WO2000078340A1

WO2000078340A1 - Sustained release preparations of physiologically active peptides

Info

Publication number: WO2000078340A1
Application number: PCT/JP2000/003946
Authority: WO
Inventors: Tadao Ohno; Bao Gang Peng
Original assignee: Riken
Priority date: 1999-06-17
Filing date: 2000-06-16
Publication date: 2000-12-28
Also published as: JP2001058955A

Abstract

Semisolid sustained release preparations containing physiologically active peptides such as cytokines which comprise: (A) a physiologically active peptide; (B) a protein with a biological origin such as albumin; and (C) a lipid with a biological origin such as lecithin or cholesterol, wherein the above components (A) to (C) are kneaded together. These preparations allow the physiologically active peptides (cytokines, etc.) to stably and continously act over a long time in vivo.

Description

Meiyo Bioactive peptide sustained-release formulation Technical field

The present invention relates to a sustained-release preparation containing a physiologically active peptide. Background art

When an immune response is elicited in a human or animal, an adjuvant is often used together with an antigen to regulate an antibody production response or a cellular immune response. Although various adjuvants are known, adjuvants generally stimulate the body's immune response nonspecifically or specifically, separately from the antigen, and thereby specific to the co-administered antigen. Substances and methods for enhancing or suppressing various immune responses. Substances used in adjuvants (such as those listed in the “Immunological adjuvant” section of the Immunology Dictionary, Tokyo Chemical Dojin, 1993) can be roughly classified into immunological adjuvants and antigen-administered adjuvants.

Examples of immunoadjuvant substances include cells such as BCG and Propionibacterium acnes, cell components such as cell wall '' trehalose dimycolate, lipopolysaccharide, lipid A fraction, beige glucan, muramyl dipeptide, and vessutin. In addition to synthetic chemicals such as levamisole, there are proteins derived from biological components such as thymus hormone, thymic humoral factor, tuftsin, and peptide substances. In addition, lymphokines, monoforceins and interferons (the bioactive peptides derived from these cells are called “siteforceins”), and the synthetic chemical substance cyclophosphamide also has adjuvant activity, so it has a broad sense. Treated as an immunoadjuvant.

Antigen administration adjuvants include incomplete Freund's adjuvant (FIA), a water-in-oil emulsion in which equal amounts of a mineral oil containing aracell A and an aqueous antigen solution are mixed. Complete Freund's adjuvant (FCA) with Mycobacterium smegmatis dead cells suspended in FIA In many cases, an immune adjuvant is mixed with FIA. In addition to these water-in-oil emulsions, an oil-in-water emulsion in which antigens are placed in mineral oil and suspended as small particles in saline, etc., and a water-in-oil emulsion in which saline is further suspended in saline There is a water-in-oil emulsion. Some ribosomes are made by combining phospholipids and cholesterol at an appropriate ratio. Aluminum hydroxide gel, alum sediment, silica, and pio power are also used as adjuvants, all of which are used in combination with immunological adjuvants (Immunology Dictionary, Tokyo Chemical Dojin, 1993).

However, the conventional adjuvant technology has the following problems.

(1) Mineral oil used in FIA is highly toxic. Therefore, the use of highly safe substances that are metabolically degraded in vivo, such as bacterial cell components, is desired.

(2) Although it has the advantage of releasing a wide variety of physiologically active substances typified by cytokines, such as the action of lipopolysaccharide derived from bacterial cells (see Masato Nakano and Masatomo Kodama, Endotoxin- -New treatment 'diagnosis' test, Chapter 6, pp. 97-138, Kodansha Sentifik, 1995), conversely only certain types of bioactive substances to induce an immune response in a specific direction The specificity of releasing is poor. For example, among the helper T cells that have been revealed by recent advances in immunology, if it is desired to differentiate and specifically induce Th 1 and Th 2 responses, The next adjuvant cannot cope.

(3) Therefore, it is desirable to administer cytokines directly as an adjuvant.Since cytokine is a protein / peptide, it is rapidly metabolized and eliminated in vivo in the order of minutes to several hours and sustained. Lack of functional action.

