JPH05310592A - Wound-healing agent and pharmaceutical preparation - Google Patents

Abstract

(57) [Summary] (Modified) [Purpose] To provide a preparation characterized by containing human cellular fibronectin. [Composition] Human cellular fibronectin is a cell line HUH
Since -6YM can be produced in a large amount without the need of a protein factor as a growth factor, a human cellular fibronectin preparation free from contamination with heterologous proteins or viruses can be produced. This human cellular fibronectin is useful as a corneal therapeutic agent, a wound healing promoter, and the like.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a hepatoblastoma-derived mutant strain HU.
The present invention relates to a wound therapeutic agent characterized by containing human cellular fibronectin produced by H-6YM (Ministry of Industrial Science and Technology No. 12890) as an active ingredient and a pharmaceutical preparation thereof.

[0002]

2. Description of the Related Art In recent years, new wound healing agents such as steroids and antibiotics have emerged one after another. However, in some cases, delayed wound healing may occur or epithelial cells regenerated at the defect may occur. Some had side effects such as abnormalities.

Generally, fibronectin is initially col
It is a glycoprotein found in plasma as d insoluble globulin, and is known to have various physiological activities such as cell-cell adhesion, migration, canceration, and wound healing. Two types are known: plasma fibronectin present in plasma and cellular fibronectin produced mainly by fibroblasts and present in tissues.

At present, fibronectin is a therapeutic agent for cornea,
Although it is being developed as a wound healing agent such as a wound healing promoter and a cosmetic, only plasma fibronectin has been used because a mass production method of cellular fibronectin has not been established.

However, in order to prepare a large amount of plasma fibronectin obtained from blood, there are problems such as securing and homogeneity of the raw material, and contamination of the purified product with raw material-derived impurities such as viruses. However, there is a demand for fibronectin that exhibits an excellent pharmacological action without side effects.

[0006]

DISCLOSURE OF THE INVENTION The present inventors have conducted extensive studies on fibronectin showing an excellent pharmacological action without side effects, and as a result, the human hepatoblastoma-derived mutant strain HUH-
The isolated human cellular fibronectin produced by 6YM (Microtechnology Research Institute No. 12890) has few side effects and an excellent pharmacological action, for example, conventional fibronectin such as cell migration activity and corneal epithelial defect treatment promoting action. The present invention has been completed by discovering that it has a pharmacological action that is possessed by and that is superior to conventional ones.

That is, the present invention provides a human hepatoblastoma-derived mutant strain H
The present invention relates to a therapeutic agent for wounds and a pharmaceutical preparation thereof, which comprises, as an active ingredient, human cellular fibronectin produced and isolated from UH-6YM (Microorganism Research Institute No. 12890). The wound healing agent of the present invention has pharmacological activities such as cell migration activity and corneal epithelial defect treatment promoting activity as shown by conventional fibronectin, and is characterized by its strong activity and weak side effects.

Therefore, the wound healing agent of the present invention is, for example, scratches, cuts, burns, burns, frostbite, skin tumors, skin dryness, keratoses, cracks, nicks, dermatitis, sores of athlete's foot, and surgery. In addition to wounds, corneal wounds, etc., as a therapeutic agent for various wounds including anal fistula, pressure sores, acne, alveolar pyorrhea,
A therapeutic agent for inflammation such as sunburn is included.

The wound healing agent of the present invention is a cell line HUH-6.
The human cellular fibronectin produced by YM and the active ingredient thereof can be used as a medicinal preparation effective for the above-mentioned various medicinal uses. The pharmaceutical preparation is usually prepared in the form of a pharmaceutical composition by mixing an appropriate pharmaceutical carrier with human cellular fibronectin and a stabilizer.

As the pharmaceutical carrier, an excipient or diluent such as a filler, a filler, a binder, a moisturizer, a disintegrant and a surface active agent which is usually used for preparing a pharmaceutical according to the use form. Any agent can be used.

