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Research Subjects

  • (1) Bioadhesive Gels for Hemostatics and Adhesion Barrier Devices
    --Clinical Study as a Hemostatic Device--
  • 1-1. Introduction

 Protein-based devices are widely used for topical hemostasis. However, there still remains risks of virus- or BSE-infection. Polysaccharide-devices are also available on the market. But, they are sometimes reported to causes inflammation. They do not stick to bleeding tissue itself, and should be held firmly until hemostasis occurs. Ideal hemostatic material would be safe, easy to use, highly efficacious, fully absorbable, and inexpensive. Unfortunately, it does not exist.

  • 1-2. Development of Novel Bioadhesive Gels for Hemostatic Device
 We developed a novel complex of poly(acrylic acid) (PAA) and poly(vinylpyrrolidone) (PVP). The complex can be swelled in water, and form a bioadhesive gel. It could be formulated as two formulations;
a flexible transparent film, and a fluffy sponge, and a soft sheet, and a powder

Four Types of Formulations

 They swelled in water to a hydrogel.
 On putting on the wet tissue, they would absorb water to a soft hydrogel,
 and adhere to tissues.
On putting on the wet tissue, they would absorb water to a soft hydrogel, and adhere to tissues.

They are highly safe:
  All the ingredients have already been approved as pharmaceutical additives.
Fully degradable in body:
  The gels will be slowly dissolved at pH 7.4.

  • 1-3. Development of Hemostatic Film
 PAA/PVP complex is generally a water non-swellable hard solid. We found that under certain particular conditions, water-swellable film of PAA/PVP complex could be obtained. The film could also be prepared on gauze, and another film can be stuck to it.

<Clinical Study>
 Clinical Studies of the Film for Hemostasis after Blood Sampling or Intravenous Injection was Performed in the Patients Taking Anticoagulant Medicines.

Female;51years old(taking warfarin) After intravenous of contrast media
Female;73years old(taking warfarin) After intravenous of contrast media

 PAA/PVP film was placed on the bleeding site. It swelled soon to a soft gel, and adhered to skin. Bleeding was completely stopped very quickly, though the patient takes anticoagulant.

  • 1-4. Development of Hemostatic Sponge
 Water-swellable soft fluffy sponge of PAA/PVP complex could be prepared by freeze-drying the PAA/PVP mixed solution under certain particular conditions.

<Clinical Study>
 Clinical studies of the sponge was carried out in dental surgery field. PAA/PVP sponge was placed in the socket after teeth extraction. It swelled to a soft gel, and filled the socket. Even when the patient takes anticoagulant, bleeding was efficiently stopped quickly. No adverse side effect was observed in the clinical study.

Male,87years old taking plavix

  • 1-5. Effect of the Bioadhesive Gels as an Adhesion Barrier Devices
 As was mentioned above, water-swellable gel in formulations of film and sponge was prepared from PAA and PVP under certain particular conditions. They form an adhesive gels on a wet skin or tissues. The gels are slowly dissolved at pH 7.4. After implanting subcutaneously or intraperitoneally, they completely disappear in days.
Possibility of the film or sponge as an adhesion barrier was then examined.

Adhesion Barrier Effect of the Film

 Without the film, the cecum stack firmly to peritoneum or other intestines, whereas the secum treated with the film did not adhere to other tissues.

  • (2) Synthetic Gene Therapy System:

  • 2-1. Introduction
 Since serious side effects have been reported in several patients treated by viral gene therapy, great efforts have been focused on developing the highly efficient non-viral vector systems as a safer alternative to viruses. Certain polycations can mediate the gene transfection into cultured cells, but in vivo transfection efficiency is very poor.
 Major obstacles to the efficient in vivo transfection would be (1) the adverse interaction of the complex with biocomponents, and (2) too large size of the complex particles to be delivered to the target cells.
  • 2-2. Development of Novel Efficient Synthetic Gene Delivery Systems
 We have developed a protective anionic polysaccharide-coating on the DNA complex particles which re-charged the particles to negative. The polyanion-coated DNA complexes showed very little interaction with blood components. Cytotoxicity was also evidently reduced by the polyanion-coating.
 The polysaccharide-coating was also found to protect the DNA/PEI complexes against aggregation and inactivation through lyophilization-and-rehydration procedures. It allows us to prepare the concentrated very small DNA complex particles (< 70 nm) suspension by preparing the complexes at highly diluted conditions, followed by lyophilized-and-rehydrated to a small volume.
 Those formulations achieved high reporter gene expression level in tumor after intravenous- or intratumoral-injection.

Efficient Gene Delivery System to Tumour Cells

  • 2-3. Antitumor Effect of the Synthetic Gene Delivery Systems
 Small complex was then made of the plasmid encoding GM-CSF gene. They showed apparent therapeutic effect in the tumor-bearing mice after intratumoral injection.

Therapeutic Effect of Small DNA Complex on Tumor Bearing Mice

<Animal Clinical Study>
 Small complex was prepared with DNA coding GM-CSF, a immuno-stimulating cytokine. Clinical effect of the DNA complex was examined on primary tumor in dogs, and cats. Those synthetic gene delivery systems showed remarkable anti-tumor efficacy on the animals.

Anti-tumor Efficacy of the DNA-GM-CSF Complex in dog

  • 2-4. Gene Medicine with Higher Anti-Tumor Efficacy
 Immune escape of the tumor cells is one of the main obstacles hindering the immunotherapy. We have developed a novel strategy to overcome the immuno-escape mechanism of the tumor cells. Small DNA complexes mentioned above were then made of the DNAs harboring the virus- or bacteria-specific pathogenic protein genes. Transfection of the pathogenic antigen genes would induce the antigen presentation on the tumor cell surfaces, which would be recognized by antigen-presenting cells (APCc) as a "danger signal", and the immune systems should be stimulated.
 Transfection of the tumor-bearing mice with these pathogenic genes showed an evident anti-tumor activity. Co-transfection of the cytokine-genes, such as GM-CSF or IL-2, with the pathogenic antigen-gene showed highly superior anti-tumor therapeutic effect.

<Animal Clinical Study>
 Animal clinical study on primary tumor-bearing dogs is now in progress in Japan and China. Evident suppression of the tumor growth was already confirmed in some cases.

Treatment of Tumor with Pathogenic Genes

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