Miho Maeda

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.

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.

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.

Potential of Atelocollagen-Mediated Systemic Antisense Therapeutics for Inflammatory Disease

To study the possibility of using atelocollagen as an oligonucleotide (ODN) delivery carrier in vivo, the activity of formulated antisense ODN targeted against the intercellular adhesion molecule-1 (ICAM-1) mRNA was investigated in an allergic dermatitis model in mice. The allergic dermatitis was elicited in one ear of animals sensitized by treatment with 2,4-dinitrofluorobenzene. Antisense ODN was given to the animals as a single intravenous injection of formulation containing atelocollagen. Antisense activity was determined by measurement of ear thickness, histopathology, and immunohistochemistry 24 hr after the initiation of the dermatitis. Antisense activity was found to increase according to the concentration of atelocollagen in the formulation. The effect mediated by the ODN formulated with 0.05% atelocollagen was more than 50 times greater than that provided by ODN infusion, although the levels of ODN formulated with atelocollagen dropped below that of the 24-hr infusion group within 30 min. The formulated ODN could suppress inflammatory progression by treatment at 8 hr after the ear challenge when inflammation had already commenced at the challenged site. Moreover, antisense activity was noted even when the formulated ODN was injected 3 days before the initiation of inflammation. These data demonstrate that atelocollagen can enhance antisense activity remarkably and that the sustainable antisense activity mediated by the formulation of ODN with atelocollagen could completely change the strategy of antisense therapeutics.

New drug delivery system for water-soluble drugs using silicone and its usefulness for local treatment: application of GCV-silicone to GCV/HSV-tk gene therapy for brain tumor.

Controlled release of a water-soluble low-molecular-weight drug from silicone and its usefulness as a local therapeutic drug were studied. For application to ganciclovir/helpes simplex virus thymidine kinase (GCV/HSV-tk) suicide gene therapy for brain tumor, two kinds of GCV-containing silicone formulations were prepared for evaluation. In vitro, GCV release from matrix-type formulation consisting of a single matrix was characterized by Fickian diffusion, while covered-rod-type formulation, in which the side surface of the outer layer was covered with 100% silicone, exhibited a near-zero-order release pattern. In an in vivo study using a rat 9L glioblastoma model, administration of GCV-silicone formulation into brain tumor yielded sustained intracerebral GCV concentration for 4 days after administration, with excellent antitumor effect equal to or better than that of daily intraperitoneal administration of aqueous solution of GCV, at a dose less than 1/100 of the total dose of solution for intraperitoneal administration. Furthermore, GCV was undetectable in blood, suggesting that decrease in systemic adverse reactions can be expected with intracerebral administration of GCV-silicone formulation.

Rewiring of regenerated axons by combining treadmill training with semaphorin3A inhibition

BackgroundRats exhibit extremely limited motor function recovery after total transection of the spinal cord (SCT). We previously reported that SM-216289, a semaphorin3A inhibitor, enhanced axon regeneration and motor function recovery in SCT adult rats. However, these effects were limited because most regenerated axons likely do not connect to the right targets. Thus, rebuilding the appropriate connections for regenerated axons may enhance recovery. In this study, we combined semaphorin3A inhibitor treatment with extensive treadmill training to determine whether combined treatment would further enhance the “rewiring” of regenerated axons. In this study, which aimed for clinical applicability, we administered a newly developed, potent semaphorin3A inhibitor, SM-345431 (Vinaxanthone), using a novel drug delivery system that enables continuous drug delivery over the period of the experiment.ResultsTreatment with SM-345431 using this delivery system enhanced axon regeneration and produced significant, but limited, hindlimb motor function recovery. Although extensive treadmill training combined with SM-345431 administration did not further improve axon regeneration, hindlimb motor performance was restored, as evidenced by the significant improvement in the execution of plantar steps on a treadmill. In contrast, control SCT rats could not execute plantar steps at any point during the experimental period. Further analyses suggested that this strategy reinforced the wiring of central pattern generators in lumbar spinal circuits, which, in turn, led to enhanced motor function recovery (especially in extensor muscles).ConclusionsThis study highlights the importance of combining treatments that promote axon regeneration with specific and appropriate rehabilitations that promote rewiring for the treatment of spinal cord injury.