The form of the pharmaceutical composition is not particularly limited as long as it effectively contains human cellular fibronectin and a stabilizer, and examples thereof include tablets, powders, granules,
Although it may be a solid formulation such as a pill, it is usually suitable to prepare an injection formulation such as a liquid formulation, a suspension formulation and an emulsion. Further, this is preferably a dried product which can be made into a liquid form by adding an appropriate carrier before use, and particularly preferably a freeze-dried preparation. Any of these can be prepared according to a conventional method.

There is no particular limitation on the stabilizer to be added to the above-mentioned pharmaceutical composition, and ordinary protein stabilizers can be used.
For example, albumin, amino acid, saccharide, surfactant and the like can be exemplified.

More specifically, examples of the stabilizer include albumins such as human serum albumin (HSA), ordinary L-type amino acids such as glycine, alanine, valine and cysteine, glucose, mannose, galactose and fructose. Sugars, mannitol, inositol, sugar alcohols such as xylicourt, sucrose, maltose, disaccharides such as lactose, dextran, polysaccharides such as hydroxypropyl starch, polyoxyethylene glycol sorbitan alkyl ester type, polyoxyethylene alkyl ether type, Examples thereof include sorbitan monoacyl ester-based and fatty acid glyceride-based surfactants, but are not limited thereto.

These can be used alone or in combination of two or more, and it is preferable to mix albumins and saccharides. More preferably, a combination of human serum albumin and sucrose is preferable.

The addition amount of these is not particularly limited, but about 0.01 mg or more, preferably about 0.1 to 100 mg of albumin, and about 0.1 to 100 mg of albumin separately for 1 mg of human cellular fibronectin. About 0.1 mg or more, preferably about 1 mg to 1000 m
It is appropriate that the amount of the surfactant is about 0.0001 mg or more, preferably about 0.001 mg to 0.1 mg.

The buffer solution that can be used as a carrier in the above is not particularly limited, but examples thereof include citric acid-sodium phosphate, citric acid-sodium citrate, acetic acid-sodium acetate, and phosphoric acid. PH 4 of disodium-phosphate-sodium, citric acid-borax, etc.
-8, more preferably, each buffer solution having a pH of 6-8 can be exemplified.

The dose of the wound healing agent thus obtained is
The dosage form is appropriately selected depending on the administration method, dosage form, purpose of use, symptom of the patient to whom it is applied, etc., but it is not constant, but generally, the dosage form contains about 0.00001 to 10% by weight of the active ingredient. Is prepared. For example, in the case of eye drops, it is adjusted to about 10 μg / ml to about 10 mg / ml, in the case of solution preparations, about 10 μg / ml to 100 mg / ml, and in the case of ointments, it is adjusted to about 10 μg / g to about 100 mg / g. However, the present invention is not limited to these.

As a particularly preferred specific example of a pharmaceutical preparation containing human cellular fibronectin as an active ingredient, a freeze-dried preparation having the following contents can be exemplified. The content of each component of the freeze-dried preparation is usually human cellular fibronectin 0.1-1.0 mg / ml human serum albumin 0.1-10 mg / ml sucrose 5-100 mg / ml phosphate buffer 0.01. What is contained in the range of -0.5 M pH 6-8 is particularly excellent in stability, solubility, storability and the like. It is desirable to administer these preparations in such an amount that the amount of the active ingredient contained therein will be about 0.1 μg to about 60 g per adult per day. The administration need not be once a day and can be divided into 3 to 6 times a day. More specifically, as an eye drop, 3 to 6 times a day,
About 10 µl to about 200 µl can be instilled once, or about 100 mg to about 10 g can be applied as an external preparation 1 to 6 times a day.

The above-mentioned various forms of pharmaceutical preparations are administered in a suitable administration route depending on the form, for example, injection-form pharmaceutical preparations are administered intravenously, intramuscularly, subcutaneously, intracutaneously, intraperitoneally, etc. The preparation can also be topically administered to the eye or nasal cavity as an eye drop or a nasal drop.

Human cellular fibronectin, which is an active ingredient of the therapeutic agent of the present invention, is a novel substance and can be produced, for example, by the method developed by Ito et al. Ito et al., HUH-6 clone 5 (Japanese, isolated from human hepatoblastoma).
Cancer Research Resources
Cloning was repeated from Bank, cell number JCRB0401), and a study was conducted. As a result, a human hepatoblastoma-derived mutant strain HUH-6YM (hereinafter referred to as cell line HU), which can be cultured in a protein-free medium and can be subcultured without limit.
It is called H-6YM. ) Was established and established as a cell line.

