Protein transduction into the mouse otocyst using arginine-rich cell-penetrating peptides

The mouse otocyst, an anlage of the inner ear, is an attractive experimental target for developing treatment modalities for congenital inner ear diseases and for studying inner ear development. Poly-arginine (6-12 residues) is a cell-penetrating peptide and can be used to deliver cargo into cells. Here, we achieved transutero delivery of enhanced green fluorescent protein (EGFP) fused to a nine-arginine peptide into mouse embryonic otocysts. The EGFP signal was detected both in the lining cells of the otocysts and in their vicinity at 18 h post injection. Mice injected with EGFP fused to a nine-arginine peptide had normal auditory and vestibular functions. These data suggest that protein transduction using poly-arginine may be a useful alternative strategy to commonly used gene delivery methods for delivering therapeutically relevant molecules to the developing inner ear.     

Purification and characterization of hemolysin from Prevotella oris

We observed hemolytic activity in culture supernatant of Prevotella oris. Results from growth-phase experiments show that hemolysin production increased during the logarithmic growth phase and decreased during the stationary phase. The hemolysin produced by P. oris was purified from the culture supernatant by ultrafiltration, diethylaminoethyl (DEAE) and carboxymethyl (CM) ion-exchange chromatography, and gel filtration chromatography; further, we investigated the purified hemolysin characteristics, including its ability to lyse human, horse, sheep, and rabbit erythrocytes. The purified hemolysin was observed as a single, 16-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The specific activity was obtained by concentrating the purified hemolysin by 9200 fold. Although hemolysin was inactivated by heat treatment, ethylenediaminetetraacetic acid (EDTA), l-cysteine, dithiothreitol (DTT), and 2-mercaptoethanol enhanced its activity. Further, treatments using trypsin, MgCl₂, CaCl₂, and cholesterol did not affect its hemolytic activity. A pH of 6.0 was optimal for inducing the hemolysin activity. To the best of our knowledge, this is the first report describing the purification and characterization of hemolysin produced by P. oris.     

Site-specific drug delivery to the middle region of the small intestine by application of enteric coating with hypromellose acetate succinate (HPMCAS)

Enteric coatings that deliver drugs to specific regions of the small intestine were examined. Hypromellose acetate succinate (HPMCAS) with different values of succinoyl group contents was used. Decreasing the succinoyl group content resulted in an increase in the pH at which HPMCAS started to dissolve. Drug-containing granules with or without enteric coating were prepared and their in vitro dissolution in a simulated intestinal fluid of pH 6.8 was examined. Granules coated with HPMCAS having the succinoyl group content of 6.2% showed a lag time of about 30 min, although drug release from granules without coating was completed within 20 min. The time lag and dissolution rate were extended and reduced, respectively, as the succinoyl group content was decreased. Rat experiments indicated that enteric-coated granules disintegrated and the bulk of the drugs was immediately released when the granules reached a specific site of the small intestine where the pH corresponded to the pH at which the enteric coating agent started to dissolve. Similar results were observed in monkey experiments. It was suggested that HPMCAS with the succinoyl group content of about 5% was suitable as an enteric coating agent for delivering drugs to the middle-to-lower region of the small intestine.     

Physiologically Based Pharmacokinetics of Cyclosporine A: Reevaluation of Dose–Nonlinear Kinetics in Rats

The disposition kinetics of Cyclosporine A (CyA) in rat, based on measurement in arterial blood, appeared dose-linear over a wide iv dose range (1.2–30mg/kg). Physiologically based pharmacokinetic (PBPK) analysis, however, demonstrated that this was an apparent observation resulting from counterbalancing nonlinear factors, such as saturable blood and tissue distribution, as well as clearance (CL_b ). A PBPK model was successfully developed taking into account these multiple nonlinear factors. Tissue distribution was distinctly different among various organs, being best described by either a linear model (muscle, fat; Model 1), one involving instantaneous saturation (lung, heart, bone, skin, thymus; Model 2), noninstantaneous saturation (kidney, spleen, liver, gut; Model 3), or one with saturable efflux (brain; Model 4). Overall, the whole body volume of distribution at steady state for unbound CyA (Vu_ss ) decreased with increasing dose, due at least in part to saturation of tissue-cellular cyclophilin binding. Clearance, essentially hepatic, and described by the well-stirred model, was also adequately characterized by Michaelis–Menten kinetics, K_m 0.60 g/ml. In model-based simulations, both volume of distribution at steady state (V _ss,b ) and CL_b varied in a similar manner with dose, such that terminal t _1/2 remained apparently unchanged; these dose responses were attenuated by saturable blood binding. CyA concentration measured in arterial blood was not always directly proportional to the true exposure, i.e., unbound or target tissue concentrations. The PBPK model not only described comprehensively such complicated PK relationships but also permitted assessment of the sensitivity of individual parameters to variation in local nonlinear kinetics. Using this approach, dose-dependent CyA uptake into brain was shown to be sensitive to both active and passive transport processes, and not merely the affinity of the active (efflux) transporter at the level of the blood–brain barrier.     