(4) In order to solve the above-mentioned problems, there has been proposed a method in which cytokines are frequently administered to a living body or administered in a liposome so that metabolic inactivation is unlikely (Adler et al., Cancer Biother, 10: 293-306, 1995). However, while the ribosome-encapsulated peptide is stable, the unencapsulated peptide is discarded without being used when purifying the ribosome, or diffuses rapidly in an aqueous solution in which the ribosome is suspended. However, it is quickly lost in the body. Also, the volume outside the ribosome is Since the volume is much larger than the volume in the ribosome, there is a problem that a cytokine molecule cannot be efficiently used in producing a liposome formulation.

(5) Therefore, a method of incorporating microbeads by incorporating cytokine with another polymer, processing it into a sustained-release dispersant so that the site force in is released slowly, and then administering it to a living body. (Pardoll, et al., US Patent 5, 861, 159) ₀ However, the process of solidifying with micro-beads with a cross-linking agent so that it does not easily dissolve and providing sustained release is complicated. It is. In addition, the safety of the new macromolecule generated by the cross-linking agent must be proven when administered to humans. Furthermore, when a synthetic polymer is used, an organic solvent is often used, and there is also a problem that the kinetics to be included are inactivated and washed. Attempts have been made to produce synthetic polymers with biodegradable substances in order to avoid the safety issues of synthetic polymers.At this stage, although polymers containing lactic acid are promising, acids are decomposed during the decomposition process. There is also a problem that acid kinetics are likely to be deactivated due to acid generation. Moreover, it is technically difficult to simultaneously and stably cover multiple types of sitekinetics. Disclosure of the invention

An object of the present invention is to provide a sustained-release preparation of a physiologically active peptide. More specifically, it is an object of the present invention to provide a sustained-release preparation that allows a bioactive peptide represented by a cytokine to act stably and continuously in vivo for a long time. It is also an object of the present invention to provide a sustained-release preparation which can be used as an effective adjuvant preparation for immunostimulation, which solves the above problems.

The inventors of the present invention have made intensive efforts to solve the above-mentioned problems. When kneaded with a semisolid composition containing a biologically-derived lipid, bioactive peptides can be released in vivo over a long period of time, which is extremely useful as a safe and highly effective sustained-release preparation. Was found. In addition, by blending cholesterol as a bio-derived lipid, It has also been found that the release rate of a physiologically active peptide from the above preparation can be controlled. The present invention has been completed based on these findings.

That is, the present invention provides a sustained-release preparation containing a physiologically active peptide, comprising the following components:

(A) bioactive peptides;

(B) a biological protein; and

(C) Biogenic lipid

And a semisolid preparation prepared by kneading the above components (A) to (C).

According to a preferred embodiment of the present invention, the sustained-release preparation wherein the bioactive peptide is one or two or more peptides selected from the group consisting of a cytokin, a growth factor, a hormone, and a chemokine; Wherein the sustained-release preparation is one or more proteins selected from the group consisting of albumin, a plasma protein fraction, and a serum protein fraction; instead of a biological protein, or The sustained-release preparation containing a biologically-derived saccharide together with the above-mentioned sustained-release preparation, wherein the biologically-derived saccharide is heparin; The biologically-derived lipid is a phospholipid, a fatty acid, a neutral fat, a cholesterol, and a fat-soluble vitamin. The sustained-release preparation of the present invention is one or more lipids selected from the group consisting of:

Further, according to another preferred embodiment, (D) the sustained-release preparation further comprising an immunoadjuvant substance; the sustained-release preparation wherein the immunoadjuvant substance is tuberculin; the release rate of the bioactive peptide as a biological lipid. The sustained-release preparation containing a lipid having a property capable of controlling the amount of the physiologically active peptide (eg, cholesterol, saturated fatty acid, or neutral fat containing saturated fatty acid); The above-mentioned sustained-release preparation containing cholesterol; the above-mentioned sustained-release preparation containing a biogenic amine E in an amount capable of preventing oxidation of lipids as a biologically derived lipid; a group containing a biodegradable polymer substance and / or a biologically derived extracellular matrix The sustained-release preparation supported on a material; the biodegradable polymer substance is collagen, gelatin, elastin, laminin, heparin, chondroitic The sustained-release preparation, which is one or more polymer substances selected from the group consisting of sulfuric acid, polylactic acid, polyethylene glycol, and borilla doco-glycolide; an extracellular matrix derived from a living body Is an extracellular matrix selected from the group consisting of umbilical cord washing residue tissue, small intestinal submucosal tissue, and vascular washing residue tissue; the above-mentioned sustained-release preparation; wherein the base material is a cloth, sheet, tube, or capsule; Certain of the sustained release formulations are provided. BEST MODE FOR CARRYING OUT THE INVENTION

The preparation of the present invention comprises: (A) a biologically active peptide; (B) a biologically-derived protein and / or a carbohydrate having a stabilizing effect on the biologically active peptide; and (C) a biologically-derived lipid. Which is a semi-solid preparation that can release bioactive peptides in a sustained manner. In a preferred embodiment, the composition further comprises (D) an immunoadjuvant substance. In another preferred embodiment, the biologically-derived lipid contains cholesterol in an amount capable of controlling the release rate of the physiologically active peptide and biubimin E in an amount capable of preventing oxidation of Z or lipid.

As a bioactive peptide, a site force factor, a global factor, a hormone, a chemokine, or the like can be used. The type of the peptide is not particularly limited, and may be any of an oligopeptide containing about 10 amino acid residues from the peptide or a polypeptide containing about 10 or more amino acid residues. Biologically active peptides may be modified with sugar chains, lipids, metals, etc., and the term “bioactive peptide” used in the present specification is interpreted as being limited to biologically active peptides consisting only of peptide chains. Not be. Examples of site force-in include, for example, Inuichi Leukin-2, granulocytes; macrophage colony stimulating factor, Inuyu Leukin-1, Inuichi Leukin-4, Interleukin-6, Inuichi Leukin-7 Inleukin-8, interleukin-12, interleukin-18 and the like can be used, and as a factor, for example, fibroblast growth factor can be used.

As a hormone, for example, insulin or the like can be used. Examples thereof include macrophage-derived chemokines, EBI1 ligand chemokines, stromal cell-derived factor 1, secondary lymphoid tissue chemokines, B-cell attractant chemokines-1 and the like. However, the bioactive peptides contained in the sustained-release preparation of the present invention are not limited to those exemplified above. The sustained-release preparation of the present invention may contain two or more physiologically active peptides.

As the biological protein, for example, a protein having a hydrophobic portion can be used, and more specifically, serum albumin, a plasma protein fraction, a serum protein fraction, or the like can be used. For example, proteins contained in body fluids, particularly serum proteins, are suitable, and it is desirable to use proteins having no antigenicity. These proteins may be used in an appropriate combination. As the carbohydrate derived from the living body, for example, heparin or the like can be used, and two or more carbohydrates may be appropriately used in combination. As used herein, the term "biologically-derived protein" or "biologically-derived carbohydrate" refers to a substance present in a living body, a recombinant produced by a method such as genetic engineering, or a physiological function. It is used as a concept that includes a modification of a part of the molecular structure such as the amino acid sequence or sugar chain structure within a range that does not impair the composition.

As the biologically-derived protein or lipid, preferably, a human-derived protein or carbohydrate can be used. Biogenic proteins and saccharides may be used in combination. As the biological protein or the biological lipid, it is desirable to select a substance that stabilizes a physiologically active peptide contained in the preparation of the present invention. Biologically-derived proteins or lipids having a stabilizing effect on bioactive peptides can be appropriately selected by those skilled in the art by temporarily measuring the residual ratio of the bioactive peptides contained in the preparation. It is.

