JP4790195B2 - Tissue regeneration agent - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a tissue regeneration agent containing hepatocyte growth factor and basic fibroblast growth factor.
[0002]
[Prior art]
Many cells are involved in the wound healing process of the skin, and the interaction is terminated through bleeding coagulation, inflammation, proliferation, and remodeling.
In the healing process, the following four biological phenomena are involved: 1) the self-repair function of the living body by the recruitment of various cells to the wound site, and 2) the complex mechanism of cell growth control by cytokines and growth factors. The ingenuity, 3) the extracellular matrix, the mechanism of collagen production, and 4) the great potential for modification of these series of phenomena are also involved.
However, even after undergoing such a natural healing process, humans often leave scars more or less, and leave regular and functional failure.
[0003]
In recent years, research on the role of cytokines in wound healing in ulcer wounds has been promoted, and many research results on clinical applications have been reported. Cytokines are low molecular weight proteins with various physiological activities, as is well known, and it is becoming clear that they are secreted from cells and play an important role in various pathological conditions. It has been elucidated that it is deeply involved in the progression of healing, especially in recent years, because various recombinant human cytokines have been obtained in large quantities due to the development of genetic engineering. It attracts a lot of attention from that point of view.
Research reports on the effects and effects of cytokines on ulcer wounds, particularly refractory ulcers, have already been made. For example, bFGF, PDGF, KGF, etc. have entered the clinical application stage, of which bFGF (basic fibroblast growth factor) Has been actually available in Japan since June 2001. When bFGF is used for ulcer wounds, it has been confirmed that it quickly closes the wound and has a granulation-proliferating effect.
However, these trials of clinical application are related to the treatment of ulcer wounds, and there are few reports on research using suture wounds, and basic researches such as the effectiveness of various cytokines after sutures of incisions have been made. Not.
[0004]
In addition, hepatocyte growth factor (hereinafter abbreviated as HGF), a kind of cytokine, is a ligand of c-Met receptor tyrosine kinase, which has mitogenic, motogenic and anti-apoptotic effects on various cells. It is known that it also has angiogenic and angioprotective actions, and clinical research is being conducted in anticipation of angiogenic effects.
In addition to the angiogenic effect, HGF has been attracting attention as it may suppress scar formation, which is fibrosis of the wound, and it has been shown that administration of HGF could prevent fibrosis after injury in the liver, lung, kidney, etc. It has been reported that the effect can be performed more efficiently by gene therapy using genes.
However, there have been no reports on the effects on skin wounds.
[0005]
There are no other therapeutic agents that are effective in preventing scarring of wounds damaged by trauma or surgery, and traditionally employ fine sutures that require advanced techniques to treat the wound surface, or wound healing. Surgical measures such as carrying out skin grafting for the treatment of subsequent scars have been taken, but it is not always satisfactory, and the development of an effective regenerative agent for human tissues including the skin has been awaited. .
[0006]
[Problems to be solved by the invention]
The problem to be solved by the present invention is to provide a therapeutic agent effective to repair and regenerate tissue damaged and damaged by trauma or surgical operation in a well-ordered and functional manner without leaving scars.
[0007]
[Means for Solving the Problems]
The present inventors have found that HGF significantly suppresses apoptosis expressed after stitching of the wound compared to the control group, suppresses excessive proliferation of granulation tissue in wounds such as the wound, and finally fibrosis Was confirmed to be effective in promoting healing of the wound and bringing it closer to regeneration. Further, the present invention was completed by confirming that the use of a combination of HGF and bFGF further reduced the scarring of the wound and had a remarkable effect on wound healing and tissue regeneration.
That is, the present invention is as follows.
(1) A tissue regeneration agent comprising a combination of hepatocyte growth factor and basic fibroblast growth factor.
(2) The tissue regeneration agent according to (1), wherein hepatocyte growth factor and basic fibroblast growth factor are combined in such a manner that there is a time difference in action expression.
(3) The tissue regenerative agent according to (2), which is combined in such a manner that the basic fibroblast growth factor is acted first.
