Kazuhiro Debari

Self-etching dentin primers containing phenyl-P.

The dentin bonding efficacies of two commercial dentin bonding systems and experimental self-etching dentin primers composed of methacryloxyethyl hydrogen phenyl phosphate (Phenyl-P) and either hydroxyethyl methacrylate (HEMA) or glyceryl methacrylate (GM, 2,3-dihydroxypropyl methacrylate) were examined. The wall-to-wall polymerization contraction gap width of a commercial light-activated resin composite in a cylindrical dentin cavity and the tensile bond strength to a flat dentin surface were measured. Changes in dentin hardness were determined by Micro Vickers Hardness measurement, and an SEM observation was performed after priming. Formation of a contraction gap was completely prevented by the application of Phenyl-P diluted in HEMA or GM solution combined with a commercial dentin bonding agent, although gap formation was evident in nearly half of the specimens with both commercial dentin bonding systems. The mean tensile bond strengths of the tested groups varied from 16.3 to 20.7 MPa, and there were no significant differences between groups. Based on the measurement of Micro Vickers Hardness and SEM observation after priming, a slight reduction in dentin hardness was observed. However, this reduction in dentin hardness due to self-etching priming did not significantly correlate with either contraction gap width or tensile bond strength.

Physicochemical and pharmacological characterization of α-tocopherol-loaded nano-emulsion system.

The main purpose of the present study is to develop a novel nano-emulsion (NE) formulation of alpha-tocopherol (alpha-TC) with enhanced oral bioavailability and pharmacological effects. Three NE formulations of alpha-TC at different loading amounts (10%, 30% and 50%) were prepared by a mechanochemical method. Physicochemical properties of NE formulations were characterized with a focus on the morphology by transmission electron microscopy (TEM), droplet size distribution and zeta-potential by dynamic light scattering (DLS), and long-term stability. According to the TEM images and DLS data, mean diameters of NE droplets ranged from 80 to 400nm, in proportion to the amount of loaded alpha-TC. Although all NE formulations of alpha-TC were found to be negatively charged with the zeta-potential of ca -40mV, NE formulations at alpha-TC content of 30% or higher exhibited severe aggregation of droplets in NE formulations during long-term storage. After oral administration of 10% alpha-TC-loaded NE formulation (30mg alpha-TC/kg) in rats, higher alpha-TC exposure was observed with a 2.6-fold increase of bioavailability as compared to the control mixture of oil and alpha-TC. In streptozotocin-induced diabetic rats, oral administration of the alpha-TC-loaded NE formulation (30mg alpha-TC/kg) exhibited a significant reduction of lipoperoxidant in several organs, especially the liver; however, the control mixture was less effective. With these findings, the NE approach might be efficacious to improve the oral bioavailability and anti-oxidative activities of alpha-TC.

Mishandling of the Therapeutic Peptide Glucagon Generates Cytotoxic Amyloidogenic Fibrils

AbstractPurpose. Some therapeutic peptides exhibit amyloidogenic properties that cause insolubility and cytotoxicity against neuronal cells in vitro. Here, we characterize the conformational change in monomeric therapeutic peptide to its fibrillar aggregate in order to prevent amyloidogenic formation during clinical application. Methods. Therapeutic peptides including glucagon, porcine secretin, and salmon calcitonin were dissolved in acidic solution at concen- trations ranging from 1 mg/ml to 80 mg/ml and then aged at 37°C. Amyloidogenic properties were assessed by circular dichroism (CD), electron microscopy (EM), staining with β-sheet-specific dyes, and size-exclusion chromatography (SEC). Cytotoxic characteristics were determined concomitantly. Results. By aging at 2.5 mg/ml or higher for 24 h, monomeric glucagon was converted to fibrillar aggregates consisting of a β-sheet-rich structure with multimeric states of glucagon. Although no aggregation was observed by aging at the clinical concentration of 1 mg/ml for 1 day, 30-day aging resulted in the generation of fibrillar aggregates. The addition of anti-glucagon serum significantly inhibited fibrillar conversion of monomeric glucagon. Glucagon fibrils induced significant cell death and activated an apoptotic enzyme, caspase-3, in PC12 cells and NIH-3T3 cells. Caspase inhibitors attenuated this toxicity in a dose-dependent manner, indicating the involvement of apoptotic signaling pathways in the fibrillar formation of glucagon. On the contrary to glucagon, salmon calcitonin exhibited aggregation at a much higher concentration of 40 mg/ml and secretin showed no aggregation at the concentration as high as 75 mg/ml. Conclusions. These results indicated that glucagon was self-associated by its β-sheet-rich intermolecular structure during the aging process under concentrated conditions to induce fibrillar aggregates. Glucagon has the same amyloidogenic propensities as pathologically related peptides such as β-amyloid (Aβ)1-42 and prion protein fragment (PrP)106-126 including conformational change to a β-sheet-rich structure and cytotoxic effects by activating caspases. These findings suggest that inappropriate preparation and application of therapeutic glucagon may cause undesirable insoluble products and side effects such as amyloidosis in clinical application.

