JP4578067B2 - Topical pharmaceutical composition - Google Patents

Description

[0001]
[Technical field to which the invention belongs]
The present invention relates to topical pharmaceutical compositions. More specifically, the present invention relates to a topical pharmaceutical composition containing macrophage migration inhibitory factor (MIF) and a biodegradable material.
The invention also relates to a wound protection dressing comprising a topical pharmaceutical composition.
The topical pharmaceutical composition and dressing of the present invention can be used for treatment of wounds by sustained release of MIF.
[0002]
[Prior art]
There are 65,000 patients who have undergone skin and mucous membrane damage or loss, such as wounds, burns, frostbite, UV irradiation, trauma, skin ulcers, bullous skin diseases, and “bed sores”, which is a problem of long-term inpatient care. Count people. These include surgical wounds, resection wounds, deep wounds with dermal and epidermal damage, ocular tissue wounds, dental tissue wounds, oral wounds, diabetic ulcers, skin ulcers, elbow ulcers, arterial ulcers, veins Congestion ulcers, heat exposure, or burns resulting from chemicals, etc., associated with uremia, malnutrition, vitamin deficiencies, and associated with systemic treatment with steroids, radiation therapy, antitumor drugs and antimetabolites Also included are abnormal wound healing conditions such as complications. In addition, “bed slip” is a symptom caused by the fact that the renewal rate of epithelial cells is basically decreased due to aging.
[0003]
Wound healing consists of a series of processes in which injured tissue is repaired, specific tissue is regenerated, and new tissue is reorganized. Wound healing consists of three main phases: a) inflammatory phase (0-3 days), b) cell growth phase (3-12 days), and c) remodeling phase (3-6 months). The wound healing process is established by a series of complicated phenomena. The following physiological reactions occur on the wound surface. (1) The blood vessel breaks down and platelets collect and stop bleeding. In addition, hemostasis is caused by vasoconstriction. (2) Simultaneously, platelet-derived growth factor and epidermal growth factor are released from platelets. (3) The contracted blood vessels expand and the permeability of the capillaries increases, and leukocytes migrate into the tissue to eliminate bacteria. (4) Macrophages enter the wound and remove necrotic tissue. (5) Macrophages release various growth factors (factors that help angiogenesis, fibroblast activation, collagen production, etc.). (6) Cover the wound surface with epithelial cells. (7) Produce scaffolds such as collagen by fibroblasts. (8) Capillaries grow.
[0004]
The vital phenomenon of skin tissue regeneration / reconstruction will not be successful unless the above complex physiological reactions progress in a regular manner. The role played by fibroblasts in the regeneration and remodeling of the dermis and subcutaneous tissue of the skin is extremely important, and it suppresses fibroblast division, proliferation, migration, and differentiation, and the collagen fibers in fibroblasts Inhibiting the production of elastic fibers, reticulated fibers, and various extracellular matrix scaffolds must be avoided in order to facilitate the regeneration and reconstruction of skin tissue. Thus, fibroblasts and blood vessels play a central role in the wound healing process. Like epidermal cells, fibroblasts undergo a process of division, proliferation, migration, and differentiation, and the collagen fibers, elastic fibers, reticulofibers, and various extracellular matrix components are regularly ordered to a state close to that of healthy tissues in the dermis and subcutaneous tissues. Regeneration and remodeling of the skin tissue will not be successful unless it is neatly reconstructed, that is, if all of the extracellular matrix components are reliably regenerated and reconstructed quickly.
[0005]
Conventionally, in wound treatment, antibiotics and the like suppress the bacterial growth in the affected area and wait for natural healing, collagen, chitin, chitosan and other highly biocompatible extracellular matrix or artificial skin engraftment, natural For example, a method of administering a granulation or epidermal formation-promoting substance by a product extract has been used.
[0006]
As a method using antibiotics, antibiotics, antibacterial agents, antiviral agents, anti-inflammatory analgesics and the like are locally applied to the wound surface in order to prevent secondary infections.
[0007]
As a method for engrafting artificial skin as a model of skin, for example, keratinocytes cultured on a multilayer sheet (Non-patent Document 1), fibroblasts are cultured in a collagen gel, After gel contraction, epidermis keratinocytes were seeded and cultured on the gel (Patent Document 1), nylon mesh was seeded with fibroblasts and cultured, and mesh pores were caused by fibroblast secretion. When seeded, seeded and cultured with epidermal keratinocytes (Non-patent Document 2), or seeded and cultured fibroblasts on collagen sponge, then layered film-like collagen sponge, and further keratinized There is a method using seeded and cultured cells (Patent Document 2).
[0008]
In addition, artificial dermis for self-extracted hair follicle transplantation consisting of a wound contact layer composed of a composite matrix of a mammal-derived fibrotic atelocollagen and a modified atelocollagen derived from a mammal having a helix content of 0 to 80% and a moisture permeation regulating layer (patent Reference 3) Tissue-containing artificial dermis that treats all-layer skin defect wounds temporarily (patented) with artificial dermis in which a silicone layer is laminated on one side of a collagen sponge layer having a recess or hole for receiving skin tissue Document 4), an artificial dermis structure characterized by containing a neutralized chitosan / collagen mixed sponge (Patent Document 5) is known.
[0009]
However, since the preparation of such artificial skin requires a long manufacturing process, an expensive facility is required to prepare the preparation, such as control of microbial contamination.
[0010]
On the other hand, it is said that there is room for debate on the grounds for therapeutic effects regarding traditional wound treatments such as disinfection and gauze coating, which are methods for protecting the wound surface (Non-patent Document 3). In order to solve the clinical problems of such wound treatment, occlusive dressing agents such as hydrophilic hydrocolloid materials are used as coating agents that cover the entire wound surface, maintain oxygen permeation, and prevent bacterial invasion. is there.