(4) The tissue regeneration agent according to any one of (1) to (3), wherein the hepatocyte growth factor is an embodiment of a nucleic acid encoding a gene of hepatocyte growth factor or an expression vector thereof.
(5) The tissue regeneration agent according to (1), wherein the tissue is a cut tissue.
(6) A package containing a combination comprising a hepatocyte growth factor and a basic fibroblast growth factor and a description describing the use of the combination for tissue regeneration.
(7) The package according to (6), wherein the use is used in such a manner that there is a time difference in the expression of the action of hepatocyte growth factor and basic fibroblast growth factor.
(8) The package according to (6), wherein the hepatocyte growth factor is an embodiment of a nucleic acid encoding a gene of a hepatocyte growth factor or an expression vector thereof, and the basic fibroblast growth factor is a protein.
(9) Inhibitor of scar formation at the wound site containing hepatocyte growth factor as an active ingredient
(10) The scar formation inhibitor of a wound site according to (9), wherein the hepatocyte growth factor is a nucleic acid encoding a gene of hepatocyte growth factor or an expression vector thereof.
(11) A tissue regeneration method comprising administering to a mammal a combination of hepatocyte growth factor and basic fibroblast growth factor.
(12) The tissue regeneration method according to (11), wherein hepatocyte growth factor and basic fibroblast growth factor are administered in such a manner that there is a time difference in action expression.
(13) The tissue regeneration method according to (11), wherein the tissue is a cut tissue.
(14) A method for suppressing scar formation at a wound site, comprising administering a hepatocyte growth factor to a mammal.
[0008]
As described above, there has not been a therapeutic agent that can cure and regenerate wounds damaged by trauma or surgical operation, in particular, scarring at the incision site so that it can be satisfactorily and functionally satisfied, The development of an effective regenerative agent for human tissues including the skin has been awaited, and the present inventors have now made the maximum possible repair ability of the living body when HGF is administered to a wound. It was confirmed that it was possible to repair a wound in the skin and to be an effective therapeutic agent capable of preventing scar formation.
In addition, the present inventors realized narrow scar formation from an early stage while suppressing the decrease in strength due to the suppression effect of fibrosis by HGF by combining HGF and bFGF, particularly when both are added at a high concentration. It was found that by combining both of them, such as healing in a state that can be said to be almost tissue regeneration, there is a synergistic effect on tissue regeneration.
HGF has an action to suppress cell infiltration, fibrosis and apoptosis, whereas bFGF has an action to promote cell infiltration and proliferation of fibroblasts and promote early apoptosis. Regardless of the substance, it is considered that the combined use of both actions worked synergistically to induce an efficient wound healing response.
By the administration of bFGF, regeneration of the wound was promoted from an early stage even if the dose of HGF was low, and it became possible to reduce the dose of HGF.
In the present invention, one of HGF and bFGF is a protein, and the other is administered in the form of a nucleic acid encoding a protein gene to control the timing and time of action, thereby enabling more rational treatment. It was. That is, the present invention is a comparison with a therapeutic agent using a single cytokine in that the progression of the reaction can be realized as an efficient theoretical treatment in a chain reaction in a series of wound repair reaction processes. It is a period.
In addition, when HGF is administered as a nucleic acid encoding a protein gene, if bFGF is used in combination, the gene can be efficiently introduced. Even if the gene is administered as a cDNA plasmid, it can be administered at a relatively low dose. It became clear that an effect can be expressed from an early stage.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
HGF and bFGF used in the present invention are widely known substances, and are also available as commercial products (for example, bFGF commercial product “Trafermin (genetical recombination): Kaken Pharmaceutical Co., Ltd.”). is there. The mode may be any of natural type or genetically modified type, or a precursor protein thereof, natural type or genetically modified type of HGF and bFGF in which one or more constituent amino acids are substituted, deleted, or inserted. Alternatively, it may be a nucleic acid encoding a gene of each protein (cDNA or cDNA plasmid... In the present invention, hereinafter collectively referred to as “gene”), and one of them is a protein. The other may be a gene. Genes can be administered as plasmids alone or in the form of complex plasmids combined with liposomes as expression vectors. In the present invention, examples of the expression vector used for increasing the efficiency of gene introduction include any expression vector such as a viral vector. Preferably, it is an expression vector for mammalian cells.