This cell line was HUH-6YM, and was FE
It has been deposited with the Institute of Mechanical Engineering at RM P-12890.

The biological properties of HUH-6YM are summarized below. It is a mutant strain derived from human hepatoblastoma-derived cell line HUH-6 clone5. It grows in a basal medium consisting of amino acids, vitamins, sugars and inorganic salts, and can be subcultured without limitation. That is, it is a cell line that does not require a proteinaceous factor as a growth factor. It produces a significant amount of human cellular fibronectin.

Next, a method for producing human cellular fibronectin by culturing the cell line HUH-6YM will be described.

The cell line HUH-6YM is cultured in a petri dish as a seed culture. Repeat passaging to ensure the required cell number in the next continuous culture. The medium used is
A commercially available e-RDF medium is suitable, but the medium is not limited to this, and any medium prepared according to its components can be used. Further, a proteinaceous factor is not particularly required as an additive component, but may be added as long as it does not adversely affect cells.

The culturing conditions are not particularly limited, but the culturing temperature is 36 to 37 ° C., and the gas phase condition is preferably air containing about 5% CO ₂ in the seed culturing with a petri dish or the like. .. However, especially in continuous culture, CO
_No need for prepared air for _2nd grade.

The method of continuous culture is not particularly limited, but a general method using a roller bottle can be used. In this case, the medium is replaced at an appropriate time according to the number of cells in the roller bottle.

Cell line HUH-obtained by such a method
The 6YM culture supernatant serves as a raw material for the next purification.

For the purification of cellular fibronectin, a method generally used for protein purification can be used, and one example thereof is as follows.

First, an appropriate protease inhibitor was added to the culture supernatant, and then the mixture was subjected to centrifugation to remove impurities such as cell debris, followed by concentration by the ultrafiltration method to obtain the cellularity in the culture supernatant. Raise fibronectin to appropriate concentration. This is to make the subsequent purification more efficient. The concentrated supernatant is PBS (-) (Phosphate Bu
Equilibration with a fed saline) and load on a gelatin affinity column. After that, PBS (-)
Elute with a buffer solution in which 6M urea is dissolved to obtain the elution fraction. The purity of cellular fibronectin in this fraction is 95% or higher according to the assay by the electrophoretic method, but in order to further increase the purity, it can be increased by using another method such as a general ion exchange method. Although it can be purified, cellular fibronectin usually has a very low solubility in a buffer solution such as PBS (-), and therefore it is necessary to perform the separation operation in the presence of urea.

After that, urea is removed, and a buffer solution exchange with a phosphate buffer solution containing 5% sucrose is carried out in order to prepare a cellular fibronectin solution having a certain concentration or more. As a method for exchanging with a buffer solution, a general method such as a membrane dialysis method or a diafiltration method using ultrafiltration can be used.

The human cellular fibronectin thus obtained is obtained as a mixture of a dimer and a monomer. Structural features of cellular fibronectin include:
According to analysis by SDS polyacrylamide gel electrophoresis under reducing and non-reducing conditions, its molecular weight is larger than that of the plasma fibronectin preparation, and it reacts with a commercially available anti-cellular fibronectin antibody.

The properties of human cellular fibronectin produced using HUH-6YM are shown below. Molecular weight: SDS polyacrylamide gel electrophoresis in reducing and non-reducing states, dimer 470 kd, monomer 24
The dimer-to-monomer ratio is 2: 5 kd and 225 kd.
It is 1. Isoelectric point: pH 5-6 Immunological specificity Commercially available anti-cell fibronectin antibody (manufactured by Sigma, M
ONOCRONALANTI-CELLULAR FI
BRONECTIN, Product No. F614
0) and also an anti-human fibronectin antibody (clone 30-8, manufactured by Takara Shuzo). In addition, the produced human cellular fibronectin has the biological effects of fibronectin such as cell adhesion promoting activity, gelatin bead aggregating activity, and cell spreading activity which have already been reported.