Phospholipids, fatty acids, neutral fats, cholesterol, fat-soluble bimin, and the like can be used as the biologically-derived lipid, and these may be used in an appropriate combination. By using Biyumin E as a biological lipid in combination with other lipids, Deterioration of lipids due to oxidation reaction of unsaturated fatty acids in lipids can be prevented. The amount of vitamin E to be added is not particularly limited. For example, it is preferable to add about 0.05 g of vitamin E to 1 g of lecithin. By adding other fat-soluble bimins as lipids in addition to biminmin E, an additive or synergistic effect of the biologically active peptide may be expected in some cases. The hardness and dissolution rate of semisolid preparations can be controlled by blending bio-derived lipids such as cholesterol, saturated fatty acids, and neutral fats containing saturated fatty acids, alone or in combination with other lipids. The release rate of the bioactive peptide from the preparation can be controlled. A preparation containing cholesterol as a biologically-derived lipid for controlling the release rate of a bioactive peptide is a preferred embodiment of the present invention. The formulation of the present invention may contain a physiologically acceptable organic solvent such as ethanol in addition to water.

Although the method for producing the preparation of the present invention is not particularly limited, it can usually be produced according to the following method. Add an organic solvent such as ethanol to the bio-derived lipid, if necessary, and dissolve by heating. It is generally not necessary to add an organic solvent if the lipid can be kept in a liquid state at room temperature or 37 ° C, but if it cannot be kept in a liquid state, it is necessary to use the minimum necessary organic solvent. It is desirable to add. For example, 0.5 ml of ethanol is added to 2 g of chicken egg-derived lecithin, and the mixture is heated to 45 ° C. After 4 hours, a liquid composition in which lecithin is dissolved can be obtained. It is preferable to use ethanol as the organic solvent.

A biologically-derived protein is added to a lipid or a mixture of a lipid and ethanol, and the mixture is sufficiently kneaded to produce a semi-solid composition having reduced lipid fluidity. The protein can be mixed in the form of an aqueous solution or a powder. The semi-solid composition preferably exhibits, for example, a grease state. The method for preparing the semi-solid composition is not particularly limited, and any method available to those skilled in the art may be used. For example, the injection inner cylinder can be reciprocated more than ten times using an adjuvant mixing tool in which two injection cylinders are connected to each other with a thin tube so as to be sufficiently kneaded. By adding a bioactive peptide to the kneaded product in this state and further kneading it, the sustained-release preparation of the present invention can be produced. it can. Alternatively, a physiologically active peptide may be mixed with the protein in advance and kneaded with the fat.

The amount of the protein added to the biological lipid is not particularly limited, but is preferably in the range of 0.1% (w / w) to 15% (w / w) based on the lipid. For example, it is preferable to use serum albumin for the cytokine Cytoichi Leukin-2 (IL-Z). 10 ⁵ may be added with 25% human serum albumin solution 1 ml against IL- 2 international units, but the amount of the albumin is not limited to the amount described above. Alternatively, instead of serum albumin, a heat-treated plasma protein fraction or whole serum protein may be used.

An example of using a biologically derived carbohydrate includes, for example, using heparin for fibroblast growth factor (FGF). The combination ratio of FGF to heparin can be determined by referring to the quantitative ratio commonly used when adding these substances to cultured animal cells. For example, it is preferable that the FGF is about 0.1 / g for 1 mg of heparin, but the amount of heparin is not limited to the above. When using a biologically derived carbohydrate, it is desirable to use a biologically derived protein at the same time, since the carbohydrate is generally not compatible with lipids. When serum albumin is added to the mixture exemplified above, it is preferable to use 1 g or more of serum albumin per 1 mg of heparin.

An immunoadjuvant substance may be added to the sustained-release preparation of the present invention. In the preparation of this embodiment, the effect of the bioactive peptide and the effect as an immunoadjuvant can be expected at the same time. Examples of the immunoadjuvant substance include cells such as BCG and Propionibacterium acnes, cell components such as cell wall ′ trehalose dimycolate, lipopolysaccharide, lipid A fraction, betaglucan, muramyl dipeptide, and the like. In addition to synthetic chemicals such as vestin, levamizole and the like, thymic hormone, thymic humoral factor, protein or peptide derived from biological components such as tuftsin, cyclophosphamide and the like can be mentioned. Bergrin is preferred as the immunoadjuvant. These immunoadjuvants are effective against biological lipids. The mixing ratio can be appropriately selected, but generally, it is desirable that the amount of the biological lipid is 30% (w / w) or more.

