Keiji Fujioka

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.

Novel delivery system for proteins using collagen as a carrier material: the minipellet

It is critical to develop effective delivery systems for biologically active peptides and proteins since most of them are rapidly destroyed by the body. In this study, interferon (IFN) was used as a model protein drug and matrix type formulations were prepared with natural biodegradable polymers, albumin, gelatin and collagen. The release of IFN from albumin and gelatin was much faster than that of collagen in vitro. The release of IFN from the collagen matrix varied with various processing conditions and were explained based on the density of collagen matrix. IFN was constantly released with a cylindrical solid dosage form prepared by extrusion and air-drying of a 30% (w/w) collagen solution of a high concentration in vitro. Pharmacokinetic studies in mice showed that the serum IFN concentration was maintained for a long time with this solid formulation called minipellet. The advantages of the minipellet are ( 1 ) carrier material is a biodegradable natural protein; (2) it is manufactured under mild processing conditions without any organic solvent or heating process; and (3) it is easily administered in the same way as conventional injections. Therefore, it is applicable to various kinds of biologically active peptides and proteins and expected to facilitate their potential therapeutic use.

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.

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.

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.

Long-acting delivery system of interferon : IFN minipellet

Abstract The optimal composition for an interferon (IFN) minipellet which released IFN with a high bioavailability and a long half-life was designed. Minipellets are cylindrical solid dosage forms using collagen as a biodegradable carrier material. A monolithic collagen matrix releases the incorporated drugs over long periods of time. Minipellets of various composition containing IFN were prepared and IFN release from minipellets was examined. Human serum albumin (HSA) added to the collagen matrix was shown to promote and maintain IFN release. In vivo experiments demonstrated that serum IFN concentrations were maintained at elevated levels for 7–10 days after administration of IFN minipellets containing 30% (w/w) HSA in the matrix. Pharmaco-kinetic studies showed that the IFN minipellet had good dose-dependency and reproducibility in serum drug level profiles. The IFN minipellet is expected to offer a significant advantage in IFN therapy because the peak IFN serum concentrations are attenuated, allowing prolonged effective dosing and reducing side effects. The increasing popularity of IFN treatment calls for the development of a formulation and a method of administration that reduces the burden on the patients. Therefore, a long-acting IFN delivery system requiring weekly subcutaneous administrations seems to be highly useful in a clinical setting.

DISTRIBUTION OF NERVE GROWTH FACTOR IN CAT BRAINS FOLLOWING TOPICAL APPLICATION OF SOLUTION OR MINIPELLET

The distribution of nerve growth factor (NGF) was studied after topical application of NGF solution or NGF Minipellet into the caudate nucleus of cat brains. Each aliquot of NGF solution or a piece of NGF Minipellet contains 200 micrograms of mouse 2.5S beta-NGF. The concentrations of NGF in various areas of the brain were determined by enzyme-linked immunosolvent assay (ELISA). The tissue concentrations of NGF were very high at 6 h after injection of NGF solution (124.0 +/- 2.46 to 2144.0 +/- 16.03 ng g-1), but quickly decreased on day 1. The tissue concentrations of NGF were low at 6 h after implantation of NGF Minipellet, but were considerably increased on day 1 (11.22 +/- 3.36 to 72.04 +/- 20.45 ng g-1), and slowly decreased during the following 6 to 14 days. The present study demonstrates the temporal and spatial profiles of NGF distribution in the brain of middle-sized animals after topical application. Injection of NGF solution resulted in abrupt, but very transient, elevation of tissue NGF. Implantation of NGF Minipellet maintained the tissue NGF at biologically effective levels for 6 to 14 days.

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.

Profile of rhBMP-2 release from collagen minipellet and induction of ectopic bone formation

For a cylindrical controlled‐release formulation using collagen as a carrier, called the minipellet (MP), which contains rhBMP‐2, the relationship between the diameter of MPs and rhBMP‐2 release profiles was investigated, and its effect in inducing bone formation was evaluated. Samples with three different diameters were tested for each of the following formulations: MP without additives, MP with 10% (w/w) glutamic acid (Glu) and 20% (w/w) alanine (Ala), and MP with 20% (w/w) Glu and 20% (w/w) Ala. The results of the in vitro release test and the amount of rhBMP‐2 remaining in the MPs after subcutaneous implantation into mice were compared among different samples. It was found that the addition of Glu accelerated release of rhBMP‐2 effectively. Release was accelerated as the diameter of MP became smaller and the amount of Glu added increased. The amount of calcium formed in 3 weeks after subcutaneous implantation into mice was dose‐dependent. The amount of calcium formed per unit rhBMP‐2 dose tended to increase as the diameter of MP became smaller and the amount of Glu added became greater; calcification was thus associated with release rate. These results indicate that MPs with smaller diameters induce bone formation more efficiently. For use in the treatment of fracture, etc., MP is considered to be a suitable dosage form, which can be administered noninvasively.