JPH0873373A - Therapeutic agent for intractable skin deficiency wound - Google Patents

Abstract

PURPOSE: To obtain the subject treating agent, containing a fibronectin-based polypeptide having a specific amino acid sequence as an active ingredient, effective against intractable skin deficiency wounds caused by local insufficient blood circulation and having high safety. CONSTITUTION: This treating agent contains a polypeptide having an amino acid sequence of the formula (hereinafter referred to as C-274), preferably in a content of 0.0001-10wt.% as an active ingredient. The C-274 is preferably obtained by converting a codon AAA of Lys<1513> of the 5th residue from the C-terminal in a polypeptide of 279 amino acid residues (Pro<1239> -Met<1517> ) into a termination codon TAA, preparing a plasmid pTFD707 capable of coding the C-274, then transducing the resultant plasmid into Escherichia coil and subsequently producing Escherichia coil JM 109/pTF7221 (FERM BP-1915) containing a plasma pTFD707 transduced thereinto. Furthermore, the C-274 is preferably administered frequently as an external preparation in 5μg to 1g dose in the case of an adult at a time.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an intractable skin defect wound therapeutic agent containing a functional polypeptide of fibronectin.

[0002]

2. Description of the Related Art Fibronectin (hereinafter abbreviated as FN)
Is a high-molecular-weight multifunctional glycoprotein present in various tissues of animals, cell surfaces, blood, etc.
It exhibits physiological actions such as spreading, migration, differentiation, proliferation, and phagocytosis, and is involved in tissue repair, cancer metastasis suppression, biological defense and the like. FN is an adhesive glycoprotein composed of a polypeptide having a molecular weight of about 250,000, and forms a dimer or a tetramer by an S-S bond near the C terminus. Further, the molecule has a domain structure having various functions, has a binding activity for collagen, heparin, fibrin, etc., and has a function of adhering and spreading cells. Among these domains, the R-G-D-S sequence has been clarified as the minimum required unit for the basic structure of the cell adhesion domain [Nature, Vol. 309, Nos. 30-33].
Page (1984)], SEQ ID NO: 1 of the sequence listing containing this sequence.
The method for producing the polypeptide represented by
No. 397 publication. Regarding the usage of this polypeptide, a cancer metastasis inhibitor (Japanese Patent Laid-Open No. 12774/1993)
2), an angiogenesis inhibitor (JP-A-3-173828).
JP-A-3-291238), skin cosmetics (JP-A-3-197411), anti-inflammatory agents (JP-A-3-291235), anti-cancer agents (JP-A-4-291). No. 59734), periodontal ligament and alveolar bone tissue regeneration and repair accelerator (Japanese Patent Laid-Open No. 4-74132). However, its use as a therapeutic agent for recovering intractable skin defect wounds such as diabetic skin ulcer and severe pressure ulcer is not known. In recent years, various types of wound dressing materials have been developed, which are synthetic materials, biomaterials, or a combination of both materials, and are applied to the treatment of burns, trauma, and the like. Furthermore, for the purpose of imparting a high function, a physiologically active substance,
Attempts to incorporate these into wound dressings have received attention. There are states of no infection and symptoms of infection, or states of no intervening necrotic tissue on the wound surface, and it is important to select a wound dressing material having an optimal function depending on the application case.

[0003]

DISCLOSURE OF THE INVENTION Problems such as diabetic skin ulcers and severe pressure ulcers are difficult to cure, and development of therapeutic agents applicable to these intractable skin defect wounds is desired. An object of the present invention is to provide a therapeutic agent having an effect on intractable skin defect wounds.

[0004]

Means for Solving the Problems The present invention will be summarized. The present invention relates to an intractable skin defect wound therapeutic agent, which comprises a polypeptide having an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing as an active ingredient. Is characterized by.

In order to develop a therapeutic agent for a refractory skin defect wound, it is necessary to create an animal experimental model of the skin defect wound, but chronic poor granulation tissue such as that seen in diabetic skin ulcers and severe pressure ulcers. Wounds cannot be exactly reproduced in animal experiments. However, the present inventor newly created a poor granulation tissue model that most closely resembles the tissue wound clinically, and as a result of screening using this model, the polygran acid having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing was found. It was found that the peptide shows a healing effect accompanied by remarkable wound contraction,
The present invention has been completed.