Sustained release of low-dose ganciclovir from a silicone formulation prolonged the survival of rats with gliosarcomas under herpes simplex virus thymidine kinase suicide gene therapy

A silicone formulation of ganciclovir (GCV-pellet) was developed to enhance the cytotoxic effects of herpes simplex virus thymidine kinase suicide gene therapy. The effectiveness of this drug delivery system was assessed in a rat 9L gliosarcoma model. The GCV-pellets (1 mm in length and in diameter) used in this experiment contained a total amount of 0.15 mg of GCV. In vitro experiments demonstrated that GCV was gradually released over a period of 7 days. Five days after stereotactic tumor inoculation into the right caudate nucleus, a herpes simplex virus type 1 (HSV-1) vector expressing herpes simplex virus thymidine kinase (HSV-tk) (T1, 2×106 pfu) was administered at the same location. The survival rate of the group treated with the GCV-pellet was compared with that of the T1 group injected intraperitoneally (IP) with GCV (30 mg/kg/day for 7 days). The GCV-pellet-treated group had a significantly prolonged survival (a median of more than 80 days) compared with the GCV IP group (a median of 65 days) and with control groups (P<0.05). The control groups (untreated or receiving only the virus vector) had a survival of 35–38 days. The survival rate of the GCV-pellet group over 80 days was 75%, and all the rats that survived more than 80 days and did not show tumors upon histological examination of the brain were deemed cured. No toxic effects or immunological reactions were observed histologically around the pellet in brain sections from the rats treated with the GCV-pellet. After GCV-pellet inoculation into the tumor, drug concentrations were kept at 1–10 μg/g tissue for 3–4 days. When the same dose of GCV (0.15 mg) in aqueous solution was injected into the tumor, GCV concentrations reached a peak of 0.5 mg/g tissue after 30 min and decreased below measurable level within 12 h. After IP injections of 3 mg GCV, GCV concentrations in the tumor reached a peak of 5.7 μg/g tissue after 30 min and also decreased below measurable level within 12 h. This sustained release of a low and effective GCV dose with the silicone formulation significantly prolonged survival in combinations with HSV-tk expression if compared to IP administration of GCV. Histological examination suggests that the treatment appears to be safe.

Novel sustained release formulation using collagen as a carrier material. (1)-Dosage form design of interferon minipellet.

We have developed a novel sustained release formulation, minipellet, which is applicable to various kinds of biologically active proteins. In this study, dosage form design of interferon (IFN) minipellet was investigated. First, in order to select suitable carrier material, matrix type formulations were prepared with natural biodegradable polymers, human serum albumin (HSA), gelatin and atelocollagen, and the release profiles of IFN from these polymers were compared. IFN was released slowly from the sample made of atelocollagen but rapidly from the samples made of HSA and gelatin. The release of IFN from the atelocollagen films, prepared by drying the atelocollagen solution, varied with the atelocollagen concentration before drying. This suggested that IFN release was controlled by the density of atelocollagen matrix. So, in order to obtain the higher matrix density of atelocollagen, we newly designed a cylindical dosage formulation prepared by extrusion and air-drying of an atelocollagen solution with high concentration and named minipellet. IFN was constantly released from minipellet in vitro. Pharmacokinetic studies in mice showed that the serum IFN concentration was maintained for a long time with minipellet. To determin the optimal minipellet composition, IFN minipellets containing different amount of HSA were prepared and IFN release profile from them in dogs was evaluated, The IFN concentration increased gradually after the administration of IFN minipellet containing 30% (w/w) HSA, reached Cmax after 24h, and decreased gradually thereafter with a detectable level for 10 days. The IFN minipellet is expected to offer a significant advantage in IFN therapy because the attenuated peak IFN concentrations in serum may reduce the side effects and the sustained release may reduce the frequency of administration.