Masaya Kawase

Novel transdermal drug delivery system with polyhydroxyalkanoate and starburst polyamidoamine dendrimer.

In search of an efficient transdermal drug delivery system (TDDS), a polyhydroxyalkanoate (PHA)-based system with a polyamidoamine dendrimer was examined. Tamsulosin was used as the model drug. The dendrimer was found to act as the weak enhancer. By adding the dendrimer, the dendrimer-containing PHA matrix achieved the clinically required amount of tamsulosin permeating through the skin model. This is also the first report of the application of PHA and dendrimer to the TDDS.

Mechanism of Enhancement Effect of Dendrimer on Transdermal Drug Permeation through Polyhydroxyalkanoate Matrix

The possible application of polyhydroxyalkanoate (PHA) in transdermal drug delivery systems (TDDSs) for tamsulosin was previously reported. PHAs containing the drugs, ketoprofen, clonidine and tamsulosin showed good adhesiveness to the skin model used, that is, shed snake skin, and dispersed well all model drugs tested. The model drugs hardly permeated through snake skin in solution form. However, these drugs permeated well through snake skin from the PHA matrix. It was previously reported that the addition of a dendrimer, a polymeric permeation enhancer, is effective for the TDDS for tamsulosin to establish an effective clinical TDDS. The effect of dendrimer addition was examined in TDDSs for ketoprofen and clonidine. The dendrimer added did not show an enhancement effect on the TDDSs for the two drugs. To investigate the mechanism of the enhancement effect of a dendrimer on the tamsulosin TDDS, X-ray analyses were performed. With dendrimer addition, drug crystallization in PHA was promoted. The crystal in PHA had highly ordered and changed its space group. These findings are very important for exploiting high-performance PHA-based TDDSs.

Effects of the counterion on dielectric spectroscopy of a montmorillonite suspension over the frequency range 105-1010 Hz

Dielectric measurements were carried out on suspensions of montmorillonite clay exchanged with three different counterions: sodium, ammonium, and tetramethylammonium (TMA). Only two dielectric absorption peaks could be identified for the clay sample with the TMA counterion, whereas three peaks were found for the two inorganic counterions. The dielectric process observed at around 10 GHz is due to the orientation of bulk water molecules, judging from the relaxation time and relaxation strength. The relaxation strength of the process occurring at around 10 MHz was compared with the coefficient of adiabatic compressibility obtained from ultrasound velocity measurements. The increase in the relaxation strength with decreasing compressibility indicates that the process at around 10 MHz is caused by the orientation of bound water molecules on the clay samples. The relaxation strength of the process occurring at around 10 MHz for the TMA sample was remarkably small. Furthermore, the network structure of the bound water molecules can be characterized by a property peculiar to the TMA sample, taking into account the value of its Cole-Cole parameter. Results for the relaxation strength of the process occurring at around 100 kHz were compared with those for electrophoretic mobility. This comparison revealed that discrimination between bound ions and ions in the diffuse double layer is important, and both the relaxation and electrophoretic results could be satisfactorily explained by surface polarization of the clay.

Morphological regulation and aggregate formation of rabbit chondrocytes on dendrimer-immobilized surfaces with D-glucose display.

The present study describes dendrimer-immobilized surfaces with D-glucose display, used to regulate the morphology of rabbit chondrocytes by changing the generation number and density of the dendrimer, and to clarify the relation between cell morphology and chondrogenic expression on the prepared surfaces. When the generation number increased, the frequency of round-shaped cells increased, whereas, cell stretching was appreciably suppressed. Further suppression of stretching was significant on the G4-LD surface, on which the density of the immobilized dendrimer was lowered, and this phenomenon was thought to be responsible for the sparse display of D-glucose on the surface. The time-lapse observations revealed that the G4-LD surface caused repeated morphological changes of stretching and contracting, together with fluctuations in cell roundness. By the cytoskeletal staining of F-actin, the immature stress fibers were recognized in both round and stretched cells on the G4-LD surface. It was also found that N-cadherin expression was promoted on this surface, thus supporting the idea that an increase in cell-cell contacts may maintain chondrocytic phenotypes. These results suggest that a G4-LD surface can provide a culture environment to promote cell-cell interactions by N-cadherin-mediated adhesion during cell aggregation, thereby facilitating chondrocytic phenotypes of cells.

Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis.

A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recombination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyte apoptosis. It also significantly extended the life span of hepatocytes cultured conventionally on polystyrene plates. Liver-specific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases.