The content of the above-mentioned polypeptide in the therapeutic agent for the intractable skin defect wound of the present invention may be arbitrary.
The therapeutic agent may be contained in an amount of 0.00001 to 30% by weight, preferably 0.0001 to 10% by weight.

The therapeutic agents of the present invention are water, physiological saline, PB.
Although it may be directly applied to the wound surface in a form dissolved or dispersed in S, ointment or the like, when the hydrous wound dressing material is applied to the wound surface, a solution containing the above polypeptide is applied to the dressing material. The method of impregnation is most effective.

Examples of the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing include, for example, JP-A-1-8
0900, 1-206998, 2-97397.
No. 2-152990 and No. 2-311498.

A method for producing the polypeptide represented by SEQ ID NO: 1 in the sequence listing (hereinafter referred to as C-274) is shown in the above-mentioned Japanese Patent Laid-Open No. 2-97397 and will be specifically described below. . In this specification, the shoulder numbers attached to amino acids refer to EMBL DATA BAN.
The number of amino acid residues from the N terminus assigned to the FN amino acid in (K) is shown. C-274 (Pro ¹²³⁹ -Asp ¹⁵¹² )
Have already filed a patent application for a 279 amino acid residue polypeptide (Pro ^1239- Met ¹⁵¹⁷ ) (Japanese Patent Laid-Open No. 1-2069).
No. 98), in which the C-terminal 5 amino acid residues are deleted. As a method for preparing C-274 (Pro ¹²³⁹ -Asp ¹⁵¹² ) by genetic engineering, it is convenient to use the plasmid pTFD707 encoding the above-mentioned 279 amino acid residue polypeptide (Pro ¹²³⁹ -Met ¹⁵¹⁷ ). By converting the codon AAA of Lys ¹⁵¹³ , which is the fifth residue from the C terminus of the 279 amino acid residue polypeptide, to the termination codon TAA, C-274 (Pro ¹²³⁹ -Asp
¹⁵¹² ) can be prepared. This base conversion can be performed by introducing a site-specific mutation. C-274 (Pro ¹²³⁹ -As
Escherichia coli into which a plasmid expressing ^p1512 ) was introduced
It was named and displayed as scherichia coli JM 109 / pTF 7221 and was already in the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology.
Deposited as 915 (FERM BP-1915). This cell adhesion polypeptide C-from the recombinant
The purification of 274 is performed as follows, for example. The bacterial cell pellet is suspended in a buffer and sonicated to separate a soluble fraction and an insoluble fraction. The latter is further solubilized with a buffer containing 7M urea. The soluble fraction was collected, and Sepharose 4 bound with an antibody specific to the cell adhesion domain of FN
Apply to column B and perform affinity purification. A buffer having a pH of around 2.3 is used for elution. The cell adhesion active polypeptide C-274 can be obtained by collecting the target fractions by immunoblotting. If necessary, it can be further purified by FPLC or HPLC.

The above polypeptide may or may not have a sugar chain. The dose is determined depending on the administration method, age of the patient, body weight, symptom, and therapeutic purpose, but in general, for adults, 5 μg to 1 g per dose is preferable. The therapeutic agent of the present invention is preferably administered locally, and frequently administered as an external preparation.

The polypeptide used in the present invention has a size of 100 m.
It is a polypeptide that shows no toxicity even when g / kg is intravenously administered to mice, and is highly safe even when administered locally.

In addition to the above, the form of application may be in the form of, for example, a suspension, emulsion, paste or the like.
It may also be applied by being contained in a wound dressing. As a material of the wound dressing material, known materials such as hyaluronic acid, collagen and chitin can be used, and the wound dressing material may be molded into a film shape, a sponge shape or the like and used. It may also be used in combination with cultured skin containing skin cells. The application form is not limited to these. In the therapeutic agent for treating intractable skin defects of the present invention, raw materials usually used for this purpose, for example, surfactants, alcohols, humectants, thickeners, excipients, preservatives, antioxidants, chelates. Agents, pH adjusters, fragrances, dyes and the like can be added within a range that does not impair the function of C-274.