[0011]
However, this type of wound dressing may cause a large wound on the tissue when a part of the tissue is in close contact with the coating and the agent is peeled off. Conversely, there is a film dressing agent that is made of a hydrophobic polyurethane film and uses a translucent transparent film that allows oxygen and water vapor to pass through. However, this type of wound dressing has the disadvantage that it cannot manage the absorption of wound exudates. For this reason, there is an alginate dressing, which is a dressing made of components extracted from seaweed and used for wound management, particularly for exudate management. For example, dressing (Airstrip®, Smith & Nephew Limited) that combines a hygroscopic pad and a non-adhesive film with a sponge-like pad and a waterproof adhesive bandage, or an adhesive layer consisting of a polyurethane film with high water vapor permeability and an adhesive layer Sex dressing (Op-SiteR, Smith & Nephew Limited; BioclusiveR, Johnson &Johnson; TegadermR3M), and recently dressing using non-adhesive porous film (MelolinR, Smith & Nephew Limited) is in contact with the wound surface. Yes. The common performance of these wound dressings is that they absorb or store wound exudate through the micropores in the inner microporous membrane, making the wound more recoverable and scratching when peeled compared to conventional wound dressings. The point is not to leave. These prior arts are characterized by passive use of natural skin regeneration or remodeling capabilities.
[0012]
As a method for administering granulation and epidermal formation promoting substances, as herbal tissue regeneration / reconstruction stimulating substances, hormones, vitamins, α-hydroxycarboxylic acid, γ-oryzanol, saponins and other herbal extracts Various substances such as placenta extract, plant lectin, mushroom extract, animal-derived protein, especially basic and acidic fibroblast growth factor have been used, but cells useful for the process of skin tissue regeneration and remodeling The required local concentration of the polypeptide having an activating action has not been fully studied.
Even if it is possible to regenerate epithelial tissue, it is difficult to predetermine and control optimal conditions for polypeptides such as growth factors that regenerate epithelial cells in the wound site in order to reconstruct complete skin. there were.
[0013]
For topical application of polypeptides and drugs consisting of polypeptides, the amount of substance released in a low dose topical formulation such as rIL-2 is about 1-100 nmole / m ² Body surface area / day (Body surface area 1.5m ² Polypeptide formulations that are 42 μg to 4 mg / day in terms of conversion have been published and various polypeptides (IL-2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, and 15 , TNF-α, TNF-β, nerve growth factor, CD40, Fas, CD27, and CD30 ligand, IFN-α, IFN-β, IFN-γ, IL-8, macrophage inhibitory protein, and Lantis, active fragments thereof, And pharmaceutically acceptable analogs and derivatives, and substances having cytokine activity selected from the group consisting of mixtures thereof (Patent Document 6), but controlled release of trace amounts of cell growth factors, etc. No topical formulation is known. Also, no sufficient formulation is known for maintaining an effective polypeptide concentration.
[0014]
In order to actively heal wounds, a group of molecules involved in the physiological reaction of regeneration and remodeling is known as a method of utilizing wound healing mechanism by regeneration or remodeling of epidermal tissue using growth factors . For example, EGF, TGF-β1, TGF-α, FGF, VEGF, PDGF-BB, TGF-β1, PDGF-AB, IGF, KGF, PDGF, TGF-β2, TGF-β3, FGF-2 basic fibroblast Examples include cell growth factor (also referred to as basic fibroblast growth factor; hereinafter referred to as “bFGF”), U-PA, t-PA, integrin, adhesion factor and the like (Non-patent Document 4). In recent years, clinical application of bFGF in the treatment of wounds (the medical drug “Fiblast Spray” was launched in 2001 and is the world's first regenerative medicine in the field of regenerative medicine that proliferates human tissues and cells) and has an angiogenic effect. Application of bFGF to the used blood vessel repair or the like has also been carried out (Non-patent Document 5). These are administered with bFGF to promote wound accumulation and angiogenesis of fibroblasts, and epithelium as a scaffold for epithelial reconstruction. The purpose is to regenerate the organization.
[0015]
In addition, for the purpose of providing a re-epithelialization promoter for application to a wound site to promote epidermis regeneration, a pectin as an active ingredient (Patent Document 7), a fibroblast containing a plant extract Growth promoter (patent document 8), keratinocyte growth promoter thought to contribute to promoting regeneration of human skin stratum corneum, injury healing agent applied to human or animal skin damaged by burns or wounds (patent Document 9) is known. Further, EGF (epidermal growth factor) as a growth factor of epithelial cells has been confirmed to have a clinical effect for practical use (Non-patent Document 6). A feature of the effect of EGF is that fibroblasts proliferate together with epithelial cells, and is therefore suitable for treatment of deep trauma to connective tissues. However, since EGF has a canceration-inducing action, it cannot be put to practical use. TGF-α, which is structurally related to EGF, also has the activity of promoting the proliferation of epithelial cells, but this property is an obstacle to practical use because it has a canceration-inducing action. Factors such as GM-CSF (Granular Macrophage-Colony Stimulating Factor) have been discovered as other epithelial cell growth promoting factors (Non-Patent Documents 7 to 9).
[0016]
By the way, the epidermis of the skin is composed of five layers of a basal layer, a spinous cell layer, a granular layer, a transparent layer, and a stratum corneum from the lowermost layer to the uppermost layer. Then, keratinocytes existing on the basement membrane repeat proliferation and differentiation and move to the uppermost layer. Therefore, keratinocytes are essential cells for epidermal regeneration. Therefore, the provision of a keratinocyte growth promoter is an effective means for skin regeneration. As a technique aiming at such a thing, there is an invention of an epithelial cell growth promoter comprising HGF (hepatocyte growth factor) of Nakamura et al. (Patent Document 10). However, the demonstration of the effect is limited to the proliferation and motility of cultured epithelial cells and is insufficient, and the concentration range in which HGF acts on epithelial cells is very low, up to 7.5 ng / ml. It has a drawback that it is difficult to control the effective amount of HGF locally because it binds strongly with extracellular acidic mucopolysaccharides such as.
[0017]
In addition, FGF (fibroblast growth factor) mainly promotes the proliferation of fibroblasts, and therefore, it was impossible to selectively propagate only epithelial cells.
[0018]
VEGF (vascular endothelial growth factor) is a vascular endothelial cell-specific growth factor (Non-patent Document 10) and is known to act on skin-derived vascular endothelial cells (non-patent document 10). Patent Document 11). In general, VEGF production is induced by environmental factors such as ischemia in the vascular endothelium and is considered to have physiologically significant significance. It is known that VEGF-producing cells in the skin are epidermal keratinocytes (Non-patent Document 11). However, VEGF is required during the inflammatory phase (0-3 days) with angiogenesis and macrophage migration early in wound healing, but VEGF is also known to act on blood vessels to increase its permeability. Therefore, when VEGF is administered during the remodeling period (3 days to 6 months), edema does not subside and can be counterproductive.
