JPH0656692A - Wound therapeutic agent comprising tcf-ii as active ingredient - Google Patents

Abstract

PURPOSE:To obtain the subject therapeutic agent, comprising a new antitumor protein tumor cytotoxic factor (TCF)-II as an active ingredient and usable as a therapeutic agent for various skin dam such as an incised wound, a decubitus, an operational wound, a burn or traumatic dermal deficiency by a parenteral method such as local injection, an ointment or surface application. CONSTITUTION:The objective wound treating agent is obtained by transplanting a human fibroblast IMR-90 (ATCC CCL 186) in a culture medium containing 5% calf serum in a container, culturing the fibroblast while rotating the container for 7 days, then peeling a cell with trypsin, collecting the cell in the bottom of the container, charging a ceramic having 5-9 mesh particle size thereinto, continuing the culturing, recovering the total amount of the culture medium at an interval of 7-10 days, supplying a fresh culture medium thereto, continuing the production for 2 months, concentrating the recovered culture solution through a membrane filter, carrying out the chromatographic purification with Concanavalin A (Con A)-Sepharose(R) and heparin-Sepharose(R), etc., providing the TCF-II, using the resultant TCF-II as an active ingredient and formulating the active ingredient into a pharmaceutical preparation such as a parenteral injection or an ointment. Thereby, this wound therapeutic agent usable for an incised wound, a decubitus, an operational wound, a burn, traumatic dermal deficiency, etc., by a parenteral method such as local injection or an ointment is obtained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a therapeutic agent for wounds containing the protein TCF-II produced by human cells as an active ingredient.

[0002]

BACKGROUND ART β-interferon is widely known as a tumor cytotoxic factor produced by human-derived fibroblasts. The substances produced by fibroblasts are disclosed in JP-A-58-146293 and JP-A-61-33120.
No. 6/1872, 62-103021
And Japanese Patent Laid-Open No. 64-10998. In the process of studying antitumor proteins derived from human fibroblasts, the present inventors discovered a novel antitumor substance that is completely different from these proteins reported so far, and further investigated the cDNA encoding this protein. The cloning was successful, the entire amino acid sequence was confirmed, and the usefulness was confirmed. This novel antitumor protein and its gene are disclosed as WO90 / 10651. This novel antitumor protein is named TCF-II. It was confirmed that this TCF-II has both strong antitumor activity and normal cell growth activity, and is a member of a diverse family of HGF which is a growth factor for liver parenchymal cells. TCF-II is 78000 ± 2000 or 74000 ± 2000 in molecular weight measurement by SDS electrophoresis.
And shows a common band of 52000 ± 2000 when reduced, and two bands (B chain and C chain) of A chain and 30,000 ± 2000 or 26000 ± 2000. Since TCF-II is a growth factor for liver parenchymal cells, it can be used for the purpose of liver regeneration after hepatectomy, but the effect on the wound treatment disclosed in the present invention has not been known so far. For skin wounds such as wounds and burns, depending on the symptoms, other than surgical treatment such as suturing and skin grafting, application of antibacterial agents to prevent inflammation due to bacterial infection, oral administration, or wounds. Only passive treatment methods such as application of acrinol tincture oil and coating with chitin fiber or freeze-dried pig dermis to protect the surface have been performed. In addition, treatment by skin regeneration by proliferation of epithelial cells by administration of epidermal growth factor (EGF) has been attempted, but it has not been established as a treatment method.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have TCF-
Focusing on the usefulness of II, we have examined its use as an antitumor agent and as a marker for diagnosis of diseases. But T
It has not been confirmed that CF-II has a wound healing effect. From the process of studying the action of TCF-II on the liver, the present inventors found that not only TCF-II caused proliferation of hepatic parenchymal cells but also proliferation of epithelial cells and fibroblasts, resulting in wounds and burns. Found to promote the recovery of.
An object of the present invention is to provide a wound healing agent containing TCF-II as an active ingredient.

[0004]

The method for obtaining TCF-II as the active ingredient according to the present invention is as follows. TCF
-II can be derived from cells obtained by the method disclosed in WO90 / 10651 described above. Further, it may be produced by a gene recombination operation by a microorganism or another cell based on the gene sequence described in the publication. As a method for producing TCF-II by genetic engineering, those produced by the method filed by the present inventors and published as WO 92/01053 can be used.
Further, even those having different sugar chains depending on the host cell and the microorganism, or those having no sugar chain attached can be used. However, since sugar chains are related to the rate of metabolism in the living body, those to which sugar chains are bound are desirable.