As described above, by using the cell line HUH-6YM and continuously culturing a large amount of cells in a protein-free medium, human cellular fibronectin can be produced very advantageously.

Surprisingly, this HUH-6YM
The fibronectin produced by Escherichia coli was found to be cellular fibronectin, and it was found that this cellular fibronectin is highly soluble in sucrose solution.

[0035]

[Means for Solving the Problem] As a result of earnest research, a method for producing large-scale culture of human cellular fibronectin was established. Therefore, the present inventors have examined various biological activities using the above-mentioned human cellular fibronectin, the efficacy was observed in the corneal defect treatment promotion experiment using a rabbit corneal epithelium defect model, It was confirmed that the specific activity was higher than that of human plasma fibronectin.

Furthermore, when the cell migration activity was measured by the modified Boyden chamber method, it was confirmed that the above-mentioned cellular fibronectin had a higher cell migration activity than plasma fibronectin.

In the freeze-drying of an aqueous solution containing fibronectin, a freeze-dried preparation containing human cellular fibronectin produced from the cell line HUH-6YM as an active ingredient is highly stable, and particularly disaccharide and human are used as stabilizers. If albumin is added, the dissolution time in distilled water for injection after lyophilization is shortened, the insoluble matter remains and the cloudiness disappears, and the stability of the cellular fibronectin against lyophilization increases synergistically. It was confirmed.

The present invention was completed based on the above findings, and relates to a therapeutic agent for wounds containing human cellular fibronectin and a pharmaceutical preparation thereof.

[0039]

INDUSTRIAL APPLICABILITY The present invention provides a human cellular fibronectin preparation. This preparation has a biological activity similar to that of plasma fibronectin, but its cell migration activity is higher than that of plasma fibronectin, so that it has an advantage that the effective dose for therapeutic use is small.

Furthermore, since the human cellular fibronectin used in the present invention is purified from the culture supernatant of a strictly controlled human cell line, there is little risk of contamination with unknown impurities.

The present invention will be specifically described with reference to the following Reference Examples and Examples.

[0042]

[Reference example] [I. Production of substances】

As a seed culture, the cell line HUH-6YM (F
ERM P-12890) was subcultured using e-RDF medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.). Culture at 37 ℃, 5% CO
_The air containing ₂ was used for one month.

The continuous culture for obtaining the culture supernatant is 175
1 × 10 ⁸ cells were seeded per 0 cm ² roller bottle (Falcon) and cultured at 37 ° C. in the atmosphere. Up to 3 weeks after seeding, the whole amount of the medium of 500 ml per roller bottle was exchanged every 2 to 3 days thereafter, and the obtained culture supernatant was collected. Continuous culture is about 3
I went there for a month. (Fig. 1)

The culture supernatant was diluted with 5 mM sodium ethylenediaminetetraacetate (EDT) as a protease inhibitor.
After the addition of A), continuous centrifugation was performed at 1500 g and a flow rate of 2
Impurities such as cell fragments were removed by 0 L / min (domestic H600-S) and the supernatant was collected.

Next, a 10-fold concentrated solution of the above supernatant was prepared using an ultrafiltration membrane having a molecular weight cutoff of 100 k (manufactured by Filtron, model OS100CO5).

This was loaded on a gelatin Sepharose 4B column (Pharmacia) equilibrated with PBS (-), washed with PBS (-) and equilibrated. Then PBS
Elution was performed with a buffer solution in which 6M urea was dissolved in (-).

The purity of cellular fibronectin in this eluate was 95% or more as a result of the assay by SDS-polyacrylamide gel electrophoresis under reducing conditions.

The eluate was collected and an ultrafiltration membrane having a molecular weight cut off of 100 k (manufactured by Filtron, model OS-100CO) was used.
First, the eluate was concentrated using 1), and then the solvent was exchanged with a 0.1 M sodium phosphate buffer containing 5% sucrose by the diafiltration method.

A human cellular fibronectin preparation was obtained by the above method.