The preparation of the present invention can be locally administered to mammals including humans as a semi-solid, but the preparation of the present invention is supported on a substrate containing a biodegradable polymer substance and / or a biologically derived extracellular matrix. This facilitates handling with instruments such as tweezers, and allows the drug to be easily implanted subcutaneously or in a body cavity for administration. Examples of the biodegradable polymer substance include collagen, gelatin, elastin, laminin, heparin, chondroitin sulfate, polylactic acid, polyethylene glycol, and polylactide doco-glycolide. May be used in combination. As the extracellular matrix derived from the living body, for example, umbilical cord washing residue tissue, small intestinal submucosal tissue, or vascular washing residue tissue can be used. The shape of the substrate is not particularly limited, but is preferably, for example, formed into a cloth, a sheet, a tube, or a capsule. For example, it is possible to prepare a sheet-like preparation by applying 0.7 ml of the above semi-solid preparation to a 1 xlx O.2 cm collagen fiber sheet and overlaying a collagen fiber sheet of the same size on top of this. it can. The preparation in this form is suitable for a form such as implantation administration in a living body.

The preparation of the present invention can release bioactive peptides such as cytokines in a sustained manner, avoids strong systemic side effects caused by large-dose administration at a time, and places the bioactive peptides near the local administration site. Allows long-term effects on mitigation. In addition, the components that make up the formulation are degraded in the body after a long period of time and disappear, leaving no side effects or physical obstacles. In particular, when eliciting an immune response mediated by cytokines, the method is particularly useful for directing the reaction in a specific direction. Further, in the preparation of the present invention, it is easy to make a plurality of bioactive peptides the same preparation, and it is also possible to produce a preparation in combination with an immunoadjuvant substance.

For example, from the group of lymphocyte helper T cells, the population that occupies the majority of Th1 cells produces a large amount of inferior ferrona, while the population that occupies the majority of Th2 cells produces the inferior ferrona. Leukin-4 (IL-4) is produced in large quantities. As a result, the former is a killer A cell-mediated immune response in which the T cell population proliferates is elicited, and in the latter case, a humoral immune response in which antibody production occurs is elicited. This reaction requires both Leukin-2 (IL-2), which maintains the viable growth of τ cells, but both cytokines are expensive and preparations containing two types of cytokines simultaneously. It is not desirable from a price point of view. In such a case, the use of the preparation of the present invention in which an inexpensive bacterial component adjuvant and infusion reagent is combined can strongly induce a cellular immune response while creating an environment for producing IL-2. Is possible. In addition, the use of the preparation of the present invention in which an inexpensive cell component adjuvant and Ito 4 are used makes it possible to strongly induce a humoral immune reaction while creating an environment for producing IL-2. . Example

Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to the following Examples.

Example 1: Sustained-release preparation of interleukin-2 (IL-2) -1

When IL-2 was kneaded with lecithin, the main component of biological phospholipids, it was examined whether or not IL-2 was sustainedly released while maintaining its activity.

[Method]

1. Preparation of lecithin solution

Lecithin (Sigma Chemical Co., USA), which is commercially available as a reagent, is in an individual form at room temperature. Take 2 g of this in a test tube, add 3 ml of vitamin E (Sigma Chemical Co., USA) diluted to 200 mg / ml with ethanol, heat at 45 ° C to dissolve, (Pore size 0.22 / m, Millipore Co., USA) to remove bacteria.

2. Preparation of IL-2 solution

Human recombinant IL- 2 (manufactured by Shionogi & Co., Ltd., 350,000 units containing vial) were dissolved by adding sterile distilled water so that the 10 ⁵ international units (IU) / ml.

3. Preparation of cholesterol solution 1 ml of ethanol was added to 10 m of cholesterol powder (Sigma Chemical Co., USA) and dissolved.