TCF-II can be further concentrated and purified by a conventional isolation and purification method. For example, a precipitation method using an organic solvent, salting out, gel filtration chromatography, affinity chromatography using a monoclonal antibody, an electrophoresis method and the like can be mentioned. Among these purification methods, affinity chromatography using a monoclonal antibody can be performed using the monoclonal antibody filed by the present inventor as Japanese Patent Application No. 3-177236.

The purified TCF-II thus obtained can be freeze-dried or frozen. The wound treatment agent of the present invention can be administered as an injection near the wound surface. In addition, it can be applied directly to the wound area, or can be applied to fats, fatty oils, lanolin, paraffin, waxes, resins, glycols, higher alcohols, glycerin, water, emulsifiers, suspending agents, etc.
It is also possible to add -II and mix them to make the whole even and apply as an ointment or the like. In addition, plasters, aerosols,
It can be used as a liniment agent or the like, or can be administered by immersing it in sterilized gauze, pig freeze-dried dermis used for wound surface protection, or chitin fiber. Also, if necessary,
It can be used in combination with antibiotics, antibacterial agents, and bactericides. Furthermore, it can be administered to the site to be treated during a surgical operation such as suturing or skin grafting. The wound healing agent of the present invention can be used for the treatment of various cutaneous injuries such as pressure ulcers, surgical wounds, burns, and traumatic skin defects in addition to ordinary cuts.

TCF-, which is contained in the wound healing agent of the present invention.
The dose of II is determined according to the wound condition of the patient to be administered, but 100 to 30000 μg as purified TCF-II per adult on the wound surface, preferably 500 to 300
A formulation containing 0 μg can be administered to the wound surface 1 to 7 times a week. In addition, long-term administration is possible depending on the patient's symptoms.

The present invention will be described in more detail below with reference to examples.

Example 1 Purification of TCF-II The method disclosed in WO90 / 10651 and the method of Higashio et al. (Higasio, K.et.al. BBRC, vol.170,397-404,1)
990) and cultured the cells to obtain purified TCF-II. Human fibroblast IMR-90 (ATCC CCL 186)
3 × 10 ⁶ cells were transferred to a roller bottle containing 100 ml of DMEM containing 5% calf serum, and 0.5 to 2 revolutions /
The culture was continued for 7 days while rotating at a rotation speed of min. When the total number of cells reached 1 × 10 ⁷ , the cells were detached with trypsin to collect the cells on the bottom surface of the bottle, and 100 g of 5-9 mesh ceramic (manufactured by Toshiba Ceramics Co., Ltd.) was sterilized and charged, and left standing for 24 hours. And cultured. After that, 500 ml of the above culture solution was added and the culture was continued. The entire amount of the medium was collected every 7 to 10 days, and fresh medium was supplemented. In this way, the production was continued for 2 months, and 4 l of the culture solution was collected per roller bottle. The specific activity per culture medium thus obtained was 32 u / ml. Culture solution 75
0l is a membrane filter manufactured by Amicon (MW 60
00 cut) processing to concentrate UF and CM Sephadex C
-50 (Pharmacia), ConA Sepharose (Pharmacia), MonoS column (Pharmacia), Heparin Sepharose (Pharmacia)
4 steps of chromatographic purification with specific activity 52480
00u / mg of purified TCF-II was obtained.

[0009]

Example 2 Production of Recombinant TCF-II According to the method disclosed in WO92 / 01053, T
The cells in which the CF-II gene has been incorporated are cultured and purified TCF
-II was obtained. The transformed Namalwa cells were cultured to obtain 20 liters of the culture solution. This culture solution was used for CM-Sephadex C-50 chromatography, Con-A Sepharose CL-6B chromatography,
Approximately 11 mg after processing in order of HPLC equipped with MonoS column.
Active TCF-II was obtained.

[0010]

Example 3 Production Example of TCF-II Formulation In this example, a production example of a recombinant TCF-II injection formulation obtained by the method of Example 2 was shown. This injection preparation can be used for injection near the affected area, intravenous, subcutaneous, or intramuscular injection, and it can be directly immersed by sterilizing gauze used for the purpose of protecting the wound surface, porcine freeze-dried dermis, or chitin fiber. It can also be used to administer to the affected area. (1) TCF-II 20 μg Human serum albumin 100 mg The above composition was dissolved in 0.01 M PBS at pH 7.0 to make a total volume of 20 mg.
After sterilization, the solution was dispensed in 2 ml aliquots, lyophilized and sealed.

