DNA/Man-PLL complex was assessed in mice using 32P-labeled DNA ([32P]DNA). After intravenous injection of [32P]DNA/Man-PLL, the radioactivity in plasma fell rapidly and was recovered mainly in the liver nonparenchymal cells. The amount in the liver reached more than 80% of the dose. Radioactivity observed in kidney, lung, and spleen was very low compared to that in the liver. Then, the in vivo gene expression after intravenous injection of DNA/Man-PLL was examined by a CAT assay. Highest CAT activity was detected in the liver, but no activity was detected in the lung, kidney, and spleen. These results clearly indicate that a cell-specific gene delivery system can be developed by regulating the biodistribution of DNA/carrier complex through the control of its physicochemical properties.

Evaluation of Factors Affecting Gastrointestinal Absorption of a Novel Anticoagulant FX-93 for Development of Oral Formulation

To find out factors causing the low bioavailability of FX-93, a novel anticoagulant, its solubility, membrane permeability, and the effect of bile salt on the absorption of FX-93 were investigated. The solubility of FX-93 under physiological conditions ranged from 0.3 to 18.3 mg/mL and the dose number was calculated to be 0.02-0.27, suggesting that the intrinsic solubility of FX-93 should not be a limiting factor for oral absorption. Apparent permeability of FX-93 across Caco-2 cell monolayer suggested that its fraction of dose absorbed would range between 30% and 40% in humans. Furthermore, FX-93 was substantially absorbed from each segment of rat intestine. However, the decrease in the gastrointestinal transit rate significantly decreased maximum plasma concentration and area under the plasma concentration-time curve of FX-93 after oral dosing in dogs, suggesting that FX-93 absorption would be suppressed by some components in the small intestinal lumen. An in situ rat administration study indicated that bile significantly decreased the intestinal absorption of FX-93 by two-thirds, which could be attributed to the decrease in FX-93 solubility by the interaction with bile or bile acid. Nuclear magnetic resonance spectroscopy analysis suggested that FX-93 would interact with bile salt between the naphthalene ring of FX-93 and steroidal backbone of bile salt.

Aminoalkylmethacrylate copolymer E improves oral bioavailability of YM466 by suppressing drug-bile interaction

The aim of this study was to find out polymeric compounds that can inhibit the interaction between YM466, a novel anticoagulant, and bile to improve its oral bioavailability. In vitro ultrafiltration method using extract gall powder was useful to detect the formation of insoluble complex of YM466 with bile and also used to select a polymer that can inhibit the interaction between YM466 and bile. The in vitro studies revealed that aminoalkylmethacrylate (AAM) copolymer E, a polymethacrylate, dose-dependently inhibited the interaction between YM466 and bile and that this polymer could interact with bile salt, but not with YM466, possibly by electrostatic and/or hydrophobic interactions. The coadministration of AAM copolymer E with YM466 to rats dose-dependently increased the plasma concentration of YM466 and it was found that the oral dose of the polymer three times of YM466 (polymer to drug ratio in weight, P-D ratio, 3) significantly increased AUC0-1 h of YM466 to 2.6-fold of that of YM466 alone. Considering the condition of therapeutic use of YM466 and the maximum tolerated dose of the polymer, the formulation of P-D ratio 3 would be clinically practical and promising from the viewpoint of safety.