Akihiko Sano

Biomaterials for gene delivery: atelocollagen-mediated controlled release of molecular medicines.

Over the last decade, increasing attention has been paid to the development of systems to deliver drugs for long periods at controlled rates. Some of these systems can deliver drugs continuously for over one year. However, little effort has been given to developing systems for the controlled release of nucleic acids. Recently, a novel gene transfer method which allows prolonged release and expression of plasmid DNA in vivo in normal adult animals was established. In this system, a biocompatible natural polymer such as collagen or its derivatives acts as the carrier for the delivery of DNA vectors. The biomaterial carrying the plasmid DNA was administered into animals and, once introduced, gradually released plasmid DNA in vivo. A single injection of plasmid DNA/biomaterial produced physiologically significant levels of gene-encoding proteins in the local/systemic circulation of animals and resulted in prolonged biological effects. These results suggest that the biomaterials carrying plasmid DNA may enhance the clinical potency of plasmid-based gene transfer, facilitating a more effective and long-term use of naked plasmid vectors for gene therapy. Furthermore, the biomaterials can be removed surgically, minimizing the effect of gene products if some unexpected side effects should be observed after application. The application of these systems to expand the bioavailability of molecular medicine, including antisense oligonucleotides and adenovirus vectors, and to aid in stem cell transplantation in the context of DNA-based tissue engineering will be discussed.

Protein release from collagen matrices.

The effective delivery of protein drugs is an important research subject in the field of pharmacology, and to prolong the effect of protein drugs, many studies are being conducted to control the release of proteins from various carrier materials. Collagen is one of the most useful candidates for this purpose, and many studies have been reported; pharmaceutical formulations containing collagen in gel, film and sponge form are used to incorporate low-molecular-weight compounds such as antibiotics and carcinostatics, and the release of these compounds is controlled by the concentration of the gel as well as the shape and degree of crosslinking of the matrix. However, it is still difficult to retain protein drugs in the collagen. In this article, we report on the controlled release of protein drugs using collagen which exhibits good biocompatibility as a carrier, focusing on a new drug delivery system, the Minipellet, which we have developed.

Microstructure and release characteristics of the minipellet, a collagen-based drug delivery system for controlled release of protein drugs

We have developed the minipellet, a matrix-type system for the sustained delivery of protein drugs using collagen as a biodegradable drug carrier. In this study, we analyzed the microstructure and release profile of the minipellet containing human serum albumin (HSA) as a model drug.The findings suggest that the minipellet has a structure in which collagen fibers are strongly oriented in the direction of extrusion from the nozzle in the molding process of the minipellet, and that HSA exists as fine particulate clusters which are homogeneously distributed among the collagen fibers in the minipellet. During release, the HSA clusters dissolve and HSA is retained within the collagen matrix as a solution.The results of release experiments indicate that HSA release from the minipellet is mainly controlled by diffusion in the collagen matrix, and that sustained release is achieved by the dense structure of the collagen matrix which is formed in the manufacture process. In addition, more detailed study suggests that the minipellet has unique directional release behavior caused by its microstructure.

Development of a new drug delivery system for protein drugs using silicone (II)

In order to achieve a zero-order release of protein drugs, we have developed a new drug delivery system using silicone, which is named the covered-rod-type formulation. Preparation of the covered-rod-type formulation was conducted under mild conditions without heat treatment or the use of organic solvents. The covered-rod-type formulation released human serum albumin (HSA) or interferon (IFN) at a constant rate for 30-100 days in vitro without significant initial burst. When the IFN covered-rod-type formulation was implanted in nude mice, the serum IFN concentration was maintained at a constant level during the period of observation, i.e., 28 days. The covered-rod-type formulation enabled precise control of the release of the protein drugs and would be expected to increase the duration of the drug effect and to reduce the frequency of administration and side effects.

Targeted conversion of the transthyretin gene in vitro and in vivo

Familial amyloidotic polyneuropathy (FAP) is the common form of hereditary generalized amyloidosis and is characterized by the accumulation of amyloid fibrils in the peripheral nerves and other organs. Liver transplantation has been utilized as a therapy for FAP, because the variant transthyretin (TTR) is predominantly synthesized by the liver, but this therapy is associated with several problems. Thus, we need to develop a new treatment that prevents the production of the variant TTR in the liver. In this study, we used HepG2 cells to show in vitro conversion of the TTR gene by single-stranded oligonucleotides (SSOs), embedded in atelocollagen, designed to promote endogenous repair of genomic DNA. For the in vivo portion of the study, we used liver from transgenic mice whose intrinsic wild-type TTR gene was replaced by the murine TTR Val30Met gene. The level of gene conversion was determined by real-time RCR combined with mutant-allele-specific amplification. Our results indicated that the level of gene conversion was approximately 11 and 9% of the total TTR gene in HepG2 cells and liver from transgenic mice, respectively. Gene therapy via this method may therefore be a promising alternative to liver transplantation for treatment of FAP.

