Seitaro Kamiya

Improving frequency of thrombosis by altering blood flow in the carrageenan-induced rat tail thrombosis model.

The carrageenan-induced tail thrombosis model in rats and mouse is useful for evaluating compounds having antithrombotic effects in drug discovery projects as an in vivo model. However, the frequency of thrombosis is low, and the signs of thrombosis are not constant under the standard conditions. In this study, we modified this model on the basis of hypercoagulability, endothelial injury, and alteration in the normal blood flow. The simple ligation to alter blood flow in rats significantly improved the frequency and signs of thrombosis after intravenous kappa-carrageenan injection. In particular, 1mg/kg kappa-carrageenan injection in combination with 10 min ligation at the tail increased the frequency of thrombosis to almost 100% in rats. These results suggest that this modified model has great advantages over the previously used conditions for evaluating the effects of antithrombotic and thrombolytic agents.

Preparation of griseofulvin nanoparticle suspension by high-pressure homogenization and preservation of the suspension with saccharides and sugar alcohols.

Aim: We have attempted to micronize drug particles with a particle size of less than 100 nm and maintain the particle size of their suspension to improve the solubility and bioavailability of poorly water-soluble drugs. Furthermore, the method of freeze-drying nanoparticles was applied to maintain particulate nature of nanoparticles containing various saccharides and sugar alcohols for a long time. Method: Griseofulvin (GF)–lipid nanoparticle suspension is prepared using GF and a lipid by high-pressure homogenization. The particle size of the obtained GF–lipid nanoparticle suspension is maintained constant by freeze-drying. Result: The mean particle size of GF–lipid nanoparticles prepared by high-pressure homogenization is approximately 60 nm. The mean particle size remains less than 100 nm for 1 month. The GF–lipid nanoparticle suspension containing xylitol, trehalose, or sucrose is freeze-dried to maintain the particulate nature. The mean particle size of the rehydrated suspension is lower than that of the rehydrated suspension containing erythritol or lactose. In particular, it is new knowledge to have found that an aggregation is minimized by adding xylitol which is sugar alcohol. The minimum concentration of xylitol, trehalose, and sucrose required to maintain a constant particle size by rehydration is 3%, 3%, and 5% (w/v), respectively.

Transcutaneous immunization by a solid-in-oil nanodispersion

We have successfully achieved transcutaneous immunization without the use of any skin pre-treatment or immune-stimulant adjuvant by applying a solid-in-oil (S/O) nanodispersion: an oil-based nanodispersion of antigens coated with hydrophobic surfactant molecules. This finding indicates that the S/O nanodispersion has great promise for effective transcutaneous vaccination.

In vivo Evaluation Method of the Effect of Nattokinase on Carrageenan-Induced Tail Thrombosis in a Rat Model

Thrombosis is characterized by congenital and acquired procatarxis. Nattokinase inhibits thrombus formation in vitro. However, in vivo evaluation of the therapeutic efficacy of nattokinase against thrombosis remains to be conducted. Subcutaneous nattokinase injections of 1 or 2 mg/ml were administered to the tails of rats. Subsequently, ĸ-carrageenan was intravenously administered to the tails at 12 h after nattokinase injections. The mean length of the infarcted regions in the tails of rats was significantly shorter in rats administered 2 mg/ml of nattokinase than those in control rats. Nattokinase exhibited significant prophylactic antithrombotic effects. Previously, the in vitro efficacy of nattokinase against thrombosis had been reported; now our study has revealed the in vivo efficacy of nattokinase against thrombosis.

Physical properties of griseofulvin-lipid nanoparticles in suspension and their novel interaction mechanism with saccharide during freeze-drying.

Size reduction of drug particles to the nanoscale is important in improving the dissolution rate of poorly water-soluble drugs. The aim of this study was to investigate the physicochemical properties of griseofulvin (GF)-lipid nanoparticles and the interactions between GF-lipid nanoparticles and various saccharides during freeze-drying. The phase transition temperature of the GF-lipid nanoparticle suspension was 56.8 degrees C, whereas that of the lipid nanoparticle suspension alone was 57.9 degrees C, indicating that the GF crystals were incorporated into the lipid phase. The mean particle size of a rehydrated suspension of xylose-containing freeze-dried GF-lipid nanoparticles was about 220 nm. However, the mean particle size on the rehydration of nanoparticles containing mannose (monosaccharide), fructose (disaccharide), lactose (disaccharide), or raffinose (trisaccharide) was about 60 nm, suggesting that these saccharides prevented aggregation during the freeze-drying process. Powder X-ray diffraction revealed that xylose existed in the crystalline state in the freeze-dried nanoparticles, whereas the other saccharides existed in amorphous states. Thus, the crystallization of the saccharide was found to be strongly correlated with the aggregation property of the nanoparticles. In the case of freeze-dried xylose, the nanoparticles were squeezed out as the saccharine crystal lattice arranged itself regularly. Then, the ejected nanoparticles were aggregated. In contrast, in the case of the other freeze-dried saccharide, the saccharide remained incorporated with the GF-lipid nanoparticles because its crystal lattice was arranged irregularly. Thus, the particle size was maintained.

The Effect of Lactic Acid Bacteria-fermented Soybean Milk Products on Carrageenan-induced Tail Thrombosis in Rats.