[0019]
In Patent Document 11, as a material technique for regenerating a tissue organ in vivo, undifferentiated mesenchymal cells, bone marrow cells, peripheral blood stem cells, neural stem cells, hepatic stem cells (small hepatocytes, oval cells), embryos Cells such as sex stem cells (ES cells), EG cells, muscle satellite cells, osteoblasts, elephant buds, chondroblasts, epithelial germ cells, cholangioblasts, and basic fibroblast growth factor (bFGF), Platelet differentiation growth factor (PDGF), insulin, insulin-like growth factor (IGF), hepatocyte growth factor (HGF), glial-induced neurotrophic factor (GDNF), neurotrophic factor (NF), hormone, cytokine, bone morphogenetic factor ( BMP), a material for in vivo regeneration of tissue organs composed of cell growth factors such as transforming growth factor (TGF) has been disclosed, and a combination of these cells and MIF There is no mention of topical treatment formulations consisting of
[0020]
Macrophage migration inhibitory factor (MIF) is a culture supernatant of guinea pig lymphocytes that has been sensitized and activated with antigen, and can prevent migration of guinea pig macrophages from capillaries in vitro. It was named as a migration inhibitory factor (Non-patent Documents 13 and 14).
[0021]
Human-MIF is also known to be a polypeptide that inhibits the migration ability of macrophages. Conventionally, MIF derived from human cells is known, but is only described as a mixture with other proteins in biological fluids. MIF belongs to the lymphokine group, which comprises a biologically active soluble polypeptide and is secreted from these cells when lymphocytes and monocytes or macrophages are stimulated by antigens, mitogens, and the like. Lymphokines, as protein mediators, play an important role in the immune regulation of cellular immunity, inflammation and effector mechanisms. Other examples of lymphokines include immune interferon (γ-interferon), interleukins 1 and 2, and macrophage activating factor (MAF). These lymphokines control the differentiation, activation and proliferation of various cell types of the immune system.
[0022]
Currently, MIF is a single substance that is clearly distinguished from γ-interferon macrophage activator (MAF) and other lymphokines. It was revealed that the protein encoded by human MIF cDNA has a molecular weight of 12.3 kD and is composed of 116 amino acid residues (Non-patent Document 15). It was announced that a protein having a primary structure of MIF binds to an affinity gel in which glutathione (GSH) is immobilized (Non-patent Document 16), and high-purity human MIF is utilized by utilizing the specific binding ability to glutathione. It was revealed that the human MIF was a homodimer having a molecular weight of 12.5 kD and an isoelectric point of pH 5.0 (Non-patent Document 17). Further, it has been discovered by recent research that MIF, which inhibits the migration ability of macrophages, shows chemotaxis in a rat epithelial cell in a concentration-dependent manner and promotes its migration (Non-patent Document 18).
[0023]
Human-MIF induces monocytes and inactive tissue macrophages to differentiate into inflammatory macrophages and plays a critical role in the early stages of the inflammatory response (delayed type hypersensitivity response). While endotoxin shock is exacerbated by MIF released from the pituitary gland, it is recognized that symptoms are reduced by anti-MIF antibody (Non-patent Document 19).
Since this MIF activity was first observed, numerous publications have reported the isolation and identification of putative MIF molecules (see, eg, Non-Patent Documents 20-32).
[0024]
A drug composition using MIF-3, which is considered to be a polypeptide closely related to MIF, is disclosed in Patent Document 11. However, since the disclosed MIF-3 is not strongly expressed in the skin, even related polypeptides have no physiological rationality for administration to the skin. In addition, their proposal has been limited to the hypothesis that increasing the number of macrophages and increasing the wound healing rate has not been demonstrated. The dose is only defined as 10 μg / kg body weight as a systemic dose, and it does not demonstrate how much MIF-3 is specifically required in the wound local area. Furthermore, it does not even mention the most important concentration range. Moreover, there is no suggestion that epithelial cell migration is an important phenomenon in the wound healing process.
[0025]
Non-Patent Document 33 describes that wound healing is delayed in MIF-deficient mice. However, there is no mention of how much MIF is administered to promote wound healing.
[0026]
As a technology to provide a skin substitute by combining materials, human fibroblasts producing substances effective for wound healing are used, and by identifying the effective production amount of substances effective for wound treatment, A cultured skin substitute having stable wound healing power is known (Patent Document 12). Specific examples of the product effective for wound healing include, for example, vascular endothelial growth factor (VEGF), interleukin-6 (IL-6) or type I collagen, fibronectin, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), interleukin-1 (IL-1) and the like are mentioned, but the possibility of utilizing MIF has not been shown.
[0027]
The use of a hydrogel composition for sustained-release delivery of a polypeptide growth factor has been disclosed for the purpose of sustained-release administration of a growth factor to a wound site (Patent Documents 13 and 14). Patent Document 13 uses a composition in which a hydrogel selected from sodium carboxymethylcellulose and polyacrylic acid, a polyoxyethylene-polyoxypropylene block copolymer and the like contains an angiogenic growth factor (for example, bFGF or VEGF). Have been disclosed to be useful in treating conditions characterized by ischemia. Patent Document 14 discloses that by adding a small amount of polyanion to a crosslinked gelatin gel, release of basic fibroblast growth factor can be suppressed, and sustained release of basic fibroblast growth factor can be achieved over a longer period of time. Possible polyanion addition crosslinked gelatin gel formulations are disclosed. However, these are not mentioned with respect to MIF.
[0028]
Patent Document 15 discloses a wound healing promoter using a biodegradable matrix characterized by containing a cytokine, a cell growth factor or a stimulant, and includes interferon-γ, tumor necrosis factor-α, interleukin-1α, Examples of interleukin-1β, acidic fibroblast growth factor, basic fibroblast growth factor, epidermal cell growth factor, vascular endothelial growth factor, and stimulators include thrombin and platelet activating factor. There is no mention of MIF.