The promoter contained in the expression vector used in the present invention is operably linked to the HGF and bFGF genes and is a functional promoter in mammalian (preferably human) cells. This promoter is inducible or constitutive and may be tissue specific if desired. In addition, it is known that the speed of gene expression varies depending on the type of the promoter. For example, early immidiate promoter, early promoter and late promoter have early expression of the gene under the control. Is different. Therefore, when one or both of HGF and bFGF are administered to a mammal as a gene, the speed and persistence of the protein expression can be regulated by appropriately selecting the type of these promoters. .
[0010]
In the present invention, HGF and bFGF can be made into appropriate preparations by conventional methods. The preparation may be a solid preparation such as a powder or granule, but a solution such as a solution, emulsion or suspension is advantageous for application to a wound site, and injection by subcutaneous injection, application, spraying or gene gun. It is preferable to administer by such a method.
As needed in the formulation, appropriate pharmaceutically acceptable carriers such as excipients, binders, solvents, solubilizers, suspending agents, emulsifiers, tonicity agents, buffers, stabilization It is formulated by blending agents, soothing agents, preservatives, antioxidants, colorants and the like.
[0011]
Examples of excipients include lactose, sucrose, D-sorbitol, starch, pregelatinized starch, corn starch, D-mannitol, dextrin, crystalline cellulose, gum arabic, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, methylcellulose, serum albumin, etc. Is mentioned.
As binders, pregelatinized starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl Examples include pyrrolidone and polyvinyl alcohol.
Solvents include purified water, physiological saline, Ringer's solution, ethanol, propylene glycol, glycerin, polyethylene glycol, macrogol and other hydrophilic solvents, olive oil, peanut oil, sesame oil, camellia oil, rapeseed oil, fatty acid monoglyceride, fatty acid Examples include oil solvents such as diglycerides, higher fatty acid esters, and liquid paraffin.
Examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate and the like. .
[0012]
As suspending agents, stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose , Hydroxyethyl cellulose, hydroxypropyl cellulose, polysorbates, polyoxyethylene hydrogenated castor oil, gum arabic, bentonite and the like.
Examples of the emulsifier include gum arabic, gelatin, lecithin, cholesterol, egg yolk, bentonite, bee gum, cetanol, glyceryl monostearate, methylcellulose, sodium carboxymethylcellulose, stearic acid and the like.
Examples of isotonic agents include sodium chloride, potassium chloride, glucose, fructose, mannitol, sorbitol, lactose, saccharose, glycerin, urea and the like.
Examples of the buffer include sodium citrate and glycerin.
Examples of the preservative include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, and the like.
Stabilizers include polyethylene glycol, sodium dextran sulfate, amino acids, human serum albumin and the like.
Examples of soothing agents include glucose, calcium gluconate, procaine hydrochloride and the like.
Examples of the antioxidant include sulfite and ascorbic acid.
Examples of the colorant include tar dyes, caramel, bengara, titanium dioxide, and FD & C dyes such as FD & C Blue No. 2 and FD & C Red No. 40 manufactured by Ellis & Everard.
[0013]
In order to obtain sustained-release preparations, polysaccharides such as alginic acid, hyaluronic acid, chitin, carboxymethyl starch, carboxymethylcellulose, proteins such as gelatin, collagen, albumin, fibrin, polyalanine, polyglycolic acid, polypropylene carbonate A carrier such as a synthetic polymer is appropriately used.
[0014]
When HGF and bFGF are used as a combination agent, a combination agent in which both components are combined in a single preparation may be used, or each preparation may be prepared separately and combined appropriately at the time of use.