[Pharmacological Test] 1) Preparation of Poor Granulation Tissue Wound Surface A model of bad granulation tissue was prepared as follows to evaluate a therapeutic agent for a poor granulation tissue wound with delayed healing. A full-thickness skin defect wound with a diameter of 30 mm is prepared on the back of a 5-week-old SD rat, a polystyrene ring having an outer diameter of 30 mm and an inner diameter of 25 mm is sutured and fixed along the periphery of the wound, and an epoxy adhesive is applied onto the ring. A nylon net having a diameter of 35 mm or a GORE-TEX film (Gore Co., Ltd.) was adhered and fixed, gauze was applied to this, which was protected with a stainless net and fixed with an elastic tape. This nylon mesh or GORE-TEX membrane is fixed without adhering to the wound surface and can maintain air permeability, providing an environment close to a semi-open wound. In this state, one rat was sacrificed one by one after 3, 6 and 9 days, and the state of the wound surface was observed histologically. When the nylon mesh was applied to the full thickness skin defect wound in a non-contact manner, the exudate was absorbed on the outer gauze without being temporarily stored on the wound surface. In addition, the central part of the nylon came into contact with the wound surface, resulting in mechanical irritation to the wound surface due to friction caused by the movement of the animal. According to the histological observation by HE staining, when it was coated for 3 days, it showed a tissue structure showing a strong inflammatory reaction, and when it was coated for 6 days or more,
The strong inflammatory response persisted and the formation of granulation tissue with a dense matrix structure was initiated. On the other hand, when the Gore-Tex membrane was non-contactly coated on the full-thickness skin defect wound, no evidence of absorption of the exudate by the outer gauze was observed. The GORE-TEX membrane has the property of being permeable to air but impermeable to exudate, so that it was possible to provide an environment in which exudate was temporarily stored on the wound surface. In the case of coating for 3 days, the tissue structure exhibiting a weak inflammatory reaction was exhibited, and in the case of coating for 6 days, the inflammatory reaction was weak and the formation of granulation tissue having a loose matrix structure accompanied by edema was observed. However, when it was coated for 9 days or more, it changed into a tissue structure having a dense matrix structure. As a result, the case where the GORE-TEX membrane was applied in a non-contact manner for 6 days was suitable for the preparation of a model of poor granulation tissue wound with edema, and the conditions for applying the GORE-TEX membrane for 6 days were selected to An evaluation test of the therapeutic agent was conducted.

2) Evaluation of C-274 on the wound surface of granulation tissue A hyaluronic acid sponge was applied to the wound surface of the granulation tissue prepared by non-contact application of a Gore-Tex membrane to a rat back full-thickness skin defect wound for 6 days. Was impregnated with 1 ml of a C-274 aqueous solution (120 μg / ml), and a covering material was applied on it to fix the periphery by suture, and gauze was applied on the periphery to fix the bandage with an elastic tape. Here, the case not impregnated with the C-274 aqueous solution was used as a control group. The coating material was removed on the 5th day and the wound surface was observed (5th day of application). 1m newly
1 hyaluronic acid sponge impregnated with C-274 or hyaluronic acid sponge not impregnated with C-274 was applied, and the dressing was fixed in the same manner on the 5th day, and the coating material was removed again to observe the wound surface (application) Day 10). 1 ml newly
The hyaluronic acid sponge impregnated with C-274 or the hyaluronic acid sponge not impregnated with C-274 was applied, the bandage was fixed in the same manner, and the coating material was removed on the fifth day to observe the wound surface (application day 15 Eye). Hyaluronic acid sponge was excellent in water retention and was suitable for application of C-274 aqueous solution. When the coating material was replaced every 5 days, the hyaluronic acid sponge was not visually observed to remain. Hyaluronic acid itself tends to promote wound healing considerably, but on day 10 and day 15 of application, C-274-containing hyaluronic acid sponge shows wound healing with more remarkable wound contraction. Was done. The results are shown in Table 1.

[0015]

[Table 1] Table 1 ──────────────────────────────────── Covering material Covering material Applicable Number of days 0 days 5 days 10 days 15 days ──────────────────────────────────── Wound area (cover Ratio with the wound area before) Hyaluronic acid containing C-274 9.62cm ² 4.95cm ² (51.5%) 2.33cm ² (24.2%) 1.51cm ² (15.7%) Sponge hyaluronic acid 9.62cm ² 6.73cm ² (70.0% ) 5.05cm ² (52.5%) 2.90cm ² (30.1%) Sponge ────────────────────────────────── ──

[0016]

EXAMPLES Next, the present invention will be described in detail with reference to examples, but the scope of the present invention is not limited to the examples.