Sustained release of human growth hormone (hGH) from collagen film and evaluation of effect on wound healing in db/db mice

Collagen films containing human growth hormone (hGH) were prepared and the release of hGH from these films and their effect on healing of full-thickness wounds in db/db mice were evaluated. The release profiles of hGH from the collagen films varied with composition and preparation conditions. The film prepared by air-drying of the mixture of hGH and collagen solution released hGH continuously over 3 days both in vitro and in vivo. By application of collagen film containing 3 mg of hGH twice at an interval of 6 days to wounds, area of wounds on day 21 was significantly reduced compared with that of non-treated wounds. Application of hGH alone at the same dose had no significant effect on wound healing. The maximum serum hGH concentration after single administration of the hGH collagen film was lower than that with hGH alone, and hGH persisted in serum over 3 days. These results suggest that hGH collagen film may be a useful topical formulation for the treatment of wounds.

Continuous antigen delivery from controlled release implants induces significant and anamnestic immune responses.

Two continuous delivery injectable silicone implants were tested to determine if they were capable of delivering vaccines in a single shot. The Type A implant delivers antigen in vitro over a 1-month-period and the Type B over several months. Vaccination studies in sheep were designed to compare the responses induced by the Type A and B implants, Alzet mini-osmotic pumps and conventional antigen delivery. A model antigen, avidin, was used along with IL-1beta or alum as adjuvants. Sheep were immunised with various formulations and the titre and isotype of the antigen specific antibodies monitored. The Type B implant induced antibody (Ab) titres of greater magnitude and duration than soluble vaccines or the Type A implant with adjuvant, but only if IL-1beta was included in the formulation. Both implants induced antibodies of IgG1 and IgG2 isotype. A memory response to soluble antigen challenge was induced by the Type B+IL-1beta implant, which was predominantly of an IgG1 isotype.

BDNF atelocollagen mini-pellet accelerates facial nerve regeneration

We investigated the effect of BDNF mini-pellet on the GAP-43 mRNA expression and functional status of facial nerve in a rat model of facial nerve transection and immediate repair. The facial function started to recover at 17 days in the placebo group and 14 days in the BDNF group. BDNF group had shorter period of increased GAP-43 mRNA expression than the placebo group. Topically applied BDNF may accelerate the facial nerve regeneration.

Atelocollagen‐Mediated Systemic DDS for Nucleic Acid Medicines

Abstract:  The goal of our research is to provide a practical platform for drug delivery in oligonucleotide therapy. We report here the efficacy of an atelocollagen‐mediated oligonucleotide delivery system applied to systemic siRNA and antisense oligonucleotide treatments in animal disease models. Atelocollagen and oligonucleotides formed a complex of nanosized particles, which was highly stable against nucleases. The complex allowed oligonucleotides to be delivered efficiently into several organs and tissues via intravenous administration. In a tumor metastasis model, the complex successfully delivered siRNA to metastasized tumors in bone tissue and inhibited their growth. We also demonstrated that a single intravenous treatment of the antisense oligodeoxynucleotide complex suppressed ear dermatitis in a contact hypersensitivity model. These results indicate the strong potential of the atelocollagen‐mediated drug delivery system for practical therapeutic technology.

Collagen minipellet as a controlled release delivery system for tetanus and diphtheria toxoid

The use of biodegradable polymer matrices as a single-dose vaccine delivery system was investigated using tetanus toxoid (TT) and diphtheria toxoid (DT). BALB/c mice were immunized with TT or DT in different formulations including individual, in minipellet and aluminum hydroxide (alum), and the antibody responses were monitored for 48 weeks. Antigens entrapped in minipellet elicited higher antibody responses compared to those obtained with individual antigens and antigens adsorbed to alum and the antibody levels remained elevated over 48 weeks. In addition, minipellet formulations induced the same subclasses of antibodies induced by alum formulations. These results raise the possibility to obtain optimal and long-lasting immune responses by a single administration of minipellet formulations.