Porcine enamel matrix derivative enhances trabecular bone regeneration during wound healing of injured rat femur

To elucidate the effects of enamel matrix derivative (EMD: Emdogain®) on bone regeneration in rat femurs after drill‐hole injury, defects in bone were filled with either EMD or its carrier, PGA, as control. On postoperative days 4 to 28, dissected femurs were examined by means of various morphological approaches. In both experimental groups, formation of trabecular bone, which was immunostained for bone sialoproteins (BSP), had occurred in the medullary cavities at cylindrical bone defects on Day 7 postoperatively. Cuboidal osteoblasts were clearly observed on these newly‐formed BSP‐positive bone trabeculae. On Days 7 and 14, many multinucleated giant cells, which strongly expressed cathepsin K, had appeared on these bone trabeculae, indicating active bone remodeling. In these bone trabeculae, Ca and P weight % and Ca/P ratio were similar to those of cortical bone, and there was no significant difference between the PGA‐ and EMD‐applied groups. Bone volume fraction of newly‐formed bone trabeculae on Day 7 postoperatively was significantly higher in the EMD‐applied group than in the PGA‐applied controls. Because of active bone remodeling and the marked decrease of bone volume, on Days 14 and 28 postoperatively, however, there was no longer a significant difference in trabecular bone volume fraction between the experimental groups. Our results suggest that EMD possesses an osteo‐promotive effect on bone and medullary regeneration during wound healing of injured long bones. Anat Rec 264:438–446, 2001.

Novel crystalline solid dispersion of tranilast with high photostability and improved oral bioavailability.

Tranilast (TL) is an anti-allergic agent and widely used in the clinical treatment of bronchial asthma, atopic rhinitis, atopic dermatitis and keloids. However, therapeutic potential of TL could be partly limited because of its poor solubility, bioavailability, and photostability. To overcome these drawbacks, crystalline solid dispersion of TL (CSD/TL) was prepared by wet-milling technique with aim of improving physicochemical and pharmacokinetic properties. Physicochemical properties of the formulations prepared were characterized by laser diffraction and dynamic light scattering for particle size analysis, scanning electron microscope for morphological analysis, and powder X-ray diffraction and differential scanning calorimetry for crystallinity assessment. TL particles in CSD/TL appeared to be crystalline with diameter of 122 nm, and CSD/TL exhibited marked improvement in the dissolution behavior as compared to crystalline TL. Under irradiation of UVA/B (250 W/m(2)), solution and amorphous solid dispersion of TL were found to be highly photodegradable, whereas high photochemical stability was seen in CSD/TL. After oral administration of CSD/TL, enhanced TL exposure was observed with increase of C(max) and AUC by 60- and 32-fold, respectively, as compared to crystalline TL. According to these observations, taken together with dissolution and pharmacokinetic behaviors, crystalline solid dispersion strategy would be efficacious to enhance bioavailability of TL with high photochemical stability.

AN ULTRASTRUCTURAL EVALUATION OF THE EFFECTS OF CYSTEINE-PROTEINASE INHIBITORS ON OSTEOCLASTIC RESORPTIVE FUNCTIONS

This study was designed to evaluate the effects of specific and potent cathepsin inhibitors on osteoclastic resorptive functions in vitro by means of a novel ultrastructural assay system. Mouse bone marrow cell-derived osteoclasts were suspended on dentine slices and cultured for 48 hours in the presence of either E-64 (a generalized cysteine proteinase inhibitor) or Z-Phe-Phe-CHN2 (a selective cathepsin L inhibitor). After the removal of cultured osteoclasts, co-cultured dentine slices were examined using electron microscopy: backscattered (BSEM), scanning (SEM), and atomic force (AFM). In morphometric analyses of BSEM images, there were no significant differences in the areas of demineralized dentine surfaces between control and inhibitor-treated groups, suggesting that cathepsin inhibitors had no effect on dentine demineralization by cultured osteoclasts. However, in SEM and AFM observations, both inhibitors remarkably reduced to the same extent, the formation of deep resorption lacunae on dentine slices that had resulted from degradation of matrix collagen. In addition, Z-Phe-Phe-CHN2 treatment produced deeper, ring-like grooves with little collagen exposure in shallow resorption lacunae. These results strongly suggest that (1) cathepsins released by osteoclasts are involved in the formation of deep resorption lacunae, and (2) cathepsin L plays a key role in bone resorption.