[0029]
Other carriers for sustained release of growth factors include those having the property of being decomposed and absorbed in vivo, such as polylactic acid, polyglycolic acid, and a copolymer of lactic acid and glycolic acid. In Patent Document 16, neovascular inducers are vascular endothelial growth factor (VEGF), bFGF, acidic fibroblast growth factor (aFGF), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF- β), at least one kind of neovascular inducer selected from the group consisting of osteonectin, angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2), and the device substrate is a hydrogel (polyvinyl alcohol) , Polyvinyl pyrrolidone, polyethylene glycol, poly-2-hydroxyethyl methacrylate, poly-2-hydroxyethyl acrylate, polyacrylamide, polyacrylic acid, polymethacrylic acid, etc. A neovascular bed forming device is disclosed. In addition, poly-ε-caprolactone, copolymers of ε-caprolactone and lactic acid or glycolic acid, polycitric acid, polymalic acid, poly-α-cyanoacrylate, poly-β-hydroxybutyric acid, polytrimethylene oxalate , Polytetramethylene oxalate, polyorthoester, polyorthocarbonate, polyethylene carbonate, polypropylene carbonate, poly-γ-benzyl-L-glutamate, poly-γ-methyl-L-glutamate, poly-L-alanine, etc. Molecules, polysaccharides such as starch, alginic acid, hyaluronic acid, chitin, pectic acid and derivatives thereof, or proteins such as gelatin, collagen, albumin and fibrin are known as carriers.
[0030]
[Patent Document 1]
U.S. Pat. No. 4,485,096
[Patent Document 2]
JP-A-6-292568
[Patent Document 3]
JP 2000-262610 A
[Patent Document 4]
Japanese Patent Laid-Open No. 10-80438
[Patent Document 5]
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[Patent Document 6]
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[Patent Document 7]
JP 2002-226378 A
[Patent Document 8]
Japanese Patent Laid-Open No. 10-36279
[Patent Document 9]
JP 2003-52366 A
[Patent Document 10]
JP-A-7-179356
[Patent Document 11]
Japanese National Patent Publication No. 10-500301
[Patent Document 12]
Japanese Patent Laid-Open No. 2002-200161
[Patent Document 13]
Special table 2002-524425 gazette
[Patent Document 14]
JP-A-8-325160
[Patent Document 15]
Japanese Patent Laid-Open No. 11-246420
[Patent Document 16]
JP 2000-178180 A
[Non-Patent Document 1]
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[Non-Patent Document 2]
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[0031]
[Problems to be solved by the invention]
The problem to be solved by the present invention is to provide a topical pharmaceutical composition that is safe for a living body, effectively releases a drug to a wound, and stably contains the drug.
[0032]
[Means for Solving the Problems]
The present inventors have focused on a macrophage migration inhibitory factor as a growth factor that causes neither edema nor canceration-inducing action, and have found that the above-mentioned problems can be solved by using this as a local medicine, resulting in the completion of the present invention. .
That is, the present invention is a topical pharmaceutical composition containing a macrophage migration inhibitory factor and a biodegradable material.
Moreover, this invention is a wound protection dressing containing the said pharmaceutical composition.
[0033]
Here, the inventors have shown that macrophage migration inhibitory factor (MIF) is particularly strongly expressed in human epithelial cells compared to other tissues (Shimidzu T et al. FEBS Letter, 381: 199-202, 1996), MIF production in the skin is increased by skin damage and ultraviolet irradiation, and MIF is a cell migration promoting factor (Shimidzu T et al. J Invest Dermatol. 112: 210-215,1999; BBA 1500 (2000) 1-9), cells We focused on growth factors and angiogenic factors as well as regulatory factors (Shimidzu T et al. BBRC. 264: 751-758, 1999). On the other hand, we are a mediator known to be specifically released from anterior pituitary cells in response to LPS during endotoxin shock (Bernhagen et al., 1993, J. et al. Cell. Biochem. Supplement 17B, Abstract E306), recognizing that MIF circulating in the whole body is extremely important to control at the lowest possible concentration. Recent studies have also revealed the activity of MIF as an antiregulator of anti-inflammatory effects of glucocorticoids (both endogenously released and therapeutically administered). The fact that the intrinsic activity of glucocorticoids that exhibit a strong anti-inflammatory response is inhibited by MIF recognizes that the endogenous MIF response is the cause or aversion factor of various inflammatory diseases and symptoms ( Donnelly and Bucala, Molecular Medicine Today 3: 502-507, 1997).
[0034]
Therefore, the idea of promoting local tissue regeneration by using MIF as a local sustained release agent is an original idea of the present inventors that has been cut off from the extension of the idea of directly using the pharmacology of MIF so far. .
[0035]
DETAILED DESCRIPTION OF THE INVENTION
In general, when a part of skin tissue falls off or is lost due to disease or trauma, epidermal cells and fibroblasts that live around the fallen tissue divide and proliferate and move to the defective part. This phenomenon is defined as regeneration of epidermal cells or epidermal tissue.
Thereafter, the regenerated epidermal cells that have migrated to and moved to the defect part start to adhere to each other, stop dividing and proliferating, and form epidermal tissue again. This phenomenon is defined herein as the reconstruction of epidermal cells or epidermal tissue (epidermis).
In the present specification, epithelial cells are a general term for cells covering the internal and external separation surfaces, and mean cells other than fibroblasts. Epithelial cells include, for example, epidermal cells, keratinocytes, and mucosal epithelial cells. In addition, a life phenomenon in which regeneration and remodeling of epithelial cells or epidermal tissue occurs is generally called epithelialization.
[0036]
Examples of the MIF used in the present invention include human-MIF and mouse-MIF, and human-MIF is more preferable. Note that human-MIF and mouse-MIF are specified as proteins having accession numbers of 31334 and 31221, respectively.
[0037]
As a method for producing MIF used in the present invention, for example, in human-MIF, an expression vector in which a region encoded by human-MIF cDNA is amplified in vitro by the polymerase chain reaction (PCR) method and the gene is inserted. Can be produced by introducing the protein into a host cell (Non-patent Document 17). The amplified DNA was inserted into the NdeI and BamHI fragments of T7 promoter expression plasmid pET3, and transfected E. Coli BL21 (DE3) LysS transformed with this plasmid was cultured, and human-MIF was cultured from the culture supernatant. (J. Nishihira et al., “Biochemical and Biophysical Research Communication”, 185: 1069-1077 (1992)).
[0038]
As a method for isolating MIF, MIF or a recombinant MIF-containing solution, for example, a cell extract of a human cell or a cell culture supernatant nutrient solution is optionally subjected to a sterilization filtration step (0.45 μm), followed by Can be isolated by two steps: a) and / or b).
That is, as step a), using specific binding of human-MIF to glutathione, it is passed through an affinity gel as a carrier having glutathione, for example, S-hexyl glutathione affinity column R, and contacted with the carrier. It removes unbound proteins and other foreign substances and selectively cleaves human-MIF bound to the antibody.