(2) TCF-II 40 μg Tween 80 1 mg Human serum albumin 100 mg The above composition was dissolved in physiological saline for injection to prepare a total volume of 20 ml, and after sterilization, dispensed in 2 ml aliquots and freeze-dried. Sealed. (3) TCF-II 20 μg Tween 80 2 mg sorbitol 4 g Dissolve the above composition in 0.01 M PBS at pH 7.0 to make a total volume of 20
After sterilization, the solution was dispensed in 2 ml aliquots, lyophilized and sealed.

(4) TCF-II 40 μg Tween 80 2 mg Glycine 2 g The above composition was dissolved in physiological saline for injection to prepare a total volume of 20 ml, and after sterilization, 2 ml each was dispensed into vials, and freeze-dried and sealed. . (5) TCF-II 40 μg Tween 80 1 mg sorbitol 2 g glycine 1 g The above composition was dissolved in physiological saline for injection to prepare a total amount of 20 ml, and after sterilization, 2 ml each was dispensed into a vial and freeze-dried and sealed.

(6) TCF-II 20 μg sorbitol 4 g human serum albumin 50 mg The above composition was dissolved in 0.01 M PBS at pH 7.0 and the total amount was 20
After sterilization, the solution was dispensed in 2 ml aliquots, lyophilized and sealed. (7) TCF-II 40 μg Glycine 2 g Human serum albumin 50 mg The above composition was dissolved in physiological saline for injection to prepare a total amount of 20 ml, and after sterilization, 2 ml each was dispensed into a vial and freeze-dried and sealed. (8) TCF-II 10mg Human serum albumin 100mg The above composition was dissolved in 0.01M PBS at pH 7.0 and the total amount was adjusted to 20mg.
After sterilization, the solution was dispensed in 2 ml aliquots, lyophilized and sealed.

[0014]

Example 4 Formulation Example of TCF-II Ointment (1) TCF-II 1000 mg Purified lanolin 20 g White petrolatum 80 g TCF-II was mixed with a small amount of purified water, and this was added little by little to lanolin and mixed. Then, white petrolatum was added to this mixture little by little and kneaded to obtain TCF-II.
An ointment was produced. (2) TCF-II 1000 mg Macrogol 400 5 ml Macrogol ointment 100 g TCF-II was mixed with a small amount of purified water, and this was added little by little to Macrogol 400 and mixed. Then, the mixture was kneaded while adding Macrogol ointment little by little to produce TCF-II ointment.

[0015]

INDUSTRIAL APPLICABILITY The present invention provides a wound healing agent containing TCF-II as an active ingredient. Hereinafter, the effects of the present invention will be described by showing experimental examples confirming the therapeutic effect of TCF-II.

[0016]

[Experimental example] Test method Eight-week-old male Wistar rats (Charles River Japan) 1 group of 8-9 backs were shaved, and the skin of about 1.5 cm left and right was symmetrical so as to be symmetrical with respect to the midline. Made an incision. In order to prevent suppuration, penicillin was applied to the incision and one suture was made at the center of the incision. 0.1 to give a concentration of 1 mg / ml
% TCF-II dissolved in pyrogen-free PBS containing human serum albumin was applied directly to the incision twice a day immediately after the skin incision, the thread was removed on the 4th day, and the skin from which the rat had been killed was exfoliated on the 6th day. A sample in which the wound part was cut into strips was prepared. Both ends of this sample were pulled to measure the tension until the wound was opened. Measured values are FD pickup (TB-611T made by Nihon Kohden) and amplifier for strain pressure (AP-made by Nihon Kohden)
It was recorded on a polygraph (RM-6200 manufactured by Nihon Kohden Co., Ltd.) via the 601G). In the control group, only the solvent (PBS containing 0.1% HSA) was applied in the same manner, and a sample was similarly prepared and measured. Test results Table 1 below shows the measured values of maximum tension when the wound area opens.

[0017]

[Table 1] ^** P <0.01 (Wilcoxon test) Numerical values ± standard error value As shown in Table 1, the adhesive force on the wound surface of the TCF-II-administered group was about 1.65 times that of the control. The wound healing effect of TCF-II was confirmed.

Claims (1)

[Claims] 1. A therapeutic agent for wound containing TCF-II as an active ingredient.