Simple and rapid analysis of tocilizumab using HPLC-fluorescence detection method.

We developed a novel assay using high-performance liquid chromatography (HPLC) with fluorescence detection for the determination of tocilizumab (TCZ), after it has undergone a facile and rapid pretreatment. TCZ belongs to the same subclass as IgG1 (Immunoglobulin G subclass 1), and we could separate TCZ from IgG1 without antigen-antibody reactions, with the novel detection method. The separation of these antibodies was achieved by pretreatment with an organic solvent containing a base, such as trimethylamine and triethylamine. The effect of these bases on the separation of TCZ is related to the hydrophobicity of the base rather than the electrostatic charge. The results indicated that the surface charge of antibodies changed because of the structural change, even though the difference in the amino acid sequences of the antibodies was very low. Our method is available for the separation of the antibody subclasses, and it would be useful to assay TCZ in blood.

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.

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

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.

[Preparation and physicochemical study of the preservation of nanoparticles].

The importance of nanoparticle formulation is increasingly recognized in supporting pharmaceutical development. Thus, maintaining nanoparticles in a constant state is a major issue. A method involving lyophilization with the addition of saccharides can be used to maintain the steady state of nanoparticles. In this study, trisaccharides, tetrasaccharides, and pentasaccharides were added to nanoparticle suspensions, followed by rehydration of the samples, which had been either dried normally or freeze-dried. The particle size after rehydration was measured. In addition, each powder was measured using a powder X-ray diffractometer and thermal analysis device to investigate the correlation between the nanoparticles' aggregation and the crystal form of saccharides. The diameter of the nanoparticles was maintained when it was freeze-dried, while particle aggregation occurred when normally dried samples were used. In addition, crystalline saccharide was not observed in the freeze-dried group, but did appear in the normally dried group.

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.

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).

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.

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, κ-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.

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.

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.

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.

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 and stabilization of nifedipine lipid nanoparticles.

Design methods of nanoparticle formulations are divided into a break-down method and a build-up method. Furthermore, 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. In this study, we investigated the preparation of nifedipine (NI) nanoparticles without using any organic solvent. NI nanoparticles with a mean particle size of approximately 50 nm could be prepared without organic solvent by a combination of roll mixing and high-pressure homogenization. The X-ray diffraction peak of the sample prepared by roll mixing was present at an identical position (2theta) to that of NI crystals, showing that no peak shift was induced by interaction with lipid. These findings clarified that most NI remained as crystals in lipid. To maintain the particle size of the nanoparticles in suspension for a long time, a method of adding gelatin powder to the NI-lipid nanoparticle suspension, dissolving the mixture by heating, and then solidifying by cooling was investigated. The mean particle size of the sample was about 55 nm, and that after heat-liquefaction of the NI-lipid nanoparticle suspension gelated at 5 degrees C for 24h was also about 55 nm, showing that the nanoparticle condition was retained.

Physical characteristics of freeze-dried griseofulvin-lipids nanoparticles.

We have attempted to prepare micronized drug particles and to maintain the micronized state long-term in order to improve the solubility of a practically insoluble drug, griseofulvin (GF). GF nanoparticles (GFNPs) prepared by high-pressure homogenization were micronized to about 45 nm (mean particle size). GFNPs were subjected to transmission electron microscopy (TEM) observation. The mean particle size remained at about 50 nm for 3 months at 25 degrees C. However, increased to about 300 nm in 6 months. After 5% (w/v) sugar was added, GFNP was freeze-dried and rehydrated. The mean particle size of the rehydrated GFNPs was about 160 nm when the sugar was a monosaccharide. In contrast, it was about 55-65 nm when the sugar was a disaccharide. The powder X-ray diffraction pattern of the monosaccharide-containing freeze-dried GFNPs revealed a crystal state of sugar. On the other hand, that of the disaccharide-containing freeze-dried GFNPs indicated an amorphous state of sugar. From this result, it is considered that the high viscosity of an amorphous sugar prevents GFNPs from aggregating through retardation of molecular movement. Freeze-dried GFNPs stored for six months at 25 degrees C showed a good re-dispersibility. After the rehydration the mean particle size of GFNPs was 55-65 nm. It was found that freeze-dried GFNPs containing sucrose, maltose or trehalose were stable for longer periods than GFNP suspensions.