Long-term Voice Handicap Index after type II thyroplasty using titanium bridges for adductor spasmodic dysphonia

Objectives

To determine the long-term functional outcomes of type II thyroplasty using titanium bridges for adductor spasmodic dysphonia (AdSD) by perceptual analysis using the Voice Handicap Index-10 (VHI-10) and by acoustic analysis.

Methods

Fifteen patients with AdSD underwent type II thyroplasty using titanium brides between August 2006 and February 2011. VHI-10 scores, a patient-based survey that quantifies a patient's perception of his or her vocal handicap, were determined before and at least 2 years after surgery. Concurrent with theVHI-10 evaluation, acoustic parameters were assessed, including jitter, shimmer, harmonic-to-noise ratio (HNR), standard deviation of F0 (SDF0), and degree of voice breaks (DVB).

Results

The average follow-up interval was 30.1 months. No patient had strangulation of the voice, and all were satisfied with the voice postoperatively. In the perceptual analysis, the mean VHI-10 score improved significantly, from 26.7 to 4.1 two years after surgery. All patients had significantly improved each score of three different aspects of VHI-10, representing improved functional, physical, and emotional well-being. All acoustic parameters improved significantly 2 years after surgery.

Conclusions

The treatment of AdSD with type II thyroplasty significantly improved the voice-related quality of life and acoustic parameters 2 years after surgery. The results of the study suggest that type II thyroplasty using titanium bridges provides long-term relief of vocal symptoms in patients with AdSD.     

Three-dimensional DSA imaging and 3-dimensional measurement of cross section for internal carotid artery stenosis]

We have performed rotational DSA for internal carotid artetry (ICA) stenosis and examined cross sectional imaging of the stenosis. Then, we compared the area stenosis rate (ASR) with stenosis rate by NASCET method and with results of duplex carotid ultrasonography. Of consecutive 451 patients who underwent digital subtraction angiography, 28 patients with ICA stenosis were selected for this study. Imaging data were transmitted to a workstation, and three-dimension (3-D) images were prepared, and cross sectional images of the highest-grade stenotic portion were obtained. ASRs were calculated [1-(the area of highest stenotic portion of ICA/the area of distal ICA)] x 100, which were compared with stenosis rates by NASCET method, as well as peak systolic velocity ratios (PSVR) of ICA to common carotid artery (CCA) determined by duplex carotid ultrasonography (USG). Cross sectional images in all patients were made except for restless patients, thereby morphology of the stenosis was feasible and measurements of cross section and diameter were possible. ASR and stenosis rate by NASCET method showed a very high correlation, and ASR was obtained by formula of (12.886 + 1.037 x stenosis rate by NASCET method). In patients with distorted stenosis, the stenosis rate was overestimated by NASECT method. ICA/CCA PSVR could predict stenosis to some extent, and in particular, all the patients with ICA/CCA PSVR of 3.1 or greater were found to have high grade stenosis. However duplex carotid USG failed to detect stenosis in a patient with high-grade stenosis at high position. In conclusion, as to ICA stenosis, 3-D image could show the stenosis precisely, and was considered to be useful as a routine examination.     

Effect of administration site in the gastrointestinal tract on bioavailability of poorly absorbed drugs taken after a meal.

Food-drug interactions may reduce the bioavailability of drugs taken after meals (negative food effect). In order to develop pharmaceutical technologies that overcome this problem, the effect of administration site within the gastrointestinal tract on the bioavailability of several model drugs was examined in rats. Bioavailability after oral administration to fed animals was one-fifth to one-tenth of that in the fasted animals because of interactions between drugs and large amounts of food components remaining in the stomach. This strong negative food effect was reduced when drugs were administered directly into any site of the small intestine. Bioavailability was maximized when the drug administration site was the middle small intestine. On the other hand, intracolonic administration did not result in the reduction of the negative food effect. Site-specific drug delivery to the middle small intestine could be a useful approach for reducing the negative food effect on drug absorption with maximized bioavailability.     

Effects of packaging forms on the stability of vitamin B1 and vitamin C in TPN admixtures

In order to reduce a microbial contamination and needle stick injuries that are associated with a mixing procedure in home parentera nutrition(HPN), nutrition(TPN)solution bags pre-mixed with trace elements may be provided in a form of outer packaging. On the other hand, a packaging form used to enclose the TPN bag after admixture may significantly affect the stability of vitamins. With a focus on possible decrease in vitamin B1 and C content, we investigated the effects of the packaging form. As a result, the TPN bag, which is packed in a light-resistant outer wrap of oxygen-barrier film with an oxygen absorbent under reduced pressure, suppressed a decrease in vitamin content most. However, the decrease in vitamin C content was observed when there was a long time-lag between a preparation and a packaging. We thought it was desirable to pack the TPN bag promptly after the preparation.