For commercial purposes, the HGF and bFGF preparations can be packaged together with a written statement such as a certificate describing the use and usage method. In the description, for example, an administration method described later is described.
The administration means and timing when the two components are made as separate preparations may be any of simultaneous administration by the same administration means, time difference administration by the same administration means, simultaneous administration by different administration means, and time difference administration by different administration means. In relation to the timing of action of the two components in FIG. 1, it is preferable to administer the bFGF action first and then administer in such a manner that the HGF action is expressed for the following reasons.
[0015]
That is, the wound healing process after tissue damage is first performed by rapid removal of destroyed cells by apoptosis, and then the regeneration field is formed by proliferation and infiltration of inflammatory cells and fibroblasts. However, the cells that played a role after the end of regeneration are thought to disappear quickly from the spot. Apoptosis that is involved in this process is a phenomenon that is deeply involved in the development of the living body, maintenance of homeostasis, reproduction, and progression of cancer. In a series of repair / regeneration reactions that require tissue modification, it is considered that they also play a major role.
[0016]
In fact, the present inventors have confirmed that apoptosis is enhanced 4 to 7 days after the operation and then decreased. It is presumed that the role played by apoptosis is necessary for removing damaged cells, and that the cells that have played a role in the reconstruction of the wound are rapidly eliminated from the wound.
Since the present inventors have confirmed that bFGF promotes apoptosis early in the 4th day after the operation, in order for the wound healing mechanism to proceed efficiently, bFGF must be used early. It is desirable to have HGF act during the subsequent maturation of the scar.
Therefore, in both cases where both are proteins or both are genes, administration is preferably performed in such a manner that HGF acts after bFGF acts at the wound site.
Even when either one is a protein and the other is a gene, administration is preferably performed in such a manner that HGF acts after bFGF acts at the wound site.
In general, when a gene is administered to a mammal, it is known that it takes some time for the protein encoded by the gene to be expressed in the mammal. When one of them is administered as a gene, it is necessary to administer the other protein in consideration of the time delay.
[0017]
When HGF is administered as a protein and bFGF is administered as a gene, it is preferable to administer HGF at or after the time when bFGF is expressed as a protein. When bFGF is administered as a protein and HGF is administered as a gene, it is effective to administer bFGF at or before the time when HGF is expressed as a protein. In the case of selecting a combination in which one is a protein and the other is a gene, the latter, in which bFGF is administered as a protein and HGF is administered as a gene, is theoretically preferable in relation to the wound healing mechanism described above.
The administration of the gene is preferable from the viewpoint of the expected effect itself and cost-effectiveness compared to the administration of the protein itself, and the protein encoded by the gene is expressed continuously, resulting in a longer period of time. It is effective in that the action can be expected.
As a method of giving a time difference to the onset of action, a method of administering both with a time difference, a method of administering one as a gene and the other as a protein, Xu Examples thereof include a method of administration as a releasable preparation, or an appropriate combination thereof.
In addition, the relationship between the administration time and the time of onset of action is, for example, when HGF is a gene and bFGF is a protein, there is a time difference effective for the onset of action even if it is administered at the same time. Depending on the type of preparation used, the condition of the wound site, etc., it is preferable to administer the action with a time difference of 1 hour to 7 days.
[0018]
The dose varies depending on the type and condition of the wound, the age of the patient, the mode of administration of HGF and bFGF, etc., and is not particularly limited, but HGF is usually 1 μg / kg to 20 mg / kg per day for adults. Preferably, 10 μg / kg to 10 mg / kg, and bFGF is usually 0.5 μg / kg to 10 mg / kg, preferably 1 μg / kg to 5 mg / kg per day for an adult. Preferably, it is administered in 1 to 3 divided doses.
In the combination of HGF and bFGF, the dosage criteria are the same, and the ratio of both components is 0.5-50 parts by weight of bFGF, preferably 1-20 parts by weight with respect to 100 parts by weight of HGF.