In step b), contact is made with a carrier having a monoclonal antibody specific for human-MIF, unbound proteins and other foreign substances are removed, and human-MIF bound to the antibody is selectively separated. To separate.
[0039]
That is, the solution containing human-MIF is stabilized from the cells or cell culture supernatant by, for example, extraction, filtration and / or addition of protease inhibitors.
Such a human-MIF solution can be contacted directly in step a) and / or step b) with glutathione (GSH) and / or antibody bound to the carrier, but preferably about 6 kD or smaller molecular weight Pre-purified by ultrafiltration on a membrane with a separation limit of, concentrated, optionally dialyzed and optionally further purified by chromatography, for example DEAE-cellulose or Sephadex.
[0040]
In step a) and / or step b) human-MIF is separated from other proteins and foreign substances contained in the solution, in this case specific binding of human-MIF to glutathione and / or human A separation action based on forced interactions between antibodies specific for -MIF and recognized antigenic determinants on human-MIF is used. For this purpose, the MIF-containing liquid is brought into contact with a carrier having monoclonal antibodies specific for glutathione (GSH) and / or human-MIF according to immunoaffinity chromatography methods known per se, and unbound proteins and other foreign substances are contacted. Remove the sexual substance and selectively cleave and isolate glutathione (GSH) and / or antibody-bound human-MIF (CPD et al., “Journal of Biological Chemistry”, 258: 4659- 4662 (1983)). Use an affinity gel as a carrier with glutathione (GSH), such as an S-hexyl glutathione affinity column, and / or a suitable carrier based on minerals or organics, such as silicates, cross-linked agarose, dextran, or suitably functional A monoclonal antibody specific for human-MIF or a derivative thereof is added to a polyacrylamide in a purified form. For example, a carrier containing an activated ester functional group, such as an N-hydroxysuccinimide ester group, is suspended in an aqueous buffer, mixed with a solution of a monoclonal antibody, and unreacted reactive sites on the carrier are Block with primary amine, eg ethanolamine. The carrier is suspended in a suitable aqueous solvent, such as a salt solution, such as a NaCl solution, or a buffer, such as a phosphate buffered NaCl solution, NaHCO 3 solution or 3- (N-morpholino) propane sulfonic acid solution, and human-MIF is suspended. Contact with the contained solution. The chromatogram column is packed and the human-MIF containing liquid is introduced and pumped through the carrier, optionally with pressure. Unbound proteins and other contaminants are washed away with an aqueous solution, such as a buffer and / or salt solution having a pH of about 5 to about 9, such as a NaCl solution. Human-MIF bound to the antibody on the carrier is mixed with a suitable aqueous solution, such as a buffer in the pH range of about 2 to about 5, such as a glycine buffer, or various mixtures or salt solutions, such as the pH of a concentrated NH4 SCN solution. Elute by gradient. The resulting purified human-MIF-containing solution was optionally neutralized and purified by methods known per se, for example by chromatography on Sephadex, electrodialysis, electrophoretic concentration and / or vacuum concentration. Human-MIF can be isolated.
In addition, other MIFs such as mouse-MIF can be produced and used in a simple manner using the same method as human-MIF.
[0041]
The biodegradable material used in the present invention is not particularly limited as long as it is a material that can be dissolved, decomposed, metabolized, or absorbed by the action of cells, microorganisms, or living organisms, and specifically, polyvinyl alcohol, starch. Or starch-based polymers, polysaccharides such as cellulose, pullulan, curdlan and xanthan gum, polyamino acids such as polyglutamic acid and polylignin, gelatin, collagen, cellulose, lignin, alginic acid, hyaluronic acid, chitin, chitin chitosan, aliphatic polyester, Polylactic acid, polyethylene glycol, poly-ε-caprolactone, copolymer of ε-caprolactone and lactic acid or glycolic acid, polycitric acid, polymalic acid, poly-α-cyanoacrylate, poly-β-hydroxybutyric acid, polytrimethylene oxa Rate, polytetramethylene oxalate Salts, polyorthoesters, polyorthocarbonates, polyethylene carbonate, polypropylene carbonate, poly-γ-benzyl-L-glutamate, poly-γ-methyl-L-glutamate, poly-L-alanine gelatin, collagen, albumin, fibrin, etc. However, the present invention is not limited thereto.
[0042]
Among these, polylactic acid / glycolic acid copolymer (“PLGA”), which is decomposed by hydrolysis of ester bond after exposure to water to produce non-toxic lactic acid and glycolic acid monomers, is a preferred example. Can be mentioned. PLGA is a copolymer prepared by a polycondensation reaction between lactic acid and glycolic acid. Various ratios of monomer ratios can be used to adjust the crystallinity and hydrophobicity of the PLGA polymer. The higher crystallinity of the polymer results in slower dissolution. For the preparation and properties of PLGA, see DKGilding and AMReed, `` Biodegradable polymers for use in surgery-poly (glycolic) / poly (lactic acid) homo and copolymers: 1 '' Polymer Volume 20, pages 1459-1464 (1981). ,Are listed.
[0043]
As another preferred example of the biodegradable material, gelatin can be mentioned, and among these, a crosslinked gelatin can be more preferred.
Cross-linked gelatin can be prepared by cross-linking an oil-in-water emulsion obtained by dropping a gelatin solution into oil or fat with a cross-linking agent such as glutaraldehyde. The crosslinked gelatin is washed and sieved to produce crosslinked gelatin microspheres that are particularly preferable as a biodegradable material (K. Kawai et al; Biomaterials 21 (2000), 489- 499). In this case, since the biodegradable material becomes fine particles (average diameter when wet is less than 32 μm), it can be injected subcutaneously and intracutaneously around the wound when used in a topical pharmaceutical composition.
[0044]
The method for producing the topical pharmaceutical composition of the present invention is not particularly limited as long as a composition containing MIF and a biodegradable material can be obtained, but the biodegradable material is impregnated with a solution in which MIF is dissolved. The method of making it preferable is. For example, when cross-linked gelatin microspheres are used as the biodegradable material, the topical pharmaceutical composition of the present invention containing MIF stably can be prepared by contacting this with a MIF solution. Also, lyophilization is available for stabilization of MIF sustained release formulations (Carpenter, JF, Pical, MJ, Chang, BS, and Randolph, TW, Pharm. Res., 14: (8) 969 (1997)).