Formulation Example 1 12 mg of C-274 was added to PBS, and the total amount was 100 m.
After lysing this as l, it is sterilized and filtered using a Millipore filter GS type. 1 ml of this filtrate was freeze-dried in a 10 ml vial, and C-274 was added to 1 vial.
A lyophilized product containing 120 μg of was obtained.

Formulation Example 2 A water-soluble epoxide compound Denacol EX810 (manufactured by Nagase Kasei) was added to a 1% aqueous solution (pH 5.9) of hyaluronic acid (manufactured by Kibun Food Chemifa) having a molecular weight of 1.8 to 2.2 million to prepare repeating units of hyaluronic acid (two 1 for the molecular weight of (sugar)
/ 10 mol amount was added and thoroughly stirred, and then 90 ml was poured into a polystyrene container (10 cm x 18 cm), and 50
After standing still in a dryer set at 0 ° C. for 5 hours, it was rapidly frozen in a −80 ° C. freezer and vacuum dried to produce a sponge-like sheet. After cutting into a predetermined size, it was sterilized with ethylene oxide gas to prepare a hyaluronic acid sponge. A C-274 aqueous solution was added to the hyaluronic acid sponge before use to prepare a C-274-containing hyaluronic acid sponge.

Formulation Example 3 For the production of a sponge sheet, atelocollagen (manufactured by Koken) prepared by enzymatically treating cowhide-derived collagen was used. A 1% aqueous atelocollagen solution was adjusted to pH 4 and stirred with a homogenizer at 15000 rpm for 3 minutes to form a cream, and 60 ml of this solution was poured into a polystyrene container (10 cm x 18 cm) and allowed to stand in an ammonia atmosphere for 1 hour to gel. It was After washing with running water for one day and night, this was rapidly frozen at −80 ° C. and vacuum dried to prepare a sponge-like sheet, and further, intermolecular crosslinking was introduced by irradiation with ultraviolet rays. After cutting into a predetermined size, it was sterilized with ethylene oxide gas to prepare a collagen sponge. C-274 on the collagen sponge
An aqueous solution was added at the time of use to prepare a C-274-containing collagen sponge.

[0020]

INDUSTRIAL APPLICABILITY According to the present invention, a therapeutic agent containing C-274 effective for a refractory skin defect wound caused by local poor circulation can be provided. C-274 is a biologically active polypeptide derived from a living body, has high safety, can be supplied in large amounts by genetic engineering, and can be a potent therapeutic agent for skin defect wounds.

[0021]

[Sequence list]

SEQ ID NO: 1 Sequence length: 274 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Sequence: Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met Arg 1 5 10 15 Val Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn Phe Leu 20 25 30 Val Arg Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu Leu 35 40 45 Ser Ile Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu 50 55 60 Pro Gly Thr Glu Tyr Val Val Ser Val Ser Ser Val Tyr Glu Gln 65 70 75 His Glu Ser Thr Pro Leu Arg Gly Arg Gln Lys Thr Gly Leu Asp 80 85 90 Ser Pro Thr Gly Ile Asp Phe Ser Asp Ile Thr Ala Asn Ser Phe 95 100 105 Thr Val His Trp Ile Ala Pro Arg Ala Thr Ile Thr Gly Tyr Arg 110 115 120 Ile Arg His His Pro Glu His Phe Ser Gly Arg Pro Arg Glu Asp 125 130 135 Arg Val Pro His Ser Arg Asn Ser Ile Thr Leu Thr Asn Leu Thr 140 145 150 Pro Gly Thr Glu Tyr Val Val Ser Ile Val Ala Leu Asn Gly Arg 155 160 165 Glu Glu Ser Pro Leu Leu Ile Gly Gln Gln Ser Thr Va l Ser Asp 170 175 180 Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu 185 190 195 Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr Arg 200 205 210 Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 215 220 225 Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys 230 235 240 Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg 245 250 255 Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 260 265 270 Thr Glu Ile Asp

Claims (1)

[Claims] 1. A therapeutic agent for intractable cutaneous defect, comprising a polypeptide having an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing as an active ingredient.