[0019]
【Example】
The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples. In particular, in the examples, examination was performed using rat skin, but the same reaction can be similarly applied to other organs and tissues such as lung, liver, bone, cartilage, muscle and nerve.
[0020]
Example 1
BFGF and HGF gene (pCArat HGF plasmid shown in FIG. 3 provided by Professor Toshikazu Nakamura, Osaka University School of Medicine) administered to a full-thickness skin incision made on the back of a rat and treated with conventional suture and bFGF With respect to the quality of scars in wounds treated with the HGF gene, histopathological changes were examined over time from the viewpoint of fibrosis. Sixty rats were treated in 9 groups and examined by the method described below, and the effects of bFGF and HGF genes on the full thickness skin incision were examined over time. In creating the wound, first, the back is shaved and disinfected with alcohol containing hibiten, followed by three full-thickness incision wounds on the fascia including the meat-like membrane 2 cm long under intravenous anesthesia with Nembutal. did. The wound thus created was subcutaneously sutured with 5-0 PDS thread, and further skin sutured with 5-0 PDS thread. At that time, of the 2 cm length, subcutaneous sutures were performed in two places with a layer of fleshy membrane, and skin sutures were performed in three places so as to include the entire skin layer. Nine groups that compared the histopathological changes caused by the addition of bFGF were the group in which only 5-0 PDS thread skin was sutured after subcutaneous suture with 5-0 PDS thread (control group), and immediately after the wound was sutured in the same manner. Group of 0.1 μg bFGF (total amount 0.3 μg) per 1 cm wound wound locally (bFGF 0.1 μg group), and 1.0 μg per 1 cm wound (total amount 3 μg) after suture as in the control group. A group in which bFGF was subcutaneously injected (bFGF 1.0 μg group), a group in which 1 μg of HGF gene (total amount of 3 μg) per 1 cm of wound was injected subcutaneously immediately after the wound was similarly sutured (HGF gene 1 μg group), and a control group Similarly, a group in which 10 μg of HGF gene per 1 cm wound (total amount: 30 μg) was subcutaneously injected locally after suture (HGF gene 10 μg group), and further, HGF gene and bFGF Nine groups plus four groups that combine doses of high and low were compared separately to the (each group 6 mice). The histological examination was performed on days 1, 4, 7, 14, and 28 by killing with an overdose of anesthetic and collecting skin. The diagnostic imaging was measured with Macintosh G4 Cube using NIH image.
[0021]
In this study using suture wounds, bFGF alone promoted apoptosis from the first day of surgery to the fourth day and rapidly matured after the operation, and healed as a relatively narrow scar at the fourth week. In addition, only the use of the HGF gene that suppresses the fibrosis of the wound ultimately causes the scar of the wound to be minor as in the bFGF treatment group, but inflammatory cell infiltration and appearance of fibroblasts are clearly mild immediately after the operation. Wound healing was delayed. By adding bFGF to the wound, cell infiltration was observed from the early stage of the operation. On the other hand, the wound was healed from an early stage but healed with a narrow scar compared to the control group. This effect is significant not only at the 4th week examined in this experiment but also at the 2nd week, and the combined effect of bFGF was clear from the point that the time of the onset of the effect was accelerated. In addition, the degree of scar formation in the wound exceeds all of the individual results, and it is more efficient while shifting the different reactions of cleaning and rebuilding the wound performed on the wound surface rather than just a synergistic effect. It became clear that it was due to proceeding to. In other words, bFGF acts immediately after being administered as a protein, and HGF acts as a gene, so that expression is slightly delayed and acts continuously, so that both of them efficiently perform scrap and build. It is considered that the scar width is extremely narrow, and it is possible to achieve a good result even though the skin is almost regenerated. As a result of measuring the width of the scar on the histopathological image, the width of the scar was significantly narrowed by HGF alone and bFGF alone as compared with the control group, but the effect of the combination of both was 2 weeks after the operation. The value was significantly low at 4 weeks. From the viewpoint of dose, it was found that the combined use of bFGF produces an effect comparable to that of 10 μg of HGF even with 1 μg or less of HGF, and has the effect of promoting gene introduction and protein expression in bFGF. It was confirmed that there was. That is, it has been clarified that bFGF significantly promotes the effect of the HGF gene as a tissue regeneration agent that breaks the conventional common sense by the combined use of the two.