[0045]
According to the topical pharmaceutical composition of the present invention, MIF is dissolved, disintegrated, or exchanged with a body fluid together with an elution or biodegradable material, so that MIF is released in small amounts, and an effective amount of MIF is released to the wound. Can be contained stably. Therefore, the topical pharmaceutical composition of the present invention can be used as a compounding component for external preparations such as ointments, creams, liquids, etc. in addition to injections and dressings.
[0046]
The dosage of the topical pharmaceutical composition of the present invention is not particularly limited as long as it is an amount that produces an effect on wound healing. ² It is preferably administered so that the amount of MIF is 1 to 100 μg, more preferably 5 to 50 μg, more preferably 10 to 30 μg. Further, the daily release amount of MIF is not particularly limited as long as it is effective in healing the wound. ² In contrast, it is preferably used by adjusting so as to release 10 to 5000 ng per day, more preferably 100 to 1000 ng, still more preferably 300 to 700 ng.
A method for controlling the local concentration of MIF and / or the daily release amount of MIF includes controlling the degradability of the biodegradable material. For example, in the case of cross-linked gelatin, cross-linking such as glutaraldehyde is performed. The release amount can be controlled by changing the blending ratio of the agent and changing the water content of the crosslinked gelatin.
[0047]
Examples of the application site of the topical pharmaceutical composition of the present invention include wounded skin and / or skin near the wound. In this case, the vicinity of the wound is not particularly limited as long as the MIF released from the topical pharmaceutical composition of the present invention acts on the wound and exhibits an effect on wound healing, but for example, 1-30 mm from the wound site. List remote sites.
[0048]
The wound protective dressing of the present invention is not particularly limited as long as it is a dressing containing the topical pharmaceutical composition of the present invention. For example, a dressing prepared by injecting the topical pharmaceutical composition of the present invention into artificial skin, etc. Can be mentioned.
Examples of the artificial skin used in the dressing of the present invention include a chitin chitosan-based covering fabric (for example, Beschitin WR or Besquitin FR manufactured by Aventis Pharma) or a wound dressing mainly composed of a chitosan derivative (succinyl chitosan) ( Examples thereof include Urezac CR manufactured by Katakura Chikkarin Co., Ltd., or a composite of collagen sponge and silicone polymer (for example, Pelnac® manufactured by Gunze Co., Ltd.).
The dressing of the present invention is, for example, a surface area of 1 cm of the above artificial skin. ² It can be prepared by impregnating the topical pharmaceutical composition solution of the present invention so as to contain 0.5 to 500 μg of MIF and drying.
As a method of using the dressing of the present invention, it can be used in the same manner as a normal dressing. For example, artificial skin dried by impregnating the above MIF is attached to a wound surface as a primary dressing, It is possible to apply gauze or the like as a secondary dressing from the top and fix it with a bandage, and replace the secondary dressing daily.
[0049]
The topical pharmaceutical composition of the present invention may contain the following ingredients as other auxiliary ingredients.
For the purpose of preventing pigmentation, whitening agents such as arbutin, vitamin C and its derivatives, kojic acid placenta extract, glutathione, and yukinoshita extract, UV blocking agents such as titanium oxide (TiO2), talc (MgSiO2), carmine ( FeO2), bentonite, kaolin, zinc oxide (ZnO) and the like can be blended.
For the purpose of anti-inflammation, anti-inflammatory agents such as glycyrrhizic acid derivatives, glycyrrhetinic acid derivatives, salicylic acid derivatives, hinokitiol, zinc oxide and allantoin can be blended.
For the purpose of promoting blood circulation, nonyl acid wallenylamide, capsaicin, gingerone, cantalis tincture, ictamol, caffeine, tannic acid, α-borneol, tocopherol nicotinate, inositol hexanicotinate, cyclandrate, cinnarizine, trazoline, acetylcholine, verapamil , Blood circulation promoters such as cephalanthin and γ-oryzanol can be blended.
For compliance purposes, alum extract, auren extract, sicon extract, peony extract, assembly extract, birch extract, sage extract, loquat extract, carrot extract, aloe extract, mallow extract, iris Extraction component, grape extract component, Yokuinin extract component, loofah extract component, lily extract component, saffron extract component, nematode extract component, ginger extract component, hypericum extract component, onionis extract component, rosemary extract component, garlic extract component, capsicum Extract components, chimpi, touki and the like can be blended.
[0050]
In order to improve the stability of a polypeptide such as MIF to be blended, a physiologically acceptable protein degradation inhibitor may be added. As a protein degradation inhibitor, soybean trypsin inhibitor (SBTI), pancreatic secretory trypsin inhibitor (PSTI) and aprotinin (BPTI), which are proteinaceous trypsin inhibitors, can be added.
[0051]
Other additives include water-soluble polymers, preservatives, antioxidants, buffering agents, chelating agents, emulsifiers, solubilizers, ointment bases, solvents, curing agents, suspending agents, thickeners, A reversible crosslinking agent, vitamins and the like can be blended.
[0052]
Natural water-soluble polymers that can be used as thickeners include gum arabic, gum tragacanth, galactan, guar gum, carob gum, caraya gum, carrageenan, tamarind gum, xanthan gum, pectin, agar, quince seed (malmello), alge colloid (gypsum extract) ), Starch (rice, corn, potato, wheat), plant polymer such as glycyrrhizic acid, microbial polymer such as xanthan gum, dextran, succinoglucan, pullulan, animal system such as collagen, casein, albumin, gelatin Molecule and the like. Semi-synthetic water-soluble polymers include starch polymers such as dextrin, carboxymethyl starch, methylhydroxypropyl starch, methylcellulose, nitrocellulose, ethylcellulose, methylhydroxypropylcellulose, hydroxyethylcellulose, cellulose dimethyldialkyl sulfate (12-20). ) Cellulose polymers such as ammonium, hydroxypropylcellulose, sodium carboxymethylcellulose (CMC), crystalline cellulose, cellulose powder, and alginic acid polymers such as sodium alginate and propylene glycol alginate. Examples of fully-synthesized water-soluble polymers include polyvinyl polymers such as polyvinyl alcohol, polyvinyl methyl ether, polyvinyl pyrrolidone, carboxyvinyl polymer, alkyl-modified carboxyvinyl polymer, and polyoxyethylene polymers such as polyethylene glycol 2000, 4000, and 6000. Examples include molecules, polyoxyethylene polyoxypropylene copolymer-based polymers, acrylic polymers such as sodium polyacrylate, polyethylene acrylate, and polyacrylamide, polyethyleneimine, and cationic polymers. Examples of inorganic water-soluble polymers include bentonite, aluminum magnesium silicate, laponite, hectorite, and anhydrous silicic acid.