[0051]

[II. Properties of substances] (1) Molecular weight

The molecular weights of the above-mentioned human cellular fibronectin preparations were compared by SDS polyacrylamide gel electrophoresis with standard proteins and human plasma fibronectin preparations (Iwaki Glass Co., Ltd.) under reducing and non-reducing conditions.

As a result, the molecular weight of the human cellular fibronectin preparation of the present invention was 470 kd for dimer and 2 for monomer.
It was estimated to be 45 kd and 225 kd and was not shown to have a higher molecular weight than human plasma fibronectin. (Figure 2) (2) Immunological reactivity

Anti-cellular fibronectin (Product No. manufactured by Sigma) that does not react with human plasma fibronectin but reacts only with cellular fibronectin.
The reactivity was confirmed by an immunostaining method after Western blotting using F-6140). As a result, this preparation reacted with this specific antibody. It also reacted with a human fibronectin antibody (clone 30-8, manufactured by Takara Shuzo).

From the above molecular weight and immunological reactivity, the fibronectin obtained in the present invention was judged to be human cellular fibronectin. (3) Water-soluble

It has been reported previously that cellular fibronectin has a very low solubility in buffer solutions of pH 6 to 8.

In the present invention, it was found that cellular fibronectin is easily soluble in a buffer solution containing sucrose.

Solubility in sucrose solution is shown in Table 1 below.

[0059]

[Table 1]

[0060]

Example 1 The following experiment was conducted to measure the effect of promoting the healing of corneal epithelial defects. First, 9 on the eyeball of N2W rabbit
A corneal epithelium was peeled off by injuring with a mm toy pan.
The eye drop was started 18 hours after the peeling, and the fibronectin solution was applied to one eye and PBS (-) to the other eye. As the fibronectin solution, the human cellular fibronectin obtained in Reference Example was diluted with a PBS (-) solution to give 125
[mu] g / ml, 250 [mu] g / ml, 500 [mu] g / ml and 1 mg / ml were prepared, and human plasma fibronectin solution purified from human blood as a target was diluted with PBS (-) solution to obtain 250 [mu] g / ml, 1 mg / ml. And 3
The concentration was adjusted to mg / ml. For each instillation, 100 μl of each solution was used, and the instillation was performed once every hour and was continued until 30 hours later. For the healing rate, photographs were taken at 18 hours and 30 hours, and the area of the corneal epithelial defect was measured by an image analyzer. The elapsed time and the corneal epithelial defect area at that time were plotted for each eye, the slope of the healing line was calculated by the least squares method, and the absolute value thereof was used as the healing rate (mm ² / h). Furthermore, the healing rate obtained by instilling the fibronectin solution was divided by the healing rate obtained by instilling PBS (−) of the one eye, and the value was multiplied by 100 to obtain the healing rate increase rate (%). The result is shown in FIG.

From the above results, it became clear that human cellular fibronectin is more effective when administered in a smaller amount than human plasma fibronectin. Furthermore, the effective concentration of human cellular fibronectin for corneal epithelial defects was 100 μg / ml to 1 mg / ml.

[0062]

Example 2 The cell adhesion activity and cell migration activity of human cellular fibronectin and human plasma fibronectin were measured using rabbit corneal epithelial cells. The corneal epithelial cells used in the experiment were transferred to a culture system by an enzymatic method and primary-cultured.

Cell adhesion activity is 0.1 μg / ml to 10
A human cellular and human plasma fibronectin solution was prepared so that the concentration was 0 μg / ml, and 100 μl of each solution was added to a 96-well plate at 37 ° C.
Fibronectin was left to stand for a period of time to coat it. After washing the plate with PBS (-), 1% BSA / PBS
The (-) solution was added to block the wells coated with fibronectin. After washing the plate with PBS (-), 1,000 corneal epithelial cells were added per well, and the plate was left at 37 ° C for 1 hour. The cells were fixed with a neutral formalin solution, stained with a crystal violet staining solution, and the number of cells was counted using a microscope. The result is shown in FIG.

As a result, no significant difference in cell adhesion activity was observed between human cellular fibronectin and human plasma fibronectin.