Thrombosis is characterized by congenital and acquired procatarxis. Lactic acid bacteria-fermented soybean milk products (FS-LAB) inhibit hepatic lipid accumulation and prevent atherosclerotic plaque formation. However, the therapeutic efficacy of FS-LAB against thrombosis has yet to be investigated. In this study, FS-LAB were administered subcutaneously into the tails of rats, with the subsequent intravenous administration of κ-carrageenan 12 hr after the initial injection. In general, administration of κ-carrageenan induces thrombosis. The length of the infarcted tail regions was significantly shorter in the rats administered a single-fold or double-fold concentration of the FS-LAB solution compared with the region in control rats. Therefore, FS-LAB exhibited significant antithrombotic effects. Our study is the first to characterize the properties of FS-LAB and, by testing their efficacy on an in vivo rat model of thrombosis, demonstrate the potency of their antithrombotic effect.

The physicochemical interactive mechanism between nanoparticles and raffinose during freeze-drying

New methods of preparing nanoparticles and in vivo studies of their behavior have been the subject of much study. However, there exist few studies on maintaining the nanoparticle size. In this work, we report on the interaction mechanism between raffinose and nanoparticles during freeze-drying. The mean particle size of the rehydrated freeze-dried raffinose-containing nanoparticles (170.5 nm) was similar to the initial particle size before freeze-drying (156.1 nm), indicating that the particle size was maintained. The powder X-ray diffraction of the freeze-dried raffinose-containing nanoparticles shows a halo pattern, while that of the normal-dried raffinose shows a crystalline pattern. No endothermic peak of the freeze-dried raffinose appeared, while the normal-dried raffinose had an endothermic peak at 84.0 °C. These results suggest that there exists a relationship between the nanoparticles and the raffinose, and that the relationship depends on whether the mixture is freeze-dried or normal-dried. In the case of normal drying, the raffinose molecules have space and time to arrange themselves into regular arrangement because the nanoparticles and raffinose molecules can move around freely in water. In contrast, in the case of freeze-drying, the moisture was sublimed while the raffinose molecules and nanoparticles were immobilized in the ice, thereby preventing aggregation.

Physicochemical interaction mechanism between nanoparticles and tetrasaccharides (stachyose) during freeze-drying

Abstract Nanoparticle suspensions are thermodynamically unstable and subject to aggregation. Freeze-drying on addition of saccharides is a useful method for preventing aggregation. In the present study, tetrasaccharides (stachyose) was employed as an additive. In addition, we hypothesize the interactive mechanism between stachyose and the nanoparticles during freeze-drying for the first time. The mean particle size of the rehydrated freeze-dried stachyose-containing nanoparticles (104.7 nm) was similar to the initial particle size before freeze-drying (76.8 nm), indicating that the particle size had been maintained. The mean particle size of the rehydrated normal-dried stachyose-containing nanoparticles was 222.2 nm. The powder X-ray diffraction of the freeze-dried stachyose-containing nanoparticles revealed a halo pattern. The powder X-ray diffraction of the normally dried stachyose-containing nanoparticles produced mainly a halo pattern and a partial peak. These results suggest an interaction between the nanoparticles and stachyose, and that this relationship depends on whether the mixture is freeze-dried or dried normally. In the case of normal drying, although most molecules cannot move rapidly thereby settling irregularly, some stachyose molecules can arrange regularly leading to some degree of crystallization and potentially some aggregation. In contrast, during freeze-drying, the moisture sublimed, while the stachyose molecules and nanoparticles were immobilized in the ice. After sublimation, stachyose remained in the space occupied by water and played the role of a buffer material, thus preventing aggregation.

Development of a new evaluation method for gelatin film sheets.

Gelatin film sheets are often used to form the shell in soft capsules prepared using the rotary die method. Examination of the film sheet properties is extremely important when materials other than gelatin are used. We examined the relationship between the width and the tensile strength of the gelatin film sheet in order to establish the required indexes of the gelatin film sheets. The coefficients of correlation (R) of the linear regressions 4 and 15 min after preparation of the gelatin film sheets were 0.9858 and 0.9167, respectively. These results suggest that the tensile strength decreased when the width of the gelatin film sheet increased. Subsequently, we examined the relationship between time and the tensile strength. We observed new phenomenon: the tensile strength of the gelatin sheets increased with time, following two phases. The first phase corresponded to over phase transition point at 4, 8, and 15 min and the second phase corresponded to under transition point at 15, 30, and 60 min. The R values were 0.9952 (4, 8, 15 min) and 0.9494 (15, 30, 60 min).

Preparation of Nanoparticles Including Antisolvent Drugs by the Combination of Roll Milling and High-pressure Homogenization

Description: Design methods of nanoparticle formulations are divided into break-down methods and build-up methods. The former is further divided into dry and wet processes. For drug nanoparticle preparations, the wet process is generally employed, and organic solvents are used in most formulations. Method: In this study, we investigate the preparation of nifedipine (IB) and griseofulvin (GF) nanoparticles without using organic solvent. Both IB and GF nanoparticles, with a mean particle size of approximately 50 nm, were prepared without organic solvent by employing a combination of roll milling and high-pressure homogenization. Result: The X-ray diffraction peak of the IB and GF samples prepared by roll milling was present at a position (2θ) identical to that of IB and GF crystals, indicating that no peak shift was induced by interaction with phospholipids. Conclusion: These findings demonstrate that most IB and GF nanoparticles exist as crystals in phospholipids.