[0053]
As preservatives, benzalkonium chloride, benzalkonium chloride solution, benzethonium chloride, benzoic acid, benzyl alcohol, butylparaben, cetylpyridinium chloride, chlorobutanol, chlorocresol, cresol, dehydroacetic acid, Ethylparaben, methylparaben, methylparaben sodium, phenol, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric nitrate, potassium benzoate, potassium sorbate, propylparaben, sodium propylparaben, sodium benzoate, sodium dehydroacetate, sodium propionate, sorbine An acid, thimerosal, thymol, etc. can be mentioned.
[0054]
Antioxidants include ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium formaldehyde sulfoxylate, sodium metabisulfite, sodium thiosulfate , Sulfur dioxide, tocopherol, tocopherol excipient and the like.
[0055]
Buffering agents include acetic acid, ammonium carbonate, ammonium phosphate, boric acid, citric acid, butyric acid, phosphorous acid, potassium citrate, potassium metaphosphate, potassium phosphate monobasic, sodium acetate, sodium citrate, sodium butyrate Examples thereof include a solution, dibasic sodium phosphate, and monobasic sodium phosphate.
[0056]
Examples of chelating agents include disodium edetate, ethylenediaminetetraacetic acid and salts thereof, and edetic acid.
[0057]
Emulsifiers or solubilizers include acacia, cholesterol, diethanolamine (adjunct), glyceryl monostearate, lanolin alcohol, lecithin, mono- and di-glycerides, monoethanolamine (adjacent), oleic acid (adjacent) Type), oleyl alcohol (stabilizer), poloxamer, polyoxyethylene 50 stearate, polyoxy 35 castor oil, polyoxy 40 hydrogenated castor oil, polyoxy 10 oleyl ether, polyoxy 20 cetostearyl ester, polyoxy 40 stearate, polysorbate 20 , Polysorbate 40, polysorbate 60, polysorbate 80, propylene glycol diacetate, propylene glycol monostearate, sodium lauryl sulfate, stearic acid Thorium, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, stearic acid, and trolamine, emulsifying wax, and the like.
[0058]
Examples of the ointment base include lanolin, anhydrous lanolin, hydrophilic ointment, white ointment, yellow ointment, polyethylene glycol ointment, petrolatum, hydrophilic petrolatum, white petrolatum, rose water ointment, squalane and the like.
[0059]
Solvents include ethyl oleate, isopropyl myristate, isopropyl palmitate, mineral oil, light mineral oil, myristyl alcohol, octyldodecanol, almond oil, olive oil, peanut oil, kyounin oil, sesame oil, soybean oil, squalene, glycerin, peanut oil Polyethylene glycol, propylene carbonate, propylene glycol, sesame oil, water for injection, sterile water for injection, purified water, and the like.
[0060]
Curing agents include hardened castor oil, cetostearyl alcohol, cetyl alcohol, cetyl ester wax, hardened fat, paraffin, polyethylene excipient, stearyl alcohol, emulsifying wax, white wax, yellow wax. And the like.
[0061]
Suspensions and / or thickeners include gum arabic, agar, alginic acid, aluminum monostearate, bentonite, purified bentonite, magma bentonite, carbomer 934p, carboxymethylcellulose calcium, carboxymethylcellulose sodium, carboxymethylcellulose sodium, Carrageenan, microcrystalline and carboxymethylcellulose sodium cellulose, dextrin, gelatin, guar gum, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, magnesium aluminum silicate, methylcellulose, pectin, polyethylene oxide, polyvinyl alcohol, povidone, propylene glycol alginate, dioxide Silicon It can be mentioned silicon colloidal dioxide, sodium alginate, tragacanth, xanthan gum and the like.
[0062]
Examples of the reversible crosslinking agent include those usually used. W. Those described in Green, Protective Groups in Organic Synthesis, John Wiley, and Sons (ed.) (1981) can be used. Various approaches are also listed in a review by Waldmann (Angewante Chemie Inl. Ed. Engl. 35, page 2056 (1996)).
[0063]
Furthermore, specific examples of vitamins having a cell activation effect that can be combined with the topical pharmaceutical composition of the present invention include vitamin A oil, vitamin A such as retinol acetate, vitamin B2 such as riboflavin and riboflavin butyrate, pyridoxine hydrochloride Such as vitamin B6 such as L-ascorbic acid, magnesium L-ascorbyl phosphate, sodium L-ascorbate, nicotinic acid, nicotinic acid amide, nicotinic acid benzyl ester, nicotinic acid β-butoxyethyl ester, etc. Vitamin D such as nicotinic acid, ergocalciferol, cholecalciferol, vitamin E such as α-tocopherol and tocopherol acetate, vitamin H such as vitamin P and biotin, royal jelly, photosensitizer, cholesterol derivative, calf blood Activation of extracts, etc. And the like can be given.
[0064]
【Example】
Hereinafter, the present invention will be further described with reference to examples. However, the present invention is not limited to the examples.
[0065]
<Production Example 1: Production of human-MIF>
Human-MIF was produced by in vitro gene amplification of the region encoded by human-MIF cDNA by polymerase chain reaction (PCR) method, and introducing the expression vector into which the gene was inserted into the host cell. The amplified DNA was inserted into the NdeI and BamHI fragments of T7 promoter expression plasmid pET3, and transfected E. Coli BL21 (DE3) LysS transformed with this plasmid was cultured, and human-MIF was cultured from the culture supernatant. Isolated.
[0066]
<Production Example 2: Production of biodegradable material>
The biodegradable material as a carrier for sustained release was prepared by dropping 10 ml of a gelatin solution concentration of 10% by weight preheated at 40 ° C. for 1 hour into 350 ml of olive oil stirred at 450 revolutions per minute to form a water-in-oil emulsion. Cross-linked gelatin was prepared by cross-linking in the presence of glutaraldehyde. Next, 100 ml of acetone was added, and the mixture was further stirred at 300 rpm for 1 hour. The resulting microspheres were washed 5 times with acetone, then twice with isopropyl alcohol, and centrifuged (treatment at 5000 rpm for 5 minutes at 4 ° C.) to disperse the precipitate with isopropyl alcohol. Microspheres were collected from this solution with a 32 micron sieve, centrifuged repeatedly, and air dried at 4 ° C. to obtain crosslinked gelatin microspheres.