4. Preparation of semi-solid grease-like preparation

While stirring 0.5 ml of the above-mentioned lecithin solution with a vortex mixer, an aqueous solution in which 100 jl of a 25% human albumin solution (Baxter Co., USA) was added to 100 μl of the IL-2 solution was dropped and mixed. In Experiment-1, 30 jul (A), 60/1 (B), or 100-1 (C) was added to the cholesterol solution. In this case, the total volume varies with the volume of ethanol. In Experiment 2, the amount of cholesterol added was 300 μg (D) or 600 jug (E), and in each case, the volume of the ethanol solution was adjusted so that the total volume was 700 l. did. The mixture was kneaded with an adjuvant mixing tool in which two syringes were connected to each other with a thin tube facing each other, and were sufficiently mixed. The composition became a thin, milky, semi-solid grease.

5. Carrying by biodegradable sheet

This semisolid grease-like preparation was applied to a fibrous collagen sheet (trade name: Integran, manufactured by Koken Co., Ltd.) cut into 1 cmxl cmx0.2 cm so as to have a thickness as uniform as possible. Another fibrous collagen sheet of the same size was covered from above and sandwiched in a sign-shaped manner to obtain a sheet-shaped preparation.

6. IL-2 release experiment

One sheet-shaped preparation prepared in step 5 above was placed in a 60-cell plastic dish for cell culture (Falcon), and RPMI-I was added with fetal serum at 10% (v / v). 5 ml of 1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) was added, and the mixture was incubated overnight at 37 ° C. in a carbon dioxide gas incubator. After 24, 48, 72 hours and 5 days, the medium was completely changed.

7. Measurement of IL-2 release

The concentration of IL-2 in the medium that is released and accumulated in a physiologically active state is measured by the bioassay method using CTLL-2 cells (M. Ishiyama, et. Al., Tatanta, 44, 1299, 1997). did. The amount of IL-2 released per day is calculated based on the amount of IL-2 accumulated in 5 ml of medium. And expressed as a percentage of 10 ⁴ IU initially added.

[Result]

Table 1 shows the results. The release rate of IL-2 was clearly changed by the amount of cholesterol dissolved in ethanol added to the semisolid grease-like preparation. In Experiment-1, when the amount of cholesterol added was 1,000 ug (the amount of ethanol added was 100 グループ 1) (Group C), the maximum free amount was observed at 24.8% on the first day. On the second day, it dropped sharply to 8.2% and on the third day to 1.4%. Such a rapid decrease in release pattern was also observed when cholesterol was added at 300 g (ethanol was added at 30 zl) (Group A), and the maximum release at day 1 was 9.4%. On the day, it was about 1/4. However, when the amount of cholesterol added was 600 µg (the amount of ethanol added was 60〃1) (Group B), the amount of IL-2 released on day 1 was small, 2.5%. On the 2nd, 3rd, and 5th days, the amount of liberation per day was relatively stable with no significant change in the 1% range, although there were fluctuations up and down.

Such a relatively stable tendency to release IL-2 was also observed in Experiment-2. The amount released per day at 600 〃g (group E) is smaller than that at cholesterol addition at 300 添加 g (group D), as in Experiment-1. And Group D were relatively stable compared to Group D. From the above results, it is clear that a desired release rate can be achieved by adjusting the amount of cholesterol and the amount of ethanol. For example, in the formulation of (B), a low concentration of a bioactive peptide can be made to act for a long time, while in the formulation of (C), a high concentration of a bioactive peptide is made to act first, and then a low concentration of a bioactive peptide is made. Suitable for long-lasting action of the peptide. Table 1 Experiment-1 experiment-2 groups (A) (B) (C) (D) (E) Cholesterol content (〃g) * 300 600 1000 300 600 Amount of free IL-2 per day

1 Statement (%) 9.4 2.5 24.8 17.4 6.2

2 Said 2.4 1.3 8.2 3.3 5.8

3 Big 1.7 1.7 1.4 2.2 3.1

5 Saying 0.5 1.0 0.3 0.6 0.8

* Amount of cholesterol in grease-like preparation coated on fibrous collagen sheet cut into 1 cm x lcm x 0.2 cm Example 2: Sustained release formulation of IL-2-2

We prepared IL-2 preparations with the lowest possible alcohol concentration in the grease-like preparations, and examined their sustained release.