Application of hydrotropy to transdermal formulations: hydrotropic solubilization of polyol fatty acid monoesters in water and enhancement effect on skin permeation of 5‐FU

Objectives  A hydrotropic formulation containing a percutaneous enhancer was developed for the transdermal formulation of a water‐soluble drug and the solubilizing mechanisms of a percutaneous enhancer in water by a hydrotropic agent were investigated. The enhancement effect was also compared with the hydrotropic formulation and the other formulations using ethanol, propylene glycol or mixed micelles.

Structure-activity relationships of anthraquinones on the suppression of DNA-binding activity of the aryl hydrocarbon receptor induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Anthraquinones are widely present in plant kingdom, and clinically used as laxatives. Environmental contaminants, dioxins, develop various adverse effects through transformation of a cytosolic aryl hydrocarbon receptor (AhR). We investigated the effects of 18 anthraquinones and 7 of their structurally related compounds on transformation of the AhR estimated by its DNA-binding activity in the cell-free system. 1,4-Dihydroxyanthraquinone (quinizarin), 1,5-dihydroxyanthraquinone (anthrarufin), 1,8-dihydroxyanthraquinone (danthron), and 5-hydroxy-1,4-naphthoquinone (juglone) strongly suppressed DNA-binding activity of the AhR induced by 0.1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), with their IC(50) values around 1 muM. On the other hand, anthraquinone, 2,6-dihydroxyanthraquinone (anthraflavic acid), and 2-hydroxy-1,4-naphthalendione (lawsone) showed moderate effects. Quantitative structure-activity relationships analysis demonstrated that hydroxyl groups at C1 or C4 but not C3 position of anthraquinone structure are critical for the suppressive effects. In addition, all compounds except lawsone had no agonistic effect. The suppressive effects of anthraquinones in a cultured cell system were also confirmed. In human hepatoma HepG2 cells, chrysophanol, danthron, and rhein also suppressed the DNA-binding activity in a dose-dependent manner, although aloe-emodin showed a moderate effect. The findings of this study may be useful for the design of the novel antagonists of the AhR.

Unusual Accumulation of Demethylspheroidene in Anaerobic-Phototrophic Growth of crtA-Deleted Mutants of Rhodovulum sulfidophilum

Rhodovulum sulfidophilum produces carotenoids in the spheroidene pathway. Spheroidene monooxygenase, CrtA, catalyzes the conversion of spheroidene to spheroidenone. crtA-deleted mutants of R. sulfidophilum did not produce spheroidenone and demethylspheroidenone. In these mutants, the ratio of demethylspheroidene to spheroidene increased with exposure to light. One mutant exhibiting a spheroidene-predominant phenotype did not grow under anaerobic-light conditions and was devoid of bacteriochlorophyll a, even under semiaerobic-light conditions There was no difference in the growth of the mutants under aerobic-dark conditions. These data suggest that demethylspheroidene is important for photosynthesis in R. sulfidophilum.

Polyethyleneimine/chitosan hexamer-mediated gene transfection into intestinal epithelial cell cultured in serum-containing medium

In search of an efficient nonviral vector, polyethyleneimine (PEI)-based vectors were examined. In general, the transfection efficiency of nonviral vectors is suppressed by serum. Here we show that PEI based vectors, particularly, the chitosan hexamer-PEI vector, could perform efficient gene transfection into intestinal epithelial cells (IEC-6) in the presence of serum. The conjugation order of the two polymers with a plasmid (first, chitosan hexamer; second, PEI) was found to be an important factor in enhancing transfection efficiency.

Cloning and Molecular Analysis of Poly(3-Hydroxyalkanoate) Biosynthesis Genes in Pseudomonas aureofaciens

Pseudomonas aureofaciens grown on octanoate or gluconate synthesized medium-chain-length polyhydroxyalkanoates (mcl-PHAs). To clone the PHA synthase gene(s) (phaC), the genomic library of P. aureofaciens was constructed using a cosmid vector. The recombinant cosmids that clone phaC were detected by the complementation with a PHA-negative mutant, P. putida GPp104. The resulting recombinant cosmid, named pVK6, contained a 13-kbp DNA insert. Genetic analysis of the pha locus in pVK6 revealed the presence of six ORFs, genes encoding two PHA synthases, 1 and 2 (phaC1 and phaC2), PHA depolymerase (phaZ), two PHA granule-associated proteins (phaF and phaI), and an unknown protein (phaD). The heterologous expression of pha genes from P. aureofaciens was confirmed. P. putida GPp104 regained the ability to accumulate PHA on introduction of pVK6. Wild-type strains P. oleovorans and P. fluorescens, which were unable to accumulate PHA when grown on gluconate, acquired the ability to accumulate PHA from gluconate when they possessed pVK6.