The obtained crosslinked gelatin microspheres had an isoelectric point of 5.0, an average particle size of about 70 μm, and a water content of about 95%.
[0067]
<Production Example 3: Production of MIF knockout mouse>
MIF knockout mice (MIF KO mice) were prepared by homologous recombination with a mouse gene in a vector in which a neomycin resistance gene was inserted into the MIF gene (see Homma N et al. Immunology, 2000, 100: 84-90). Then, it was confirmed by immunoblotting that no MIF protein was expressed.
Next, the wound healing effect of the prepared MIF KO mice and wild type BALB / c mice was confirmed.
Specifically, 6-8 weeks old MIF KO mice and wild-type (WT) BALB / c mice made ulcers of 4 mm x 4 mm size on the back skin, and ulcers were observed over time and compared with BALB / c mice. investigated. The results are shown in FIG.
This confirmed that wound healing was delayed in MIF KO mice.
In addition, the skin was peeled from 1-day-old MIF KO mice and WT mice, and fibroblasts were cultured.The MTS assay was performed on each of the second-generation cultured fibroblasts to stimulate lipopolysaccharide (LPS). The difference of the cell proliferation effect by the presence or absence was investigated. The results are shown in FIG.
Thus, in MIF KO mice, fibroblasts were not proliferated by LPS stimulation on days 3 and 5, whereas in BALB / c fibroblasts of wild type (control) mice, cell proliferation was observed by LPS stimulation. I understand that.
In addition, the migration ability of cultured fibroblasts from MIF KO mice and WT mice was evaluated by making a wound on the cultured cell petri dish and observing the fibroblasts migrating there over time. The results are shown in FIG.
This shows that the migration ability of fibroblasts is low in the MIF KO mouse.
[0068]
<Example 1: Production of topical pharmaceutical composition (MIF sustained release agent)>
36 μg of human-MIF obtained in Production Example 1 (hereinafter simply referred to as “MIF”) was dissolved in 20 μl of PBS solution, and 2 mg of the crosslinked gelatin microspheres obtained in Production Example 2 was added to impregnate MIF. Thereafter, the pellet collected by centrifugation at 2000 rpm was allowed to stand at room temperature for 30 minutes to obtain a crosslinked gelatin microsphere containing MIF.
[0069]
<Example 2: Production of MIF sustained-release solution>
6 ml of PBS was added to the cross-linked gelatin microspheres containing MIF prepared in Example 1 to obtain a MIF sustained-release solution containing 6 μg of MIF in 1 ml.
Next, the MIF release amount of the MIF sustained-release agent solution was measured by measuring the absorbance, and it was confirmed that MIF was hardly released even after 48 hours in vitro (FIG. 4).
[0070]
<Example 3: Wound healing experiment using MIF sustained-release solution>
An ulcer was created in the MIF KO mouse obtained in Production Example 3, and the wound healing effect by injecting a MIF sustained-release agent into the periphery of the ulcer was examined.
Specifically, ulcers were created on the backs of 5 MIF KO mice, each with a size of 5 × 5 mm, using a scalpel to form a wound (Day 0). On the next day (Day 1), the MIF sustained-release agent solution produced in Example 2 was injected at 10 sites around the ulcer approximately 3 mm away from the ulcer by 50 μl each (total amount of 3 μg MIF was injected). . Therefore, wound 1cm ² This means that 12 μg was injected.
As comparative examples, the same experiment was carried out on 50 μl of MIF-only 6 μg / ml PBS solution not combined with cross-linked gelatin microspheres injected at 10 locations and no injection (control).
The wound healing effect was determined by observing the reduction rate of the ulcer by measuring the maximum diameter of the ulcer. The result is shown in FIG. 5A.
From this, it was clarified that, on the 6th day after wound creation, those injected with the topical pharmaceutical composition of the present invention had a significant healing effect compared to those injected with MIF alone and those without injection. It was.
Moreover, about the mouse | mouth which injected the topical pharmaceutical composition of this invention, and the mouse | mouth which injected only MIF, the skin of the injection part vicinity was extract | collected 10 days after wound preparation, and presence of MIF protein was confirmed by the Western blot. The result is shown in FIG. 5B.
Thus, in mice injected with MIF alone, the presence of MIF protein cannot be confirmed, whereas in mice injected with the topical pharmaceutical composition of the present invention, MIF remains in the vicinity of the wound, and the sustained release effect continues. Can be confirmed.
Furthermore, it was confirmed that this sustained-release agent releases the entire amount of MIF in about 21 days. Therefore, about 143 ng of MIF was gradually released per day (total amount of 3 μg of MIF / 21 days), and the wound was 1 cm. ² In contrast, 572 ng of MIF was gradually released per day.
[0071]
<Example 4: Production of wound protective dressing>
Artificial dermis (manufactured by Pelnac® Gunze Co., Ltd., outer layer consisting of a silicone polymer layer and inner layer consisting of a collagen sponge layer with a pore size of 70-110 μm) was prepared, and the final MIF content was 2.5 μg / cm ² Then, a cross-linked gelatin microsphere solution containing MIF was injected evenly into the artificial dermis to prepare a wound protective dressing.
[0072]
【The invention's effect】
According to the present invention, it is possible to obtain a topical pharmaceutical composition that releases MIF little by little, realizes effective MIF release to a wound, and stably contains MIF.
[Brief description of the drawings]
FIG. 1 is a diagram showing wound healing in MIF KO mice.
FIG. 2 shows an evaluation of fibroblast proliferation.
FIG. 3 is a diagram showing an evaluation of fibroblast migration ability.
FIG. 4 is a diagram showing an in vitro sustained release effect.
FIG. 5 is a diagram showing the wound healing effect by injecting a MIF sustained-release agent using MIF KO mice.

Claims (4)

A topical pharmaceutical composition for wound healing comprising macrophage migration inhibitory factor and cross-linked gelatin microspheres . The topical pharmaceutical composition according to claim 1, wherein the macrophage migration inhibitory factor is administered so as to be 1 to 100 µg per 1 cm ² of wound. The topical pharmaceutical composition according to claim 1 or 2, wherein 10 to 5000 ng of macrophage migration inhibitory factor is released per day per 1 cm ² of wound. A wound protective dressing comprising the topical pharmaceutical composition according to any one of claims 1 to 3.

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