Next, the cell migration activity was measured by the modified Boyden chamber method. First, 0 in a 24-well plate
TCM-199 to a concentration of ~ 100 μg / ml
Human cellular and plasma fibronectin solutions diluted with medium were added at 0.5 ml per well. A chemotaxis chamber was placed in the well, and (2 × 10 ⁵ cells / ml) as a cell suspension was added to one chamber in an amount of 0.
2 ml each was added. Then, the cells were cultured in a 37 ° C. incubator in the presence of 5% CO ₂ for 6 hours. The chemotaxis chamber was taken out, the cells on the inner membrane were peeled off with a cotton swab, and washed with PBS (−). The cells on the lower surface of the chamber were fixed with a neutral formalin solution and then stained with crystal violet. The number of cells in 5 fields of view of the stained cells is measured under a microscope of 100 magnification, and the value multiplied by 100 is defined as the number of migrated cells. The result is shown in FIG.

From these results, it was confirmed that the cell migration activity of human cellular fibronectin was higher than that of human plasma fibronectin.

From the results of Examples 1 and 2, it is considered that human cellular fibronectin has a lower effective amount for promoting healing of corneal epithelial defect than human plasma fibronectin because human cellular fibronectin has high cell migration activity. Be done.

Further, even in epithelial defects in other tissues such as skin, human cellular fibronectin can be expected to have a more effective healing promoting effect than human plasma fibronectin.

[0069]

Example 3 Preparation and re-dissolution of human cellular fibronectin freeze-dried preparation Human cellular fibronectin 1.0 mg /
ml, sucrose 50 mg / ml, 0.1 M phosphate buffer (pH 7.4) of human cellular fibronectin stock solution 1 ml, and human serum albumin 1.0 mg
/ Ml, 1 ml of human serum albumin solution consisting of 0.1 M phosphate buffer (pH 7.4) was added (total 2 m
l), human cellular fibronectin 0.5 mg / ml,
Sucrose 25 mg / ml, human serum albumin 0.5 mg
A human cellular fibronectin solution consisting of 0.1 M phosphate buffer (pH 7.4) / ml was prepared. Next, this human cellular fibronectin solution was lyophilized by a conventional method to obtain a human cellular fibronectin lyophilized preparation. The freeze-dried preparation of human cellular fibronectin had a good appearance (white mass), and the redissolvability of fibronectin after addition of 2 ml of distilled water was also good.

The following table shows other examples, the appearance after freeze-drying and the re-solubility after adding 2 ml of distilled water.

[0071]

[Table 2]

[Brief description of drawings]

FIG. 1 shows that the cell line HUH-6YM
It is a figure which shows the change with respect to the culture period of the concentration of human cellular fibronectin produced in the culture supernatant.

FIG. 2: SDS of the human cellular fibronectin of the present invention
It is a figure which shows the result of having applied to polyacrylamide gel electrophoresis. A human plasma fibronectin preparation was used as a comparative control.

FIG. 3 is a diagram showing an effect of promoting healing of corneal epithelial defect. The vertical axis represents the rate of increase in healing rate, and the horizontal axis represents the concentration of each fibronectin in the eye drop solution.

FIG. 4 is a diagram showing corneal epithelial cell adhesive activity. The vertical axis represents the number of adherent cells, and the horizontal axis represents the concentration of fibronectin coated on the plate.

FIG. 5 is a diagram showing cell migration activity. The vertical axis shows the number of migrated cells, and the horizontal axis shows the fibronectin concentration used in the experiment.

Continued Front Page (72) Inventor Taka Sakamoto 2-19-15 Mize, Otsu City, Shiga Prefecture

Claims (3)

[Claims] 1. A human hepatoblastoma-derived mutant strain HUH-6YM
A wound healing agent characterized by containing human cellular fibronectin produced as an active ingredient, by Microbiology Research Institute No. 12890. 2. A therapeutic agent for cornea containing the active ingredient according to claim 1. 3. A human hepatoblastoma-derived mutant strain HUH-6YM
A pharmaceutical preparation characterized by containing human cellular fibronectin produced as an active ingredient, and further containing a stabilizer.