[Method]

1. Preparation of lecithin solution

To 2 g of lecithin (Sigma Chemical Co., USA), 0.1 g of vitamin E (Sigma Chemical Co., USA) and 0.5 ml of ethanol were added, and the mixture was dissolved by heating to 45 ° C, and Millipore filter ( The bacteria were removed by passage through a 0.22 μm pore size, Millipore Co., USA).

2. Preparation of IL-2 solution

25% human albumin solution as human recombinant IL- 2 (manufactured by Shionogi &) becomes ^{10 5 IU / ml (Baxter Co.} , USA) was dissolved in.

3. Preparation of cholesterol solution

50 mg of cholesterol powder (Sigma Chemical Co., USA) was dissolved in ethanol to make 2 ml. 4, Preparation of semi-solid grease preparation

To the lecithin solution 600, 24 μl of ethanol and 100 jl of the IL-2 solution were added, and the two syringes were kneaded and mixed sufficiently with an adjuvant mixing tool connected by a thin tube facing each other. This mixture became a translucent / semi-solid grease-like composition which was hardly emulsified. In addition, a semisolid grease-like preparation was prepared in which cholesterol solution 24-1 was added instead of ethanol 24.1. This was also a semi-transparent and semi-solid grease-like composition that was hardly emulsified. The former does not contain cholesterol, while the latter contains 600 mg of cholesterol.

5. Loading on biodegradable sheet and IL-2 release experiment and measurement of IL-2 release amount The case of Example 1 was followed.

[Result]

In Example 2, the alcohol concentration was lower than that in Example 1 to a final concentration of 19.3%. The release rate of IL-2 on day 1 further decreased, and as shown in Table 2, the addition of cholesterol released IL-2 at an almost stable rate over 5 days. Table 2 Cholesterol content (jug) * 0 600

Free IL-2 per day

Day 1 (%) 1.67 1.25

2 Statement 0.83 1.00

Day 3 0.83 1.25

Day 5 0.16 0.63

* Amount of cholesterol in grease-form preparation applied to fibrous collagen sheet cut into 1 cm xl cm x 0.2 cm Example 3: Investigation of sustained release of IL-2 sustained release preparation in vivo

In Examples 1 and 2 of the above Examples, a sustained release of IL-2 in a medium outside the body was shown, but it was examined whether or not this shows a sustained release also in the body.

[Method]

1. Preparation of lecithin solution '' Preparation of cholesterol solution

Example 2 was followed. However, lecithin used was made by Wako Pure Chemical.

2. Preparation of IL-2 solution

Human recombinant IL- 2 solution (manufactured by Shionogi & Co.) so as to have a 10 ⁷ IU / ml 25 ° human albumin solution (Baxter Co., USA) was dissolved in.

3. Preparation of semi-solid grease preparation

According to Example 2, add 24〃1 of cholesterol solution and 100〃1 of IL-2 solution to 600〃1 of lecithin solution, and knead well with an adjuvant mixing tool in which two syringes are connected to each other with a thin tube facing each other. Mixed.

4. Support by biodegradable sheet

Apply 0.1 ml of this semi-solid grease-like preparation to a fibrous collagen sheet (trade name: Integran, manufactured by Koken Co., Ltd.) cut into 0.5 cm x 0.5 cm x 0.2 cm so that the thickness becomes as uniform as possible. Was. Another sheet of fibrous collagen sheet of the same size was covered from above and sandwiched in a sandwich to form a sheet-shaped preparation.