[0022]
The results are shown in FIGS. 1 and 2 (in the figure, the horizontal axis represents the number of days after surgery, and the vertical axis represents the width of the scar (unit: mm)).
FIG. 2 shows extracted results of the HGF and bFGF high-dose single groups and the combination group in FIG.
As is apparent from these figures, the width of the scar gradually increased after the operation in the control group, whereas the width of the scar was significantly narrowed in the group to which the bFGF protein and HGF gene were administered. Furthermore, the combined effect of both bFGF protein and HGF gene further enhanced the effect, and the scar width in the high-dose treatment group of both was about 1/10 of that of the control group.
[0023]
【The invention's effect】
In the scar formation inhibitor at the wound site of the present invention, HGF, which is an active ingredient, suppresses excessive proliferation of granulation tissue in wounds, especially in cuts, mitigates fibrosis, and makes healing of cuts close to regeneration. It is useful because it can be cured. In addition, the tissue regeneration agent combining HGF and bFGF is presumed that bFGF acts on wound healing at the initial stage of injury, and HGF acts on suppression of excessive proliferation and fibrosis of granulation tissue in the subsequent scar maturation stage. However, the action and effect are more remarkable than expected even when compared with the results of using HGF and bFGF alone, respectively, and can be referred to as a synergistic effect.
[Brief description of the drawings]
FIG. 1 shows HGF and bFGF low dose and high dose administration each alone, HGF low dose and bFGF high dose, HGF high dose and bFGF low dose, HGF high dose and bFGF high dose, The change of the scar width of the upper dermis up to 4 weeks after the operation is shown.
FIG. 2 shows the change in the width of the upper dermis scar up to 4 weeks after surgery by high-dose bFGF administration, high-dose HGF administration, and a combination administration thereof.
FIG. 3 shows the structure of a plasmid (pCArat HGF plasmid) used for HGF gene administration.

Claims (11)

A wound skin tissue regenerating agent comprising a combination of hepatocyte growth factor and basic fibroblast growth factor in such a manner that the basic fibroblast growth factor acts first. The wound skin tissue regeneration agent according to claim 1, wherein the hepatocyte growth factor is a nucleic acid encoding a gene for hepatocyte growth factor or an expression vector thereof. The wound skin tissue regeneration agent according to claim 1, wherein the hepatocyte growth factor is a nucleic acid encoding a gene of hepatocyte growth factor or an expression vector thereof, and the basic fibroblast growth factor is a protein. The wound skin tissue regeneration agent according to any one of claims 1 to 3, wherein the hepatocyte growth factor is a sustained-release preparation. The wound skin tissue regeneration agent according to any one of claims 1 to 4, wherein the wound is a cut. The wound skin tissue regenerating agent according to any one of claims 1 to 5, which suppresses scar formation. Combination comprising hepatocyte growth factor and basic fibroblast growth factor, use of the combination for wound skin tissue regeneration, and aspect in which basic fibroblast growth factor is first expressed by action A package containing a statement describing use in A mode in which the basic fibroblast growth factor is activated first is the administration of the basic fibroblast growth factor first, and the hepatocyte growth factor is administered after 1 hour to 7 days. The package according to claim 7, wherein The package according to claim 7, wherein the hepatocyte growth factor is a nucleic acid encoding a gene of hepatocyte growth factor or an expression vector thereof, and the basic fibroblast growth factor is a protein. A scar formation inhibitor for wound skin , which is a combination of hepatocyte growth factor and basic fibroblast growth factor in such a manner that the basic fibroblast growth factor acts first. The wound skin scar formation inhibitor according to claim 10, wherein the hepatocyte growth factor is a nucleic acid encoding a gene of hepatocyte growth factor or an expression vector thereof.

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