Liposomal formulations of glucagon-like peptide-1: Improved bioavailability and anti-diabetic effect

Glucagon-like peptide-1 (GLP-1), an incretin hormone, is recognized to be potent drug candidate for treatment of diabetes, however its clinical application has been highly limited, because of rapid enzymatic degradation by dipeptidyl-peptidase IV. To protect GLP-1 from enzymatic degradation and improve pharmacological effects, liposomal formulations of GLP-1 were prepared using three types of lyophilized empty liposomes such as anionic, neutral and cationic liposomes. Electron microscopic and dynamic light scattering experiments indicated the uniform size distribution of GLP-1-loaded liposomes with mean diameter of 130-210 nm, and inclusion of GLP-1 did not affect the dispersibility and morphology of each liposome. Of all liposomal formulations tested, anionic liposomal formulation exhibited the highest encapsulation efficiency of GLP-1 (ca. 80%). In intraperitoneal glucose tolerance testing in rats, marked improvement of hypoglycemic effects were observed in anionic liposomal formulation of GLP-1 (100 nmol/kg) with 1.7-fold higher increase of insulin secretion, as compared to GLP-1 solution. In pharmacokinetic studies, intravenous administration of anionic liposomal formulation of GLP-1 (100 nmol/kg) resulted in 3.6-fold higher elevation of serum GLP-1 level as compared to GLP-1 injection. Upon these findings, anionic liposomal formulation of GLP-1 would provide the improved pharmacokinetics and insulinotropic action, possibly leading to efficacious anti-diabetic medication.

Ameloblast Modulation and Changes in the Ca, P, and S Content of Developing Enamel Matrix as Revealed by SEM-EDX

Freeze-dried rat incisors were examined by high-resolution scanning electron microscopy (SEM) combined with energy-dispersive x-ray microanalysis (EDX) for determination of the correlation between the morphology of the enamel organ and the concentrations in the adjacent developing enamel matrix of calcium (Ca), phosphorus (P), and sulfur (S), as well as the CalP ratio. In SEM examination of the freeze-dried enamel organ, it was possible to identify the stages of enamel secretion, transition, and maturation, and furthermore to identify ruffle-ended and smooth-ended maturation ameloblasts. EDX analysis of the outer layer of forming and maturing enamel was carried out from the apical to the incisal end at interval points of approximately 50 μm. Ca and P concentrations increased gradually and continuously from the secretion zone to the end of the maturation zone, but never showed a steep rise in any of the zones examined. Maturing enamel overlaid by either ruffle-ended or smooth-ended maturation ameloblasts showed similar Ca and P concentrations. Throughout the outer enamel layer, the CalP molar ratio was fairly constant. Sulfur concentration began to decrease in the zone of enamel secretion, and was no longer detected in the middle of the maturation zone.

Effects of Chemically Modified Tetracycline, CMT‐8, on Bone Loss and Osteoclast Structure and Function in Osteoporotic States

ABSTRACT: We examined the effects of a nonantimicrobial tetracycline analogue, CMT‐8, on bone loss and osteoclasts in ovariectomized (OVX) rats. Three‐month‐old female rats were OVX, and, one week later, distributed into three groups: sham‐operated non‐OVX controls, untreated OVX controls, and CMT‐8‐treated OVX rats. After 145 days of daily drug administration (p.o.), the femurs were dissected and examined histologically. Ovariectomy markedly decreased trabecular and cortical bone volume in the metaphyses compared to sham‐operated controls. Treating the OVX rats with CMT‐8 produced a significant inhibition of trabecular and cortical bone loss and induced new bone formation, in which connectivity of the trabecular struts was increased by bridging the adjacent longitudinal bone trabeculae. Ultrastructurally, CMT‐8 reduced ruffled border formation in osteoclasts, while it caused no structural impairment in osteoblasts. To further evaluate the effects of CMT‐8 on the resorbing activity of osteoclasts, osteoclasts were cultured on dentine slices pretreated with CMT‐8 at concentrations of 2, 10, or 50 μg/ml, and resorption lacuna formation on the dentine surface was found to be reduced, dose‐dependently, by the bound CMT‐8. Our results suggest that CMT‐8 therapy effectively inhibits post‐ovariectomy bone loss not only by inducing new bone formation, but also by inhibiting osteoclastic bone resorption, and that CMT‐8 binding to bone may provide a prolonged release delivery of this anti‐resorptive therapy.

Is increased fat content of hindmilk due to the size or the number of milk fat globules

BackgroundIt is known that the fat content of breast milk is higher in hindmilk than in foremilk. However, it has not been determined if this increased fat content results from an increase in the number of milk fat globules (MFGs), an increase in the size of MFGs, or both. This study aims to determine which factor plays the most important role.MethodsThirteen breastfeeding mothers were enrolled in the study and we obtained 52 samples from 26 breasts before (foremilk) and after (hindmilk) a breastfeeding session. The fat content was evaluated by creamatocrit (CrCt) values. MFG size was measured with the laser light scattering method. We compared CrCt values and MFG size between foremilk and hindmilk.ResultsAlthough the CrCt values were higher in the hindmilk (8.6 ± 3.6%) than in the foremilk (3.7 ± 1.7%), the MFG size did not change (4.2 ± 1.0 μm and 4.6 ± 2.1 μm, foremilk and hindmilk, respectively). There was no relationship between the changes in CrCt versus MFG size from foremilk to hindmilk.ConclusionThe results indicate that the increase in fat content results mainly from the increased number of MFGs, which may be released into the milk flow as the mammary lobe becomes progressively emptied.