5. IL-2 release experiment

A mouse (strain C57BL / 6J, 7-10 week-old female) was treated with 1 ml of nebula for mouse anesthesia (1 mg / ml in Dalbecco's phosphate buffered saline). Anesthetized by intraperitoneal injection of 0.6-0.8 ml per animal. One sheet-shaped preparation prepared in 4. above was implanted subcutaneously on the back of one mouse. The wound on the skin was sutured. One day later, 1 ml of RPMI-1640 medium containing 10% (v / v) of fetal bovine serum was added in a syringe with an 18G injection needle adjacent to and directly in contact with this installed preparation. Injection, with the needle stabbed, pinch the subcutaneous bulge caused by the injection and the bulge due to the installation product with two fingers, gently rub 50 times (about 1 minute), and release into the installation product IL-2 was also diffused into the medium to be sufficiently distributed. Immediately thereafter, about 2001 of the medium was extracted from the medium injection site. The swelling due to the medium injection had disappeared by the next day. Sampling from the site where the preparation was placed by this medium injection was performed once a day for a total of 5 days. The sample was centrifuged at 12,000 rpm for 5 minutes in a micro high-speed centrifuge to remove bacteria, and the supernatant was removed.

6. Measurement of IL-2 release

The activity of IL-2 was quantified in the same manner as in Example 1 with the centrifuged supernatant of the medium extracted in step 5 as the measurement target.

[Result]

Table 3 shows the local free IL-2 concentration of IL-2 from day 1 to day 5 for each mouse. It was found that IL-2 was continuously released locally for at least 3 days, and sustained release was maintained in the body.

Table 3 Local free IL-2 concentration for each mouse (U / ml) Average (U / ml)

1 after 25, 26 25.5

2 After 13, 13, 47 25.7

3 days later 2, 27, 17 15.3

4 After 0.1, 0.1

5 After 0.2, 0, 0 0.1

(Note: The number of mice per measurement point is 2-3)

Industrial applicability

The formulation of the present invention administered to a living body gradually dissolves upon contact with a body fluid and continuously releases the kneaded physiologically active peptide. The release rate of the physiologically active peptide depends on the amount of cholesterol and / or solvent (eg, ethanol) added to the drug product. And the desired release rate can be easily achieved. In addition, the kneaded physiologically active peptide is stably retained in the preparation, so that it can be stored for a long period of time, and when it is processed into a preparation such as a sheet form, it is excellent in handling at the time of administration.

Claims

The scope of the claims

1. A sustained-release preparation containing a physiologically active peptide, comprising the following components:

(A) bioactive peptides;

(B) a biological protein; and

(C) Biogenic lipid

A preparation in a semi-solid state, wherein the components (A) to (C) are kneaded.

2. The sustained-release preparation according to claim 1, wherein the physiologically active peptide is one or more peptides selected from the group consisting of cytokines, growth factors, hormones, and chemokines.

3. The sustained-release preparation according to claim 1, wherein the biological protein is one or more proteins selected from the group consisting of albumin, plasma protein fraction, and serum protein fraction. .

4. The sustained-release preparation according to any one of claims 1 to 3, wherein the sustained-release preparation contains a biologically-derived saccharide in place of or in addition to the biologically-derived protein.

5. Any one of claims 1 to 4, wherein the biological lipid is one or more lipids selected from the group consisting of phospholipids, fatty acids, neutral fats, cholesterol, and fat-soluble bimin. Or the sustained-release preparation according to item 1.

6. The sustained-release preparation according to claim 5, wherein the lipid derived from a living body is biminmin E.

7. The sustained-release preparation according to any one of claims 1 to 6, further comprising (D) an immunoadjuvant substance.

8. The sustained-release preparation according to any one of claims 1 to 7, comprising a lipid having a property capable of controlling the release rate of the bioactive peptide as a biological lipid.

9. The sustained-release preparation according to any one of claims 1 to 8, which is supported on a substrate containing a biodegradable polymer substance and / or a biologically-derived extracellular matrix.

10. The method according to claim 9, wherein the base material is in the form of a cloth, a sheet, a tube, or a capsule. The sustained-release preparation according to the above.

11. The sustained-release preparation according to claim 9, wherein the biodegradable polymer is gelatin or collagen.