Development of Squalene-Based Oil-in-Water Emulsion Adjuvants Using a Self-Emulsifying Drug Delivery System for Enhanced Antigen-Specific Antibody Titers.

Introduction: A recombinant protein cannot induce sufficient immune response by itself. Various substances, including cytokine and mineral, have been used as adjuvants to enhance the immunogenicity and efficacy of vaccines; however, most of them induce excessive immune responses or exhibit cytotoxicity. In this study, a self-emulsifying drug delivery system (SEDDS), an isotropic mixture of oil, surfactant, and solvent, was designed for oil-in-water emulsions as a non-toxic adjuvant to increase immune response to antigens. Methods: Squalene-based oil-in-water emulsions were prepared by SEDDS to assess its value as an adjuvant. Fifteen emulsions (F1-F15) were prepared by stirring two types of surfactants (Span® 85 and Kolliphor® RH40), and squalene and carboxymethyl cellulose (CMC) were added at different ratios. The physical properties and viscosity of the 15 emulsions were evaluated by measuring droplet size, zeta potential, and polydispersity index. The toxic effect of emulsions was assessed by acute toxicity test in mice. Mice were immunized twice with 1:1 mixtures of antigen and adjuvant (15 emulsions, phosphate-buffered saline, and commercial alum-based adjuvant). Antigen-specific antibody titers from immunized mice serum were measured by an indirect enzyme-linked immunosorbent assay. Results: All emulsions exhibited droplet sizes ranging from 322 to 812 nm and maintained zeta potential values between -30 mV to -10 mV for 4 weeks, indicating good physical stability as a vaccine adjuvant. Additionally, all emulsions were non-toxic, and they induced humoral immunity at a similar level compared to commercial alum-based adjuvant in the first immunization. However, 12% squalene-based oil-in-water emulsion containing 0.5% of ultra-high viscosity CMC (F15) showed significantly higher immune response than a commercial adjuvant in the second immunization. Conclusion: Squalene-based oil-in-water emulsions could be conveniently prepared using SEDDS technique and are non-toxic and stable at room temperature storage. Moreover, squalene-based oil-in-water emulsions show enhanced immune induction with antigen; hence, they can possibly be used as effective adjuvants.

Improvement of physical properties of calcium phosphate cement by elastin-like polypeptide supplementation.

Calcium phosphate cements (CPCs) are synthetic bioactive cements widely used as hard tissue substitutes. Critical limitations of use include their poor mechanical properties and poor anti-washout behaviour. To address those limitations, we combined CPC with genetically engineered elastin-like polypeptides (ELPs). We investigated the effect of the ELPs on the physical properties and biocompatibility of CPC by testing ELP/CPC composites with various liquid/powder ratios. Our results show that the addition of ELPs improved the mechanical properties of the CPC, including the microhardness, compressive strength, and washout resistance. The biocompatibility of ELP/CPC composites was also comparable to that of the CPC alone. However, supplementing CPC with ELPs functionalized with octaglutamate as a hydroxyapatite binding peptide increased the setting time of the cement. With further design and modification of our biomolecules and composites, our research will lead to products with diverse applications in biology and medicine.

Smooth muscle relaxation activity of an aqueous extract of dried immature fruit of Poncirus trifoliata (PF-W) on an isolated strip of rat ileum.

We demonstrated that an aqueous extract of dried immature fruit of Poncirus trifoliate (PF-W) produces relaxation of intestinal smooth muscle using the ileac strips of a rat. Furthermore, the underlying mechanism of its relaxant activity was investigated. PF-W was prepared using the standard extraction protocol. A 1.5 - 2 cm long rat ileac strip was placed in an organ bath with Tyrode's solution and smooth muscle contractility was recorded by connecting it to a force transducer. Various compounds were added to the organ baths, and changes in muscular contractility were measured. PF-W concentration-dependently induced relaxation of rat ileac strips that were contracted both spontaneously and via acetylcholine treatment. Various potassium channel blockers did not inhibit the relaxation by PF-W. No difference in the effect of PF-W was observed between ileac strips treated with low (20 mM) and high concentrations (60 mM) of KCl. PF-W inhibited the contraction of rat ileac strips induced by extracellular calcium. PF-W acts as a potent smooth muscle relaxant, implicating its possible action as a rapid acting reliever for abdominal pains and a cure for intestinal convulsion. Considering that PF-W also exhibits prokinetic activity, its use in various gastrointestinal disorders seems promising.

An improved prediction of the human in vivo intestinal permeability and BCS class of drugs using the in vitro permeability ratio obtained for rat intestine using an Ussing chamber system.

The Biopharmaceutics Classification System (BCS) was developed to facilitate estimation of the in vivo pharmacokinetic performance of drugs from human intestinal permeability and solubility. However, the measurement of human in vivo intestinal permeability, unlike that of solubility, is problematic and inefficient. Thus, rat in vitro intestinal permeability results obtained via the Ussing chamber technique are often used instead. However, these data could be unreliable due to difficulty in maintaining the viability of the dissected intestinal membrane in the Ussing chamber. Therefore, a more efficient method to obtain a reliable in vitro permeability is mandatory. Here, we propose a new approach by introducing a novel factor called the permeability ratio (PR). Basically, PR is a rat in vitro intestinal permeability obtained from the Ussing chamber, which is then corrected by the permeability of lucifer yellow, a paracellular permeability marker. To prove the validity of the method, 12 model drugs representing different BCS classes were tested, and the correlation with human in vivo intestinal permeability was high. More importantly, the new method perfectly classified all 12 model drugs. The results indicate that PR is a reliable factor with high correlation to human in vivo intestinal permeability, which can further be used to accurately predict the BCS classification.

Fluorescence detection of Zabofloxacin, a novel fluoroquinolone antibiotic, in plasma, bile, and urine by HPLC: the first oral and intravenous applications in a pharmacokinetic study in rats.

PURPOSE: To develop an HPLC method using fluorescence detection for the pharmacokinetic evaluation of levels of zabofloxacin, a novel broad spectrum fluoroquinolone antibiotic, in the plasma, bile and urine of rats. METHODS: A simple reversed-phase HPLC method using a C18 column with fluorescence detection was developed and validated for the simultaneous determination of zabofloxain and enrofloxacin as an internal standard. The plasma sample was treated with methanol for protein precipitation, and treatment of the bile and urine samples included deproteinization and extraction using chloroform. The applicability of the developed assay method to pharmacokinetic studies of zabofloxacin in rats was examined. Zabofloxacin was intravenously and orally administered to rats at a dose of 20 mg/kg. RESULTS: The limits of quantification (LOQ) was determined to be 50 ng/mL for the plasma with acceptable linearity ranging from 50 to 25,000 ng/mL (R>0.999), and 0.5 µg/mL for the bile and urine samples with acceptable linearity ranging from 0.5 to 100 µg/mL (R>0.999). The validation parameters for zabofloxacin were found to be acceptable according to FDA assay validation (2001). While zabofloxacin in plasma and urine has been stable in all tested handling conditions, it has been unstable in bile during freeze-thaw cycles for 24 h at room temperature. Following intravenous and oral administration of zabofloxacin to rats at a dose of 20 mg/kg, concentration was quantifiable in plasma for up to 8 h. The bioavailability of zabofloxacin was 27.7%, and it was excreted into bile and urine at about 8% each per oral administration. CONCLUSIONS: These observations suggest that a validated assay can be used in pharmacokinetic studies of zabofloxacin in small animals. Due to the limited stability of zabofloxcin in rat bile, freeze-thaw cycles or prolonged handling at room temperature is not recommended. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.