Scaling up nano-milling of poorly water soluble compounds using a rotation/revolution pulverizer.

We previously reported that a rotation/revolution pulverizer (NP-100) could mill a small amount of a drug (0.1 g) into nanoparticles in several minutes. In this investigation, scale up from the milligram to the kilogram scale of the nano-milling process by the rotation/revolution pulverizer was studied. Phenytoin was used as a model drug with low solubility in water. After confirming the improvement of the phenytoin bioavailability by milling to nanoparticles using NP-100, scaling parameters were evaluated using NP-100 and the middle scale model of NP-100 (ARV-3000T). A theoretical equation for the specific collisional energy was adapted for wet milling; this suggested that the relative centrifugal acceleration of revolution (revolution G) and the drug concentration in the suspension were the two most important parameters. The results obtained using NP-100 and ARV-3000T correlated well when these two parameters were identical. These results were applied to the large scale model of NP-100 (ARV-10KT), where 2 kg (1 kg x 2) of phenytoin nanoparticles were obtained in 60 min. The results from PXRD and DSC indicated that the milled phenytoin by ARV-3000T and ARV-10KT maintained its crystallinity. These results suggest nano-milling using a rotation/revolution pulverizer will be widely applicable to the development of nano-medicine.

Nano-pulverization of poorly water soluble compounds with low melting points by a rotation/revolution pulverizer.

We report a method for pulverizing poorly water soluble compounds with low melting points to nanoparticles without producing an amorphous phase using a rotation/revolution pulverizer. Fenofibrate, flurbiprofen, and probucol were used as crystalline model compounds. They were suspended in a methylcellulose aqueous solution and pulverized with zirconia balls by the rotation/revolution pulverizer. Beeswax, an amorphous compound, was also examined to investigate whether nano-pulverization of a compound with a low melting point was possible. Beeswax was suspended in ethyl alcohol cooled with liquid nitrogen and pulverized with zirconia balls by the rotation/revolution pulverizer. By optimizing the pulverization parameters, nanoparticles (D50 < 0.15 microm) of the crystalline compounds were obtained with narrow particle size distributions at a rotation/revolution speed of 1000 rpm and a rotation/revolution ratio of 1.0 when the vessel was 0 degrees C. Amorphous fenofibrate and flurbiprofen were not detected by differential scanning calorimetry or powder X-ray diffraction, whereas small amounts of amorphous probucol were detected. Beeswax was pulverized to nanoparticles (D50 = 0.14 microm) with ethyl alcohol cooled with liquid nitrogen. Fine nanoparticles of these poorly water soluble compounds with low melting points were obtained by controlling the rotation/revolution speed and reducing the vessel temperature.

Glucagon-like peptide-1 receptor agonist ameliorates renal injury through its anti-inflammatory action without lowering blood glucose level in a rat model of type 1 diabetes.

AIMS/HYPOTHESIS: Glucagon-like peptide-1 (GLP-1) has various extra-pancreatic actions, in addition to its enhancement of insulin secretion from pancreatic beta cells. The GLP-1 receptor is produced in kidney tissue. However, the direct effect of GLP-1 on diabetic nephropathy remains unclear. Here we demonstrate that a GLP-1 receptor agonist, exendin-4, exerts renoprotective effects through its anti-inflammatory action via the GLP-1 receptor without lowering blood glucose. METHODS: We administered exendin-4 at 10 µg/kg body weight daily for 8 weeks to a streptozotocin-induced rat model of type 1 diabetes and evaluated their urinary albumin excretion, metabolic data, histology and morphometry. We also examined the direct effects of exendin-4 on glomerular endothelial cells and macrophages in vitro. RESULTS: Exendin-4 ameliorated albuminuria, glomerular hyperfiltration, glomerular hypertrophy and mesangial matrix expansion in the diabetic rats without changing blood pressure or body weight. Exendin-4 also prevented macrophage infiltration, and decreased protein levels of intercellular adhesion molecule-1 (ICAM-1) and type IV collagen, as well as decreasing oxidative stress and nuclear factor-κB activation in kidney tissue. In addition, we found that the GLP-1 receptor was produced on monocytes/macrophages and glomerular endothelial cells. We demonstrated that in vitro exendin-4 acted directly on the GLP-1 receptor, and attenuated release of pro-inflammatory cytokines from macrophages and ICAM-1 production on glomerular endothelial cells. CONCLUSIONS/INTERPRETATION: These results indicate that GLP-1 receptor agonists may prevent disease progression in the early stage of diabetic nephropathy through direct effects on the GLP-1 receptor in kidney tissue.

Adherence of oral streptococci to an immobilized antimicrobial agent.

An antimicrobial agent, 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride, was immobilized on silica. Interaction between the material (termed) OAIS) and various oral bacterial species were then studied. Seven species of Streptococcus and two Actinomyces were investigated for their ability to adhere to this biomaterial. Cell-surface hydrophobicity and zeta-potential were examined as well. Analysis of extracted hydrophobic proteins which adhered to OAIS revealed that the adherence of these micro-organisms was closely related to the hydrophobicity of their cell surfaces. The results of zeta-potential assays indicated that negative charge on the cell surface inhibited adherence to OAIS. Gel electrophoresis revealed that OAIS could absorb cell-surface hydrophobic proteins from all bacterial species tested. Preadsorption of hydrophobic components on the cell surface inhibited adherence of the Strep. mutans strain to OAIS in a dose-dependent manner. The results indicate that OAIS adsorption of these oral bacteria was dependent on the degree of hydrophobicity of their surfaces. A major component of this adherence was hydrophobic cell-surface proteins.

Adhesion in vitro of oral streptococci to porcelain, composite resin cement and human enamel.

We investigated the initial adhesion in vitro of oral streptococci to porcelain inlays. Four strains of streptococci, Streptococcus mitis NCTC12261, Streptococcus oralis ATCC9811, Streptococcus sanguis ATCC10556 and Streptococcus sobrinus OMZ176, were used in this study. The disc specimens were made of porcelain, composite resin cement and human enamel. These specimens, with or without a saliva-coat, were immersed in a suspension of each streptococci strain at 37 degrees C for 1 hr, and the numbers of cells adhering to specimens were counted after staining. The saliva-coat significantly decreased the numbers of adhering cells in all strains tested (t-test, p < 0.05). The adhesion to the porcelain and resin cements could be explained by a thermodynamic approach, although the adhesion to enamel could not be explained. These results indicated the possibility that the mechanism is different in initial adhered strains of streptococci between enamel and dental restorative materials.

Effect of hesperidin in vitro on root dentine collagen and demineralization.

OBJECTIVES: Caries progress might be controlled when collagen matrix could be preserved after demineralization. The aim of this pH cycling study was to investigate the effect of hesperidin, a citrus flavonoid antioxidant, on dentine collagen and remineralization in dentine lesion, and compared with that of chlorhexidine. METHODS: The pH cycling was employed on bovine root dentine by demineralization for 14 h, incubation in testing solutions (hesperidin or chlorhexidine) for 2 h and remineralization with bacteria-derived collagenase for 8 h, for 8 days. Calcium release was measured by means of an atomic absorption spectrophotometer, and degraded collagen matrix by collagenase was investigated by assaying hydroxyproline. The lesion depth and mineral loss was evaluated by means of transverse microradiography. RESULTS: The effect of testing solutions had a significant difference on the results of chemical analyses (p<0.0115 for calcium release; p<0.0008 for degradated collagen). The lesion depth and mineral loss were reduced in the lesions where were incubated with hesperidin and chlorhexidine. The remineralization in deep lesions was found when the matrix was incubated in hesperidin, whilst no mineral uptake in deep lesion when incubated in chlorhexidine. CONCLUSION: Hesperidin preserved collagen and inhibited demineralization, and enhanced remineralization even under the fluoride-free condition.

Bactericidal effect of dentin primer containing antibacterial monomer methacryloyloxydodecylpyridinium bromide (MDPB) against bacteria in human carious dentin.

The aim of this study was to investigate the bactericidal effect of a dentin primer incorporating the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) against bacteria in human dentinal carious lesions. To evaluate the antibacterial activity of MDPB against anaerobes, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) against obligate anaerobes and facultative anaerobic strains of lactobacilli were determined. Bacteria were recovered from carious dentin samples obtained from the teeth of patients, and the bactericidal activities of the experimental primer containing 5% MDPB and three commercially available primers were compared by counting the number of viable cells after contact with diluted solutions of each primer for 30 s. MDPB showed strong antibacterial activity against anaerobes, the MIC and MBC values ranging from 3.9 to 31.3 micrograms/ mL-1 and 15.6-125 micrograms/ mL-1, respectively. Experimental primer containing MDPB was the most bactericidal among the materials tested (ANOVA, Fisher's PLSD test, P < 0.05) and was able to kill the bacteria completely even when diluted 40 times, while the three commercial products exhibited little activity at 40 times dilution. These results indicate that incorporation of MDPB into dentin primer could be beneficial for eliminating the residual bacteria in cavities.

Confocal laser scanning microscopic analysis of early plaque formed on resin composite and human enamel.

The purpose of this study was to analyse quantitatively the early bacterial plaque formed on resin composite and human enamel in vivo, using a confocal laser scanning microscope. Test pieces of resin composite and human enamel were retained at the buccal surfaces of the upper first molars of three volunteers for 4, 8 and 24 h to allow plaque formation. Then, the specimens were immersed in propidium iodide in phosphate-buffered saline to stain adherent bacteria and observed with a confocal laser scanning microscope. The ratios of the area occupied by microorganisms to the whole area of the optical field were calculated using a photo-image analysis system. The thickness of the plaque was also measured. Quantitative analysis revealed that the resin composite showed significantly higher bacterial adherence than human enamel throughout the test period. A difference was noticed in the morphology of the bacteria between the two groups. Our findings suggest that resin composite shows higher bacteria adherence during early plaque formation compared with human enamel. In addition, the present findings may suggest a presence of the difference in bacterial composition of plaque in both specimens.